Productive infection of primary human endothelial cells by pig endogenous retrovirus (PERV)

Abstract: The potential risk of viral transmission in the setting of xenotransplantation has gained major attention. Different porcine cell types have been shown to release retroviral particles, which are infectious for human cell lines in vitro. However, there are only a few data on whether PERV (pig endogenous retrovirus) is able to infect primary human cells. In this study we have analyzed endothelial cells, vascular fibroblasts, mesangial cells, mononuclear cells, hematopoetic stem cells and bone marrow stromal cells for PERV transmission. We now provide evidence for primary human endothelial cells, vascular fibroblasts, and mesangial cells to be susceptible to PERV transmission. PERV infection was productive in endothelial cells and mesangial cells. Our data confirm and extend former reports concerning the PERV infection of human cells. The PERV infection of different primary human cells represents further significant evidence for a viral risk during xenotransplantation. In this context, special attention should be directed towards productive infection of human endothelial cells: in the setting of xenotransplantation this cell type will have close contact with porcine cells and PERV particles.     

Combinedin vitro effect of marijuana and retrovirus on the activity of mouse natural killer cells

Both marijuana and retroviruses impair natural killer (NK) cell functions, but no data on their simulataneous effects are available. Similarities to human AIDS induced by Friend leukemia complex (FLO and its helper Rowson-Parr virus (RPV) provides a mouse model to study drug-virus action. Leukemia susceptible BALB/c and resistant C57BL/6 mice were infected, then at time intervals their nylon wool-separated splenocytes were exposed to tetrahydrocannabinol (THC) for 3h. Natural killer cell activity against Yac-1 cells was assayed by 51Cr-release for 4 and 18h. Recovery of splenocytes was found to be suppressed by FLC, but in BALB/c only by RPV. After a transient enhancement in C57BL/6 by FLC, NK cell activity of both mice became suppressed early (2 to 4 days), normalized subsequently and enhanced late (11 to 14 days) postinfection. A moderate increase in BALB/c, no change in C57BL/6 were induced by low (1-2.5 g/ml) THC doses. NK cell activity of BALB/c became suppressed exponentially by higher (5-10 g/ ml) THC doses in 18h as compared to 4h assays, while its proportional and moderate impairment was seen in C57BL/6. The magnitude of NK cell activity of infected mice was determined by THC: enhancement or impairment followed those of untreated, infected counterparts on the level of THC-treated cells. Low doses hardly, high doses additively influenced NK cells of infected BALB/c. THC slightly affected very early and late enhancement in NK cell activity of FLC infected C57BL/6, but augmented RPV induced suppression late in 18h assays. Genetic factors similar to endotoxin resistance, altered cytokine profile might determine these effects. Similar phenomena in humans might result in earlier manifestation of AIDS.     

survivin基因RNAi逆转录病毒载体设计与构建方法实验研究

目的　设计和构建survivin基因的表达si RNA逆转录病毒重组载体,探讨胶质瘤分子病因及用于基因治疗的可行性。方法　利用在线软件si Direct设计干扰survivin基因靶序列,合成回文DNA序列退火后克隆至线性化p SUPER质粒载体,重组质粒载体双酶切电泳鉴定和测序分析,再转染phoenix细胞产生病毒转染低分化SHG4 4 - 9胶质瘤细胞株。利用NIH3T3细胞测定病毒滴度。Western blot测定转染后SHG4 4 - 9细胞survivin表达量。结果　p SU PER表达si RNA重组质粒载体经双酶切电泳鉴定和DNA测序分析,证实插入6 0 bp序列与原序列一致,位置正确。测定p SUPER.retro- S1、p SU PER.retro- S2病毒滴度值分别为5 .5×10 5CFU/ m l和5 .75×10 5CFU/ ml,干扰效率分别为70 .5 %和接近10 0 .0 %。结论　survivin基因的表达si RNA逆转录病毒重组载体的构建成功,不但为研究胶质瘤分子病因和基因治疗提供了有用工具,而且也为研究高表达survivin的其它肿瘤构建了新的平台。     

绿色荧光蛋白和酪氨酸羟化酶在骨髓基质细胞中的表达

目的研究反转录病毒介导的绿色荧光蛋白(EGFP)基因和酪氨酸羟化酶(TH)基因在原代培养的骨髓基质细胞(bMSCs)中的表达情况。方法体外分离培养大鼠四肢骨的bMSCs,原代培养8~10 d后,同时利用含EGFP和TH基因的反转录病毒上清依次转染bMSCs并进行抗性筛选,7~10 d可得到稳定表达EGFP的bMSCs,在稳定转染EGFP后10 d进行TH病毒上清的转染,7~10 d可得到稳定表达TH和EGFP的bMSCs。结果原代培养bMSCs 3~4 d后生长至70%~80%汇合,病毒转染EGFP后第2天,细胞呈现弱荧光,经G418筛选2~3 d后出现阳性细胞,约8~10 d生长融合,阳性率60%~70%,第2~5代阳性细胞比率无明显变化。TH阳性率也达60%以上。结论利用反转录病毒载体可有效地将EGFP和TH基因转至bMSCs中,并能在体外稳定表达传代。这种表达EGFP和TH的bMSCs对于研究bMSCs的体内迁移分化及帕金森病(PD)的基因治疗具有重要意义。     

Association of genetic polymorphisms and autoimmune Addison’s disease

Autoimmune Addison’s disease (AAD) is a complex genetic disease that results from the interaction of a predisposing genetic background with as yet unknown environmental factors. The disease is marked by the appearance of circulating autoantibodies against steroid 21-hydroxylase. Mutations of the autoimmune regulator gene are responsible for the so-called autoimmune polyendocrine syndrome type I (APS I), of which AAD is a major disease component. Among genetic factors for isolated AAD and APS II, a major role is played by HLA class II genes: HLA-DRB1^*0301-DQA1^*0501-DQB1^*0201 and DRB1^*04-DQA1^*0301-DQB1^*0302 are positively, and RB1^*0403 is negatively, associated with a genetic risk for AAD. The MHC class I chain-related gene A allele 5.1 is strongly and positively associated with AAD. Other gene polymorphisms contributing to genetic risk for AAD are MHC2TA, the gene coding for class II transactivator, the master regulator of class II expression, cytotoxic T lymphocyte antigen-4, PTPN22 and the vitamin D receptor.     

Implication of Human Endogenous Retrovirus Envelope Proteins in Placental Functions

Human endogenous retroviruses (ERVs) represent 8% of the total human genome. Although the majority of these ancient proviral sequences have only retained non-coding long terminal repeats (LTRs), a number of “endogenized” retroviral genes encode functional proteins. Previous studies have underlined the implication of these ERV-derived proteins in the development and the function of the placenta. In this review, we summarize recent findings showing that two ERV genes, termed Syncytin-1 and Syncytin-2, which encode former envelope (Env) proteins, trigger fusion events between villous cytotrophoblasts and the peripheral multinucleated syncytiotrophoblast layer. Such fusion events maintain the stability of this latter cell structure, which plays an important role in fetal development by the active secretion of various soluble factors, gas exchange and regulation of fetomaternal immunotolerance. We also highlight new studies showing that these ERV proteins, in addition to their localization at the cell surface of cytotrophoblasts, are also incorporated on the surface of various extracellular microvesicles, including exosomes. Such exosome-associated proteins could be involved in the various functions attributed to these vesicles and could provide a form of tropism. Additionally, through their immunosuppressive domains, these ERV proteins could also contribute to fetomaternal immunotolerance in a local and more distal manner. These various aspects of the implication of Syncytin-1 and -2 in placental function are also addressed in the context of the placenta-related disorder, preeclampsia.     

Innovation in Chemistry,Manufacturing, and Controls—A Regulatory Perspective From Industry

This review describes the landscape of novel modalities such as cell and gene therapies, viruses, other novel biologics, oligomers, and emerging technologies, including modern analytics. We summarize the regulatory history and recent landmark developments in some major markets and examine specific chemistry, manufacturing, and controls (CMC) challenges, including suggestions for exploration of potential science-based approaches in support of regulatory strategy development from an industry perspective. In addition, we evaluate the economic factors contributing to patient access to innovation and discuss the impact of regulation. There is a desperate need for a consistent form of regulation where global approaches to regulatory strategies can be harmonized, and specific CMC challenges can be dealt with using the appropriate science and risk-based tools. Although these tools are well described in current guidance documents, the specifics of applicability to complex novel modalities can still result in differing regulatory advice and outcomes. The future goals for efficiently regulating innovative modalities and technologies could be aided by more regulatory harmonization, regulatory education, and industry cooperation through consortia, enabling industry to supply key information to regulators in a transparent yet well-defined manner, and utilizing mutually understood risk-benefit analyses to produce drugs with appropriate safety, efficacy, and quality characteristics.