Separation of Simulants of Biological Warfare Agents from Blood by a Miniaturized Dielectrophoresis Device

Separation of simulants of biological warfare agents from blood using dielectrophoresis (DEP) was demonstrated in a miniaturized DEP device. The device was fabricated by laminating five different layers (all 40 mm×40 mm) including a polycarbonate substrate, a pressure sensitive acrylic adhesive (PSA) layer, a patterned polyimide layer with a flip-chip bonded dielectrophoresis chip (DEP chip), a PSA layer with microfluidic channel, and a glass cover plate. The DEP chip consisted of repetitive interdigitated electrodes with characteristic dimension of 50 m. This device was employed to separate different simulants of biological warfare agents (BWA), namely Bacillus cereus (B. cereus), Escherichia coli (E. coli) and Listeria monocytogenes (L. monocytogenes), from blood, individually or simultaneously. PCR amplification, which was inhibited by blood components in pre-separation samples, successfully revealed bands in post-separation samples containing single or multiple BWA. Up to 97% efficiency of separation was achieved as demonstrated by culturing post-separation E. coli cells. The DEP device described here can potentially be used to reduce sample complexity for detection of infectious disease pathogens and biological warfare agents.     

Insect Cell Culture in Microfluidic Channels

Microfluidic channels were constructed out of polydimethylsiloxane (PDMS) and used as culture vessels for ovary cells from the fall armyworm, Spodoptera frugiperda, (Sf9). PDMS allows cells to be visually inspected and provides excellent permeability to oxygen and carbon dioxide. Cells were grown in static culture conditions and observed every 24 hours for seven days. The growth rate in microchannels of varying volume (2.8 l to 0.6 l) was significantly slower in two sets of experiments (P<0.05 and P<0.001) than in a 25 ml tissue culture flask.     

Cell Culture in 3-Dimensional Microfluidic Structure of PDMS (polydimethylsiloxane)

In this paper, a device with 3-dimensional microfluidic structure composed of two stacked layers of PDMS (polydimethylsiloxane) is fabricated for mammalian cell culture. This microdevice is tested with Hepatocarcinoma liver cells (Hep G2 cells). The purpose of this study is to understand to what extent cell culture in a PDMS microdevice is available. The experimental protocols for Hep G2 cell culture in the microdevice, such as sterilization steps, collagen pre-coating, etc. have been investigated and established. The oxygen supply could be achieved thanks to the high gas permeability of the PDMS material without any external oxygen supplying system. The cells could be kept in good condition for several days with the present set-up as far as the culture medium is periodically changed. Morphological observations of the cells have shown that they could successfully attach, spread and grow until they reached the confluence over the microfluidic structure. By measuring the glucose consumption and albumin production, the activity of the cells was monitored, and those values had increased gradually along the term of the culture. Those encouraging results illustrate the good cell response to the microfluidic structure, in other words, the culture environment made of PDMS material. In future work, this culture system will be extended to non-cancerous liver cells like normal hepatocytes or endothelial cells.     

雪旺细胞分离技术的研究

作者用酶消化的方法分离雪旺细胞（Schwanncell，SCs），比较了匀浆与否，及不同浓度的胰酶、胶原酶、胰酶／胶原酶混合液对不同材料所获分离SCs数量的影响。发现：1．不同酶的消化效果有一定的差异，其中胶原酶最好，胰酶／胶原酶混合液次之。2．浓度高的酶较浓度低者分离的细胞多。3．匀浆后用酶消化较不匀浆者效果好。4。匀浆后用酶消化，SCs数于20分钟时达到高峰；若不匀浆，酶消化30-40分钟SCs数才达到高峰。5．从脊神经节分离获得的SCs较坐骨神经者多。     

The Irre Cell Recognition Module (IRM) Proteins

Abstract: One of the most challenging problems in developmental neurosciences is to understand the establishment and maintenance of specific membrane contacts between axonal, dendritic, and glial processes in the neuropils, which eventually secure neuronal connectivity. However, underlying cell recognition events are pivotal in other tissues as well. This brief review focuses on the pleiotropic functions of a small, evolutionarily conserved group of proteins of the immunoglobulin superfamily involved in cell recognition. In Drosophila, this protein family comprises Irregular chiasm C/Roughest (IrreC/Rst), Kin of irre (Kirre), and their interacting protein partners, Sticks and stones (SNS) and Hibris (Hbs). For simplicity, we propose to name this ensemble of proteins the irre cell recognition module (IRM) after the first identified member of this family. Here, we summarize evidence that the IRM proteins function together in various cellular interactions, including myoblast fusion, cell sorting, axonal pathfinding, and target recognition in the optic neuropils of Drosophila. Understanding IRM protein function will help to unravel the epigenetic rules by which the intricate neurite networks in sensory neuropils are formed.     

高速枪弹压力波对肝细胞的损伤作用及机理探讨

为了从致伤理角度了解压力波对肝细胞究竟有无作用及机理，本文采用培养肝细胞在体外用高速枪弹的压力波致伤并结合高速摄影技术，观察压力波对肝细胞的作用情况。结果发现伤后肝细胞有明显损伤：ＡＴＰ含量减少，ＬＰＯ升高，上清液中ＧＰＴ水平增高。压力波大，细胞损伤越明显，压力波引起细胞损伤的机制与它对细胞的直接剧烈牵拉破损作用及引发自由基反应有关。     

基于微流控芯片的姜黄素诱导MCF-7细胞凋亡的研究

目的探讨应用微流控芯片实现高内涵药物筛选（high content screening,HCS）的可行性。方法本文将微流控芯片技术与HCS技术相结合,通过自行设计、制作聚二甲基硅氧烷（polydimelhylsiloxane,PDMS）-玻璃微流控芯片,并在芯片上实现人乳腺癌MCF-7细胞培养、脂质体转染、药物姜黄素刺激等操作,最后通过显微成像技术进行检测。结果姜黄素可以诱导MCF-7细胞凋亡,并呈浓度依赖性,同时获得了细胞在凋亡过程中一些生物信息的改变：随着姜黄素浓度的增加,细胞凋亡比例、EndoG—GFP重定位比例增大,膜通透性增加,细胞核固缩变小。结论上述微流控芯片可以为HCS技术提供良好的研究平台。     

沉默 NEK2 通过调节 Wnt 信号通路抑制非小细胞肺癌的细胞增殖

目的 探讨沉默有丝分裂相关激酶 2 ( never in mitosis related kinase 2, NEK2) 对非小细胞肺癌 (non-small cell lung cancer, NSCLC) 细胞生物学行为的影响及其可能机制。 方法 收集 27 对 NSCLC 和邻 近的正常组织, 免疫组化检测组织 NEK2 表达, 收集临床病理数据。 生物信息学分析研究 NEK2 在肺腺癌 中的表达和预后价值。 通过 NCI-H1299 和 A549 细胞体外转染 siRNA 敲低 NEK2, 通过免疫印迹及 qPCR 实 验验证敲低效果, CCK-8 评估细胞增殖能力, 流式细胞术评估细胞凋亡及周期, 蛋白印迹测定 Wnt 信号通 路相关蛋白表达。 结果 在肺腺癌组织中的 NEK2 表达显著高于癌旁组织, ⅡB-ⅢB 级组显著高于ⅠA-ⅡA 级组, 从 TCGA 数据库获得的数据也显示肺腺癌患者的 NEK2 明显高于相应的对照组, 并与预后不良显著 相关。 转染 NEK2 siRNA 显著降低了 NCI-H1299 和 A549 细胞的增殖, 细胞阻滞在 G0 / G1期, 细胞凋亡率增 加。 此外, 转染 NEK2 siRNA 的 NCI-H1299 和 A549 细胞中 Wnt 和 β-catenin 的表达水平显著下调, 而 p-GSK3β 的表达水平上调。 结论 siRNA 干扰 NEK2 的表达可抑制非小细胞肺癌细胞的增殖, 其机制可能是 通过抑制 Wnt / β-catenin 通路的促进细胞凋亡和周期阻滞。     

多种物理方法分离脂肪源基质血管组分细胞的比较

文题释义： 脂肪源基质血管组分：是指来源于脂肪组织的基质血管组分细胞,它广泛存在于脂肪组织中,主要包含脂肪来源干细胞、内皮祖细胞、造血干细胞、抗炎细胞、T细胞等混合细胞成分。由于它相对较易获取且对供体不会造成过大的损伤,同时可以避免细胞培养等优点,被认为是干细胞医学良好的细胞来源。 物理方法：酶解法为国际公认的富集基质血管组分的方法,但加入了外源性消化物质,国际上不允许用于临床试验,故使用不加入外源消化物质的物理方法获取基质血管组分即成为了研究的主要方向。 背景：脂肪源基质血管组分和脂肪源性干细胞在组织工程中的应用受到越来越多科研工作者的关注。当前,分离脂肪源基质血管组分的方法主要有酶解法和推注法,但这两种方法都存在着不容忽视的缺点。 目的：寻找一种更加高活性、安全、简便的制备脂肪源基质血管组分的方法。 方法：以无任何处理的脂肪组织为阴性对照,酶解法为阳性对照,通过细胞量、存活率、细胞碎片、细胞活性、增殖率等指标来比较酶解法、普通推注法、改良推注法、玻璃珠破碎法(简称玻璃珠法)及内置式超声波破碎法(简称内置超声波法)的差异。酶解法及普通推注法为目前分离脂肪源基质血管组分细胞普遍使用的方法;改良推注法是在普通推注法的基础上进行改良后得到的方法;玻璃珠法是利用玻璃珠震荡产生的剪切力,在脂肪颗粒中加入玻璃珠后在2 500 r/min的条件下震荡9 min以制备基质血管组分细胞;内置超声波法则是利用空化效应,在25 W的功率下对脂肪组织处理36 s以获得基质血管组分细胞。 结果与结论：①5种方法获得的基质血管组分细胞的大小差异无显著性意义(P > 0.05);②阴性对照组细胞活性最低,酶解法细胞活性最高,酶解法、玻璃珠法及内置超声波法的细胞活性高于改良推注法和普通推注法(P < 0.05);③酶解法、玻璃珠法及内置超声波法的细胞存活率、细胞增殖率高于改良推注法和普通推注法   (P < 0.05);④酶解法、玻璃珠法及内置超声波法细胞碎片比例、细胞凋亡率要远远低于普通推注法和改良推注法(P < 0.05);⑤结果表明,玻璃珠法和内置超声波法富集基质血管组分细胞的效果优良,但玻璃珠法加入了外源物质进行处理,增加了污染的风险,而内置超声波法尽管将超声探头插入脂肪组织中,但只要将超声探头彻底灭菌,即可将污染风险降到最小。总的来说,内置超声波法和玻璃珠法优于普通推注法及改良推注法。 ORCID: 0000-0003-1692-6038(张玲华) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Isolation of myeloid-derived suppressor cells subsets from spleens of orthotopic liver cancer-bearing mice by fluorescent-activated and magnetic-activated cell sorting: similarities and differences

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that commonly expand during tumor development and that play a critical role in suppression of immune responses. MDSCs can be classified into two groups: Mo-MDSCs and G-MDSCs. These cells differ in their morphology, phenotype, differentiation ability, and immunosuppressive activity, and inhibit immune responses via different mechanisms. Therefore, identifying an effective method for isolating viable Mo-MDSCs and G-MDSCs is important. Here, we demonstrated the differences and similarities between fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) in sorting G-MDSCs and Mo-MDSCs. Both MACS and FACS could obtain G-MDSCs and Mo-MDSCs with high viability and purity. A high yield and purity of G-MDSCs could be obtained both by using FACS and MACS, because G-MDSCs are highly expressed in the spleen of tumor-bearing mice. However, Mo-MDSCs, which comprise a small population among leukocytes, when sorted by MACS, could be obtained at much greater cell number, although with a slightly lower purity, than when sorted by FACS. In conclusion, we recommended using both FACS and MACS for isolating G-MDSCs, and using MACS for isolation of Mo-MDSCs.     

Modelling host tissue degradation by extracellular bacterial pathogens.

Extracellular bacterial pathogens such as Pseudomonas aeruginosa are able to penetrate into host tissues (given an initial breech in the outer barrier, e.g. a wound) through the action of exo-toxins and degradative exo-enzymes. A mathematical model of this process is presented which, in the absence of significant immune response, predicts the progression of the bacteria into the tissue as a travelling wave whose velocity can be determined explicitly in terms of the model parameters. Simple in vitro experiments in protein-based matrices are performed which yield results consistent with this behaviour. A complementary in vitro experimental system with distinct qualitative behaviour is also studied, giving further insight and confidence in the modelling approach.     

Effects of the homeopathic preparation Engystol on interferon-gamma production by human T-lymphocytes

There is a growing interest in complementary medical practices, but few studies have investigated mechanisms behind the possible benefits. The effects of the homeopathic preparation Engystol on interferon-γ producing T-lymphocytes were studied in vitro. Lymphocytes were isolated from 30 healthy human volunteers and the percentage of interferon-γ producing cells was analysed by fluorescence activated cell sorting. Cells were treated with NaCl (control) or Engystol at concentrations from undiluted to 2%. All concentrations of Engystol increased the percentage of interferon-γ producing lymphocytes significantly, from a mean of 20.9% ± 10.5% to over 24%. There was no dose-dependence of the effect at the concentrations tested.     

免疫磁珠分选系统在分离大鼠骨髓干细胞群中的应用

采用免疫磁珠分选系统(MACS)分离大鼠骨髓Thy-1.1 干细胞群。收集大鼠胫骨和股骨的骨髓细胞,Percoll密度梯度离心分离单个核细胞,Thy1.1单抗标记,间接MACS分离纯化Thy-1.1 干细胞群,流式细胞仪检测其纯度和分选前后CD34 百分比,台盼兰拒染法检测细胞活力,计算回收率。结果表明分选后的Thy-1.1 细胞纯度达94.2%,回收率64.7%;分选后细胞活力为99.6%,与分选前99.8%无明显差异;分选前CD34 百分比为1.2%,与分选后1.3%无差异。MACS能有效分选大鼠骨髓Thy-1.1 干细胞群,所得细胞纯度高,细胞活力保持好。大鼠Thy-1.1抗原与CD34分子无明显相关性。     

Substance P primes lipoteichoic acid- and Pam3CysSerLys4-mediated activation of human mast cells by up-regulating Toll-like receptor 2

We investigated the distribution of natural killer (NK) cell subsets, their activating and inhibitory receptors, and their cytolytic potential, in primary human immunodeficiency virus (HIV)-infected (PHI) individuals at baseline and during 1 year of follow-up with or without antiretroviral therapy, and compared the results with those obtained in treatment-naïve, chronically HIV-infected (CHI) individuals, and HIV-seronegative (HN) healthy individuals. The proportion of the CD56^dim and CD56^bright subsets decreased with disease progression, whereas that of the CD56⁻ CD16⁺ subset increased. In the CD56^dim subset, the proportion of cells with natural cytotoxicity receptors (NCRs) decreased with disease progression, and their cytolytic potential was reduced. Conversely, the CD56^bright subset was characterized by a high proportion of NCR-positive, killer cell immunoglobulin-like receptor (KIR)-positive NKG2A⁺ cells in both CHI and PHI individuals, which was associated with an increase in their cytolytic potential. During the 1 year of follow-up, the PHI individuals with high viraemia levels and low CD4⁺ T-cell counts who received highly active antiretroviral therapy (HAART) had a similar proportion of NK subsets to CHI individuals, while patients with low viraemia levels and high CD4⁺ T-cell counts who remained untreated had values similar to those of the HN individuals. Our results indicate a marked perturbation of the NK cell compartment during HIV-1 infection that is multifaceted, starts early and is progressive, primarily involves the CD56^bright subset, and is partially corrected by effective HAART.     

The frequency of phospholipase A2 binding of basophilic granulocytes does not decrease during bee‐venom‐specific immunotherapy

BACKGROUND: The major allergenic component of bee venom is phospholipase A2 (PLA2). METHODS: In this study, PLA2 was used to analyze and enrich PLA2-binding cells from peripheral blood by high gradient magnetic cell sorting. RESULTS: In normal donors, the frequency of allergen (PLA2)-binding cells among peripheral blood mononuclear cells (PBMC) as determined by flow cytometry is below 0.1%, whereas in bee-venom-allergic patients, PLA2-binding cells are readily detectable at frequencies of up to 2.3%. In severely bee-venom-allergic patients, many basophilic granulocytes are present, as defined by anti-CD9, CD25, and CD38 mAb, comprising up to 95% of the PLA2-binding cells. From blood of allergic and normal donors, about equal absolute numbers of allergen-binding CD19/21-positive B cells can be enriched. Severe anaphylactic reactions (Mueller grade IV) and failure of or adverse reactions during immunotherapy are associated with high numbers of circulating allergen-binding basophils. Interestingly, in the patients studied, the number of PLA2-binding basophilic granulocytes did not markedly change during rush immunotherapy and up to 6 months of maintenance immunotherapy. CONCLUSIONS: The specific and reproducible enrichment of PLA2-binding cells provides a new tool for the analysis and monitoring of effector cells in bee-venom-allergic patients with immediate-type hypersensitivity.     

Feedback Inhibition of B Cell Differentiation by Monomeric Immunoglobulin

Polyspecific monomeric immunoglobulin (Ig) isolated from either a commercial source (pooled, > 2000 donors), or an autologous donor was capable of inhibiting both B cell proliferation, induced by T dependent mitogens or T cell factors and B cell differentiation, induced by similar stimuli. These effects appear to be directed at the B cell itself since inhibition of differentiation is detectable when monomeric Ig is added to cultures of B cell lines in the presence of B cell differentiation factor (BCDF). The inhibition of B cell differentiation does not appear to relate to inhibition of B cell proliferation, as no detectable change is seen in thymidine incorporation or cell number in these cultures. Furthermore, the effect of monomeric Ig appears to relate to an early event in B cell differentiation, as there is no effect of IgSRK on spontaneously secreting B cell lines and maturation to cytoplasmic Ig containing cells is markedly impaired. Therefore, monomeric Ig secreted by B cells may serve as an immunoregulator of further Ig secretion.     

同种特异T细胞疫苗诱导移植物存活期延长的研究——Ⅱ.抗独特型T细胞免疫反应

同种特异T细胞疫苗(TCV)免疫诱导出同种免疫反应低下,同种移植物存活时间显著延长,推测其作用机制可能是同种特异TCV免疫诱导机体抗同种特异TCV(独特型)T细胞的上调.实验证实了“抗TCV-T细胞”的存在.以同种特异TCV免疫动物可诱导出对TCV特异的细胞增殖反应.TCV免疫小鼠淋巴细胞能特异杀伤TCV细胞.将TCV免疫小鼠脾细胞作为调节细胞,观察到它能显著地抑制同种MLR,表明它是一“抑制性T细胞”.这些结果提示,同种特异TCV免疫可诱导机体免疫网络中独特型-抗独特型的上调,产生了同种反应T细胞的独特型T细胞,从而保护同种移植物免受同种反应性T细胞的攻击使同种移植物存活时间显著延长.     

EBV LMP2A-specific T Cell Immune Responses Elicited by Dendritic Cells Loaded with LMP2A Protein

Type Ⅱ Epstein-Barr virus （EBV） associated malignancies such as nasopharyngeal carcinoma and non-Hodgkin＇s lymphomas consistently express latent membrane 2A （LMP2A） proteins, which have been suggested to be an ideal target for immunotherapy. In previous studies we have demonstrated that using LMP2A protein loaded dendritic cells, the most powerful antigen processing cells in the body can elicit specific and robust anti-tumor cellular immune response in vitro. In this paper, we further investigated the T cell profile of the anti-tumor immune response. We found that LMP2A specific CD4＋ and CD8＋ T cells could be stimulated by LMP2A protein loaded dendritic cells （DCs）. The Thl type immune response is dominant in the immune response mediated by LMP2A specific CD4^＋ T cells. The CD8^＋ cytotoxic T cells can lyse LMP2A bearing cells effectively and specifically. The CD8^＋ cytotoxic T cells can also secrete high level of intracellular IFN-γ, which indicates these cells are EBV-LMP2A specific cytotoxic T cells. Altogether, our studies proved that LMP2A protein loaded DCs can elicit anti-tumor cellular immune responses efficiently. This study provides a rationale for the DC-based immunotherapy against EBV-LMP2A expressing malignancies.     

Control of Cell Proliferation by Embryonal-Origin Factors

Embryogenesis can be paralleled and contrasted with cancerous cell proliferation; both embryogenesis and cancer are associated with extremely rapid cell proliferation. However, unlike cancer, embryogenesis is characterized by a delicate balance of proliferative and anti-proliferative processes. We have found two chromatographically separated fractions derived from human embryonal neural tissue extracts that significantly suppress the proliferation of human breast cancer cells. The reduction in cell number was time dependent, with maximal inhibition (70%) observed after 4 days of incubation while maintaining cell viability. The anti-proliferative effect was also evidenced by decreased [³H]-thymidine incorporation. Significant inhibition of proliferation of osteosarcoma, fibrosarcoma, and Balb/c 3T3 cell lines was also obtained with a low concentration of the active fractions. Embryonal factors inhibited mouse and rat cell lines, indicating cross-species effectiveness. The SDS-PAGE of the biologically active ? 10.7 kDa region revealed several protein bands, while the biologically active ? 4.5 kDa fraction contained only weakly stainable bands. Thus, the embryo contains factors that control the proliferation of malignant cells. These potent and possibly novel compounds should be investigated for their potential therapeutic role in cancer and other proliferative disorders.