Selective gene transfer to tumor cells by recombinant Newcastle Disease Virus via a bispecific fusion protein

Much interest exists presently in development of vectors for gene therapy of tumors based on RNA viruses because these viruses replicate in the cytoplasm and do not integrate into DNA. The negative stranded paramyxovirus, Newcastle Disease Virus (NDV) from chicken has the additional advantages of preferential replication in tumor cells and of oncolytic and immunostimulatory properties. We here describe the bispecific fusion protein alphaHN-IL-2 which binds to NDV, inhibits its normal cell binding property and introduces a new binding specificity for the interleukin-2 receptor (IL-2R). We demonstrate selective gene transfer to tumor cells expressing IL-2R via the bispecific fusion protein when using recombinant NDV carrying as marker gene the enhanced green fluorescence protein (NDFL-EGFP). Hemadsorption (HA) and neuraminidase activities (NA) of the HN protein of NDV were shown to be blocked by alphaHN-IL-2 simultaneously and the absence of HA-activity of modified NDV was confirmed in vivo. Retargeted virus-binding to IL-2R positive tumor cells was not sufficient for the process of cellular infection. It required in addition membrane fusion via the viral F-protein. By modification of recombinant NDV with a bispecific molecule, our results demonstrate a novel and safe strategy for selective gene transfer to targeted tumor cells.     

新城疫病毒对血管生长的抑制作用

 目的 研究鸡新城疫病毒对血管的生长抑制作用。方法 采用小鼠肺癌转移模型观测鸡新城疫病毒（NDV）对癌细胞转移的影响及组织内微血管的生长情况；用体外药物敏感实验（MTT）检测NDV在不同剂量和作用时间下对人血管内皮细胞ECV304细胞的增殖抑制效应；用PI和Hoechst33342进行荧光染色检钡4NDV作用后细胞有无凋亡改变及其形态学变化。结果 NDV在体内可以有效抑制小鼠发生癌转移时组织内微血管的生成，在体外可以抑制ECV304细胞的增殖并导致较为轻微的凋亡现象。结论 体内外实验均表明NDV可以抑制肿瘤发生时微血管的生长，这一作用通过抑制血管内皮细胞的增殖而非直接的细胞毒作用实现，该作用机制可能与某种特定病毒蛋白有关，尚待进一步研究。     

新城疫病毒HN基因构建的核酸疫苗抗肿瘤作用研究

目的 :探讨HN基因在NDV抗肿瘤上发挥的作用。方法 :以pVAX1为表达载体构建了含NDVHN基因的pVHN核酸疫苗 ,以脂质体介导方法在体外转染OS732细胞 72h后 ,用 3,5 二羟基甲苯法测定OS732细胞膜唾液酸含量的变化。 6周龄BALB/c鼠左后肢皮下接种 2× 10 6个S180肿瘤细胞 ,分别于肿瘤接种后的第 7天 (肿瘤直径为 2～ 3mm)和第 17天瘤内注射 10 0 μg核酸重组体 ,同时设pVAX1对照组和单纯荷瘤对照组。荷瘤鼠经治疗后框窦采血 ,分离血清 ,测定血清唾液酸含量。取脾 ,采用FACS方法测定淋巴细胞亚群数量的变化。结果 :pVHN体外转染OS732细胞后 ,能显著降低细胞表面唾液酸含量 (P <0 .0 5 )。pVHN应用于荷瘤鼠显著降低血清中唾液酸含量 (P <0 .0 5 ) ,引起CD8+ T淋巴细胞亚群数量的增加 (P <0 0 5 )。结论 :单独HN基因在体外转染能够降低肿瘤细胞表面唾液酸含量 ,体内表达后引起T淋巴细胞亚群数量改变。     

Enhancement of Anti-tumor Activity of Newcastle Disease Virus by the Synergistic Effect of Cytosine Deaminase

This study was conducted to investigate enhancement of anti-tumor effects of the lentogenic Newcastledisease virus Clone30 strain (NDV rClone30) expressing cytosine deaminase (CD) gene against tumor cells andin murine groin tumor-bearing models. Cytotoxic effects of the rClone30-CD/5-FC on the HepG2 cell line wereexamined by an MTT method. Anti-tumor activity of rClone30-CD/5-FC was examined in H22 tumor-bearingmice. Compared to the rClone30-CD virus treatment alone, NDV rClone30-CD/5-FC at 0.1 and 1 MOIs exertedsignificant cytotoxic effects (P<0.05) on HepG2 cells. For treatment of H22 tumor-bearing mice, recombinantNDV was injected together with 5-FC given by either intra-tumor injection or tail vein injection. When 5-FCwas administered by intra-tumor injection, survival for the rClone30-CD/5-FC-treated mice was 4/6 for 80 daysperiod vs 1/6 , 0/6 and 0/6 for the mice treated with rClone30-CD, 5-FC and saline alone, respectively. When5-FC was given by tail vein injection, survival for the rClone30-CD/5-FC-treated mice was 3/6 vs 2/6 , 0/6 and0/6 for the mice treated with rClone30-CD, 5-FC or saline alone, respectively. In this study, NDV was used forthe first time to deliver the suicide gene for cancer therapy. Incorporation of the CD gene in the lentogenic NDVgenome together with 5-FC significantly enhances cell death of HepG2 tumor cells in vitro, decreases tumorvolume and increases survival of H22 tumor-bearing mice in vivo.     

Antitumor effects of Newcastle Disease Virus in vivo: local versus systemic effects

Newcastle Disease Virus (NDV) has interesting anti-neoplastic and pleiotropic immune stimulatory properties. The virus preferentially replicates in and kills tumor cells and appears to be safe and to varying degrees effective in phase II-clinical studies in the US and in Europe. Here we have compared various lytic and non-lytic strains of NDV with regard to their antitumor effects after local or systemic application. As tumor models we used human metastatic melanoma xenotransplants in nude mice and murine metastatic colon carcinoma (CT26), renal carcinoma (Renca) and lymphoma (ESb) cell lines. Intra or peri-tumoral application of NDV or NDV infected tumor cells showed more pronounced antitumor activity than systemic application even when in the latter case much higher dose ranges were used. In the CT26 colon carcinoma model the non-lytic strain Ulster showed stronger antitumor activity than the lytic strain 73T. In the human MeWo melanoma xentransplant model strong antitumor bystander effects were observed by 20% admixture of melanoma cells pre-infected in vitro with NDV (either strain Ulster or Italien). Virus therapy of pre-established human melanomas by intra-tumoral injection of NDV was effective with the lytic strain Italien but not with the non-lytic strain Ulster. Systemic anti-metastatic effects were never observed with NDV alone in contrast to previous results obtained with NDV modified tumor vaccines.     

新城疫病毒HN基因对肝癌细胞SMMC7721的细胞毒性研究

目的:探讨新城疫病毒HN基因对肝癌细胞SMNC7721的杀伤作用及其机制.方法:以脂质体介导方法在体外转染含新城疫病毒HN基因的质粒pVHN于细胞SMMC7721 24 h后,采用MTT法检测细胞活性;3,5-二羟基甲苯测定其唾液酸含量的变化;丫啶橙/溴化乙锭(AO/EB)染色,荧光显微镜观察细胞形态学改变;以及FCM检测病毒HN基因和HLA-A,B,C的表达.结果:体外转染pVHN能显著地降低细胞表面唾液酸含量(P＜0.05),有效地杀伤肿瘤细胞SMMC7721;荧光显微镜观察可见典型的细胞死亡形态学改变;实验组细胞与对照组比较HN表达差异显著,HLA-A,B,C表达上调.结论:HN基因在体外能够显著降低肿瘤细胞表面唾液酸含量,同时,使其高表达HN抗原,上调HLA-A,B,C表达,从而,可能增强了肿瘤细胞的抗原性和免疫识别,诱导了细胞SMMC7721死亡.     

新城疫病毒对癌细胞的杀伤作用

 目的　探讨新城疫病毒NDV对癌细胞的直接杀伤作用。方法　将NDV与各组细胞的混悬液一起加入细胞培养板中三孔法,同时设不加NDV的对照孔,经8、16、24、36和48小时五个时相观测细胞生长情况和病毒复制情况。结果　成纤维细胞和对照组细胞生长良好,而实验孔的各组癌细胞逐渐减少,出现融合、裂解。48小时癌细胞组的上清液病毒效价较高而成纤维细胞的上清液病毒效价为0。结论　NDV对癌细胞有较强的亲和力,可在癌细胞内大量复制,具有直接杀伤癌细胞的作用,而对正常细胞无明显的杀伤作用。     

新城疫病毒抗癌作用及其应用的研究进展

新城疫病毒（Newcastle disease virus,NDV）为反义负链RNA病毒，能选择性的杀伤人类肿瘤细胞。应用NDV免疫肿瘤细胞进行各种体内外实验研究，均有不同程度的抗肿瘤效应。用NDV对癌症病人进行临床治疗则可延长肿瘤病人的存活期，肿瘤复发率较低。作用机制包括其直接对肿瘤细胞产生细胞毒作用；增强机体的各种细胞及体液免疫；诱导多种细胞因子的产生。其中T细胞的杀肿瘤作用增强及活化巨噬细胞数量增加是主要的抗瘤因素。     

新城鸡瘟病毒治疗肉瘤180小鼠的实验研究

肉瘤１８０（Ｓ_１８０）细胞注入到小鼠腹腔中制得荷瘤鼠模型，用新城鸡瘟病毒（ＮＤＶ）Ｂ１株治疗，观察１个月。青年鼠（７周龄）生存率达７６．７％，而老龄鼠（１年龄）和未经治疗的青年鼠均１００％死亡。病毒在Ｓ１８０腹水中基本不繁殖。进一步观察发现，经ＮＤＶ治疗的荷瘤鼠，７周龄鼠血清干扰素增高、ＮＫ细胞活性增强，而１年龄鼠中则未观察到此现象。７周龄鼠治疗后第４天可检测出抗病毒抗体，第８天时效价达１：１６００。用ＮＤＶ体外感染巨噬细胞也可诱生出较高浓度干扰素。结果提示，干扰素增高、ＮＫ细胞活性增强可能与抗肿瘤疗效有关，而抗病毒抗体与疗效间的关系，尚需进一步研究。     

定位表达的新城疫病毒HN蛋白对荷瘤小鼠的抗肿瘤免疫作用

背景与目的 新城疫病毒HN蛋白是新城疫病毒产生溶瘤作用的重要免疫原.在前期体外实验基础 上,比较定位表达细胞不同部位的HN蛋白体内抗肿瘤免疫作用.方法 通过构建荷瘤小鼠,瘤内注射定位表达于 细胞不同部位的新城疫病毒HN蛋白,即胞浆型(Cy-HN)、跨膜型(M-HN)、分泌型(Sc-HN)重组真核表达质 粒,比较荷瘤小鼠的肿瘤生长速度,脾淋巴细胞增殖反应和细胞毒T细胞活性.结果 瘤内注射跨膜型重组真核表达 质粒的荷瘤小鼠肿瘤生长缓慢,与瘤内注射胞浆型和分泌型重组真核表达质粒的荷瘤小鼠相比有统计学差异(第18 天:P=0.022;第21天:P<0.01),同时,该组荷瘤小鼠的淋巴细胞增殖反应和细胞毒T细胞活性也较高[M-HN vs Cy- HN, P=0.019; M-HN vs Sc-HN, P=0.043; M-HN vs pcDNA3.1(+), P<0.01].结论 定位表达于细胞不同部位的HN蛋白体内 抗肿瘤免疫作用存在差异,跨膜型HN蛋白的重组DNA质粒能够提高荷瘤小鼠的特异性细胞免疫应答.     

新城疫病毒Lasota 弱毒株对人肿瘤细胞的体外修饰作用

 目的 研究新城疫病毒（NDV）弱毒株Lasota对体外培养的人肿瘤细胞株免疫功能的影响。方法 以不同病毒滴度作用于肿瘤细胞，采用MTT染色法检测NDV对肿瘤细胞的杀伤作用，同时用NDV处理过的小鼠黑色素B16细胞免疫小鼠，检测其对小鼠NK细胞活性影响。结果 NDV Lasota株对4种癌细胞株均具有较强的杀伤作用，用其处理小鼠黑色素瘤细胞株后，再次接种可提高小鼠体内的NK细胞活性。结论 NDV能够抑制肿瘤细胞生长，诱导其凋亡，免疫接种小鼠后，可提高小鼠的细胞免疫功能。     

新城疫病毒联合化疗对晚期非小细胞肺癌的疗效和安全性评价

  目的  探讨新城疫病毒在ⅢB、Ⅳ期非小细胞肺癌(NSCLC)化疗中的作用。  方法  2005年7月~2008年5月, 在辽宁省肿瘤医院收治的146例非小细胞肺癌患者, 随机分为实验组与对照组。实验组73例, 采用新城疫病毒联合长春瑞滨+卡铂方案化疗; 对照组73例, 单纯采用长春瑞滨+卡铂方案化疗。每28天为1个周期, 化疗4个周期。每2个周期评价疗效, 并监测治疗前后患者白细胞、血小板、T细胞亚群CD4/CD8变化。  结果  实验组和对照组的有效率分别为50.68%和41.10%(P < 0.05)。实验组不良反应较对照组轻, 差异有统计学意义(P < 0.05)。实验组治疗前后CD4/CD8分别为1.22+0.94和1.59±1.17, 差异有统计学意义(P < 0.05)。  结论  新城疫病毒联合长春瑞滨+卡铂方案治疗ⅢB、Ⅳ期非小细胞肺癌(NSCLC)疗效好, 不良反应轻, 具有提高机体的免疫功能和改善患者生活质量的作用。      

新城疫病毒诱导的抗体反应与肿瘤生长的相关分析

目的:了解新城疫病毒(NDV)接种机体产生的血凝抑制(HI)抗体反应水平与NDV在体内的抑瘤效果是否存在一定关系,从而探讨测定HI抗体能否成为一种简便可行的、既能判断NDV接种成功与否,又能估计疗效预后的监测手段.方法:以小鼠肉瘤180为实验模型,于实验开始后第4周及终止实验时,收集小鼠的眼底血标本测定NDV HI抗体,同时解剖和称量每只小鼠的移植瘤,以HI抗体为横座标,瘤重为纵座标描绘相关散点图,并计算和检验直线相关系数.结果:小鼠的HI抗体阳性率试验组第一批为90%,第二批为100%;对照组两批小鼠均为阴性;试验组小鼠HI抗体与瘤重的相关散点图呈直线趋势,相关系数为-0.6495,p＜0.002,负相关呈高度显著性.结论:测定NDV HI抗体,除能判断接种成功与否外,可能对疗效的预后估计也有一定的间接性的参考价值.     

新城疫病毒诱导的抗体反应与肿瘤生长的相关分析

目的：了解新城疫病毒（NDV）接种机体产生的血凝抑制（HI）抗体反应水平与NDV在体内的抑瘤效果是事存在一定关系,从而探讨测定HI抗体能否成为一种简便可行的、既能判断NDV接种成功与否,又有估计疗效预后的监测手段。方法：以小鼠肉瘤180为实验模型,于实验开始后第4周及终止实验时,收集小鼠的眼底血标本测定NDVHI抗体,同时解剖和称量每只小鼠的移植瘤,以HI抗体为横座标,瘤重为纵座标描绘相关散点图,并计算和检验直线相关系数。结果：小鼠的HI抗体阳性率试验组第一批为90％,第二批为100％,第二批为100％;对照组两批小鼠均为阴性;试验组小鼠HI抗体与瘤重的相关散点图呈直线趋势,相关系数为－0．6495,P＜0．002,负相关呈高度显著性。结论：NDVHI抗体,除能判断接种成功与否外,可能对疗效的预后估计也有一定的间接性的参考价值。     

Liver Pathology in Rats Treated with Newcastle Disease Virus Strains AF2240 and V4-UPM

Background: Treatment of cancer with chemo-radiotherapy causes severe side effects due to cytotoxic effects towards normal tissues which often results in morbidity. Therefore, developing anticancer agents which can selectively target the cancer cells and cause less side effects are the main objectives of the new therapeutic strategies for treatment advanced or metastatic cancers. Newcastle disease virus strains AF2240 and V4-UPM were shown to be cytolytic against various cancer cells in-vitro and very effective as antileukemicagents. Methods: 45 rats at 6 weeks of age, were randomly assigned to nine groups with 5 rats in each group, both azoxymethane (AOM) and 5-Fluorouracil (5-FU) were given to rats according to the body weight. NDV virus strains (AF2240 and V4-UPM) doses were determined to rats according to CD50 resulted from MTT assay. After 8 doses of NDV strians and 5-FU, tissue sections preparations and histopathological study of rats’ organs were done. Results: In this article morphological changes of rats’ organs, especially in livers, after treatment with a colon carcinogen (azoxymethane) and Newcastle disease virus strains have been recorded. We observed liver damage caused by AOM evidenced by morphological changes and enzymatic elevation were protected by the oncolytic viruses sections. Also we found that combination treatment NDV with 5-FU had greater antitumor efficacy than treatment with NDV or 5-FU alone. Conclusion: We noted morphological changes in liver and other rats’ organs due to a chemical carcinogen and their protection by NDV AF2240 and NDV V4-UPM seems to be most protective.     

新城疫病毒感染体外诱导胃癌细胞热休克蛋白70的表达及意义

目的 :观察NDV在体外抗胃癌细胞活性 ,研究NDV感染诱导胃癌细胞热休克蛋白 70 (HSP70 )的表达及其意义。方法 :应用倒置显微镜观察细胞形态 ,MTT法测细胞生长抑制状况 ,血凝试验检测NDV在细胞中的增殖状况 ,免疫组化染色观察细胞中HSP70表达情况 ,流式细胞术检测胃癌细胞HSP70的表达。结果 :NDV在体外可使BGC 82 3胃癌细胞形成明显的细胞病变效应及生长抑制作用 ,且这种抑制作用与肿瘤细胞被NDV感染后HSP70表达增加呈正相关。结论 :NDV的抗BGC 82 3胃癌细胞活性显著 ,其杀伤机制之一为通过感染而诱导BGC 82 3产生过多的HSP70。     

Evaluation of Ultra-Microscopic Changes and Proliferation of Apoptotic Glioblastoma Multiforme Cells Induced by Velogenic Strain of Newcastle Disease Virus AF2240

Aim: Newcastle disease virus (NDV) is a member of genus Avulavirus within the family Paramyxoviridae. Interestof using NDV as an anticancer agent has arisen from its ability to kill tumor cells with limited toxicity to normal cells.Methods: In this investigation, the proliferation of brain tumor cell line, glioblastoma multiform (DBTRG.05MG)induced by NDV strain AF2240 was evaluated in-vitro, by using MTT proliferation assay. Furthermore, Cytologicalobservations were studied using fluorescence microscopy and transmission electron microscopy, DNA laddering inagarose gel electrophoresis assay used to detect the mode of cell death and analysis of the cellular DNA content byflowcytometery. Results: MTT proliferation assay, Cytological observations using fluorescence microscopy andtransmission electron microscopy show the anti-proliferation effect and apoptogenic features of NDV on DBTRG.05MG.Furthermore, analysis of the cellular DNA content showed that there was a loss of treated cells in all cell cycle phases(G1, S and G2/M) accompanied with increasing in sub-G1 region (apoptosis peak). Conclusion: It could be concludedthat NDV strain AF2240 is a potent antitumor agent that induce apoptosis and its cytotoxicity increasing while increasingof time and virus titer.     

NDV7793体外激活的小鼠单核巨噬细胞(MΦ)对小鼠肝癌细胞的杀伤作用及其机制

 目的
初步研究NDV7793激活的小鼠单核巨噬细胞（MΦ）对小鼠肝癌Novikoff细胞的杀伤作用,并探讨其杀伤机制与TNF-
α和TRAIL的关系。 方法从腹腔分离6周龄BALB/C小鼠MΦ, 用NDV7793于体外刺激小鼠MΦ,以ELISA分别测定
NDV7793刺激小鼠MΦ后产生的TNF-α及TRAIL水平;NDV7793体外刺激MΦ后,与小鼠肝癌Novikoff细胞混合培养,
以LDH微量释放法测定小鼠MΦ对小鼠肝癌Novikoff细胞的杀伤效应。同时设立3组实验对照组：IFN-β阳性对照组
、紫外线灭活NDV（UV-NDV）对照组以及空白对照组。 结果与3个对照组相比,NDV7793在体外能提高MΦ分泌TNF-
α、TRAIL的水平;NDV体外刺激后的小鼠MΦ能杀伤小鼠肝癌Novikoff细胞。结论NDV7793在体外能激活小鼠MΦ,
小鼠MΦ被NDV7793刺激后,对小鼠肝癌Novikoff细胞的杀伤作用增强,NDV激活后的小鼠MΦ对小鼠肝癌Novikoff细
胞的杀伤机制可能与TNF-α和TRAIL有关。     

Hodgkin's Disease in the Setting of Human Immunodeficiency Virus Infection

Although Hodgkin's disease (HD) is not usually associated withcongenital or acquired immunodeficiency disorders, recent evidencewould suggest a statistically significant increase in HD amongindividuals infected with human immunodeficiency virus (HIV).In the setting of underlying HIV infection, clinical and pathologiccharacteristics of HD may differ from usual expectations. Thus,70%-100% of HIV-infected patients with HD present with systemic"B" symptoms. Likewise, disseminated, stage III or IV diseaseis reported in approximately 75%-90%. Bone marrow is a commonsite of extranodal HD, occurring in 40%-50%. Complete responserates after multiagent chemotherapy range from approximately45% to 70%, although median survival has been only in the rangeof approximately 18 months. Hematologic toxicity from multiagentchemotherapy may be substantial, even with the use of hematopoieticgrowth factor support. It is apparent that new strategies oftherapeutic intervention must be explored.     

Importance of retinoic acid-inducible gene I and of receptor for type I interferon for cellular resistance to infection by Newcastle disease virus

Newcastle disease virus (NDV) is an avian paramyxovirus with oncolytic properties which shows promising effects in the treatment of cancer. Anti-cancer effects are due to the virus ability: i) to replicate in and kill tumor cells, leading finally to their selective elimination; and ii) to induce the stimulation of antitumor activities in immune cells. NDV does not harm normal cells and has a high safety profile. In this study, we first report a direct correlation between the degree of cell resistance to NDV infection and the cellular expression of the retinoic acid-inducible gene I (RIG-I) which is a cytosolic viral RNA receptor. RIG-I plays an important role in the recognition of and response to infection by RNA viruses. We also demonstrate that impairment of the interferon (IFN) pathway through deletion of the receptor for type I IFN (IFNR1) in primary macrophages leads to NDV replication. In tumor cells, addition of exogenous IFN-α4 is shown to lead to tumor growth reduction and inhibition of viral replication. Finally, increase of the RIG-I concentration of tumor cells via plasmid transfection is shown to be associated with a stronger resistance to NDV infection. These findings shed new light on the crucial role played by the cytosolic receptor RIG-I and the plasma membrane receptor IFNR1 as key molecules to protect cells against infection by NDV.