EB病毒变异型EBER2对鼻咽癌细胞增殖和凋亡的影响

EB病毒(EBV)编码小RNA(EBERs,包括EBER1和EBER2)的致癌作用已在多种细胞系中得到证实,我们前期研究发现在EBER2基因发生6处突变的EB-8m变异型可能与鼻咽癌(NPC)的发生相关。本研究探讨变异型EBER2对NPC细胞增殖和凋亡的影响,以进一步明确EBER2基因变异在NPC发生中的作用。分别以B95-8原型和EB-8m变异型EBER2稳定转染EBV阴性NPC细胞系,MTT法和平板克隆检测细胞增殖,流式细胞仪检测细胞凋亡。与转染原型EBER2细胞及转染载体的对照细胞比较,转染变异型EBER2细胞的MTT吸光度值和平板克隆形成率增高,细胞凋亡率降低(P均小于0.05),原型和对照之间细胞增殖无差异,但原型的细胞凋亡率亦低于对照(P0.05)。上述结果表明,EB-8m变异型EBER2可通过提高NPC细胞增殖及抗凋亡能力而增强其致癌作用。     

EB病毒即刻早期BRLF1基因在淋巴瘤组织中多态性研究

目的：EB病毒（Epstein．Barrvirus,EBV）感染宿主细胞包括潜伏期和裂解期,它的即刻早期基因BRLFl编码产物Rta蛋白是EBV从潜伏期到裂解期的关键性调节因子。目前,Rta蛋白已成为治疗EBV相关肿瘤的靶蛋白,但有关BRLFl基因在EBV阳性淋巴瘤中的研究甚少,本研究旨在明确EB病毒即刻早期基因BRLFl在EB病毒阳性淋巴瘤组织中的变异规律,探讨其变异意义。方法：原位杂交筛选EB病毒阳性淋巴瘤（BL）,提取EB病毒DNA。PCR结合DNA测序检测EBV阳性淋巴瘤中BRLFl基因多态性,应用DNAStar软件对序列进行对比分析。结果：共筛选出27例EBV阳性淋巴瘤标本,在27例BL阳性标本中成功扩增BRLFl基因,与B95—8标准序列比对分析,共发现20处无义突变和19处有义突变。8例属于BR1-A亚型,18例属于BRl—C亚型,1例属于BRl-E亚型,其中以BRl-A亚型和BRl-C亚型最为常见,分别为29．6％（8／27）、66．7％（18／27）。与EBVaGC、NPC和1W相比,4种人群BRLFl亚型的分布不同,其中BRl-A亚型更多出现在健康人群中,BRl-C亚型更多出现在NPC和BL中。在Rta蛋白功能区中,二聚体区域呈高度保守,在DNA结合区域和反式激活区域均检测出序列突变,16种CTL抗原表位中,只有NAA、QKE和ERP表位发生改变,且NAA和QKE在健康人群中突变率较高。结论：山东地区EB病毒阳性淋巴瘤BRLFl基因变异类型主要为BRl-A亚型和BRl-C亚型。BRl-C亚型可能与BL相关,发生在Rta蛋白DNA结合区域和反式激活区域的突变可能对其功能产生影响,多数CTL表位序列保守提示BRLFl可以用作EBV相关淋巴瘤免疫治疗的靶基因。     

我国北方地区鼻咽癌患者EB病毒LMP1基因缺失分析

为研究我国北方地区鼻咽癌和非鼻咽癌患者Epstein-Barr病毒(EBV)潜伏膜蛋白1(LMP1)基因C端区缺失状况,探讨其在鼻咽癌发生中所起的作用.收集我国东北地区鼻咽癌石蜡组织22例,非鼻咽癌患者外周血26例,提取DNA后,采用聚合酶链反应技术(PCR)扩增LMP1基因的C末端,对其中有缺失的4例鼻咽癌进行了克隆和序列分析.结果显示:22例鼻咽癌组织标本中,有19例扩增出特异性条带,阳性率为86%.其中8例存在缺失,缺失率42%.取4例缺失样品进行序列分析,并与B95-8原型LMP1做比较,结果显示:4例鼻咽癌样品均存在30个碱基的缺失和某些位点的单点突变,并由此引起所编码的氨基酸改变.26例非鼻咽癌患者LMP1阳性扩增率为92.31%,无一例缺失.此结果表明:与广东、广西鼻咽癌高发区相比,我国北方地区鼻咽癌与非鼻咽癌患者中EB病毒LMP1基因缺失型和原型的分布有明显的地区差异.     

EBV阳性与阴性NPC细胞中AKR1B10的差异表达及生物学意义

研究EB病毒（Epstein-Barr virus,EBV）阳性与阴性鼻咽癌（Nasopharyngeal carcinoma,NPC）细胞中醛酮还原酶家族1成员B10(AKR1B10)的差异表达及生物学意义。收集NPC组织并检测EBV感染情况及AKR1B10表达水平，比较EBV阳性与阴性NPC组织中AKR1B10表达水平的差异；培养EBV阳性的NPC细胞株HK-1和C666-1、EBV阴性的NPC细胞株CNE-1及CNE-2，检测AKR1B10的表达水平。HK-1和C666-1进行分组，给予10μmol/L、20μmol/L AKR1B10抑制剂处理，20μmol/L AKR1B10抑制剂联合对照溶剂或20μmol/L β-catenin激动剂处理，检测细胞增殖水平、侵袭数目及c-myc、MMP2、MMP9的mRNA表达水平，AKR1B10、总β-连环蛋白（β-catenin）、核β-catenin的蛋白表达水平。结果显示HK-1和C666-1细胞中AKR1B10的蛋白表达水平高于CNE-1及CNE-2;10μmol/L、20μmol/L AKR1B10抑制剂组HK-1和C666-1...     

鼻咽癌病人和正常人群中EB病毒特异性T细胞对靶抗原的识别和应答

为探讨痘苗病毒表达的Eoskein-Barr病毒（EBV）核抗原1、4（EBNA1、4）和潜伏膜蛋白1、2（LMP1、2）,在不同人群的特异性T细胞杀伤9CTL）中的作用,采集EBV阴性正常人、未经治疗的鼻咽癌（NPC）病人的EBV－IgA/VCA阳性者各10人的周围血淋巴单核细胞（PBMC）,用EBV转化B淋巴细胞,建立类淋巴母细胞（LCL）,用LCL刺激自休的T淋巴细胞作为效应细胞,以LCL感染重组痘苗病毒表达的EBNA1、4和LMP1、2为靶细胞,以^51Cr释放法检测EBV特异性CTL所识别的靶抗原。结果表明,EBV－LMP1、2可能既是EBV特异性T细胞的刺激抗原,又是其识别的靶抗原。将采集的30例试验者的各5 单克隆T细胞株分别检测HLA-Ⅰ型（A、B、C）,按照不同型别寻找相对应的EBNA1、4和LMP1、2的不同合成肽,应用酶免疫吸附斑点法（Elispot）检测EBV特异性CD8^ 的CTL应答。结果显示：10例正常人中9人有特异的LMP2应答,4人有特异的EBNA4应答;10例未治疗的NPC病人中3人有特异的LMP2,2人有特异的EBNA1,3个有特异的EBNA4应答;10例未治疗的NPC病人中3人有特异的EBNA1,3人有特异的EBNA4应答,在10例EBV－IgA/VCAbj ntg k ,6人有特异的LMP2,5人有特异的EBNA4应答。所有的试验者均未发现LMP1的特异性应答。     

一氧化氮诱导食管癌细胞线粒体DNA编码基因过表达

以人食管癌细胞系EC109作为驱赶方（driver）,以被一氧化氮（nitric oxid,NO）诱导的EC109作为实验方（tester）,应用抑制消减杂交(suppression subtractive hybridization, SSH)、反向mRNA斑点印迹和RNA印迹等技术手段研究了NO诱导的食管癌细胞中基因的过表达情况.然后对过表达基因的表达序列标签（expressed sequence tag,EST）实施序列测定,并与GenBank进行BLAST同源性比较和序列突变分析.结果先后两次从69个SSH阳性克隆中共鉴定出6个线粒体DNA（mitochondrial DNA, mtDNA）编码的基因,即ND-4L、ND-4、COX-2、Lys-tRNA、ATP-8和ATP-6.表明NO可以诱导食管癌细胞mtDNA编码的基因过表达.另外,在ND-4L/ND-4基因的片段（10 736～11 449）上发现了三处同型单核苷酸置换（10 872 T→C, 11 001 A→G, 11 346 A→G）,在COX-2/Lys-tRNA/ATP-8/ATP-6基因片段（8 011～8 589）上发现了一处单核苷酸缺失（8 380 A）.氨基酸序列分析表明,在NO诱导的EC109中可能存在着一种结构异常的ATP-8肽链（一条在N端被截短的只有11个氨基酸残基的肽链,而正常的ATP-8肽链为68个氨基酸残基）.这些研究结果为深入揭示NO对肿瘤细胞的作用机制提供了新的重要线索.     

用PCR技术检测活检组织和鼻咽上皮细胞内EB病毒基因

选用Epstin-Barr病毒(EBV)基因组内部重复序列1(IR1)片断作为多聚酶链反应(Polymerase Chain Reaction,PCR)扩增引物,用于检测了31例不同病例活检组织和4例新鲜鼻咽组织经体外培养6周以上的新生上皮细胞内EBV基因,其中检出EBVDNA:高分化鼻咽癌5/5,低分化鼻咽癌4/4,何杰金氏病5/5,非何杰金氏病0/2,头颈其他肿瘤1/6,鼻咽慢性炎症0/5,正常鼻咽组织0/4;新生上皮细胞DNA抽提物;低分化鼻咽癌2/2,炎症0/1,正常人胚鼻咽上皮0/1;携带EBV基因组细胞系(Raji,B_(95-8)各1)2/2,致淋巴细胞转化之B_(95-8)病毒为10~(-4),PCR检测10~(-4)～10~(-6)均阳性,10~(-7)未检出。结果表明EBV与鼻咽癌与何杰金氏病有关,常规石蜡包埋切片仅8μm×0.1mm~2,贮存时间至三年仍可用于PCR检测EBV DNA,证实PCR是一种快速、灵敏和特异测捡EBV基因组的方法,可作为肿瘤和疚病病毒病因回顾性调直研究的有力手段。     

实时定量PCR方法检测鼻咽癌病人全血EB病毒拷贝数的实验研究

EBV在多种肿瘤中存在,并在相关肿瘤的发病中起一定的作用。本文应用实时定量PCR方法检测鼻咽癌与健康对照全血EBV拷贝数。结果表明在73例鼻咽癌病人与83例正常对照中,NPC组中EBV阳性率为46.6%,对照组阳性率为13.3%。对照组平均EB病毒拷贝数为3.9×10~4拷贝/μgDNA,高于鼻咽癌组(1.7×10~5拷贝/μgDNA)。全血EBV的感染与鼻咽癌的发生相关,而裂解状态EBV在细胞转化过程中的作用可能更大。应用实时定量PCR进行全血EBV的检测可用于鼻咽癌的分子诊断。     

EB病毒潜伏膜蛋白1多态性与鼻咽癌的关系

为了分析广东地区鼻咽癌病人和非鼻咽癌病人携带的Epstein-Barr病毒（EBV）潜伏膜蛋白1（LMP1）基因多态性,探讨LMP1基因羧基端缺失30个碱基的EBV是否与华南地区鼻咽癌高发有关。为此收集了初始的107例鼻咽癌病人和106例非鼻咽癌肿病人的漱口液,用巢式PCR扩增LMP1羧基端,观察缺失型LMP1的构成比。同时,测序分析缺失型LMP1编码区序列,了解缺失型LMP1多态性与鼻咽癌的关联度。结果：在50％的样品中可检出LMP1基因,缺失型LMP1在鼻咽癌病人和非鼻咽病人的构成比相似,约占70？％,原型和缺失型LMP1混合感染少见（0－6％）。另外,广州地区的缺失型LMP序列与上海、台湾、香港地区的缺失型LMP1基因高度同源,鼻咽癌病人是和非鼻咽癌病人携带的缺失型LMP1基因序列基本相同。此结果表明,广州地区流行的LMP1羧基端缺失30个碱基的EBV株,漱口液中检测出的缺失型与原型LMP1构成比约为7：3,在鼻咽癌病人和非鼻咽癌病人此分布情况相似。     

EB病毒BNLF—1基因研究的新进展

EB病毒广泛存在于人群中,它的潜伏感染与鼻咽癌的发生密切相关。EBV在潜伏感染的过程中表达一种潜伏膜蛋白1（LMP1）,具有致瘤性,因此编码LMP1的基因BNLF－1被认为是EBV的瘤基因。BNLF－1基因具有广泛的生物学功能,在鼻咽癌的发生发展过程中起重要作用。近年的研究表明,鼻咽癌来源的EBV与标准株EBV比较存在相当的变异,而鼻咽癌来源的LMP1在致瘤性上也明显强于标准株LMP1。本文将综述BNLF－1基因生物学功能研究方面的新进展,并简要介绍变异型LMP1研究的新成果。     

对鼻咽癌恶性转化基因Tx表达的初步研究

运用杂交组化及Ｎｏｒｔｈｅｒｎ法,对中国人鼻咽癌细胞株恶性转化基因Ｔｘ的表达进行了初步研究。结果表明该基因在鼻咽癌细胞株中表达１．３ｋｂｍＲＮＡ,但表达强度较低,在ＨＰＶ阳性或阴性人宫颈癌细胞中均无表达,ＥＢＶ阴性的Ｂ淋巴细胞系及ＨＴＬＶ－１阳性的Ｔ细胞系中为阴性,而在ＥＢＶ阳性的Ｂ９５－８及Ｒａｊｉ细胞中Ｔｘ基因表达强烈,从而提示ＨＰＶ及ＨＴＬＶ－１不增强Ｔｘ的表达水平,而ＥＢＶ可能使Ｔｘ基因活化．这为进一步研究ＥＢＶ与Ｔｘ等瘤基因协同作用提供了新的依据。     

Epstein-Barr virus types 1 and 2 differ in their EBNA-3A, EBNA-3B, and EBNA-3C genes.

The two Epstein-Barr virus (EBV) types, EBV-1 and EBV-2, are known to differ in their EBNA-2 genes, which are 64 and 53% identical in their nucleotide and predicted amino acid sequences, respectively. Restriction endonuclease maps and serologic analyses detect few other differences between EBV-1 and EBV-2 except in the EBNA-3 gene family. We determined the DNA sequence of the AG876 EBV-2 EBNA-3 coding region and have compared it with known B95-8 EBV-1 EBNA-3 sequences to delineate the extent of divergence between EBV-1 and EBV-2 isolates in their EBNA-3 genes. The B95-8 and AG876 EBV isolates had nucleotide and amino acid identity levels of 90 and 84%, 88 and 80%, and 81 and 72% for the EBNA-3A, -3B, and -3C genes, respectively. In contrast, nucleotide sequence identity in the noncoding DNA adjacent to the B95-8 and AG876 EBNA-3 open reading frames was 96%. We used the polymerase chain reaction to demonstrate that five additional EBV-1 isolates and six additional EBV-2 isolates have the type-specific differences in their EBNA-3 genes predicted from the B95-8 or AG876 sequences. Thus, EBV-1 and EBV-2 are two distinct wild-type EBV strains that have significantly diverged at four genetic loci and have maintained type-characteristic differences at each locus. The delineation of these sequence differences between EBV-1 and EBV-2 is essential to ongoing molecular dissection of the biologic properties of EBV and of the human immune response to EBV infection. The application of these data to the delineation of epitopes recognized in the EBV-immune T-cell response is also discussed.     

Genomic sequencing and comparative analysis of Epstein-Barr virus genome isolated from primary nasopharyngeal carcinoma biopsy

Whether certain Epstein-Barr virus (EBV) strains are associated with pathogenesis of nasopharyngeal carcinoma (NPC) is still an unresolved question. In the present study, EBV genome contained in a primary NPC tumor biopsy was amplified by Polymerase Chain Reaction (PCR), and sequenced using next-generation (Illumina) and conventional dideoxy-DNA sequencing. The EBV genome, designated HKNPC1 (Genbank accession number JQ009376) is a type 1 EBV of approximately 171.5 kb. The virus appears to be a uniform strain in line with accepted monoclonal nature of EBV in NPC but is heterogeneous at 172 nucleotide positions. Phylogenetic analysis with the four published EBV strains, B95-8, AG876, GD1, and GD2, indicated HKNPC1 was more closely related to the Chinese NPC patient-derived strains, GD1 and GD2. HKNPC1 contains 1,589 single nucleotide variations (SNVs) and 132 insertions or deletions (indels) in comparison to the reference EBV sequence (accession number NC007605). When compared to AG876, a strain derived from Ghanaian Burkitt's lymphoma, we found 322 SNVs, of which 76 were non-synonymous SNVs and were shared amongst the Chinese GD1, GD2 and HKNPC1 isolates. We observed 88 non-synonymous SNVs shared only by HKNPC1 and GD2, the only other NPC tumor-derived strain reported thus far. Non-synonymous SNVs were mainly found in the latent, tegument and glycoprotein genes. The same point mutations were found in glycoprotein (BLLF1 and BALF4) genes of GD1, GD2 and HKNPC1 strains and might affect cell type specific binding. Variations in LMP1 and EBNA3B epitopes and mutations in Cp (11404 C>T) and Qp (50134 G>C) found in GD1, GD2 and HKNPC1 could potentially affect CD8(+) T cell recognition and latent gene expression pattern in NPC, respectively. In conclusion, we showed that whole genome sequencing of EBV in NPC may facilitate discovery of previously unknown variations of pathogenic significance.     

Interleukin-10 and interleukin-18 promoter polymorphisms in an Italian cohort of patients with undifferentiated carcinoma of nasopharyngeal type

Purpose: Cytokines such as IL-10 and IL-18 seem to be involved in the inflammatory response of undifferentiated carcinoma of nasopharyngeal type (UCNT). The aim of this study was to evaluate the correlation between functional single nucleotide polymorphisms (SNPs) in the promoter region of IL-10 and IL-18 genes and the virological and clinical characteristics in a large case series of Caucasian patients suffering from UCNT, a tumor regularly associated with the Epstein Barr Virus (EBV). Methods: Eighty-nine patients with histologically confirmed UCNT and 130 healthy donors were included in our study. DNA was examined for the polymorphisms of IL-10 gene at positions –1082, −819, −592 by direct sequencing and IL-18 gene at position −607 and −137 by allele –specific PCR. EBV DNA serum viremia was evaluated by QC-PCR. Results: The distributions of the IL-10 and IL-18 genetic variants were not different between UCNT patients and healthy controls. The frequency of IL-10 –1082G allele, which is associated with high IL-10 expression, showed a nearly statistically significant increase in UCNT patients EBV DNA-negative as compared to healthy controls (OR=3.3 95% CI: 1.2–9.8). Subjects with C/C or C/G combined IL-18 genotypes showed an increased risk of being with Stages III-IV (OR=2.1 95% CI: 1.2–6.6). Conclusion: This study was performed to improve the definition of the pathogenetic factors implicated in UCNT by addressing the correlation between cytokine polymorphisms and clinical parameters. This is the first study investigating the possible role of the IL-18 and IL-10 polymorphisms in the development and outcome of UCNT. In our genetic analysis there is no evidence for involvement of IL-10 promoter polymorphisms alone in the genetic predisposition to this tumor. On the other hand, IL18 genetic variants may represent a genetic risk factor for tumor aggressiveness.     

DNAJC10基因在鼻咽癌中的表达及表达下调机制的研究

运用RT-PCR检测候选抑瘤基因DNAJC10在鼻咽癌组织中的表达,运用甲基化特异性PCR(MSPCR)技术、LOH和测序等技术分别检测DNAJC10基因在鼻咽癌组织中的甲基化状况、LOH和启动子突变情况.结果表明,DNAJC10基因在肿瘤组织中较对照慢性鼻咽炎组织表达明显下调(P<0.05).DNAJC10在鼻咽癌中不表现高甲基化,其LOH的缺失率为6.25%,突变率为66.7%.因此,DNAJC10基因的表达下调主要是其遗传改变(LOH和突变)所致.     

DNA of Epstein-Barr virus. IV. Linkage map of restriction enzyme fragments of the B95-8 and W91 strains of Epstein-Barr Virus.

The arrangement of EcoRI, Hsu I, and Sal I restriction enzyme sites in the DNA of the B95-8 and W91 isolates of Epstein-Barr virus (EBV) has been determined from the size of the single-enzyme-cleaved fragments and from blot hybridizations that identify which fragments cut from the DNA with one enzyme contain nucleotide sequences in common with fragments cut from the DNA with a second enzyme. The DNA of the B95-8 isolate was the prototype for this study. The data indicate that (i) approximately 95 X 10(6) to 100 X 10(6) daltons of EBV (B95-8) DNA is in a consistent and unique sequence arrangement. (ii) Both termini are variable in length. One end of the molecule after Hsu I endonuclease cleavage consists of approximately 3,000 base pairs, with as many as 10 additional 500-base pair segments. The opposite end of the molecule after Sal I endonuclease cleavage consists of approximately 1,500 base pairs, with as many as 10 additional 500-base pair segments. (iii) The opposite ends of the molecule contain homologous sequences. The high degree of homology between the opposite ends of the molecule and the similarity in size of the "additional" 500-base pair segments suggests that there are identical repeating units at both ends of the DNA. The arrangement of restriction endonuclease fragments of the DNA of the W91 isolate of EBV is similar to that of the B95-8 isolate and differs from the latter in the presence of approximately 7 X 10(6) daltons of "extra" DNA at a single site. Thus, the size of almost all EcoRI, Hsu I, and Sal I fragments of EBV (W91) DNA is identical to that of fragments of EBV (B95-8) DNA. A single EcoRI fragment, C, of EBV (W91) DNA is approximately 7 X 10(6) daltons larger than the corresponding EcoRI fragment of EBV (B95-8) DNA. Digestion of EBV (W91) DNA with Hsu I or Sal I restriction endonucleases produces two fragments (Hsu I D1 and D2 or Sal I G2 and G3) which differ in total size by approximately 7 X 10(6) daltons from the fragments of EBV (B95-8) DNA. Furthermore, the EcoRI, Hsu I, and Sal I fragments of EBV (W91) and (B95-8) DNAs, which are of similar molecular weight, have homologous nucleotide sequences. Moreover, the W91 fragments contain only sequences from a single region of the B95-8 genome. Two lines of evidence indicate that the "extra" sequences present in W91 EcoRI fragment C are viral DNA and not cellular. (i) The molecular weight of the "enlarged" EcoRI C fragment of EBV (W91) DNA is identical to that of the EcoRI C fragment of another isolate of EBV (Jijoye), (ii) The HR-1 clone of Jijoye has previously been shown to contain DNA which is not present in the B95-8 strain but is present in the EcoRI C and Hsu I D2 and D1 fragments of EBV (W91) DNA (N. Raab-Traub, R. Pritchett, and E. Kieff, J. Virol. 27:388-398, 1978).     

高分化及低分化鼻咽癌细胞株中Epstein－Barr病毒潜伏感染膜蛋白（LMP1）基因的原位杂交与克隆及序列分析

应用染色体原位杂交、ＰＣＲ扩增、克隆及核苷酸序列分析等方法,分析了ＣＮＥ１和ＣＮＥ３细胞株中的潜伏感染膜蛋白（ＬＭＰ１）基因。ＣＮＥ１是来自我国东北的高分化鼻咽癌细胞株,ＣＮＥ３是来自广西的低分化鼻咽癌细胞株。染色体原位杂交结果表明,ＣＮＥ１细胞中ＬＭＰ１基因存在于细胞核内,整合在第一号染色体上,ＣＮＥ３中ＬＭＰ１基因则随机存在于细胞核内及多条染色体上。用ＰＣＲ方法分别从ＣＮＥ１及ＣＮＥ３中扩增得到了ＬＭＰ１基因片段（外显子３）,核苷酸序列分析证明,来自ＣＮＥ１的ＬＭＰ１与来自Ｂ９５－８细胞的ＬＭＰ１核苷酸序列同源性极高,达９９．５％,而ＣＮＥ３的ＬＭＰ１基因与Ｂ９５－８的ＬＭＰ１基因同源性为９３％。     

CD4 and CD8 T cell responses to tumour-associated Epstein–Barr virus antigens in nasopharyngeal carcinoma patients

Nasopharyngeal carcinoma (NPC), an Epstein–Barr virus (EBV)-associated tumour common in Southern Chinese populations, is a potentially important target for T cell-based immunotherapy. The tumour cells are HLA class I- and II-positive and express a limited subset of EBV latent proteins, namely the nuclear antigen EBNA1 and the latent membrane proteins LMP2 and (in some cases) LMP1. To ask whether the tumour develops in the presence of a potentially protective host response or in its absence, we set out to determine the prevailing levels of CD4+ and CD8+ T cell memory to these proteins in NPC patients at tumour diagnosis. We first screened healthy Chinese donors against Chinese strain EBNA1, LMP1 and LMP2 sequences in Elispot assays of interferon-γ release and identified the immunodominant CD4+ and CD8+ epitope peptides presented by common Chinese HLA alleles. Then, comparing 60 patients with >70 healthy controls on peptide epitope mini-panels, we found that T cell memory to CD4 epitopes in all three proteins was unimpaired in the blood of patients at diagnosis. In most cases NPC patients also showed detectable responses to CD8 epitopes relevant to their HLA type, the one consistent exception being the absence in patients of a B*4001-restricted response to LMP2. We infer that NPC arises in patients whose prevailing levels of T cell memory to tumour-associated EBV proteins is largely intact; the therapeutic goal must therefore be to re-direct the existing memory repertoire more effectively against antigen-expressing tumour cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA of Epstein-Barr virus. I. Comparative studies of the DNA of Epstein-Barr virus from HR-1 and B95-8 cells: size, structure, and relatedness.

We have compared the properties of the DNA of Epstein-Barr virus (EBV) purified from HR-1 (EBV HR-1 DNA) and B95-8 (EBV B95-8 DNA) continuous lymphoblast cultures. Our data indicate that (i) the S suc of native EBV DNA relative to T4D DNA is 55S. Using the modified Burgi-Hershey relationship (5), we estimate the molecular weight of native EBV DNA is 101 (plus or minus the molecular weight of native FBV DNA by measurement of the length of 3) times 106. Estimation of the molecule relative to form II PM2 DNA yields a value of 105 (plus or minus 3) times 106. (ii) After alkali denaturation, less than 50% of EBV DNA sediments as a single band in alkaline sucrose gradients in the region expected for DNA of 50 times 406 daltons. (iii) Intact EBV HR-1 and EBV B 95-8 DNAs band at 1.718 g/cm3 and a smaller band (approximately 25% of the DNA) AT 1.720 G/CM3. (IV) EBV HR-1 DNA possesses greater than 97% of the sequences of EBV B95-8 DNA. Hybrid DNA molecules formed between (3H)EBV HR-1 DNA and EBV HR-1 DNA or EBV B95-8 DNA had identical thermal stability. EBV B95-8 DNA lacks approximately 15% of the DNA sequences of EBV HR-1 DNA. We interpret these data to mean that EBV B95-8 is derived from a parental EBV through loss of genetic complexity. This defect may be linked to the ability of EBV B95-8 to "transform" lymphocytes invitro.