人外周血树突状细胞的分离与鉴定

目的:体外分离培养并鉴定人外周血树突状细胞,并观察其抗原呈递功能。方法:实验于2005-05/2006-11在南方医科大学南方医院肿瘤中心生物治疗实验室完成。从人类白细胞抗原A2表达阳性的健康人外周血中分离获得单个核细胞。培养5h后洗涤贴壁细胞,加入含有10%人AB血清的RPMI1640培养基,及重组人粒细胞-巨噬细胞集落刺激因子和重组人白细胞介素4,于培养的第1,3,6天对树突状细胞的形态、表型进行分析,并定期检测树突状细胞的纯度与得率。抽取与以上树突状细胞不同来源的其他健康人外周血。经淋巴细胞分离液分离后,获取非贴壁细胞,用含10%人AB血清的1640培养基重悬,加入白细胞介素2继续孵育6d,作为同种异体T淋巴细胞。将树突状细胞分为两组,一组按常规方法培养6d,另一组在培养至第5天时加入黑色素瘤抗原基因A3编码的多肽继续培养24h。在经紫外线处理后的96孔板中,分别加入树突状细胞悬液1×104,5×103,2×103,1×103细胞/每孔,以自身T淋巴细胞作为对照,每孔设3个复孔,分别加入1×105淋巴细胞/每孔。评价树突状细胞刺激T淋巴细胞增殖的能力。结果:①单个核细胞体外培养至第6天,可获得大量、90.81%高纯度的树突状细胞,能够较高地表达21.8?1a、99.0%HLA-DR、63.4?80、18.9?83和80.6?86。②将诱导培养6d获得的两组树突状细胞作为刺激细胞,以不同的浓度与同种异体淋巴细胞混合,均可产生增殖反应;经过黑色素瘤抗原基因A3编码的多肽处理的各种比例的树突状细胞,较相应未经黑色素瘤抗原基因A3编码的多肽处理的树突状细胞激发淋巴细胞增殖的能力明显增强,浓度相对较高的树突状细胞刺激效果最明显,能够强烈地激发同种混合淋巴细胞增殖。结论:得到了一群较高程度表达CD83、CD86和HLA-DR分子、体外可强烈激发同种异体T淋巴细胞增殖的树突状细胞群。     

Isolation and characterization of mesenchymal cells from human fetal membranes

Bone marrow (BM) multipotent mesenchymal stromal cells (MSCs) present with multipotent differentiation potential and immunomodulatory properties. As an alternative to bone marrow, we have examined fetal membranes, amnion and chorion, of term human placenta as a potential source of multipotent MSCs. Here we show that amnion mesenchymal cells (AMCs) and chorion mesenchymal cells (CMCs), isolated by mechanical separation and subsequent enzymatic digestion, demonstrate plastic adherence and fibroblast-like morphology and are able to form colonies that could be expanded for at least 15 passages. By FACS analysis, AMCs and CMCs were shown to be phenotypically similar to BM-MSCs and, when cultured in differentiation media, they demonstrated high morphogenetic plasticity by differentiating into osteocytes, chondrocytes and adipocytes. In an attempt to isolate cells with MSC characteristics from human fetal membranes, AMCs and CMCs expressing CD271 were enriched by immunomagnetic isolation and were demonstrated to possess higher clonogenic and osteogenic differentiation potential than CD271-depleted fractions. Based on these findings, amnion and chorion can be considered as a novel and convenient source of adult MSCs.     

Isolation and functional characterization of human intestinal mucosal lymphoid cells.

Viable suspensions of human colonic mucosal lymphoid cells have been prepared by sequential treatment of tissue with dithiothreitol, EDTA in calcium- and magnesium-free salt solutions, and purified collagenase. The intestinal lymphocyte population, in comparison with that of peripheral blood, had greater numbers of bone marrow-derived cells, particularly cells bearing membrane IgA; showed spontaneous association with macrophages; underwent rapid rosette formation with sheep erythrocytes; and demonstrated increased in vitro synthesis of immunoglobulin. Total thymus-derived cells were equal in the two populations. Decreases were found in "null" cell numbers, in cells bearing membrane IgD and IgM, and in responsiveness to phytohemagglutinin. Macrophage/monocytes in the intestinal population were increased in size, granularity, motility, sustained glass adherence, and phagocytic activity. Human intestinal lymphoid cells appear to constitute a cell population that is more "mature" and/or "activated", in comparison with the lymphoid cells of peripheral blood. The method of preparation should lend itself to the study of inflammatory bowel disease, gastrointestinal cancer, and the intestinal secretory immune system.     

Isolation, characterization, and propagation in vitro of human glomerular endothelial cells

Human glomerular endothelial cells have been isolated, cloned, and characterized. They appeared as the first outgrowth from human glomeruli in the presence of platelet-derived growth factor, which was also a requirement for continuous growth. By phase microscopy they appeared as monolayers of polygonal cells. Von Willebrand's factor (VWF) was detected in the cytoplasm of all clones. Their intermediate filaments differed antigenically from that present in human umbilical vein endothelial cells. Like other endothelial cells, they demonstrated high levels of membrane-associated angiotensin-converting enzyme (ACE).     

Phenotypical and functional characterization of clinical grade dendritic cells

Dendritic cells (DC) are the professional antigen presenting cells of the immune system. Therefore, several clinical studies have been initiated in which tumor antigen-loaded DC are used as a vaccine to boost an immune response against malignant tumors in patients with cancer. A prerequisite for DC used in these vaccination studies is not only that they are grown under "Good Manufacturing Practice" but equally important that they retain their functional properties. In an extensive study, various conditions were tested to optimize the maturation and yield of DC grown for clinical use. DC grown in XVIVO-15 medium supplemented with 5% HS yielded the best results, morphologically and phenotypically. Mature DC expressed significant amounts of mature DC markers (CD83) and the costimulatory molecules CD80 and CD86. It was shown that mature and immature DC can be frozen and retain their phenotype and function after thawing. These clinical grade DC secreted high levels of the chemokines dendritic cell chemokine 1 (DC-CK1), interleukin-8 (IL-8), macrophage-derived chemokine (MDC), and thymus and activation-regulated chemokine (TARC). This implicates that these DC can attract na?ve T and B cells as well as natural killer cells and memory T cells. Finally, to test their migratory capacity in vivo, (111)In-labeled DC were injected into tumor-free lymph nodes of patients with melanoma. Autoradiographic analysis of the dissected lymph nodes indicated that these DC could migrate into the T cell area of adjacent lymph nodes. In conclusion, a culture procedure was established to generate large numbers of monocyte-derived immature and mature DC that retain their morphologic, phenotypic, and functional characteristics in vitro and can be visualized in situ.     

Localization and characterization of antigen-presenting dendritic cells in the gastric mucosa of murine and human autoimmune gastritis

BACKGROUND: Activated CD4+ T cells and inflammatory cytokines are implicated in the pathogenesis of experimental autoimmune gastritis. However, there is a paucity of information about the cells that induce them. Antigen-presenting dendritic cells (DCs) play a cardinal role in the formation and survival of activated lymphocytes. MATERIALS AND METHODS: Autoimmune gastritis was induced in neonatal BALB/c mice by thymectomy. DCs were detected in situ in the gastric mucosa from thymectomized mice and in patients with autoimmune gastritis, by immunohistochemistry and immunoelectron microscopy. The expression of MHC class II and CD86 antigens on DCs in the gastric mucosa and spleen was evaluated in dual-colour flow cytometry. RESULTS: DCs were detected in the gastric mucosa of mice with autoimmune gastritis, and the number of DCs increased as the levels of gastritis became more severe as time passed following thymectomy. Flow cytometric analyses revealed that more than 60% of the DCs in the gastric mucosa had a mature phenotype (expressed MHC class II and/or CD86 antigens) both at 4 and 16 weeks after thymectomy. Activated and mature DCs were localized in the gastric mucosa from patients with autoimmune gastritis. CONCLUSIONS: This is the first report on the localization and phenotypes of DCs in the gastric mucosa of autoimmune gastritis. The presence of mature DCs in the gastric mucosa of murine and human autoimmune gastritis, in spite of their absence in the gastric mucosa of normal mice, suggests that mature DCs play a role in the pathogenesis of autoimmune gastritis.     

人外周血内皮祖细胞的分离、培养及鉴定

背景：内皮祖细胞是成熟内皮细胞的前体细胞,具有新生血管和新生内皮化作用,在许多方面均有广泛应用前景,但其生物学特征及鉴定方法仍存争议。目的：探索从人外周血分离培养内皮祖细胞的方法并鉴定其生物学特征。方法：外周采血后应用密度梯度离心法分离成人外周血单核细胞,在内皮细胞全培养基重悬后接种于纤维连接蛋白包被的培养瓶中,体外培养扩增获取人内皮祖细胞并观察其形态变化、生长增殖潜能及细胞表面抗原表达情况,并通过细胞一氧化氮分泌功能测定及体外血管形成实验检测其功能学特征。结果与结论：体外诱导培养后,六七天形成纺锤样细胞簇,两三周黏附细胞发育形成鹅卵石样外观细胞,逐渐融合呈外生性生长。在相同培养条件下,与人主动脉内皮细胞相比人内皮祖细胞具有高的增殖潜能。人内皮祖细胞表达CD31、CD34、CD144、KDR,表现为典型内皮细胞系表型,此外细胞可摄取ac-LDL并结合UEA-I。在功能上内皮祖细胞可分泌一氧化氮并可在Matrigel中形成管腔样结构。提示通过密度梯度离心法分离人外周血单核细胞体外黏附诱导培养可获取人外周血内皮祖细胞;细胞形态、增殖能力、生物表型特征结合细胞功能学的综合性鉴定方法用于内皮祖细胞的鉴定具有一定意义。     

人胎盘贴壁细胞的分离培养及造血相关因子表达

目的：探讨人胎盘贴壁细胞(hPDAC)的分离培养及造血相关因子的表达。方法：酶消化法分离培养人胎盘组织中贴壁细胞，并鉴定其生物学特性，用RT-PCR方法检测造血相关因子表达。结果：成功分离培养出hPDAC，并证实其具有间充质干细胞特性，表达多种造血相关因子，包括SCF、FL、G-CSF、GM．CSF、M-CSF和IL-6。结论：hPDAC可为脐血CD34^ 细胞体外扩增提供潜在滋养层。     

Activation of human dendritic cells by p-phenylenediamine

Exposure to p-phenylenediamine (pPD), a primary intermediate in hair dye formulations, is often associated with the development of allergic contact dermatitis. Such reactions involve activation of the subject's immune system. The aim of these studies was to explore the relationship between pPD oxidation and functional maturation of human monocyte-derived dendritic cells in vitro. Dendritic cells were incubated with pPD and Bandrowski's base (BB) for 16 h, and expression of the costimulatory receptors CD40, CD80, CD83, CD86, and major histocompatibility complex class II intracellular glutathione levels and cell viability were measured. In certain experiments, glutathione (1 mM) was added to culture medium. Liquid chromatography-mass spectrometry (LC-MS) analysis and exhaustive solvent extraction were used to monitor the rate of [(14)C]pPD oxidation and the extent of pPD binding to cellular and serum protein, respectively. Proliferation of allogeneic lymphocytes was determined by incorporation of [(3)H]thymidine. Exposure of dendritic cells to pPD (5-50 microM), but not BB, was associated with an increase in CD40 and MHC class II expression and proliferation of allogeneic lymphocytes. Dendritic cell activation with pPD was not associated with apoptotic or necrotic cell death or depletion of glutathione. Neither pPD nor BB altered dendritic cell expression of CD80, CD83, or CD86. LC-MS analysis revealed pPD was rapidly oxidized in cell culture media to BB. Addition of glutathione inhibited BB formation but did not prevent covalent binding of pPD to dendritic cell protein or dendritic cell activation. Collectively, these studies show that pPD, but not BB, selectively activates human dendritic cells in vitro.     

Human and murine dermis contain dendritic cells. Isolation by means of a novel method and phenotypical and functional characterization.

Dendritic cells (DC) comprise a system of cells in lymphoid and nonlymphoid organs that are specialized to present antigens and to initiate primary T cell responses. The Langerhans cell of the epidermis is used as a prototype for studies of DC in the skin. We have characterized a population of DC in human dermis, one of the first examples of these cells in nonlymphoid organs other than epidermis. To identify their distinct functions and phenotype, we relied upon the preparation of enriched populations that emigrate from organ explants of dermis. The dermal cells have the following key features of mature DC: (a) sheet-like processes, or veils, that are constantly moving; (b) very high levels of surface MHC products; (c) absence of markers for macrophages, lymphocytes, and endothelium; (d) substantial expression of adhesion/costimulatory molecules such as CD11/CD18, CD54 (ICAM-1), B7/BB1, CD40; and (e) powerful stimulatory function for resting T cells. Dermal DC are fully comparable to epidermis-derived DC, except for the lack of Birbeck granules, lower levels of CD1a, and higher levels of CD36. DC were also detected in explants of mouse dermis. We conclude that cutaneous DC include both epidermal and dermal components, and suggest that other human nonlymphoid tissues may also serve as sources of typical immunostimulatory DC.     

Human dendritic cells. Enrichment and characterization from peripheral blood

Previous studies demonstrated that lymphoid tissues of mice and rats contain small numbers (less than 1 percent of nucleated cells) of dendritic cells (DC) with special cytologic, surface, and functional properties. We show here that similar DC represent 0.1-0.5 percent of human peripheral blood mononuclear cells. DC can be enriched to 20-60 percent purity by a multistep procedure analogous to that used in mice. Adherent peripheral blood mononuclear cells are cultured overnight, and the released cells are depleted of monocytes and B cells by readherence to plastic, rosetting with erythrocytes coated with anti-human IgG, and centrifugation in dense albumin columns. Enriched DC have similar cytologic features to rodent DC by light and electron microscopy. DC express HLA, and HLA-DR and the leukocyte-common antigens. They lack phagocytic capacity, receptors for antibody-coated and neuraminidase-treated erythrocytes, surface and intracellular Ig, esterase, peroxidase, and azurophilic granules. DC do not react with several monoclonal antibodies directed to phagocytes (OKM 1, “mac-1,” 63D3, and 61D3) and T cells (OKT 3, 6, 8). Unlike the mouse, human DC express complement receptors. When maintained in culture for 4 d, human DC did not give rise to either B cells or monocytes. Therefore, DC identified by cytologic criteria are distinct from other leukocytes. Enriched populations of DC have been compared to fractions enriched in monocytes, B cells, and T cells in three functional assays: stimulation of the primary allogeneic mixed leukocyte reaction, stimulation of the primary syngeneic MLR, and accessory function for the proliferation of periodate- modified T cells. In each case, the DC fraction was 10-fold or more active than other cell fractions. We conclude that DC circulate in man, and represent the principal cell type required for the initiation of several immune responses.     

Direct transfection and activation of human cutaneous dendritic cells

Gene therapy techniques can be important tools for the induction and control of immune responses. Antigen delivery is a critical challenge in vaccine design, and DNA-based immunization offers an attractive method to deliver encoded transgenic protein antigens. In the present study, we used a gene gun to transfect human skin organ cultures with a particular goal of expressing transgenic antigens in resident cutaneous dendritic cells. Our studies demonstrate that when delivered to human skin, gold particles are observed primarily in the epidermis, even when high helium delivery pressures are used. We demonstrate that Langerhans cells resident in the basal epidermis can be transfected, and that biolistic gene delivery is sufficient to stimulate the activation and migration of skin dendritic cells. RT-PCR analysis of dendritic cells, which have migrated from transfected skin, demonstrates the presence of transgenic mRNA, indicating direct transfection of cutaneous dendritic cells. Importantly, transfected epidermal Langerhans cells can efficiently present a peptide derived from the transgenic melanoma antigen MART-1 to a MART-1-specific CTL. Taken together, our results demonstrate direct transfection, activation, and antigen-specific stimulatory function of in situ transduced human Langerhans cells.     

Isolation and preliminary characterization of glycosaminoglycans in human plasma

Plasma glycosaminoglycans were isolated, after pronase digestion by precipitation with cetylpyridinium chloride. The glycosaminoglycans in plasma were found exclusively in the protein fraction precipitated by trichloroacetic acid. The concentration of glycosaminoglycans in normal plasma was 168 to 272 μg per 100 ml.

Electrophoresis of plasma glycosaminoglycans in barium acetate buffer on cellulose acetate revealed three fractions, all resistant to streptomyces hyaluronidase but susceptible to testicular hyaluronidase. Electrophoretic mobility of the major fraction was similar to dermatan sulfate and hyaluronic acid. Enzymatic assay with chondroitinases, however, indicated that the major constituent of plasma glycosaminoglycans was undersulfated chondroitin sulfate composed of equimolar amounts of unsaturated non-sulfated disaccharides and unsaturated 4-sulfated disaccharides at the disaccharide subunit levels.

The other two fractions were identical with chondroitin sulfate A and C. The quantity of chondroitin sulfate C was least. Hyaluronic acid and over-sulfated chondroitin sulfate were not demonstrated in plasma.     

Isolation and characterization of glycosaminoglycans in human plasma.

We have described methodology for the isolation and quantitation of glycosaminoglycans present in human plasma. Plasma glycosaminoglycans can be quantitatively adsorbed on a DEAE-Sephacel ion exchanger and eluted with a salt gradient as two groups: a low-charge fraction and a high-charge fraction. The low-charge fraction consists of chondroitin sulfate with a low sulfate content and the high-charge fraction consists of heparan sulfate, chondroitin sulfate, and keratan sulfate (type I). We have determined the plasma concentration of each of these glycosaminoglycans in six normal human subjects. We have established that none of the glycosaminoglycans in plasma are covalently linked to plasma proteins. All are isolated as complexes with plasma proteins in noncovalent linkages. The glycosaminoglycans in the low-charge fraction are bound with high affinity to a single plasma glycoprotein by a lectin-type bond that can be disrupted by a simple glycoside. The high-charge fraction contains three major proteins and several minor proteins associated with the glycosaminoglycans by both lectin-type and ionic bonding. The plasma proteins associated with glycosaminoglycans represent less than 0.5% of the total plasma proteins. Little is known about the physiologic role of the plasma glycosaminoglycans as components of metabolic processes. Because glycosaminoglycans have been implicated in lipid metabolism and atherosclerosis, we tested all of these compounds, isolated in free form, on the in vitro hydrolysis of triglycerides by lipoprotein lipase. Plasma heparan sulfate stimulated the rate of this reaction severalfold. All other plasma glycosaminoglycans were inactive. Thus, plasma heparan sulfate may play an important role in plasma lipoprotein metabolism.     

米非司酮对人外周血来源树突状细胞成熟和功能的影响

目的观察米非司酮对人外周血来源树突状细胞(DCs)成熟及生物学功能的影响,探讨米非司酮作为紧急避孕药的免疫学机制。方法人外周血CD14+单核细胞在体外经GM-CSF、IL-4培养6d诱导分化为未成熟DCs,加入1800 nmol/L米非司酮继续培养48h,流式细胞仪检测细胞表型,ELSIA检测DCs培养上清液中IL-12p70水平,混合淋巴细胞反应检测DCs刺激同种异体T细胞增殖的能力。结果与阴性对照组相比,米非司酮处理后的DCs,其细胞表面HLA-DR及CD83分子表达上调(t分别=15.23、7.63,P均<0.05),IL-12p70分泌增加(t=12.62,P<0.05),且刺激同种异体T细胞增殖的能力明显增强,差异均有统计学意义(t分别=9.12、13.24、6.78,P均<0.05)。结论米非司酮能诱导人外周血来源DCs成熟,促进DCs诱导的免疫应答启动。     

Isolation and characterization of beta-glucan receptors on human mononuclear phagocytes

beta-glucan receptors, with ligand specificity for yeast and fungal carbohydrate polymers, have been studied as phagocytic receptors of human monocytes. To characterize their structure, binding studies were carried out with human U937 cells and a rabbit IgG anti-Id that recognizes epitopes on monocyte beta-glucan receptors. Unstimulated U937 cells specifically bound large amounts of the anti-Id, but almost none of the control anti-isotype. At saturation, the number of anti-Id molecules bound per U937 cell was 2.6 x 10(6) with an apparent Ka of 1.9 x 10(7) M-1. Immunoprecipitates from detergent lysates of surface-radioiodinated U937 cells contained only two membrane proteins with antigenic specificity for the anti-Id, one having a mol wt of 180 kD and the other 160 kD. Both proteins were disulfide-linked and presented, after reduction, as five polypeptides of 95, 88, 60, 27, and 20 kD. Detergent lysates of unlabeled U937 cells, purified by affinity chromatography on anti-Id-Sepharose, yielded the same two nonreduced proteins and five reduction products in slab gels stained with Coomassie blue. In Western blots probed with the anti-Id, the most immunoreactive nonreduced and reduced affinity-purified products were the 160 and 20 kD molecules, respectively. Immunoblots of two-dimensional gels showed the 180 and 160 kD proteins to express a common epitope through disulfide linkage to the 20 kD polypeptide. By immunoblot analysis, U937 cell glucan-binding proteins from detergent lysates contained two cell proteins antigenic for the anti-Id that were indistinguishable from affinity-purified molecules in size and subunit composition. Studies of affinity-purified proteins from detergent lysed human monocytes were characterized by immunoblot analysis and found to be identical to U937 cell beta-glucan receptors. They consisted of two disulfide-linked proteins, with mol wt of 180 and 160 kD, and had in common a 20 kD polypeptide with the anti-Id epitope.     

Transfected human dendritic cells to induce antitumor immunity

Dendritic cells are professional antigen-presenting cells able to prime naive T lymphocytes and regulate steadily the delicate balance between tolerance and activation during the immune response. In past years several reports have shown that genetically engineered dendritic cells (DCs) can be a powerful tool for inducing an antigen-specific immune response. The use of such modified antigen-presenting cells is a real working hypothesis in preclinical studies and in clinical vaccination approaches for cancer treatment. The definition of optimal transfection conditions for preserving DC survival and functionality is necessary to design a correct immunotherapeutic protocol. Different lipid-based transfection compounds were studied for their effects on DC survival, phenotype and functional properties. All the transfection procedures were able to select DCs with a higher expression of activation and costimulatory molecules (ie MHCII-DR, CD83, CD86, CD25) than the untreated DCs. However, only two compounds (LipofectAMINE PLUS and FuGENE 6), preserved or even increased the immunopotency of DCs as antigen-presenting cells. These protocols were applied to modify DCs in order to express an epithelial tumor-associated antigen, MUC1, and such cells were able to induce in vitro a specific immune response in healthy donors.