Optimising non-viral gene delivery in a tumour spheroid model

BACKGROUND: Our current understanding of how the unique tumour microenvironment influences the efficacy of gene delivery is limited. The current investigation systematically examines the efficiency of several non-viral gene transfer agents to transfect multicellular tumour spheroids (MCTS), an in vitro model that displays a faithful three-dimensional (3D) representation of solid tumour tissue. METHODS: Using a luciferase reporter assay, gene transfer to MCTS was optimised for 22 kDa linear and 25 kDa branched polyethyleneimine (PEI), the cationic lipids Lipofectamine(trade mark) and DCChol : DOPE, and the physical approach of tissue electroporation. Confocal microscopy was used to take optical tissue slices to identify the tissue localisation of green fluorescent protein (GFP) reporter gene expression and the distribution of fluorescently labelled complexes. A MCTS model of quiescent tumour regions was used to establish the influence of cellular proliferation status on gene transfer efficiency. RESULTS: Of the polyplexes tested, 22 kDa linear PEI provided optimal gene delivery, with gene expression peaking at 46 h. Despite being the optimal vector tested, PEI-mediated transfection was limited to cells at the MCTS periphery. Using fluorescent PEI, it was found that complexes could only penetrate the outer 3-5 proliferating cell layers of the MCTS, sparing the deeper quiescent cells. Gene delivery in an MCTS model comprised entirely of quiescent cells demonstrated that in addition to being inaccessible to the vector, quiescent tumour regions are inherently less susceptible to PEI-mediated transfection than proliferating regions. This 'resistance' to transfection observed in quiescent cells was overcome through the use of electroporation. Despite the improved efficacy of electroporation in quiescent tissue, the gene expression was still confined to the outer regions of MCTS. The results suggest that limited access to central regions of an MCTS remain a significant barrier to gene delivery. CONCLUSIONS: This data provides new insights into tumour-specific factors affecting non-viral gene transfer and highlights the difficulties in delivering genes to avascular tumour regions. The MCTS model is a useful system for the initial screening of future gene therapy strategies for solid tumours.     

A novel heart‐cell line from brown‐marbled grouper Epinephelus fuscoguttatus and its susceptibility to iridovirus

A novel cell line (bmGH) was established from the heart of brown‐marbled grouper Epinephelus fuscoguttatus and its viral susceptibility was evaluated. The bmGH cells have been subcultured to passage 65 in Dulbecco's modified eagle medium:Ham's nutrient mixture F‐12 (1:1) medium (DMEM/F12) which was further supplemented with foetal bovine serum (FBS), carboxymethyl‐chitosan, basic fibroblast growth factor (bFGF) and insulin‐like growth factor‐I (IGF‐I) at 24° C. The heart cells have a fibroblastic morphology and proliferated to confluence 14 days later. The cells grew at a steady rate during subsequent subculture and had a population doubling time of 40·3 h at passage 60. Karyotype analysis showed that these cells exhibited chromosomal aneuploidy with a modal chromosome number of 48. The results of viral susceptibility characterization revealed that cytopathic effects (CPE) of bmGH cells appeared after infection by two iridoviruses, turbot reddish body iridovirus (TRBIV) and lymphocystis disease virus (LCDV). A large number of TRBIV and LCDV particles were also observed in the infected bmGH cells by electron microscope examination. All of these facts indicate that the bmGH cell line established here may serve as a valuable tool for studies of cell‐virus interactions and has potential applications in fish virus isolation, propagation and vaccine development.     

Viral phylodynamics and the search for an ‘effective number of infections’

Information on the dynamics of the effective population size over time can be obtained from the analysis of phylogenies, through the application of time-varying coalescent models. This approach has been used to study the dynamics of many different viruses, and has demonstrated a wide variety of patterns, which have been interpreted in the context of changes over time in the ‘effective number of infections’, a quantity proportional to the number of infected individuals. However, for infectious diseases, the rate of coalescence is driven primarily by new transmissions i.e. the incidence, and only indirectly by the number of infected individuals through sampling effects. Using commonly used epidemiological models, we show that the coalescence rate may indeed reflect the number of infected individuals during the initial phase of exponential growth when time is scaled by infectivity, but in general, a single change in time scale cannot be used to estimate the number of infected individuals. This has important implications when integrating phylogenetic data in the context of other epidemiological data.     

Deregulated signalling networks in human brain tumours

Despite the variety of modern therapies against human brain cancer, in its most aggressive form of glioblastoma multiforme (GBM) it is a still deadly disease with a median survival of approximately 1 year. Over the past 2 decades, molecular profiling of low- and high-grade malignant brain tumours has led to the identification and molecular characterisation of mechanisms leading to brain cancer development, maintenance and progression. Genetic alterations occurring during gliomagenesis lead to uncontrolled tumour growth stimulated by deregulated signal transduction pathways. The characterisation of hyperactivated signalling pathways has identified many potential molecular targets for therapeutic interference in human gliomas. Overexpressed or mutated and constitutively active kinases are attractive targets for low-molecular-weight inhibitors. Although the first attempts with mono-therapy using a single targeted kinase inhibitor were not satisfactory, recent studies based on the simultaneous targeting of several core hyperactivated pathways show great promise for the development of novel therapeutic approaches. This review focuses on genetic alterations leading to the activation of key deregulated pathways in human gliomas.     

Decreased antiviral immune response within the central nervous system of aged mice is associated with increased lethality of West Nile virus encephalitis

West Nile virus (WNV) is an emerging pathogen that causes disease syndromes ranging from a mild flu‐like illness to encephalitis. While the incidence of WNV infection is fairly uniform across age groups, the risk of lethal encephalitis increases with advanced age. Prior studies have demonstrated age‐related, functional immune deficits that limit systemic antiviral immunity and increase mortality; however, the effect of age on antiviral immune responses specifically within the central nervous system (CNS) is unknown. Here, we show that aged mice exhibit increased peripheral organ and CNS tissue viral burden, the latter of which is associated with alterations in activation of both myeloid and lymphoid cells compared with similarly infected younger animals. Aged mice exhibit lower MHCII expression by microglia, and higher levels of PD1 and lower levels of IFNγ expression by WNV‐specific CD8⁺ T cells in the CNS and CD8⁺CD45⁺ cells. These data indicate that the aged CNS exhibits limited local reactivation of T cells during viral encephalitis, which may lead to reduced virologic control at this site.     

MCMV不同途径感染免疫缺损性小鼠体内效应的研究

通过血管注射、腹下注射、唾液腺注射等3种不同途径将野生型小鼠巨细胞病毒(murine cytomegalovirus,MCMV)感染免疫缺损型小鼠CM17 SCID(sevele combined immunodeficiency,严重免疫缺损综合症),在感染后不同的时间内分别取唾液腺、脾、肝、肺和肾脏测定其病毒滴度,以及测定感染后SCID小鼠的死亡率．同时通过唾液腺注射RvM43突变株,测定病毒在唾液腺中的滴度．结果显示：经尾部血管途径注射的体内唾液腺、肺、脾、肝和肾脏的病毒滴度高峰期和SCID小鼠死亡时间均显著性早于经腹下注射、唾液腺注射途径、除唾液腺器官外,经唾液腺注射途径肺、脾、肝和肾脏的病毒的出现时间晚于经尾部血管和腹下注射途径．经唾液腺注射后,唾液腺中突变型RvM43在各时间点的病毒滴度及高峰出现时间与野生型相同．由此可知,从唾液腺感染小鼠后：MCMV病毒在唾液腺中的生长不受M43基因突变的影响,小鼠巨细胞病毒不同途径感染免疫缺损型小鼠CMl7 SCID的体内生物学效应有差异．因此有必要通过不同途径感染宿主来研究MCMV基因的体内功能．     

杆状病毒囊膜糖蛋白gp64基因启动子活性分析

GP64是杆状病毒在其发育循环第一时相所产生的芽生型病毒粒子（budded voirus,BV)的特异性囊膜糖蛋白。它在病毒入侵细胞过程中具有重要作用。为了探明杆状病毒gp64基因的表达调控。从所克隆的BmNPV和AcMNPVgp64基因ATG上游437-439bp启动子的序列分析发现。该启动子同时具有早期和晚期转录模体,将所构建的该启动子控制下荧光素酶报告基因（Luc)的非融合表达质粒分别转染Bm-N和Sf-21细胞进行瞬间表达分析,BmNPVgp64启动子能被允许宿主Bm-N细胞RNA聚合酶Ⅱ所识别,AcMNPVgp64启动子既能被允许宿主Sf-21又能被非允许宿主Bm-N细胞RNA聚合酶Ⅱ所识别,同时,这两个启动子的转录活性在各自的允许宿主细胞中被相应的病毒因子所反式激活2.4-4倍。将家吞核多角体病毒同源重复序列3（BmNPV homologous region-3,hr3)克隆到该启动子控制下的Luc报告基因下游,用所构建的质粒分别转染细胞,瞬间表达分析结果表达,BmNPVhr3能分别增强该启动子在Bm-N和Sf-21细胞中的转录活性13-22倍和7000-14000倍以上,同时,相应的病毒因子能反式激活插入了hr3的gp64启动子在各自允许宿主细胞中转录活性73-78倍,这暗示着BmNPVhr3除了具有病毒DNA复制原点和增强子的功能外,它在病毒的反式激活过程中起着重要的作用。