EB病毒潜伏期膜蛋白LMP2A对胃癌细胞增殖的影响

目的:构建EBV潜伏期膜蛋白基因LMP2A的腺病毒表达载体,探讨LMP2A表达对胃癌细胞的作用.方法:RT-PCR扩增获取目的基因LMP2A,采用AdEasy系统构建携带目的基因的重组腺病毒载体pAd-2A,脂质体法将重组质粒pAd-2A转染HEK293细胞包装重组腺病毒.用重组腺病毒感染靶细胞SGC(胃癌细胞),通过MTT实验、流式细胞分析、激光共聚焦检测目的基因LMP2A表达对靶细胞增殖的影响.结果:限制性酶切、PCR及测序鉴定证实,目的基因LMP2A正确插入重组质粒,HEK293细胞成功包装出具有稳定感染性的重组腺病毒,命名为vAd-2A,经测定病毒滴度为3.16×10~(12)pfu/L.MTT实验、流式细胞分析以及激光共聚焦检测结果显示,感染腺病毒vAd-2A的SGC细胞增殖旺盛(感染vAd-2A细胞vs感染vAd细胞和未感染病毒细胞,P<0.01),S期细胞比例升高(24 h:35.2%±5.1%vs 14.0%±3.4%,13.2%±4.6%,P<0.01;48 h:25.6%±4.1%vs 12.9%±2.6%,12.5%±3.2%,P<0.01),LMP2A可诱导细胞周期调节蛋白cyclinE的表达.结论:成功构建了EBV潜伏期膜蛋白基因LMP2A的重组腺病毒表达载体,并在HEK293细胞中成功包装出重组腺病毒,同时证实LMP2A可通过诱导cyclinE的表达促进SGC细胞增殖.     

Epstein-Barr virus in malignancies in renal transplant recipients in Japan

A role for Epstein-Barr virus (EBV) in the development of malignancies including lymphomas, and carcinoma of the stomach, nasopharynx, thymus and salivary gland is suggested. It is indicated that EBV evokes polyclonal-B-cell-proliferative diseases in immunocompromised hosts, such as transplant patients, which results in monoclonal malignant lymphomas. The suppression of immune functions in these patients is thought to lead to incomplete elimination of the cells expressing EBV latent infection genes. To examine the etiological role of EBV in the development of malignancies following renal transplant in Japan, 42 malignancies in 1744 cases of renal transplant were studied for the presence and type of EBV. The polymerase chain reaction revealed that 5 malignancies were positive for EBV, all type A: 2 of 2 cases of non-Hodgkin's lymphoma (NHL), 2 of 8 cases of gastric adenocarcinoma of the common type, and 1 of 2 cases of gastric plasmacytoma. In situ hybridization revealed positive signals in the nucleus of tumor cells in 2 cases of NHL and 1 of plasmacytoma. Positive signals were found in the small lymphoid cells but not in the tumor cells in 2 cases of gastric carcinoma. On the basis of these findings, a role for EBV in the development of malignancies in renal transplant patients is unlikely except for lymphoid neoplasias.Abbreviations PCB polymerase chain reaction - EBV Epstein-Barr virus - NHL non-Hodgkin's lymphoma     

EBV感染与胃癌关系的研究进展

EB病毒(Epstein-Barr virus,EBV)是引起胃癌的重要生物学因素之一,其感染与胃癌的发生和发展密切相关.随着分子生物学技术的发展和应用,研究者对与EBV感染相关胃癌(EBVaGC)的特征和发生机制进行了深入研究,发现EBV潜伏感染和细胞恶性转化是EBV致EBVaGC的重要基础,为EBVaGC的诊断、治...     

利妥昔单抗治疗异基因造血干细胞移植后EB病毒病的疗效和安全性

 目的 了解利妥昔单抗治疗异基因造血干细胞移植（allo-HSCT）后EB病毒（EBV）病的疗效和安全性。方法 回顾性分析2006年6月—2012年3月在北京大学人民医院诊断为allo-HSCT后EBV病并应用利妥昔单抗治疗的26例患者,其中临床诊断EBV病15例,活检确诊（移植后淋巴组织增殖性疾病,PTLD）11例。利妥昔单抗静脉输注375 mg/m²,每周1次。采用非霍奇金淋巴瘤（NHL）的疗效标准判定疗效,采用通用的毒性分级标准判定输注过程中的不良反应。结果共应用利妥昔单抗78例次,中位3（1~6）例次。利妥昔单抗输注过程中无严重不良反应发生。治疗后1、2、3、4、8周的累积完全缓解（CR）率分别为（11.5±6.3）%、（42.2±10.2）%、（64.4±10.0）%、（74.6±9.4）%、（87.3±7.9）%。总有效率84.6%,CR率73.1%;单个器官受累患者的CR率高于多器官受累患者（10/10比9/16,P=0.023）,临床诊断的EBV病患者CR率高于PTLD患者(13/15比6/11,P=0.095）,但差异无统计学意义。自首剂利妥昔单抗应用后的1年和2年总生存（OS）率分别为（55.7±10.2）%和（39.6±12.4）%。单器官受累患者的存活率高于多器官受累患者（8/10比5/16,P=0.041）,临床诊断的患者存活多于PTLD患者(11/15比2/11,P=0.015）。结论 利妥昔单抗治疗EBV病安全有效,建议根据临床诊断在单器官受累时即开始治疗,同时争取尽早获取病理结果。利妥昔单抗的治疗方案需要前瞻性研究提供循证医学的证据来进一步规范。     

Miliary tuberculosis in a patient with Epstein-Barr virus-associated angioimmunoblastic lymphadenopathy

 A 74-year-old woman developed angioimmunoblastic lymphadenopathy (AILD) with involvement of intra-abdominal and retroperitoneal lymph nodes. Southern blot analysis showed germline configuration of the JH genes and an oligoclonal pattern of the TcRβ genes. The immunoblasts were of B-cell phenotype and often expressed the CD30 antigen and the latent membrane protein 1 (LMP1) oncogene. Six nonsilent point mutations were identified near the 3′ end of the LMP1 gene, leading to a cluster of six amino acid changes within a protein domain needed for maximal NF-κB stimulation. After a clinical remission of 8 months the patient relapsed with generalized lymphadenopathy and died secondary to tuberculosis. The oligoclonal rearrangements of the TcRβ genes may reflect an unsuccessful cellular immune response to Mycobacterium tuberculosis or an HLA-restricted T-cell response to B-immunoblasts expressing mutated viral antigens. A positive percutaneous tuberculin test observed 6 months prior to the onset of AILD is in favor of the first possibility. Received: 9 October 1995 / Accepted: 30 January 1996     

Epstein-Barr virus association is rare in esophageal squamous cell carcinoma

Background. A critical role of Epstein-Barr virus (EBV) in carcinogenesis of nasopharyngeal squamous cell carcinoma and gastric adenocarcinoma is strongly suspected. We analyzed the possible EBV association for Japanese squamous cell carcinoma (SCC)-dominant esophageal cancer cases. Methods. We retrospectively screened 36 surgically resected esophageal cancer lesions from 36 patients maily with SCC using in situ hybridization (ISH) for EBV-encoded small RNA1 (EBER-1). EBV DNA analysis using real-time quantitative polymerase chain reaction (Q-PCR) was performed for three recent cases. Results. We found no EBER-1-positive cancer cell in any tested esophageal cancer lesion. There were many EBER-1-positive tumor-infiltrating lymphocytes in the basaloid SCC lesion and a small number of positive lymphocytes in the other five advanced SCC lesions (14.7% of SCC). One SCC lesion with a highcopy number of EBV DNA had EBER-1-positive lymphocytes. Conclusions. EBV is rarely associated with esophageal SCC, and may appear through tumor-infiltrating lymphocytes in some advanced lesions.     

Programmed death 1 monoclonal antibody helped to treat mixed chimeric and reactivation of Epstein-Barr virus in a patient with adult-onset chronic active Epstein-Barr virus infection after allogeneic hematopoietic stem cell transplantation: A case report

Rationale:Systemic forms of chronic active Epstein-Barr virus infection (CAEBV) can predispose a patient to a protracted course of fulminant hemophagocytic lymphohistiocytosis, which has a poor prognosis. Epstein-Barr virus (EBV) infection may persist even after theoretically curative hematopoietic stem cell transplantation.Patient concerns:A female patient with CAEBV underwent chemotherapy followed by allogeneic hematopoietic stem cell transplantation from her human leukocyte antigen-matched sister. Neutrophil and platelet engraftment was observed on day +12 and +10. Full donor chimerism (DC) was achieved on Day +21.Diagnoses:From day +38, EBV-DNA in the blood was persistently positive, and DC declined. We attempted empirical interventions such as withdrawal of immune suppression, multiple donor lymphocyte infusion, stem cell boost, and interferon-α treatment. However, EBV-DNA copies continued to increase aggressively, whereas DC decreased rapidly and then reached a nadir of 63.27%.Interventions:Salvage programmed death 1 (PD-1) antibody treatment was administered as salvage therapy at +69 and +84.Outcomes:EBV-DNA was negative on day +97 and was ultimately undetectable. Equivalently, a full and stable DC was obtained at +97.Lessons:We summarize a case of PD-1 antibody used as salvage treatment in a post-transplant patient with CAEBV, which was eradicated and full DC was obtained. This case suggests that the PD-1 antibody appears to be a promising option for fighting EBV and mixed DCs.     

心脏移植术后肾功能不全患者不同免疫抑制方案的临床研究

目的:回顾性分析心脏移植后出现肾功能不全的患者,进行不同的免疫制方案调整策略的结果。方法:34例在心脏移植1年后随访期出现肾功能不全的患者,以诊断标准分类,进行3种不同的免疫抑制方案调整:①、降低环孢素A剂量,记为Lo-C组;②、转换为低剂量他克莫司,记为Lo-F组;③、转换为西罗莫司,记为S组。观察各组方案调整后的血肌酐(Scr)及肾小球滤过率(GFR)变化趋势、血压、血脂、血糖变化情况及全部随访期的排斥反应发生情况。结果:33例患者存活并完成1年随访,1例死亡。排斥反应发生率分别为16.7%(2/12)vs.5.9%(1/17)vs.20%(1/5)。各组Scr及GFR在免疫抑制方案替换后均有所改善,其中Lo-C组在替换后6个月和12个月时,Scr值比较替换前差异有统计学意义(P<0.05);Lo-F组在替换后3个月、6个月和12个月时Scr及GFR比较替换前差异均有统计学意义(P<0.05);S组在替换后3个月、6个月和12个月时Scr及GFR比较替换前差异均有统计学意义(P<0.05)。各组患者高血压、糖尿病及高脂血症罹患情况,Lo-C组在替换前后无变化;Lo-F组在替换后6个月较替换前糖尿病明显增多,而高脂血症减少;S组在替换后6个月较替换前高脂血症明显增多。结论:心脏移植术后出现肾功能不全的患者,低剂量的钙调素抑制剂(CNI)或替换为西罗莫司(SLR)可改善肾功能,但相应排斥反应和代谢并发症的风险会加大,免疫抑制方案的调整仍须谨慎,并考虑个体情况和加强监测。     

Treatment of advanced rectal cancer after renal transplantation

Renal transplantation is a standard procedure for endstage renal disease today. Due to immunosuppressive drugs and increasing survival time after renal transplantation, patients with transplanted kidneys carry an increased risk of developing malignant tumors. In this case report, 3 patients with advanced rectal cancer after renal transplantation for renal failure were treated with anterior resection or abdominoperineal resection plus total mesorectal excision, followed by adjuvant chemotherapy. One patient ...     

DNA methylation in gastric cancer,related to Helicobacter pylori and Epstein-Barr virus

Gastric cancer is a leading cause of cancer death worldwide,and significant effort has been focused on clarifying the pathology of gastric cancer.In particular,the development of genome-wide analysis tools has enabled the detection of genetic and epigenetic alterations in gastric cancer;for example,aberrant DNA methylation in gene promoter regions is thought to play a crucial role in gastric carcinogenesis.The etiological viewpoint is also essential for the study of gastric cancers,and two distinct pathogens,Helicobacter pylori(H.pylori)and Epstein-Barr virus(EBV),are known to participate in gastric carcinogenesis.Chronic inflammation of the gastric epithelium due to H.pylori infection induces aberrant polyclonal methylation that may lead to an increased risk of gastric cancer.In addition,EBV infection is known to cause extensive methylation,and EBV-positive gastric cancers display a high methylation epigenotype,in which aberrant methylation extends to not only Polycomb repressive complex(PRC)-target genes in embryonic stem cells but also non-PRC-target genes.Here,we review aberrant DNA methylation in gastric cancer and the association between methylation and infection with H.pylori and EBV.     

Successful umbilical cord blood transplantation for severe chronic active Epstein-Barr virus infection after the double failure of hematopoietic stem cell transplantation

An 11-year-old boy with severe chronic active Epstein-Barr virus infection (CAEBV) underwent successful cord blood transplantation (CBT) after consecutive failure of peripheral blood and bone marrow transplantation from his HLA-mismatched mother. CB cells from an unrelated donor were infused after conditioning with total body irradiation (12 Gy), melphalan (120 mg/m(2)), and etoposide (600 mg/m(2)). Complete remission without circulating EBV-DNA has continued for 15 months after a delayed hematologic recovery. This is the first successful report of CBT for CAEBV. CB may therefore be an alternate source of stem cells for the curative treatment of CAEBV, despite the absence of EBV-specific cytotoxic T lymphocytes.     

Impact of EBV infection and immune function assay for lymphoproliferative disorder in pediatric patients after liver transplantation: A single-center experience

Background:Post-transplant lymphoproliferative disorder(PTLD)is a lethal complication after pediatric liver transplantation,but information regarding risk factors for the development of PTLD remains unclear.This study was to identify characteristics and risk factors of PTLD.Methods:A total of 705 pediatric patients who underwent liver transplantation between January 2017 and October 2018 were studied.Impact of clinical characteristics and Epstein-Barr virus(EBV)infection on the development of PTLD was evaluated.In addition,ImmuKnow assay was adopted in partial patients to analyze the immune status.Results:Twenty-five(3.5%)patients suffered from PLTD with a median time of 6 months(3–14 months)after transplantation.Extremely high tacrolimus(TAC)level was found in 2 fatal cases at PTLD onset.EBV infection was found in 468(66.4%)patients.A higher peak EBV DNA loads(>9590 copies/mL)within 3 months was a significant indicator for the onset of PTLD.In addition,the ImmuKnow assay demonstrated that overall immune response was significantly lower in patients with EBV infection and PTLD(P<0.0001).The cumulative incidence of PTLD was also higher in patients with lower ATP value(≤187 ng/mL,P<0.05).Conclusions:A careful monitoring of EBV DNA loads and tacrolimus concentration might be supportive in prevention of PTLD in pediatric patients after liver transplantation.In addition,application of the ImmuKnow assay may provide guidance in reducing immunosuppressive agents in treatment of PTLD.     

The real-time polymerase chain reaction-guided modulation of immunosuppression enables the pre-emptive management of Epstein-Barr virus reactivation after allogeneic haematopoietic stem cell transplantation

To assess a real-time polymerase chain reaction-based modulation of immunosuppression in patients with an increasing Epstein-Barr virus (EBV) viral load, we studied 79 paediatric allogeneic stem cell transplantations (allo-SCT) performed between January 1998 and December 2003. EBV reactivation was observed in 42 of 79 patients (53%) after a median time of 45 d from allo-SCT: 37 (88%) and five (12%) patients had received the graft from an unrelated and a related donor respectively (P = 0.001). Twenty-eight patients (67%) had a viral load > or =300 genomic copies x10(5) peripheral blood mononuclear cells (PBMC) and antithymocyte globulin was the only factor significantly associated with EBV reactivation (P = 0.001, RR 7.1). Among these 28 patients, immunosuppression was suspended and reduced in 17 and 11 patients respectively. Overall, post-transplant lymphoproliferative disease was diagnosed in one of 79 patients (1%). The pre-emptive modulation of immunosuppression in patients with EBV reactivation and a viral load > or =300 genomic copies x10(5) PBMC did not negatively influence transplant-related mortality, overall survival or event-free survival. In conclusion, EBV reactivation is frequent even in 'low risk' patients and the pre-emptive modulation of immunosuppression enables it to be managed safely, with no significant flare in graft-versus-host disease status.     

Hepatitis B virus infection after renal transplantation in the presence of antibody to hepatitis B surface antigen immunity

BACKGROUND AND AIM: Hepatitis B virus (HBV) infection has been known to be hampered by immunity against hepatitis B surface antigen (HBsAg). However, HBV with mutations within the common antigenic epitope of HBsAg, the "a" determinant region, can escape from humoral immunity. Moreover, HBV infection by "a" determinant mutants in chronic HBV patients has been reported after renal transplantation. In the present study, the authors investigated HBV infection after renal transplantation despite passive immunization or resolved HBV infection. METHODS: A total of 1682 patients who underwent a renal transplant between 1979 and 1998 at the Severance Hospital, Yonsei University College of Medicine, Korea, were enrolled. The sequence of the HBV genome was analyzed from two patients with antibody to HBsAg (anti-HBs) immunity. RESULTS: Of 1682 patients who were HBsAg negative before transplantation, 21 patients were found to be HBsAg positive, with elevated aspartate aminotransferase and alanine aminotransferase levels after transplantation. Interestingly, six of 21 (28.6%) patients were anti-HBs positive before the transplantation. Sequence analysis of the cloned HBV from two of six patients with anti-HBs immunity showed no evidence of significant mutations within the "a" determinant region, suggesting a wild-type of HBV. Their donors were not exposed to HBV before transplantation (all HBV markers were negative). Seven deaths of 21 patients were ascribed to HBV-related complications. CONCLUSIONS: Regardless of anti-HBs immunity, HBV infection occurred in immunosuppressed patients in a high endemic area. The molecular mechanism and clinical impact of HBV infection after renal transplantation in patients with anti-HBs immunity should be further reappraised.     

肠道T细胞淋巴瘤中EB病毒感染的研究

目的 了解我国肠道T细胞淋巴瘤（ITCL）中EB病毒（EBV）潜伏感染的状态，其亚型的感染情况，基因产物表达与EBV阳性细胞的性质。方法采用PCR检测42例ITCL中的不同EBV亚型的核抗原基因（EBNA－3C），并对其扩增产的行DNA序列分析。运用EBER1／2－RNA原位杂交证实EBV潜伏感染，IHC／ISH双重染色技术判断EBV阳性细胞性质。免疫组化检测EBV基因产物（LMP－1，EBNA－2）的表达。结果 42例ITCL中EBV的阳性率为97．6％，以A型EBV感染居多（32／38例，84．2％），B型为2／38例（5．3％），混合型为4／38例（10．5％）。EBNA－3C基因的PCR产物DNA序列中存在个别碱基的缺失和插入.EBER1/2的检出率为85．7％。EBER1／2阳性细胞同时表达CD45RO和TIA－1，且表达CD4，CD8及CD56的肿瘤细胞呈EBER1／2阳性。16／42例ITCL（38．1％）表达LMP－1。ITCL中EBV的潜伏感染模式多为I型（24／42，66．7％），Ⅱ型次之（12／42，33．4％）。结论 在我国，ITCL中存在高水平的EBV潜伏感染，且多为A型EBV感染，感染模式为Ⅰ型和Ⅱ型。部分ITCL可能与鼻NK／T细胞淋巴瘤属于同一疾病谱系。