Enhanced Antiviral Function of Magnesium Chloride-Modified Heparin on a Broad Spectrum of Viruses

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.     

免疫细胞联合腺病毒Ad205-IL-15对肝癌细胞的杀伤研究

在已有腺病毒Ad205-IL-15的基础上尝试与过继免疫细胞CIK、NK92等联合开展对肝癌细胞的杀伤性研究。首次通过荧光化学发光的原理构连出携带表达萤光素酶报告基因的肝癌细胞系Huh7-Luc、MHCC97H—Luc来检测细胞的存活率。结果表明：免疫细胞联合Ad205-IL-15对Huh7-Luc、MHCC97H—Luc两株肝癌细胞表现出了较高协同杀伤作用。证明将免疫细胞与腺病毒联合可以相互弥补各自治疗的不足之处,提高了抗肿瘤效果。同时,由于操作方便,荧光素酶报告基因测定法也为检测细胞存活率提供了新的技术手段。     

Intratumoral Canine Distemper Virus Infection Inhibits Tumor Growth by Modulation of the Tumor Microenvironment in a Murine Xenograft Model of Canine Histiocytic Sarcoma

Histiocytic sarcomas refer to highly aggressive tumors with a poor prognosis that respond poorly to conventional treatment approaches. Oncolytic viruses, which have gained significant traction as a cancer therapy in recent decades, represent a promising option for treating histiocytic sarcomas through their replication and/or by modulating the tumor microenvironment. The live attenuated canine distemper virus (CDV) vaccine strain Onderstepoort represents an attractive candidate for oncolytic viral therapy. In the present study, oncolytic virotherapy with CDV was used to investigate the impact of this virus infection on tumor cell growth through direct oncolytic effects or by virus-mediated modulation of the tumor microenvironment with special emphasis on angiogenesis, expression of selected MMPs and TIMP-1 and tumor-associated macrophages in a murine xenograft model of canine histiocytic sarcoma. Treatment of mice with xenotransplanted canine histiocytic sarcomas using CDV induced overt retardation in tumor progression accompanied by necrosis of neoplastic cells, increased numbers of intratumoral macrophages, reduced angiogenesis and modulation of the expression of MMPs and TIMP-1. The present data suggest that CDV inhibits tumor growth in a multifactorial way, including direct cell lysis and reduction of angiogenesis and modulation of MMPs and their inhibitor TIMP-1, providing further support for the concept of its role in oncolytic therapies.     

Generation of an Oncolytic Herpes Simplex Viral Vector Completely Retargeted to the GDNF Receptor GFRα1 for Specific Infection of Breast Cancer Cells

Oncolytic herpes simplex viruses (oHSV) are under development for the treatment of a variety of human cancers, including breast cancer, a leading cause of cancer mortality among women worldwide. Here we report the design of a fully retargeted oHSV for preferential infection of breast cancer cells through virus recognition of GFRα1, the cellular receptor for glial cell-derived neurotrophic factor (GDNF). GFRα1 displays a limited expression profile in normal adult tissue, but is upregulated in a subset of breast cancers. We generated a recombinant HSV expressing a completely retargeted glycoprotein D (gD), the viral attachment/entry protein, that incorporates pre-pro-GDNF in place of the signal peptide and HVEM binding domain of gD and contains a deletion of amino acid 38 to eliminate nectin-1 binding. We show that GFRα1 is necessary and sufficient for infection by the purified recombinant virus. Moreover, this virus enters and spreads in GFRα1-positive breast cancer cells in vitro and caused tumor regression upon intratumoral injection in vivo. Given the heterogeneity observed between and within individual breast cancers at the molecular level, these results expand our ability to deliver oHSV to specific tumors and suggest opportunities to enhance drug or viral treatments aimed at other receptors.     

Oncolytic adenovirus targeting LASP-1 inhibited renal cell cancer progression

Recent studies suggested that LIM and SH3 protein 1 (LASP-1) is a promising therapeutic target for renal cellcancer (RCC). This study aimed to explore the role of LASP-1 in RCC. For this purpose, LASP-1 expression in RCCtissues was analyzed by immunohistochemistry and Western blot analysis. Cell proliferation, migration, invasion, andgene expression were detected by CCK-8 assay, Transwell assay, and Western blot analysis. The results showed thatLASP-1 was highly expressed in RCC, and its expression level,t was positively correlated with lymph node metastasisand tumor, nodes, and metastases (TNM) stage. The knockdown of LASP-1 expression significantly inhibited theproliferation of RCC cells, increased the apoptosis rate, and inhibited RCC cell invasion and migration by inhibitingepithelial–mesenchymal transition. We conclude that LASP-1 promotes RCC progression and metastasis and is apromising therapeutic target for RCC.     

双靶向溶瘤腺病毒联合奥沙利铂对肿瘤细胞凋亡的研究

研究化疗药物奥沙利铂(Oxaliplatin)联合AdCN205-IL-24对人结肠癌细胞的体外杀伤作用。实验采用MTT法检测肿瘤特异性增殖腺病毒AdCN205-IL-24联合奥沙利铂对两种肿瘤细胞以及正常细胞增殖的抑制作用;Hoechest33342染色,在荧光显微镜对病毒联合化疗诱导的肿瘤细胞凋亡进行形态学观察。结果表明:AdCN205-IL-24联合奥沙利铂处理4 d后对结肠癌细胞株HT-29和SW620的增殖抑制率达到23.17%和22.98%,并且联合化疗并未增加对人正常细胞株L-02的毒性。     

携带oct4、klf4s、ox2、nanog4种转录因子的腺病毒载体的构建

构建并鉴定携带人oct4、klf4、sox2、nanog4个基因的腺病毒载体,重组出能同时表达这4种转录因子的腺病毒,为进一步诱导多能干细胞的研究提供新方法。使用口蹄疫病毒2A序列将人的oct4、klf4、sox2、nanog4个基因顺序连接,定向克隆至腺病毒载体pDC328上,并在nanog下游插入红色荧光蛋白dsRed2作为报告基因。利用酶切及PCR方法鉴定病毒载体的正确性;荧光显微镜下观察病毒感染HEK 293细胞24 h后红色荧光的表达,检测病毒功能;实时定量PCR检测细胞内4种基因的表达情况。结果表明,酶切后DNA电泳鉴定证实载体构建成功,重组后病毒PCR显示病毒携带oct4、klf4、sox2、nanog基因;荧光显微镜下观测到红色荧光的表达,实时定量PCR检测到细胞内4种基因的表达。本研究成功构建的腺病毒载体,能够介导以上4种转录因子在细胞内的同时表达,为进一步诱导多能干细胞的提供新方法。     

重组人p53腺病毒注射液治疗晚期实体肿瘤的安全性和近期疗效评价

目的: 总结重组人p53 腺病毒注射液(rAdp53)治疗的晚期实体肿瘤患者的资料, 初步评价其安全性与疗效。方法: 常规治疗失败的晚期实体肿瘤患者24 例, 其中肾癌5 例, 鼻咽癌4 例, 结直肠癌4 例, 黑色素瘤2 例, 非小细胞肺癌1 例, 食管癌1例, 贲门癌1 例, 胸腺癌1 例, 十二指肠癌1 例, 甲状腺癌1 例, 胰腺癌1 例, 子宫内膜癌1 例, 横纹肌肉瘤1 例。rAd-p53 给药方案为1 ×1012VP/次, 每周1次, 4 次为1 疗程。给药途径包括瘤内注射、支气管内喷洒、腹腔内注射、动脉灌注和静脉滴注。联合化疗18 例, 联合放疗2 例, 联合同期放、化疗1 例, 联合腹部热疗和吉非替尼1 例, 联合免疫治疗1 例,rAd-p53 单药治疗1 例。结果: 24 例患者中因早期进展而停药1 例, 接受1 疗程治疗20 例, 2 疗程治疗2例, 5 疗程治疗1 例。在可评价的21 例中, 部分缓解(PR) 5 例, 稳定(SD) 5 例, 进展(PD) 11 例, 有效率23.8%(5/21), 疾病控制率47.6%(10/21)。常见不良反应为自限性、I ~ II 度注射部位疼痛、寒颤、发热和肌肉酸痛。III 度发热2 例, 联合化疗者发生III ~IV 度骨髓抑制4 例, 骨痛加剧2 例, 一过性低血压1例。结论: 晚期实体瘤患者可耐受rAd-p53 治疗, 有必要进一步设计临床试验, 确定rAd-p53 联合常规治疗的有效性。     

Anti-tumor Immune Response Mediated by Newcastle Disease Virus HN Gene

Hemagglutinin-neuramidinase(HN) is one of the most important surface structure proteins of the Newcastle disease virus(NDV). HN not only mediates receptor recognition but also possesses neuraminidase(NA) activity,which gives it the ability to cleave a component of those receptors, NAcneu. Previous studies have demonstrated that HN has interesting anti-neoplastic and immune-stimulating properties in mammalian species, including humans. To explore the application of the HN gene in cancer gene therapy, we constructed a Lewis lung carcinoma(LLC) solid tumor model using C57BL/6 mice. Mice were injected intratumorally with the recombinant adenovirus expressing HN gene(Ad-HN), and the effect of HN was explored by natural killer cell activity assay, cytotoxic lymphocyte activity assay, T cell subtype evaluation, and Th1/Th2 cytokines analysis. The results demonstrate that HN not only can elicit clonal expansion of both CD4+ and CD8+ T cell populations and cytotoxic T lymphocyte(CTL) and killer cell response, but also skews the immune response toward Th1. Thus, vaccination with Ad-HN may be a potential strategy for cancer gene therapy.     

Inactivation of internalized and surface contaminated enteric viruses in green onions

With increasing outbreaks of gastroenteritis associated with produce, it is important to assess interventions to reduce the risk of illness. UV, ozone and high pressure are non-thermal processing technologies that have potential to inactivate human pathogens on produce and allow the retention of fresh-like organoleptic properties. The objective of this study was to determine if UV, ozone, and high pressure are effective technologies compared to traditional chlorine spray on green onions to reduce enteric viral pathogens and to determine the effect of location of the virus (surface or internalized) on the efficacy of these processes. Mature green onion plants were inoculated with murine norovirus (MNV), hepatitis A virus (HAV) and human adenovirus type 41 (Ad41) either on the surface through spot inoculation or through inoculating contaminated hydroponic solution allowing for uptake of the virus into the internal tissues. Inoculated green onions were treated with UV (240 mJ s/cm²), ozone (6.25 ppm for 10 min), pressure (500 MPa, for 5 min at 20 °C), or sprayed with calcium hypochlorite (150 ppm, 4 °C). Viral inactivation was determined by comparing treated and untreated inoculated plants using cell culture infectivity assays. Processing treatments were observed to greatly affect viral inactivation. Viral inactivation for all three viruses was greatest after pressure treatment and the lowest inactivation was observed after chlorine and UV treatment. Both surface inoculated viruses and viruses internalized in green onions were inactivated to some extent by these post-harvest processing treatments. These results suggest that ozone and high pressure processes aimed to reduce the level of microbial contamination of produce have the ability to inactivate viruses if they become localized in the interior portions of produce.