补体C3d-p28增强阿尔茨海默病DNA疫苗Th2型免疫反应的研究

目的 探讨补体C3d-p28作为分子佐剂,在阿尔茨海默病DNA疫苗免疫反应中的作用。方法 分别将重组质粒p(Aβ3-10)10,p(Aβ3-10)10-C3d-p28.3和空载体pc DNA3.1(+)用肌肉注射的方法免疫8~10周龄的雌性BALB/c鼠。质粒注射前24 h,布比卡因肌肉注射诱导轻微的肌肉变性。应用ELISA方法检测血清抗Aβ抗体的滴度、抗体分型、体外脾细胞培养上清液中IL-4和IFN-γ的含量。免疫组织化学染色法检测免疫血清与转基因鼠脑内Aβ斑的结合能力。结果 重组质粒疫苗p(Aβ3-10)10仅诱导出低滴度的抗Aβ抗体,产生了Th1/Th2混合型的免疫反应。而重组质粒疫苗p(Aβ3-10)10-C3d-p28.3诱导出较高滴度的抗Aβ抗体,体外脾细胞培养上清液中IFN-γ低和IL-4高,即引起了Th2型细胞免疫反应,同时产生的抗Aβ抗体能够与双转基因鼠APP/PS1脑中沉积的Aβ斑块结合。结论 补体C3d-p28分子佐剂能够增强抗Aβ抗体的产生并且诱发Th2型的免疫反应。     

构建SD大鼠鱼藤酮中毒模型及其线粒体DNA突变的初步探索

目的构建SD大鼠鱼藤酮慢性中毒模型,同时对SD大鼠组织标本(脑组织和肝脏)进行线粒体DNA突变的初步探索,为后续实验提供线索。方法将75只雄性SD鼠随机分为三组,实验组:0.5 mg/(kg·d)鱼藤酮皮下注射4~8周,共45只;空白组:不予任何特殊处理4~8周,共15只;对照组:相同体积的二甲基亚砜(DMSO)皮下注射4~8周,共15只。其中实验组随机分为1、2、3小组,每小组15只;空白组和对照组也分别分为1、2、3小组,每小组5只。每组内相应的1、2、3小组分别在实验的第4、6、8周处死老鼠并留取相应的组织标本(中脑组织和肝脏),存储于-80℃冰箱中待用。在实验组的每个小组内分别随机选取3对组织标本,共9对;在空白组和对照组的每个小组内分别随机选取1对组织标本,共6对。随后进行组织DNA提取,采用2对引物进行长PCR并将PCR产物进行凝胶电泳分析,同时设计并利用线粒体DNA引物,用一代测序的方法进行线粒体全基因组测序。实验中观察各组大鼠的一般情况并进行行为学测试。结果1)实验组大鼠在实验中表现出较为明显的中毒症状。在体重增长速度上与对照组和空白组存在明显差异(P0.05)。在行为学上,实验组大鼠亦表现出了较为明显的行为学异常(P0.05)。2)大鼠脑以及肝脏组织的长PCR凝胶电泳结果未发现明显的异常条带;所有测序突变(缺失、插入、点突变)均为大鼠固有突变。结论 0.5 mg/(kg·d)鱼藤酮皮下注射4~8周可导致SD大鼠鱼藤酮慢性中毒。SD幼鼠的选择、鱼藤酮剂量及作用时间、一代测序等多种因素可能是导致本次实验未检测到有意义的线粒体DNA突变的原因。因此,后续实验应做相应改进。     

Bone marrow genotoxicity of 2,5‐dimethylfuran,a green biofuel candidate

2,5‐Dimethylfuran (DMF) is being considered as a potential green transportation biofuel, but there is limited information about its toxicity and safety. We examined DMF toxicity in the bone marrow using a murine in vitro erythropoietic micronucleus assay and found that exposure to DMF (0.1 mM, 1 hr) induced an increase in micronuclei frequency compared with controls. These data suggest that DMF may be genotoxic to hematopoietic cells and that more thorough toxicological studies on DMF should be conducted to ensure public and occupational safety before it is considered a viable biofuel and produced in mass quantities. As well as specific data on DMF, our study further validates an in vitro cell culture system that captures the essential features of the in vivo mammalian micronucleus genotoxicity assay, enabling increased throughput and controlled studies on hematopoietic DNA damage response, while reducing animal sacrifice. In vitro assays, such as the in vitro micronucleus assay, will be essential as international chemical policy is increasingly utilizing green chemistry principles that require more toxicological testing. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

Combination of MIDGE-Th1 DNA vaccines with the cationic lipid SAINT-18: Studies on formulation,biodistribution and vector clearance

We have previously shown that the combination of MIDGE-Th1 DNA vectors with the cationic lipid SAINT-18 increases the immune response to the encoded antigen in mice. Here, we report on experiments to further optimize and characterize this approach. We evaluated different formulations of MIDGE-Th1 vectors with SAINT-18 by assessing their influence on the transfection efficiency in cell culture and on the immune response in mice. We found that high amounts of SAINT-18 in formulations with a w/w ratio MIDGE Th1/SAINT-18 of 1:4.8 are beneficial for cell transfection in vitro. In contrast, the formulation of HBsAg-encoding MIDGE-Th1 DNA vectors with the lowest amount of SAINT-18 (w/w ratio MIDGE Th1/SAINT-18 of 1:0.5) resulted in the highest serum IgG1 and IgG2a levels after intradermal immunization of mice. Consequently, latter formulation was selected for a comparative biodistribution study in rats. Following intradermal administration of both naked and formulated MIDGE-Th1 DNA, the vectors localized primarily at the site of injection. Vector DNA levels decreased substantially over the two months duration of the study. When administered in combination with SAINT-18, the vectors were found in significantly higher amounts in draining lymph nodes in comparison to administration of naked MIDGE-Th1 DNA. We propose that the high immune responses induced by MIDGE-Th1/SAINT-18 lipoplexes are mediated by enhanced transfection of cells in vivo, resulting in stronger antigen expression and presentation. Importantly, the combination of MIDGE-Th1 vectors with SAINT-18 was well tolerated in mice and rats and is expected to be safe in human clinical applications.     

The role of epigenetic therapies in colorectal cancer

Although developments in the diagnosis and therapy of colorectal cancer (CRC) have been made in the last decade, much work remains to be done as it remains the second leading cause of cancer death. It is now well established that epigenetic events, together with genetic alterations, are key events in initiation and progression of CRC. Epigenetics refers to heritable alterations in gene expression that do not involve changes in the DNA sequence. These alterations include DNA methylation, histone alterations, chromatin remodelers, and noncoding RNAs. In CRC, aberrations in epigenome may also involve in the development of drug resistance to conventional drugs such as 5-fluorouracil, oxaliplatin, and irinotecan. Thus, it has been suggested that combined therapies with epigenetic agents may reverse drug resistance. In this regard, DNA methyltransferase inhibitors and histone deacetylase inhibitors have been extensively investigated in CRC. The aim of this review is to provide a brief overview of the preclinical data that represent a proof of principle for the employment of epigenetic agents in CRC with a focus on the advantages of combinatorial therapy over single-drug treatment. We will also critically discuss the results and limitations of initial clinical experiences of epigenetic-based therapy in CRC and summarize ongoing clinical trials. Nevertheless, since recent translational research suggest that epigenetic modulators play a key role in augmenting immunogenicity of the tumor microenvironment and in restoring immune recognition, we will also highlight the recent developments of combinations strategies of immunotherapies and epigenetic therapies in CRC, summarizing preclinical, and clinical data to signify this evolving and promising field for CRC treatment.     

DNA transfer: Review and implications for casework

DNA-bearing cellular material can come to be present on a surface by either direct or indirect transfer. Direct transfer includes contact, but also includes activities within the vicinity of an item that may result in the transfer of DNA directly from an individual without any contact, such as speaking, coughing, and sneezing. Indirect transfer of DNA is when DNA from an individual comes to be on an item via an intermediary surface. It is important to consider indirect transfer in the evaluation of trace DNA in casework. The term ‘trace DNA’ in this review refers solely to DNA that cannot be attributed to an identifiable body fluid.This review presents and considers data from trace DNA experiments to establish whether the quantity of DNA recovered from a crime stain and/or the quality of a DNA profile obtained can be used to infer the likely mechanism of transfer. The data show that varied results are obtained from apparently similar trace DNA samples, presumably due to the many factors that affect the detection of trace DNA. The nature and effect of these varying factors and the application of the data to casework is considered generally and with specific reference to DNA transfer to skin, DNA beneath fingernails, ‘wearer DNA’, and various contamination considerations.     

Cytotoxic effects and specific gene expression alterations induced by I-125-labeled triplex-forming oligonucleotides

Abstract

Purpose: Triplex-forming oligonucleotides (TFO) bind to the DNA double helix in a sequence-specific manner. Therefore, TFO seem to be a suitable carrier for Auger electron emitters to damage exclusively targeted DNA sequences, e.g., in tumor cells. We studied the influence of I-125 labeled TFO with regard to cell survival and induction of DNA double-strand breaks (DSB) using TFO with different genomic targets and target numbers. Furthermore, the ability of TFO to alter the gene expression of targeted genes was examined.

Materials and methods: TFO were labeled with I-125 using the primer extension method. DNA triplex formation and sequence-specific DSB were demonstrated in vitro. Cell survival was analyzed by colony-forming assay and DNA damage was assessed by microscopic quantification of protein 53 binding protein 1 (53BP1) foci in the human squamous carcinoma cell line II (SCL-II). Quantitative real-time polymerase-chain-reaction (qRT-PCR) was performed to analyze gene expression alterations.

Results: The sequence-specific induction of a single DSB in a 1695 bp long DNA double stranded fragment was demonstrated in vitro. I-125-labeled TFO binding to single and multiple targets were shown to induce a pronounced decrease in cell survival and an increase of DSB. TFO targeting multiple sites differing in the total target number showed a significant different cell killing per decay that is also in good accordance with the observed induction of DSB. Single gene targeting I-125-labeled TFO significantly decreased cell survival and altered gene expression in the targeted gene.

Conclusions: I-125-labeled TFO enable specific targeting of DNA in vitro as well as in a cellular environment and thus induce sequence-specific complex DNA lesions. Therefore I-125-labeled TFO might be a very useful tool for basic DNA repair research.