银屑病患者外周血单核细胞衍生的树突状细胞的检测

研究证明,皮肤中的DC与外周血单核细胞衍生的树突状细胞(MoDC)的细胞表型一致,由此认为银屑病皮损中数量增加的DC主要由外周血单核细胞游走到局部组织并分化而成。我们最近的研究发现银屑病患者的外周血单核细胞向DC分化的能力增强。在此,我们进一步探讨银屑病患者的MoDC的吞噬功能,以正常人为对照评价它们在抗原摄取阶段的改变。寻常型银屑病患者2 8例(男17例,女11例) ,年龄18～6 8岁,病程2周至2 1年。所有患者在半年内未用系统治疗,2周内未用局部治疗。对照组为医院职工和医学院大学生共12例(男6例,女6例) ,年龄2 4～5 1岁。单核细胞…     

钙离子载体对外周血单核细胞来源的树突状细胞的影响

目的：探讨钙离子载体（calcium ionophore,CI)对外周血单核细胞来源的树突状细胞（DC）的影响。方法：分离分离献血者外周血单核细胞。分别加入重组人粒/单集落刺激因子（rhGM-CSF)100μg/L，rhGM-CSF100μg/L CI10μg/L及rhGM-CSF CI各100μg/L，体外培养40h后，于光镜及电镜下观察细胞的形态，流式细胞仪检测细胞的表面标志，MTT比色法检测上述分子对自体T细胞的刺激增殖作用。结果：外周血单核细胞在GM-CSF CI各100μg/L的条件下培养40h，就可看到典型的DC形态，其表面CD14分子表达减少。HLA-DR，CD40，CD83及CD86分子的表达明显增高，且具有明显刺激自体T细胞增殖的能力。结论：CI有显著加速GM-CSF诱导的外周血单核细胞向DC转化的作用。     

钙离子载体诱导人外周血单个核细胞向树突状细胞分化的信号转导

目的 :初步探讨钙离子载体 (calciumionophore,CI)诱导人外周血单个核细胞 (PBMC)向树突状细胞 (DC)分化的细胞信号转导途径。方法 :分离健康献血者的PBMC ,加入rhGM CSF及A2 3187各 10 0 μg/L ,部分细胞预先用W 7(10 μmol/L)或CsA(0 .5mg/L)或KT5 92 6 (1μmol/L)处理 30min后 ,再加入rhGM CSF及A2 3187各 10 0 μg/L。体外培养 4 0h后 ,于相差显微镜下观察细胞的形态 ;流式细胞仪检测细胞的表面标志 ;用MTT比色法检测上述细胞刺激同种异体T细胞的增殖作用。结果 :与 10 0 μg/L的A2 3187及rhGM CSF共同培养 4 0h的健康献血者的PBMC ,出现典型的树突状突起 ,同时CD83、CD80及CD86分子的表达上调 ,CD14分子的表达下调 ,刺激同种异体T细胞增殖的能力增强 ;而预先用W 7或CsA或KT5 92 6处理 30min后、再给予rhGM CSF及A2 3187处理的PBMC ,其形态、表面标志物及对T细胞的刺激增殖能力 ,均不同程度的受到抑制。结论 :CI诱导的PBMC向DC的分化 ,可能受控于Ca2 /钙调蛋白及其下游的多个细胞信号转导途径的调节     

类风湿性关节炎中树突状细胞亚群分析

目的 分析类风湿性关节炎患者外周血和关节液中的DC(dendritic cell,DC)亚群以及DC亚群与炎症产生的关系.方法 收集来自西南医院体检中心26例健康人外周血作为对照组,来自西南医院风湿免疫科28例RA患者的外周血及滑膜液作为实验组,其中RA患者的外周血和滑膜液来自同一 RA患者.采用人外周血淋巴细胞分离液...     

人外周血单核细胞来源树突状细胞的成熟诱导

目的：探讨诱导树突状细胞成熟的最优方法。方法：以细胞因子GM-CSF和IL-4体外诱导人单核细胞来源的树突状细胞，分别采用CD40L、LPS、TNF-α、细胞因子鸡尾酒法（TNF-α、IL-6、IL-1β、PGE2）诱导成熟，24小时后收获DCs以流式细胞仪检测其成熟表型CD80、CD83、CD86、HLA-DR和FITC-Dextran的内吞能力，ELISA法检测其IL-12的分泌，MTT法检测其刺激淋巴细胞增殖活性。结果：CD40L、LPS、TNF-α、鸡尾酒法均可诱导DCs的成熟，其中以鸡尾酒法诱导成熟的效果最优，CD83的表达率为66.91%（P〈0.05）；成熟DCsFITC-Dextran的内吞能力明显下降；成熟DCsIL-12分泌量明显高于未成熟DCs，其中鸡尾酒法诱导成熟的DCs的IL-12分泌量最高，成熟的DCs有较强的刺激淋巴细胞增殖能力。结论：细胞因子鸡尾酒法是诱导DCs成熟的最佳方法。     

人外周血树突状细胞的研究现状

人外周血树突状细胞(DC)是一种独特的细胞,其形态、组织化学、免疫细胞化学反应和超微结构均与单核细胞不同。无吞噬能力,也无单核细胞、淋巴细胞和NK细胞所特有的标志。它是T细胞免疫反应的主要辅助细胞。并参与多种自身免疫疾病、免疫缺陷病、肿瘤和某些炎症反应的病理过程。观察外周血中DC的质和量的变化,将为了解这些疾病的发生、发展和预后提供有价值的参数。     

少量人外周血体外培养鉴定单核细胞来源树突状细胞方法初探

目的:在少量人外周血条件下体外培养并鉴定单核细胞来源树突状细胞(Monocyte-derived dendritic cells,Mo DC)。方法:取健康成人少量新鲜外周血经改良密度梯度离心法分离获得单核细胞,加入重组人粒-巨噬细胞集落刺激因子(rh GM-CSF)、重组人白细胞介素4(rh IL-4)诱导Mo DC生长,并用肿瘤坏死因子α(TNF-α)刺激成熟,倒置显微镜及扫描电镜观察细胞形态;分别于培养第4天和第7天用流式进行表型鉴定、CCK-8法检测同种异体混合淋巴细胞反应鉴定抗原递呈能力。结果:Mo DC呈类圆形,聚集成团,悬浮生长,扫描电镜观察其表面有典型毛刺状突起;流式检测Mo DC高表达CD11c、CD1a,经TNF-α刺激后的Mo DC表面MHCⅡ、CD80、CD83、CD86表达均较培养4 d的Mo DC明显升高,具有统计学意义(P0.05);经TNF-α刺激后的Mo DC刺激同种异体淋巴细胞增殖能力较培养4 d的Mo DC明显升高,具有统计学意义(P0.05)。结论:用本实验方法可从少量人外周血中获得成熟Mo DC,为其在多种变态反应疾病、自身免疫性疾病及肿瘤疫苗等领域的研究提供基础保障。     

人外周血及脐血枝突状细胞的体外分离培养

取正常人外周血或脐血，经淋巴细胞液分离，取中间白膜层，培养板中进行粘附，粘附细胞加培养液和细胞因子培养，对其形态，表型和功能分别进行鉴定和测定。结果表明约经过１周左右培养，悬浮细胞表现为典型的ＤＣ形态，带有毛刺样凸起，经ＤＣ单克隆抗体染色后用流式细胞仪测定脐血７２％为ＤＣ，外周血９３％为ＤＣ，并且可以刺激同种异体淋巴细胞的增殖反应。所以通过这样的不同细胞因子组合可以从人外周血和脐血中诱导培养出大量     

人血树突状细胞与单核细胞的比较观察

对人血树突状细胞和单核细胞的光镜、透射电镜和扫描电镜观察表明：Ｇｉｅｍｓａ染色两菜态似；但树突状细胞呈Ｓ－１００蛋白阳性，Ｌｙｓｏｚｙｍｅ阴性，细胞表面光滑，有各种突起，细胞器很少。而单核细胞呈Ｌｙｓｏｚｙｍｅ阳性，Ｓ－１００蛋白阴性，细胞表面有微绒和微皱褶，细胞器发达，特别是有大量溶酶体。扫描电镜和透射电镜检查也有鉴别意义。     

人外周血树突状细胞对LAK细胞杀伤活性的影响

从人外周血中分离出树突状细胞，体外观察了其对ＬＡＫ细胞活性的影响。发现：５×１０２～１×１０４／ｍｌ树突状细胞对ＬＡＫ细胞活性起增强作用，而５×１０４～１×１０５／ｍｌ树突状细胞却抑制ＬＡＫ杀伤活性。这说明人外周血树突状细胞对ＬＡＫ细胞活性起双相性调节作用。     

Purification of human monocytes on gelatin-coated surfaces

Human peripheral blood monocytes secrete a cell membrane-associated glycoprotein, cold insoluble globulin (fibronectin). Since fibronectin binds to gelatin-coated surface, we developed a simple technique for separation of human peripheral blood monocytes from whole mononuclear cell preparation. These preparations are characterized by a high monocyte purity (more than 90%), low platelet contamination and excellent viability.     

Differential MHC class II expression on human peripheral blood monocytes and dendritic cells

Both monocytes (MO) and dendritic cells (DC) in human peripheral blood are of a plastic-adherent nature. The expression of the MHC class II sublocus products HLA-DP, -DQ and -DR on human peripheral blood transiently adherent cells (TA) was examined by an immunocytochemical staining technique. While most TA showed strong expression of molecules of the HLA-DR subtype, only a small proportion of cells (2-6%) showed strong HLA-DP or -DQ positivity. This strong expression of the HLA-DP and HLA-DQ sublocus products by a subset of TA was seen only after short-term culture; freshly isolated cells expressed comparatively low levels of these molecules. Enrichment for Fc receptor-negative or low-density cells from TA produced populations with strong HLA-DQ and -DP expression. Such co-enrichment of the strongly HLA-DQ+ and strongly HLA-DP+ cells suggests that the same cells express high levels of both types of MHC class II molecule. Immunocytochemical analysis of TA indicated that the strongly HLA-DQ+ cells, at least, were only weakly or non-reactive with the MO-specific monoclonal antibodies OKM1, UCHM1, MO2 and EB11. In addition, strongly HLA-DQ- or -DP-positive cells were poorly phagocytic in comparison with the majority of adherent cells. The apparent FcR-negative, low-density and weakly phagocytic nature of the strongly HLA-DQ/DP+ cells, combined with their lack of reactivity with several MO-specific antibodies, suggests that they may represent the DC component of TA. Such strong HLA-DQ/DP expression by DC may aid their positive identification in human peripheral blood and may be of relevance to DC function in antigen presentation.     

Flavivirus entry into cultured mosquito cells and human peripheral blood monocytes

Summary The entry modes of Japanese encephalitis (JE) and dengue-2 (DEN-2) viruses into C6/36 mosquito cells and of DEN-2 virus into human peripheral blood monocytes in vitro were studied. Inoculation of either JE or DEN-2 virions into C6/36 cells resulted in direct penetration of the virions into the cytoplasm at the cell surface in 3 stages. At stage 1, virions attached to the plasma membrane of host cells by their envelope spikes; at stage 2, the virion envelopes approximated to and eventually overlapped the host plasma membrane, and in the process the plasma membrane at the attachment sites dissolved; and, at stage 3, virions penetrated into the cytoplasm through the plasma-membrane disruptions created at the adsorption sites. Virions themselves apparently disintegrated at or near the penetration sites, for no virions were seen in the deeper cytoplasm. Coated pits did not form at the virion attachment sites, and virion-containing vesicles were not found in the cytoplasm. In the entry of DEN-2 virus into human peripheral blood monocytes, virions were found, adsorbed onto the external surface of the plasma membrane and attached to the luminal surface of macropinocytic vacuolar membranes. The latter apparently occurred as the result of ruffling and macropinocytic activities of the cells. At both sites virions penetrated into the cytoplasm through the plasma or vacuolar membrane in the same manner as they did through the plasma membrane of C6/36 cells. No evidence of viral entry by receptor-mediated endocytosis was observed. Implications of the entry mode of the mosquito cell-generated DEN-2 virus into human peripheral blood monocytes to an early process of natural, mosquito-transmitted infection is discussed.     

Morphological characteristics and immunophenotype of dendritic cells generated from monocytes of human peripheral blood

Morphological features and immunophenotype of mature dendritic cells (DC) generated from the monocytes of healthy donor blood after culture with GM-CSF and IL-4 with the addition of TNF, were studied using light and electron microscopy, as well as flow-cytometry. It was shown that DC were characterized by a number of morphological features such as: large size, eccentrically located nucleus, highly developed system of extensions, large vacuolated cytoplasm, prominent and activated Golgi complex and ribosomal apparatus. Mature DC are characterized by active surface expression of MHC and co-stimulatory molecules (MHC I, MHC II, CD40, CD80, CD86).     

Effect of PSK on the maturation of dendritic cells derived from human peripheral blood monocytes

Dendritic cells (DCs) are powerful antigen-presenting cells (APCs) that have attracted attention in recent years from the viewpoint of DC vaccine therapy against cancer. However, the existence of a strongly immunosuppressed state in cancer-bearing individuals inhibits DC maturation, which is one of the problems facing anti-cancer DC vaccine therapy. Protein-bound polysaccharide K (PSK), which is extracted from the cultured mycelium of Coriolus versicolor (Fr.) Quél, is used as an anti-cancer agent in Japan. PSK is reported to improve the immunosuppressed state and might be associated with DC maturation directly. We examined the effect of PSK on the maturation of DC derived from CD14-positive cells obtained from human peripheral blood monocytes using a negative selection method. CD14-positive cells cultured in the presence of PSK significantly increased the expression of HLA class II antigen and CD40; significantly increased the number and expression of CD80-, CD86- and CD83-positive cells; decreased Fluorescein isothiocyanate (FITC)-dextran uptake, augmented IL-12 production; augmented the allogeneic mixed lymphocyte reaction; and induced antigen-specific cytotoxicity. These results indicate that PSK promotes both the phenotypic and functional maturation of DC derived from human CD14-positive mononuclear cells. The clinical significance of the combined use of PSK in DC vaccine therapy remains for study.     

Analysis of methods for the generation of dendritic cells from human peripheral blood monocytes

Dendritic cells (DC) are highly efficient antigen-presenting cells that initiate the primary immune response. Several laboratories have developed culture systems for human DC from peripheral blood monocytes. Most of these studies have used fetal calf serum (FCS) containing culture conditions that are inappropriate for human application. GM-CSF and IL-4 were used to make immature DC. The monocyte-conditioned medium (MCM) was used to induce the final maturation of DC. Using the previously described methods, the quality of MCM has unpredictable variations. Therefore using a defined cocktail of growth factors for the generation of mature DC would be advantageous for experimental as well as clinical purposes. In this study, it is suggested that combinations of both GM-CSF/IL-4 or GM-CSF/IL-13 could be used as the first-step culture to produce immature DC, and that cytokine cocktail (GM-CSF, IL-4, IL-1beta, TNF-alpha, IL-6, PGE2) was as efficient as MCM for the second step-culture to produce fully maturated DC. Here, we have generated an easily reproducible culture system for DC that allows for the generation of large amounts of immature and mature DC, and we also now have established the method in a FCS-free system that is suitable for clinical use.     

Accelerated in vitro differentiation of blood monocytes into dendritic cells in human sepsis

Summary Sepsis-induced immune depression is characterized by infection susceptibility and monocyte early deactivation. Because monocytes are precursors for dendritic cells (DC), alterations in their differentiation into DC may contribute to defective immune responses in septic patients. We therefore investigated the ability of monocytes to differentiate into functional DC in vitro in patients undergoing surgery for peritonitis. Monocytes from 20 patients collected immediately after surgery (D0), at week 1 and at weeks 3-4 and from 11 control donors were differentiated into immature DC. We determined the phenotype of monocytes and derived DC, and analysed the ability of DC to respond to microbial products and to elicit T cell responses in a mixed leucocyte reaction (MLR). We show that, although monocytes from septic patients were deactivated with decreased responses to lipopolysaccharide (LPS) and peptidoglycan and low human leucocyte antigen D-related (HLA-DR) expression, they expressed the co-stimulatory molecule CD80, CD40 and CCR7. Monocytes collected from patients at D0 and week 1 differentiated faster into DC with early loss of CD14 expression. Expression of HLA-DR increased dramatically in culture to reach control levels, as did responses of DC to LPS and peptidoglycan. However, although patient and control immature DC had similar abilities to induce T cell proliferation in MLR, maturation of DC derived from patients did not increase T cell responses. These results show that circulating monocytes from septic patients express markers of activation and/or differentiation despite functional deactivation, and differentiate rapidly into phenotypically normal DC. These DC fail, however, to increase their T cell activation abilities upon maturation.     

Generation and characterization of ovine dendritic cells derived from peripheral blood monocytes

Dendritic cells (DCs) are professional antigen‐presenting cells with a highly immunostimulatory function and the capacity to activate naïve T cells. In recent years the rapid progress in mouse and human DC research can be mainly attributed to the generation of DCs from precursor cells in vitro, although a lack of reagents has hampered DC research in many large animal models. Here we describe the generation and characterization of ovine monocyte‐derived DCs in vitro. In addition to the characteristic morphology and non‐adherence of DCs, peripheral blood mononuclear cell monocytes cultured with ovine granulocyte–macrophage colony‐stimulating factor (GM–CSF) and interleukin‐4 (IL‐4) expressed CD11c and major histocompatibility complex (MHC) class II, but did not express CD14. High levels of endocytosis and an ability to stimulate antigen‐specific proliferation of CD4 T lymphocytes were also demonstrated.     

Purification of human monocytes: Isolation and collection of large numbers of peripheral blood monocytes

Monocytes from human peripheral blood were purified by elutriation centrifugation. Up to 1.5 × 10⁹ peripheral blood mononuclear cells could be separated in isotonic media to yield 6–8 × 10⁸ lymphocytes with 95% purity and 1.5–2.5 × 10⁸ monocytes with greater than 90% purity. The temperature at which elutriation was performed determined the purity of the monocyte fraction. Both the lymphocyte and monocyte fractions were characterized by cell sizing and histochemical staining. Most of the myeloperoxidase positive mononuclear cells (monocytes) has a modal volume of 378 μm³ while 10% were within the modal volume of the lymphocytes, 131 μm³. Thus, there are two populations of cells with histochemical properties of monocytes which are separable by size. The collection and isolation of human peripheral blood monocyte populations in large numbers will facilitate studies of their functional characteristics.