Distinct Patterns of Mitogen-Activated Protein Kinase Phosphorylation and Epstein–Barr Virus Gene Expression in Burkitt's Lymphoma Cell Lines versus B Lymphoblastoid Cell Lines

In order to understand mechanistic relationships between signaling pathways regulating mitogen-activated protein kinase (MAPK) phosphorylation and Epstein–Barr virus (EBV) reactivation, we compared MAPK phosphorylation, and EBV reactivation and latency in Burkitt's lymphoma cell lines (BLCLs) versus B lymphoblastoid cell lines (LCLs). EBV was reactivated in the BLCLs Akata and Raji, and in a LCL OB-R33 cells after cross-linking surface immunoglobulin (sIg) with anti-Ig. After stimulation with anti-Ig, MAPK phosphorylation was strongly induced in all BLCLs and in a few LCLs, but not in other LCLs. MAPK was constitutively phosphorylated in most LCLs but not in BLCLs. Expression of EBNA2 and LMP1, and LMP2A was analyzed with both immunoblotting and RT-PCR. EBNA2 and LMP1 were expressed in most LCLs and in some BLCLs. LMP2A was expressed in all BLCLs and LCLs except Namalwa cells. To test the hypothesis that LMP1 induces constitutive MAPK phosphorylation, the LMP1 expression vector was transfected into Akata cells. MAPK phosphorylation was not induced in such transfected cells. Our results indicate that BLCLs and LCLs respectively have distinct MAPK phosphorylation patterns, and that induction of MAPK phosphorylation correlates with EBV reactivation in a few cell lines but not in most of the tested cell lines.     

低分子量多肽基因多态性与多发性硬化的相关性

目的对广东地区汉族多发性硬化症(MS)患者作低分子量多肽(LMP)基因分型,探讨LMP基因与广东人群中MS遗传易感性的可能关系.方法采用聚合酶链式反应———限制性片段长度多态性技术对30例无亲缘关系的MS患者和95例无血缘关系的广东籍健康汉族人作LMP基因分型.结果LMP7和LMP2各等位基因基因型频率在MS组和正常人组间无显著差异(p均>0.05).结论从目前调查的例数,广东人群中MS与LMP基因不关联.     

Analyses of Epstein-Barr virus latent membrane protein-1 in Malaysian nasopharyngeal carcinoma: high prevalence of 30-bp deletion,Xho1 polymorphism and evidence of dual infections

Nasopharyngeal carcinoma, a malignancy associated closely with Epstein-Barr virus (EBV), is prevalent among Chinese of Southern China origin. Epidemiological studies indicate a high prevalence of EBV in Asia with viral isolates having typical characteristics of the putative viral oncogene, latent membrane protein 1 (LMP-1), such as the loss of the Xho1 restriction site in Exon 1 and the 30-bp deletion in Exon 3. The EBV LMP-1 gene from throat washings of 120 nasopharyngeal carcinoma patients and 14 healthy individuals were analyzed. Similar analyses were also carried out on 30 and 12 postnasal space biopsies from nasopharyngeal carcinoma patients and healthy individuals, respectively. The 30-bp deletion was detected in 20% of nasopharyngeal carcinoma throat washes and in 100% of nasopharyngeal carcinoma postnasal space biopsies. Interestingly, 16% of the nasopharyngeal carcinoma biopsies possessed both the deleted and the undeleted variants, suggestive of dual infections. The notion of dual infections in nasopharyngeal carcinoma was further supported by the coexistence of both "F" and "f" (BamH1F region) EBV variants in 11% of the nasopharyngeal carcinoma biopsies. All of the throat washes and biopsies from the healthy controls showed the undeleted variant. The loss of the Xho1 restriction site was found with higher frequency both in throat washes and biopsies from patients with nasopharyngeal carcinoma. The discrepancy in the frequency of the 30-bp deletion between throat washes (20%) and postnasal space biopsies (100%) was an indication that this deletion is specific for viral isolates from primary tumour sites.     

Expression of Epstein‐Barr virus‐encoded LMP1 and hTERT extends the life span and immortalizes primary cultures of nasopharyngeal epithelial cells

Cell immortalization is regarded as an early and pre‐requisite step in tumor development. Defining the specific genetic events involved in cell immortalization may provide insights into the early events of carcinogenesis. Nasopharyngeal carcinoma is common among the Southern Chinese population. Epstein‐Barr virus (EBV) infection is associated closely with nasopharyngeal carcinoma. The involvement of LMP1 (an EBV‐encoded oncogene) has been implicated in the pathogenesis of nasopharyngeal carcinoma. In this study, LMP1 expression, in combination with ectopic expression of hTERT (catalytic unit of human telomerase), was shown to extend the life span of primary cultures of nasopharyngeal epithelial cells and facilitate the immortalization of one of the cell lines (NP446). This is the first report on the successful immortalization of nasopharyngeal epithelial cells involving LMP1. The events associated with the immortalization of nasopharyngeal epithelial cells by LMP1/hTERT were characterized. Expression of c‐Myc, Bmi‐1, and Id‐1 were upregulated at an early stage of immortalization. At a later stage of immortalization, downregulation of p21 and p16 expression were observed. Upregulation of EGFR expression and activation of MAPK signaling pathway were observed in LMP1/hTERT‐immortalized nasopharyngeal epithelial cells. The LMP1/hTERT‐immortalized NP446 cells were non‐tumorigenic in immunosuppressed nude mice and retained anchorage‐dependent growth, suggesting that additional events are required for tumorigenic transformation. The ability of the EBV‐encoded LMP1, in the presence of hTERT expression, to extend the life span and immortalize primary cultures of nasopharyngeal epithelial cells supports the involvement of EBV infection and its viral products in the early stage of pathogenesis of nasopharyngeal carcinoma. J. Med. Virol. 82:1711–1723, 2010. © 2010 Wiley‐Liss, Inc.     

EBV-LMP2蛋白的表达和初步纯化

目的 应用杆状病毒表达载体表达并纯化EB病毒LMP2蛋白.方法 利用杆状病毒Bac-to-Bae杆状病毒表达系统,将EBV-LMP2基因插入到质粒pFastBacTMHT B中,获得携带EBV-LMP2基因的重组杆状病毒Bac-LMP2.重组病毒感染Sf-9细胞,表达N端携带6个组氨酸（6 X His）的LMP2融合蛋白His-LMP2.经镍离子亲和层析纯化,获得纯化蛋白.结果 SDS-PAGE及Western-Blot检测表达的蛋白相对分子质量大小与预计结果一致.HPLC分析纯化后蛋白的纯度可达86%.结论 利用杆状病毒表达系统表达EB病毒LMP2基因并经过初步纯化后,可以获得较好纯度的LMP2蛋白.     

EB病毒融合基因重组腺病毒表达载体的构建

目的构建EB病毒(EBV)潜伏膜蛋白2A(LMP2A)编码基因和即刻早期基因(BZLF1)融合基因的重组腺病毒表达载体。方法逆转录-聚合酶链反应分别获得LMP2A和BZLF1编码序列的cD-NA,采用剪接式重叠延伸(spliced overlap exetension,SOE)技术将两段基因通过多肽接头(Gly4Ser)3的DNA序列进行连接,构建融合基因Z2A。将融合基因Z2A定向亚克隆到pAdTrack-CMV质粒上,在原核细胞E.coli BJ5183中完成穿梭质粒与骨架质粒pAdEasy-1的同源重组,构建融合基因Z2A真核表达载体pAd-Z2A。经抗生素培养板筛选重组体,然后转染293细胞,获得复制缺陷型重组腺病毒vAd-Z2A。结果重组腺病毒载体经限制性核酸内切酶酶切,电泳后可观察到长31kb和4.5kb两条DNA条带,测序鉴定结果表明序列正确;从感染重组腺病毒vAd-Z2A的293细胞中检测到融合基因Z2A的表达。结论本研究成功地构建了EBV LMP2A和BZLF1融合基因Z2A重组腺病毒表达载体,为进一步研究Z2A的功能提供了实验基础。     

EBV-LMP1促CNE2细胞迁移的作用机制

目的：探讨EBV-LMP1对鼻咽癌细胞CNE2迁移表型的影响及作用机制。方法：用RV-LNSX,RV-LMP1和RV-LMP1^TRADD。逆转录病毒分别感染CNE2细胞,G418筛选后,观察和检测转基因细胞的形态学特点,在基质中的运动迁移能力以及R-钙黏蛋白（E-Cadherin）表达的变化;用pLNSX,pLNSX-LMP1和pLNSX—LMPI^TRADD质粒分别与E-Cadherin报告基因共转染293细胞,检测LMP1对E-Cadherin启动子活性的影响。结果：与CNE2和CNE2-LNSX细胞相比,CNE2-LMP1在培养过程中由扁平、鹅卵状上皮细胞形态逐渐演变为细胞间接触消失的长梭状纤维细胞形态,相对迁移明显增大（n=3,P〈0.05）。且E-Cadherin表达明显下调或缺失;随着共转染野生型LMP1（pLNSX-LMP1）剂量的增加（0．2,0、6,1．0μg）,对E／-Cadherin启动子转录活化的抑制率增加,呈明显浓度依赖性;LMPI^TRADD不改变CNE2细胞形态学及迁移表型,对E-Cadherin启动子的转录活性及蛋白表达亦无明显影响。结论：抑制E-Cadherin启动子活化可能是EBV-LMP1下调E-Cadherin蛋白表达、促CNE2细胞迁移的机制之一,位于LMP1羧基端的TRADD可能是其促迁移的主要活性部位。     

Meta-analysis of pregnancy outcomes in pooled randomized trials on a prophylactic adjuvanted glycoprotein D subunit herpes simplex virus vaccine

The primary objective of this investigation was to assess whether the AS04-adjuvanted herpes simplex virus (HSV) glycoprotein D candidate prophylactic vaccine against genital herpes disease increases the risk of spontaneous abortion associated with pregnancy conceived within the vaccination exposure window (vaccine dose received within the period starting 60 days before and ending 20 weeks post-conception day). We performed a meta-analysis of studies designed as part of the clinical development program for this vaccine, to examine the relative risk of abortion (spontaneous or elective) associated with unintended vaccination exposure during pregnancy. Nineteen studies, completed before September 2010, were eligible; 5 matched the inclusion criteria for this analysis (presence of a control arm and at least one adverse pregnancy outcome reported). All vaccinated women (N = 19,727) were included, of whom 660 reported a pregnancy during the study period. Overall, 13.3% of pregnancies in the HSV vaccine group and 11.0% in the control group resulted in spontaneous abortion; 24.2% and 20.0% resulted in elective abortion. Among 180 women with a first pregnancy conceived in the vaccination exposure window, 16.7% (HSV vaccine) and 9.5% (control) had a spontaneous abortion and 38.5% and 33.3%, elective abortion. The relative risk for spontaneous abortion associated with vaccine exposure during the risk period for abortion in the course of pregnancy was 1.7 (95% CI: 0.7–4.6). For all women receiving HSV vaccine, this relative risk was 1.3 (95% CI: 0.8–2.1). The corresponding relative risks for elective abortion were 1.2 (95% CI: 0.7–2.0) and 1.3 (95% CI: 0.9–1.8). There was no apparent relationship to dosing and no difference between groups in gestational age at the time of spontaneous or elective abortion. In conclusion there is no statistical evidence that the investigational HSV vaccine increased the risk of spontaneous or elective abortion.     

Lipofuscin causes atypical necroptosis through lysosomal membrane permeabilization

Lipofuscin granules enclose mixtures of cross-linked proteins and lipids in proportions that depend on the tissue analyzed. Retinal lipofuscin is unique in that it contains mostly lipids with very little proteins. However, retinal lipofuscin also presents biological and physicochemical characteristics indistinguishable from conventional granules, including indigestibility, tendency to cause lysosome swelling that results in rupture or defective functions, and ability to trigger NLRP3 inflammation, a symptom of low-level disruption of lysosomes. In addition, like conventional lipofuscins, it appears as an autofluorescent pigment, considered toxic waste, and a biomarker of aging. Ocular lipofuscin accumulates in the retinal pigment epithelium (RPE), whereby it interferes with the support of the neuroretina. RPE cell death is the primary cause of blindness in the most prevalent incurable genetic and age-related human disorders, Stargardt disease and age-related macular degeneration (AMD), respectively. Although retinal lipofuscin is directly linked to the cell death of the RPE in Stargardt, the extent to which it contributes to AMD is a matter of debate. Nonetheless, the number of AMD clinical trials that target lipofuscin formation speaks for the potential relevance for AMD as well. Here, we show that retinal lipofuscin triggers an atypical necroptotic cascade, amenable to pharmacological intervention. This pathway is distinct from canonic necroptosis and is instead dependent on the destabilization of lysosomes. We also provide evidence that necroptosis is activated in aged human retinas with AMD. Overall, this cytotoxicity mechanism may offer therapeutic targets and markers for genetic and age-related diseases associated with lipofuscin buildups.

Lipofuscin is an indigestible mixture of fluorescent cross-linked proteins and lipids that accumulates in the lysosomes of many nondividing cells. Its composition varies with the tissue analyzed. Retinal lipofuscin differs from other lipofuscins in that it is mostly lipid-bisretinoids with almost no proteins (1). However, retinal lipofuscin also presents a number of characteristics that make it indistinguishable from conventional lipofuscin granules. Specifically, 1) it is an indigestible material that fills irreversibly the lumen of lysosomes and affect their degradative, sensing and signaling functions (2); 2) it causes lysosomal swelling, linked to increased susceptibility to rupture (3–5); 3) it triggers NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammation, indicative of low-level lysosomal disruption (6, 7); 4) retinal lipofuscin is also an autofluorescent pigment that serves as biomarker of aging (8, 9); and 5) it is considered toxic waste (10, 11).Retinal lipofuscin accumulates in the retinal pigment epithelium (RPE), a single layer of epithelial cells that separates the neuroretina from the choroidal blood vessels. RPE cells are long lived, do not divide, and each cell is responsible for the phagocytic removal of massive amounts of waste, daily produced by its numerous overlying photoreceptors (12, 13). This incessant and endless clearance demand predisposes the RPE to experience buildups of lipofuscin with time. The lipid-bisretinoids found in retinal lipofuscin are a family of at least 27 structurally related autofluorescent lipo-pigments, among which N-retinilidene-N- retinyl ethanolamine (A2E) and all-trans-retinal dimer (ATRD) are the most abundant ones (1, 14). Lipid-bisretinoids are slowly generated in photoreceptors by the spontaneous dimerization of vitamin A aldehydes involved in the synthesis of visual pigments (15, 16). This dimerization is dramatically accelerated by mutations in the Abca4 gene that encodes a flippase that transfers retinaldehydes from photoreceptor discs to the cytosol, in which retinal dehydrogenases (RDH8 in mice) reduce them to retinols. Photoreceptor bisretinoids are transferred by phagocytosis into RPE lysosomes in which, due to their resistance to hydrolysis, accumulate permanently in lipofuscin granules (5). Their progressive accumulation with age or their fast accumulation through genetic mutations in Abca4 may lead to primary RPE cell death and secondary death of photoreceptors, a phenotype found respectively in atrophic Age-related Macular Degeneration (dry AMD) and Stargardt disease (17–19).Currently, the underlying mechanisms of retinal lipofuscin cytotoxicity are incompletely understood, and the strategies to modulate them are not effective. The dominant dogma is that RPE cell death is caused by the phototoxic decomposition of lipid-bisretinoids, leading to oxidative stress and apoptosis (20–22). However, retinal damage by ambient light was only evident in albino mice (23), and there is no evidence that antioxidants, light blockage, or anti-apoptotic drugs provide any benefit against degeneration in pigmented retinas overloaded with lipofuscin (24, 25) in which photodegradation of lipid-bisretinoids is considerably diminished (23). All these suggest the existence of light-independent cell-death mechanisms that need to be deactivated in order to protect against lipofuscin.Here, we demonstrate that lipofuscin promotes retinal degeneration via a light-independent atypical necroptotic cascade. The pathway that we describe is fundamentally different from previously reported mechanisms of RPE cell death, as it does not involve oxidative stress, apoptosis, or classical necrosomes containing Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) or Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) kinases (26) and implicates lysosomal membrane permeabilization (LMP). Importantly, light-independent lipofuscin-elicited necroptosis was prevented with necrosulfonamide (NSA), necrostatin-7 (Nec7), and arimoclomol, a drug that preserves lysosomal integrity. Summarizing, the present work provides directions to develop potential treatments for Stargardt disease that could eventually be applied to treat other pathological conditions associated with accumulation of lipofuscins.     

Polymorphism of the LMP2 gene and disease phenotype in ankylosing spondylitis: No association with disease severity

Summary Although a number of reports have now described an association between polymorphism of the LMP2 gene and disease phenotype in HLA-B27 positive individuals with ankylosing spondylitis (AS), some describe associations with acute anterior uveitis, others with juvenile onset disease, and one report provides no association. A recent study describes yet a further association with disease severity in patients with juvenile rheumatoid arthritis. We therefore hypothesized that the discrepant findings in adult disease may be a reflection of an underlying association with disease severity. Our study population consisted of 100 HLA-B27 positive Caucasians with AS of ten or more years duration. Clinical assessment of disease severity was based on a metrology index scoring five measurements, the modified health assessment questionnaire for the spondyloarthropathies, and a disease activity index consisting of a visual analog scale to score the amount of pain, stiffness and fatigue. LMP2 genotypes were assigned following polymerase chain reaction amplification from genomic DNA and restriction enzyme digestion with CfoI. Despite confirmation of a significantly higher prevalence of the LMP2 BB genotype in AAU positive (66.0%) versus AAU negative (45.2%) patients (P<0.05), we observed no association between LMP2 genotypes and any of the indices of disease severity. Furthermore, although a significant association was noted between the presence of peripheral synovitis and the functional index score (P<0.05), a history of AAU was not associated with more severe disease. Our data is thus internally consistent in demonstrating no association between LMP2 genotypes and either disease severity or peripheral arthritis, and supports the notion that polymorphism of LMP2 primarily influences the development of AAU and not some other phenotype of AS.