A Comparative Study of the JAM Test and 51Cr‐Release Assay to Assess the Cytotoxicity of Dendritic Cells on Hematopoietic Tumor Cells

Dendritic cells (DCs) are potent antigen presenting cells and possess a direct anti‐tumor cytotoxic ability. Nevertheless, the mechanism of anti‐tumor cytotoxicity by DCs and the methods for its evaluation are not fully elucidated. In order to clarify this mechanism of cytotoxicity, we examined the ability of DCs 1) to suppress [³H] thymidine (³H‐TdR) uptake by tumor cells; 2) to induce cytolysis on ⁵¹Cr‐labeled tumor cells; 3) and to induce DNA fragmentation on ³H‐TdR labeled tumor cells (JAM test). Cytolysis and DNA fragmentation are markers of necrotic and apoptotic mechanisms of cytotoxicity in vitro, respectively. DCs inhibited approximately 38.6% to 54.8% of the growth of B4D6, NB4, U937, and Daudi cells as evaluated by the uptake of ³H‐TdR. However no cytolysis was verified by ⁵¹Cr‐release assay. On the other hand, cytotoxicity rates found using the JAM test ranged from 3 to 81% depending on the cell line and the effector to target cell ratio. The discrepancy of cytotoxicity between ⁵¹Cr‐release assay and the JAM test may be due to the phagocytosis of apoptotic tumor cells or the absorption of released ⁵¹Cr by DCs surrounding the target cells. In conclusion, the JAM test was more sensitive than the 4‐h and the 10‐h ⁵¹Cr‐release assay to investigate cytotoxicity mediated by DCs toward hematopoietic tumor cell lines in vitro.     

A sensitive assay of cytotoxicity applicable to mixed cell populations

The fluorescent vital dye, Hoechst 33342, was used to stain cultured cells prior to assay of antibody dependent complement mediated cytotoxicity. The fluorescence of nonviable dye stained cells is quenched by cellular uptake of trypan blue, but trypan blue excluding cells remain intensely fluorescent. Detection by fluorescence microscopy of one viable prestained cell per 10(5) unstained cells was accurate and reliable. The technique was found to have sensitivity equal to a clonogenic assay for measuring cytotoxicity. The dye stained cell assay may be used to measure depletion of a selected cell type, when those cells are stained prior to mixing with another cell population. This technique may prove useful to study model systems for depletion of tumor cells or T-cells from bone marrow.     

肿瘤炎性微环境与树突状细胞研究进展

肿瘤免疫是近年来的研究热点,已知肿瘤微环境尤其是炎性微环境在促进肿瘤发生、发展过程中起重要作用。肿瘤浸润的树突状细胞（DC）多存在表型未成熟化、分布异常及功能障碍等现象,这可能是肿瘤诱导机体免疫耐受的重要机制之一。肿瘤炎性微环境可通过多种途径调节DC的分化和成熟,相关信号通路和分子机制正逐步得到阐明,此将为DC疫苗的研制和肿瘤免疫治疗提供新的希望。     

A real‐time digital bio‐imaging system to quantify cellular cytotoxicity as an alternative to the standard chromium‐51 release assay

Reliable measurement of cellular cytotoxicity is essential for the characterization of immune responses and for the monitoring of antibody treatment efficacy. Until now, the standard ⁵¹Cr‐release assay has remained the sole sensitive assay that measures cellular cytotoxicity. Alternative non‐radioactive assays have been developed but they do not provide accurate measurement of target cell cytotoxicity. The cost and hazard of handling radioactivity are strong incentives to find alternative solutions to ⁵¹Cr. We took advantage of the recent development of cell‐imaging multimode readers to develop a novel non‐radioactive and real‐time cytotoxic assay that demonstrates good reproducibility and sensitivity. The extent of target‐cell cytotoxicity is monitored over time by imaging and quantifying live fluorescent target cells in 96‐well plates. We have developed classical natural killer cell assays in the presence or absence of blocking antibodies and antibody‐dependent cell‐mediated cytotoxicity. We show that in these assays, cell killing occurs within the first 2 hr with half maximum killing reached after 30 min. This technology has numerous applications such as natural killer and T‐cell cytotoxicity assays and can be extended to cell survival and apoptosis measurement assays.     

树突状细胞对佐剂性关节炎作用的实验研究

本文建立了大鼠佐剂性关节炎模型，经体内应用ＷＺＤ５（大鼠树突状细胞单抗）和输注新鲜分离的同系大鼠淋巴树突状细胞，以模型鼠的关节肿胀率和关节组织的病理改变、ＣｏｎＡ刺激淋巴细胞增殖反应和循环免疫复合物等为指标，来分析树突状细胞在佐剂性关节炎发病过程中的作用。结果表明：该细胞具有促进佐剂性关节炎的作用，而ＷＺＤ５则能减少关节肿胀率、减轻病变、缩短病程。     

Effect of laminin environments and tumor factors on the biology of myeloid dendritic cells

Dendritic cells (DCs) are immune cells that surveil the organism for infections or malignancies and activate specific T lymphocytes initiating specific immune responses. Contrariwise, DCs have been show to participate in the development of diseases, among them some types of cancer by inducing angiogenesis or immunosuppression. The ultimate fate of DC functions regarding their role in disease or health is prompted by signals from the microenvironment. We have previously shown that the interaction of DCs with various extracellular matrix components modifies the immune properties and angiogenic potential of these cells.The objective of the current studies was to investigate the angiogenic and immune profile of murine myeloid DCs upon interaction with laminin environments, with a particular emphasis on ovarian cancer.Our results show that murine ovarian tumors produce several types of laminins, as determined by PCR analysis, and also that tumor-associated DCs, both from ascites or solid tumors express adhesion molecules capable of interacting with these molecules as determined by flow cytometry and PCR analysis. Further, we established that DCs cultured on laminin upregulate both AKT and MEK signaling pathways, and that long-term culture on laminin surfaces decreases the immunological capacities of these cells when compared to the same cells cultured on synthetic substrates. In addition, we observed that tumor conditioned media was able to modify the metabolic status of these cells, and also reprogram the development of DCs from bone marrow precursors towards the generation of myeloid-derived suppressor cells.Overall, these studies demonstrate that the interaction between soluble factors and extracellular matrix components of the ovarian cancer microenvironment shape the biology of DCs and thus help them become co-conspirators of tumor growth.     

树突状细胞体外定向诱导扩增及其分离纯化

目的：探讨定向诱导及纯化树突状细胞（ＤＣ）的方法，为进一步研究树突状细胞的功能、特性及临床应用提供技术方法。方法：利用免疫磁珠江（ＭＡＣＳ）分离纯化脐血ＣＤ３４细胞，在液体培养体系中加入ＦＬ、ＧＭ－ＣＳＦ、ＴＮＦα、ＩＬ－４ｅｙ ＳＣＦ，培养１２ｄ后光镜检测细胞形态及流式细胞仪分析表达标志；利用抗估状细胞单克隆抗体（Ｘ－１１）及免疫磁珠分离纯化树突状细胞，流式细胞仪检测纯化后ＤＣ的纯度。结果：经体     

树突状细胞疫苗在妇科肿瘤免疫治疗中的研究进展

树突状细胞(DC)具有摄取、加工、处理和呈递抗原以及激活初始T淋巴细胞的功能,是体内功能最强的专职抗原递呈细胞,也是免疫应答的关键细胞.近年来研究发现,体外经肿瘤抗原刺激的DC回输到病人体内,能诱导机体产生细胞毒性T淋巴细胞,从而发挥特异性肿瘤杀伤作用.根据这一发现研制的DC疫苗目前在妇科肿瘤免疫治疗中已取得重要进展,...     

含CpG基序的寡核苷酸对树突状细胞的作用及其机制

树突状细胞(dendriticcells,DC)是目前所知功能最强的抗原提呈细胞(antigenpresentingcells,APC),其最大特点在于能激活初始型T细胞(naiveTcell),是机体免疫应答的始动者,在免疫应答中起着重要的作用。DC的成熟、活化是其发挥免疫作用的先决条件,因而成为研究的焦点。目前已发现刺激DC成熟的多种因素,包括肿瘤坏死因子-α(tumornecrosis factor-α,TNF-α)、CD40-CD40L、脂多糖(lipopolysaccharide,LPS)和含有非甲基化胞嘧啶鸟嘌呤二核苷酸(cytosinephosphate-guanosine,CpG)基序的寡核苷酸(CpGmotif-containingoligodeoxynucle …     

致敏树突细胞及exosome对胃癌的免疫治疗作用研究

目的 :了解胃癌组织中树突细胞 (DC)的功能状态 ,探讨DC及其分泌的亚细胞成分exosomes用于防治胃癌的可能性 ,旨在为胃癌的防治探索新的途径。方法 :利用致敏DC及其所分泌的亚细胞成分exosomes对荷胃癌小鼠进行免疫治疗 ,观察其免疫治疗效果。结果 :荷胃癌小鼠经胃癌细胞RNA致敏的DC及其分泌的亚细胞成分exosomes治疗后 ,小鼠腹水产生率、肿瘤转移率、动物存活率、瘤体平均重量等指标均明显优于对照组 (P <0 0 1) ,局部组织中IL 12、IFN γ和IL 18的基因表达水平都明显升高 (P <0 0 1) ,肿瘤局部CD4 + 、CD8+ 细胞数量增加 (P <0 0 5 ) ,TIL细胞毒活性明显增强。结论 :胃癌RNA致敏的DC及其所分泌的亚细胞成分exosomes,能促进胃癌抗原的提呈 ,并启动和增强机体的抗瘤免疫功能 ,改善临床症状和生存质量 ,对荷胃癌小鼠具有一定的免疫治疗效果。     

Influence of autologous dendritic cells on the in-vitro expansion and functions of peripheral blood NK cells

Abstract

Context: Allogeneic reactive NK cells were previously shown to exert a graft-versus-leukemia (GVL) effect during allogeneic hematopoietic stem cell transplantation, as well as reduce the incidence of graft-versus-host disease (GVHD).

Objective: We used autologous immature DCs as feeder cells for the in-vitro expansion of NK cells and studied the function of the NK cell cultures.

Materials and methods: NK cells were cultured for 15 days in the presence of autologous, immature DCs. Fold expansion, killing activity and expression of IFN-γ, perforin and granzyme B were evaluated.

Results: The highest NK cell expansion efficiency was observed when the ratio of NK cells:DCs was 2:1 and when cells were cultured in a contact-dependent manner. The killing activity of NK cells was highest when the NK:DC ratio was 10:1. NK cell cultures exhibited a significant upregulation in the mRNA expression of IFN-γ, perforin and granzyme B when the ratio of NK cells to DCs was 10:1.

Discussion: We successfully amplified NK cells using autologous immature DCs derived from human peripheral monocytes after induction as feeder cells. The use of autologous immature DCs for ex-vivo expansion of NK cells can be clinically applied to overcome limitations, such as the small number of NK cells in peripheral blood, and the high cost of NK cell sorting. Transfusion of allogeneic reactive NK cells has been suggested as a potential adjunctive therapeutic strategy after transplantation.

Conclusion: Autologous immature DCs can be used as feeder cells for ex-vivo expansion of functional NK cells.     

PDOX与DOX对乳腺癌MCF-7细胞的杀伤作用比较及毒性研究

目的：比较PDOX与阿霉素(doxorubicin,DOX)对乳腺癌MCF-7细胞的杀伤作用,阐述PDOX可能的作用机制,同时研究PDOX对正常肝细胞LO2、肾小管细胞NRK52E的毒性。方法：用DOX及PDOX按一定的浓度梯度的分别处理乳腺癌MCF-7细胞,计算两种药物对MCF-7细胞的半数抑制浓度;用相同浓度梯度的DOX及PDOX处理人正常肝LO2细胞、大鼠肾小管上皮NRK-52E细胞,比较PDOX、DOX对LO2、NRK-52E的毒性大小。流式细胞术分析PDOX、DOX处理后MCF-7细胞的周期分布情况;采用TUNEL、Hoechst染色、对PDOX、DOX处理后的MCF-7细胞进行细胞凋亡相关形态学分析并计算凋亡细胞比例。结果：DOX、PDOX处理MCF-7细胞48 h后,对MCF-7细胞的IC50分别为0.94、3.91μM;72 h时IC50分别为0.63、1.62μM。体外实验显示PDOX对LO2、NRK-52E细胞的细胞毒性较DOX小;PDOX处理后的细胞周期被阻滞在G1/S期;PDOX及DOX处理后的细胞表现出明显的凋亡细胞特点;1.96、3.91μM DOX或者1.96、3.91μM PDOX处理48 h后,各组的细胞凋亡率分别为46.1%、61.1%、41.3%及48.1%。结论：PDOX在体外诱导MCF-7细胞凋亡的能力较相同浓度的DOX弱;PDOX对LO2、NRK-52E细胞的毒性较DOX小。     

The effects of glucocorticoid therapy on the inflammatory and dendritic cells in muscular dystrophies

Various clinical trials have documented the therapeutic benefit of glucocorticoids (GCs) in enhancing muscle strength and slowing disease progression of Duchenne and Becker muscular dystrophies (DMD/BMD). We hypothesized that GCs may have relevance to the differential anti-inflammatory effect on mononuclear inflammatory cells (MICs) and Dendritic cells (DCs) infiltrating the dystrophic muscles. In this prospective study, two muscle biopsies were obtained (before and after 6-month prednisone therapy) from 30 patients with dystrophies (DMD = 18; BMD = 6; and limb girdle muscular dystrophies (LGMD) = 6). MICs and DCs infiltrating the muscles were examined using mouse monoclonal antibodies and immunoperoxidase staining methods. Muscle strength was evaluated monthly by manual testing, motor ability and timed tests. Prednisone therapy was associated with: (i) functional improvement of overall motor disability, in upper limbs of DMD (P < 0.001) and BMD (P < 0.01) and lower limbs of DMD (P < 0.001) and BMD (P < 0.05); (ii) histological improvement such as fibre size variation (DMD, P < 0.01; BMD, P < 0.05), internalization of nuclei (DMD, P < 0.05), degeneration and necrosis (DMD and BMD, P < 0.01), regeneration (DMD, P < 0.001; BMD, P < 0.01) and endomysial connective tissue proliferation (DMD, P < 0.01; BMD, P < 0.05) and (iii) reduction of total MICs (P < 0.01) and DCs (P < 0.01). There was a positive correlation between the degree of improvement in overall motor disability and reduction of DCs numbers (In upper limbs; r = 0.638, P < 0.01 for DMD and r = 0.725, P < 0.01 for BMD, in Lower limbs; r = 0.547, P < 0.05 for DMD and r = 0.576, P < 0.05 for BMD). Such improvements and changes of MICs/DCs were absent in LGMD. In DMD/BMD, prednisone therapeutic effect was associated with reduced MICs and DCs numbers. Whether this therapeutic effect reflects targeting of the deleterious immune response produced by these cells mandates further investigations.     

Targeting dendritic cells through gold nanoparticles: A review on the cellular uptake and subsequent immunological properties

Gold nanoparticles (NPs) have been proposed as a highly potential tool in immunotherapies due to its advantageous properties including customizable size and shapes, surface functionality and biocompatibility. Dendritic cells (DCs), the sentinels of immune response, have been of interest to be manipulated by using gold NPs for targeted delivery of immunotherapeutic agent. Researches done especially in human DCs showed a variation of gold NPs effects on cellular uptake and internalization, DC maturation and subsequent T cells priming as well as cytotoxicity. In this review, we describe the synthesis and physiochemical properties of gold NPs as well as the importance of gold NPs in immunotherapies through their actions on human DCs.     

孕酮对人树突状细胞成熟和免疫功能的影响

目的：研究孕酮（P₄）对人外周血来源树突状细胞（DCs）成熟和免疫学功能的影响。方法：在人外周血来源DCs体外培养时加入两种浓度的P₄ (10-7 mol/L和10-6 mol/L)处理，光镜和电镜下观察DCs的生成情况及形态变化，以流式细胞仪分析各组细胞的免疫表型，用ELISA方法测定其分泌的IL-10和IL-12水平，［3H］-TdR掺入法检测体外混合淋巴细胞反应中DCs刺激同种反应性T细胞的增殖能力。结果：加入P4培养后, DCs树突状和膜面状伪足减少，表达低水平MHC-II分子和共刺激分子CD40、CD80和CD86，其分泌的IL-10水平升高，IL-12水平明显下降，DCs刺激同种反应性T细胞的功能显著下降。结论：P₄抑制人外周血来源DCs的成熟，对其免疫学功能具有负性调节作用。     

树突状细胞和造血干细胞移植的研究进展

树突状细胞(DC)是功能最强的抗原递呈细胞(APC),在移植免疫中,DC启动免疫排斥还是维持免疫耐受取决它们的起源以及成熟状态,成熟DC启动免疫排斥及移植物抗宿主病(GVHD)反应,未成熟(iDC)及淋巴样DC诱导免疫耐受。造血干细胞移植(HSCT)中,可以通过调控不同来源、不同分化状态的DC来预防和治疗移植物抗宿主病(GVHD),促进造血干细胞的植入。     

Effects of murine leukemia viruses on the function of dendritic cells

In asymptomatic human immunodeficiency virus-1 infection T cells respond normally to allogeneic dendritic cells (DC), but DC show reduced stimulatory capacity. By contrast in HTLV-1 infection no significant changes in allogeneic stimulation were seen but DC-stimulated activity of autologous T cells. In seeking animal models relevant to these diseases the effects of two murine leukemia retroviruses, Rauscher leukemia virus (RLV) and Moloney leukemia virus (MLV) on the function of dendritic cells and T cells in a primary mixed leucocyte reaction have been tested. Treatment by RLV in vitro suppressed the ability of DC to stimulate allogeneic T cells from healthy animals. MLV at the same concentration did not significantly affect the ability of DC to stimulate allogeneic T cells, but provoked considerable enhancement of the low level stimulation by DC in the syngeneic system. Similar results were obtained following in vivo exposure to viruses. Two pieces of evidence suggested that these effects were due to impairment of DC function and were not operating through infection of T cells. Firstly, exposure of T cells directly to virus in vitro and in vivo before stimulation with untreated allogeneic DC caused no significant alteration in T cell activity. Secondly, the impact of murine leukemia virus on DC function was not abrogated when infected DC were added to normal T cells and cultured in the presence of zidovudine. Treatment of DC by RLV caused a decrease of cluster formation with allogeneic T cells. No statistically significant influence of MLV was observed on cluster formation after 3-h of incubation in the allogeneic system. However, after 18-h incubation MLV-treated DC formed fewer clusters with T cells than untreated DC. At the same time a stimulatory effect of MLV on DC cluster formation with syngeneic T cells was found. Considerable decrease was found in major histocompatibility complex class II antigen and LFA-1 receptor expression on the DC surface in mice infected by RLV MLV induced no significant changes. These mouse retroviruses can therefore cause changes in DC function similar to those already reported using human retroviruses and may provide models for studying their effects.     

In vitro assay to test differential substrate affinities of growing axons and migratory cells

Summary An in vitro assay is presented in which different soluble substrates are arranged in narrow alternating stripes which forces growing axons and migratory cells to choose between them. The usefulness of this assay is exemplified by offering goldfish retinal axons and glial cells of the optic nerve a variety of substrates in stripes. Given a choice between substrates of unequal growth supporting activities axons and migratory cells grow in stripes, thus expressing their preference for one of the substrates. Growth in stripes was observed 1. when a substrate with growth promoting properties was next to one which did not possess these properties, 2. when the growth promoting activity of a substrate applied to both stripes was in one stripe blocked by an antibody, 3. when two different growth promoting substrates were offered.     

Expression of the class A macrophage scavenger receptor on specific subpopulations of murine dendritic cells limits their endotoxin response

Dendritic cells (DC) function at the interface of innate and acquired immunity and are uniquely sensitive to specific stimuli. Pattern recognition receptors (PRR) on these cells are critically important because of their ability to recognise and initiate responses to conserved microbial-associated molecular signatures. With the exception of Toll-like receptors (TLR), we know relatively little about the specific distribution of other PRR amongst populations of DC. Here, we describe the expression of the murine class A macrophage scavenger receptor (SR-A) and show that it is restricted to specific subpopulations of bone marrow-derived and splenic DC. Importantly, we demonstrate that the receptor significantly alters the response of DC to endotoxin. In contrast to the activities of other PRR that have so far been examined, uniquely SR-A limits the maturation response; SR-A-/- cells display enhanced CD40 expression and TNF-alpha production. We discuss the potential contributions of SR-A to DC biology in the context of the known multiple activities of this receptor.     

维甲酸对树突状细胞增殖及细胞骨架微丝的调节作用

目的观察药理剂量的全反式维甲酸对人单核细胞来源的树突状细胞增殖及细胞骨架微丝的调节作用。方法 rhGM-CSF1000U/mL、IL-4500U/mL诱导人外周血单核细胞向树突状细胞分化,在细胞分化过程中加入0.1μmol/L全反式维甲酸,相差显微镜下观察细胞的形态变化及集落形成,活细胞计数和MTT检测细胞的增殖。成熟树突状细胞经全反式维甲酸处理24h后,用FITC标记的鬼笔环肽检测细胞骨架微丝的形态和分布。结果细胞因子诱导的外周血单核细胞来源的树突状细胞具有典型的树枝状结构。在树突状细胞的分化过程中,全反式维甲酸作用组的树突状细胞的产量明显低于对照组(P0.05)。已经分化成熟的树突状细胞经维甲酸处理后细胞骨架微丝出现重排和分布不均。结论药理剂量的全反式维甲酸抑制树突状细胞的增殖和分化,并可能通过细胞骨架调节树突状细胞的功能。