Surface‐Mediated Priming During In Vitro Generation of Monocyte‐Derived Dendritic Cells

Ex vivo‐generated human dendritic cells (DC) are most commonly generated from monocytes using standard cell culture dishes. To elucidate the effect of the plastic surface during the differentiation process, we compared a standard adhesive plastic dish with four different mainly non‐adherent surfaces. Untouched monocytes were cultured for 3 days in the presence of IL‐4 and GM‐CSF. Time‐lapse videos were recorded, and the phenotype of the cells was analysed by flow cytometry. The cytokine profiles were analysed using a 25‐plex cytokine assay. The use of non‐adherent surfaces led to a significant reduction in expression of CD14 and CD38, and a significant increase in expression of CD86 compared to standard culture dishes. Expression levels of DC‐SIGN and PD‐L2 were reduced significantly on cells cultured on non‐adherent surfaces. The cytokine production was independent on the surface used. The surface‐mediated priming should therefore be considered when aiming to induce specific immune responses. This is especially important with regard to DC‐based immunotherapy, where an adjustment of the surface during the DC generation process might have highly beneficial effects.     

Characterization of Monocyte‐Derived Dendritic Cells from Immunosuppressed Renal Transplant Recipients with and without Squamous Cell Carcinomas

Renal transplant recipients (RTR) have a high risk of tumour development, especially cutaneous squamous cell carcinomas (SCC), due to long‐term immunosuppressive therapy. RTR may develop multiple lesions over short time periods, and these are often more aggressive with a higher risk of local recurrence and metastasis resulting in increased morbidity and mortality in these patients. Therefore, we took the first step towards evaluating the possibility of generating a therapeutic vaccine based on monocyte‐derived dendritic cells (moDC) for these patients. We analysed the phenotype and cytokine/chemokine profile of moDC from long‐term immunosuppressed RTR with and without previous SCC. The number of peripheral blood mononuclear cells (PBMC) isolated per ml blood as well as the efficiency of generating moDC from peripheral blood mononuclear cells (PBMC) was similar in patients and immunocompetent controls. Phenotype and cytokine/chemokine profile of the moDC from immunosuppressed patients were similar to those from immunocompetent controls, making moDC‐based immunotherapy a potential future treatment option for RTR with multiple SCC.     

ORIGINAL ARTICLE: Glycodelin A Induces a Tolerogenic Phenotype in Monocyte‐Derived Dendritic Cells In vitro

Problem Successful mammalian pregnancy requires a delicate immunological balance at the feto‐maternal interface that allows the semi‐allogeneic fetus to grow, while protecting mother and child from environmental pathogens. As in other mucosal tissues, antigen‐recognition and ‐handling by professional antigen‐presenting cells such as dendritic cells (DC) determine the course of the subsequent immune response. DC at the feto‐maternal interface help shape this immunological equilibrium. Endometrial tissue secretes high quantities of glycodelin A (GdA) during the so‐called fertile window (i.e. the time of implantation of the blastocyst). Method of study We investigated the effect of GdA on monocyte‐derived DC (moDC) regarding surface marker expression, endopinocytotic activity, cytokine profile as well as lymphoproliferative activity. Results Upon pretreatment with GdA and subsequent maturation with tumor necrosis factor‐α and interleukin (IL)‐1β, moDC displayed a reduced expression of costimulatory molecules, an unchanged major histocompatibility complex‐II expression and persistence of DC‐SIGN positive cells. GdA‐pretreated moDC had a higher endopinocytotic activity, an increased IL‐10 production and a dose‐dependent reduction in lymphoproliferative activity. GdA incubation alone did not alter the immature phenotype. Conclusion Our results suggest a model in which the human endometrium secretes high quantities of GdA during implantation and thereby helps to shape the unique immunological interaction between mother and fetus via decidual DC.     

Redox Control of Indoleamine 2,3‐Dioxygenase Expression and Activity in Human Monocyte‐Derived Dendritic Cells is Independent of Changes in Oxygen Tension

Dendritic cells (DCs) initiate adaptive immune responses to pathogens and tumours and maintain tolerance to self and innocuous antigens. These functions occur in organs and tissues exhibiting wide variations in nutrients, growth factors, redox and oxygen tension. Understanding how these microenvironmental factors influence DCs to affect immunological outcomes is of increasing relevance with the emerging success of DC‐based cellular vaccines. In a previous study, we examined whether redox, an important environmental cue, could influence DC expression of the immunosuppressive enzyme indoleamine 2,3‐dioxygenase (IDO). IDO‐competent DCs promote long‐term immune homoeostasis by limiting exaggerated inflammatory responses and directing regulatory T‐cell effector function. To alter redox, we manipulated the activity of the cystine/glutamate antiporter, which functions to maintain intracellular and extracellular redox. The results of that study showed that redox perturbation strongly induced IDO expression and activity in DCs. While this study was performed using standard cell culture techniques with DCs cultured under 5% CO₂ and 20% O₂, it is clear that DCs capture and present antigens in inflamed tissues and secondary lymphoid organs which exhibit low oxygen tension (1–5% O₂). Therefore, here we investigated whether oxygen tension influences DC expression of IDO in the context of homoeostatic and altered redox.     

成人外周血来源树突状细胞体外诱导培养及成熟调控

人体内树突状细胞具有成熟和未知成熟两种形态。前者是早期免疫应答的强有力的抗原递呈细胞。后者具有截然不同的生物学特性，且能诱导免疫耐受。本研究从成人外周血中分离前体细胞，利用单核细胞条件性培养液，培养出比较均一的未成熟及成熟阶段的树突状细胞。第一阶段：从外周血分离出能粘附塑料的单核细胞，在GM－CSF＋IL-4存在条件下培养6－7d；第二阶段，加入单核细胞条件性培养液促进树突状细胞分化成熟，仅经第一阶段培养 的细胞主要为未成熟树突状细胞，仍然表达单核细胞的表达标志CD14，具有活跃的内,骐 MHC－II分子主要分布在胞内的MIIC器官；经第二阶段培养的树突状细胞则具有成熟树突状细胞的全部特征：典型的树突状形态，悬浮生长，MHC－II分子主要分布在包膜表面，．表达树突状细胞的特异性标志CD83，刺激同种T淋巴细胞增殖的能力强，并在脱离特定细胞因子环境3d后仍能保持该性状不变。由于该途径培养成熟、未成熟树突状细胞完全受外部因素调控，可能是获得均一的成熟和未成熟树突状细胞供科研和临床应用的较好途径。     

Wnt5a Inhibits the Proliferation and Melanogenesis of Melanocytes

Wnt5a, which is a noncanonical Wnt molecule, has been shown to be involved in a variety of developmental processes and cellular functions. In this study, we used “melan-a” cells as a cell model to investigate the effects of Wnt5a on melanocyte proliferation and melanogenesis, and to elucidate the possible mechanisms involved. We infected melan-a cells with recombinant Wnt5a adenoviruses to express Wnt5a protein and to simulate the Wnt5a processing environment. MTT assay and BrdU incorporation assay revealed that Wnt5a significantly inhibited the proliferation of melan-a cells. Melanin content and tyrosinase activity assays showed that Wnt5a was an inhibitor of melanin synthesis. Furthermore, RT-PCR and Western blot showed that this suppressive effect depended on noncanonical Wnt/Ror2 pathway activation and accessed the inhibition of the canonical Wnt pathway. The above results provided a novel insight into the role of Wnt5a and its related signaling in melanocyte homeostasis.     

Passive Transfer of Tumour‐Derived MDSCs Inhibits Asthma‐Related Airway Inflammation

Myeloid‐derived suppressor cells (MDSCs), a heterogeneous population including myeloid progenitor and immature myeloid cells, are known to inhibit T cell responses. The issue of whether tumour‐derived MDSCs regulate the immune response in an asthma environment is currently unclear. Here, we have reported that tumour‐derived MDSCs shift the balance back to normal in a Th2‐dominant asthmatic environment. In an ovalbumin (OVA)‐induced mouse asthma model, injected tumour‐derived MDSCs were recruited to the lungs of asthmatic mice by CC chemokine ligand 2 (CCL2). MDSCs transferred into asthmatic mice via i.v. injection suppressed the infiltration of inflammatory cells into the lung, the Th2 cytokine, IL‐4, concentration in bronchial lavage fluid and the serum level of OVA‐specific IgE. Increased TGF‐β1 production in the lung was detected after transfer of MDSCs. The inhibitory effects of MDSCs were reversed upon treatment with an anti‐TGF‐β1 antibody, suggesting dependence of these activities on TGF‐β1. Our findings imply that tumour‐derived MDSCs inhibit the Th2 cell‐mediated response against allergen in a TGF‐β1‐dependent manner. Based on the collective results, we propose that asthma may be effectively targeted using a novel MDSC‐based cell therapy approach.     

Human Dendritic Cell Subsets for Vaccination

TProtective immunity results from the interplay of antigen (Ag)-nonspecific innate immunity and Ag-specific adaptive immunity. The cells and molecules of the innate system employ non-clonal recognition pathways such as lectins and TLRs. B and T lymphocytes of the adaptive immune system employ clonal receptors recognizing Ag or peptides in a highly specific manner. An essential link between innate and adaptive immunity is provided by dendritic cells (DCs). As a component of the innate immunc system, DC organize and transfer information from the outside world to the cells of the adaptive immune system. DC can induce such contrasting states as active immune responsiveness or immunological tolerance. Recent years have brought a wealth of information regarding DC biology and pathophysiology that shows the complexity of this cell system. Thus, presentation of antigen by immature (non-activated) DCs leads to tolerance, whereas mature, antigen-loaded DCs are geared towards the launching of antigen-specific immunity. Furthermore, DCs are composed of multiple subsets with distinct functions at the interface of the innate and adaptive immunity. Our increased understanding of DC pathophysiologywill permit their rational manipulation for therapy such as vaccination to improve immunity.     

Montelukast Inhibits Leukotriene Stimulation of Human Dendritic Cells in vitro

Background: Leukotrienes are potent inflammatory mediators which modulate immune responses and induce bronchoconstriction in susceptible individuals. Montelukast (MK) is a leukotriene receptor (CysLT1) antagonist that has been shown to prevent exacerbation of asthma. Considering the plethora of potential cellular targets for MK, specific mechanisms for its therapeutic action are still not fully understood. In vitro, we determined whether human dendritic cell function could be affected by leukotriene C(4) (LTC(4)) treatment and whether MK had potential in modulating this response. We also studied the effect of LTC(4) in the context of response to an airway virus (respiratory syncytial virus, RSV). Methods: Human monocyte-derived dendritic cells (moDCs) exposed to LTC(4), MK, or both, were cocultured with autologous T cells, with or without RSV. The effects of LTC(4) and MK on cell function were determined by ELISA and proliferation assays. Results: Both moDCs and their precursors - monocytes - express LTC(4) receptor CysLT1, making them potential targets for MK. moDCs cultured with LTC(4) release the eosinophil chemoattractant RANTES (CCL5) and induce greater T cell proliferation. Both were blocked by the presence of MK. MK treatment, albeit anti-inflammatory, did not interfere with the moDC-dependent T cell-proliferative responses induced by RSV. Conclusions: LTC(4), chronically present in the airways of asthma patients, could induce an exaggerated inflammatory response to airway infection via dendritic cell activation, which would be prevented by MK. Our study provides additional insight into the mechanisms of action of this leukotriene receptor antagonist.     

Mammal‐Derived Respiratory Lipocalin Allergens do not Exhibit Dendritic Cell‐Activating Capacity

Most mammal‐derived respiratory allergens belong to the lipocalin family of proteins. Determinants of their allergenic capacity are still unknown. Innate immune cells, in particular dendritic cells, have been shown to be involved in the allergenicity of some proteins. As recognition by dendritic cells is one of the few plausible mechanisms for the allergenicity of proteins, we wanted to investigate their role in the allergenicity of lipocalin allergens. Therefore, we first incubated human monocyte‐derived dendritic cells with immunologically functional recombinant allergens mouse Mus m 1, dog Can f 1 and 2, cow Bos d 2, horse Equ c 1 and natural Bos d 2. Then, the surface marker expression and cytokine production of dendritic cells and their capacity to promote T cell proliferation and Th2 immune deviation in naïve CD4⁺ T cells were examined in vitro. We found that near to endotoxin‐free lipocalin allergens had no effect on the activation, allostimulatory capacity or cytokine production of dendritic cells. The dendritic cells could not induce immune deviation in naïve CD4⁺ T cells. In contrast, lipopolysaccharide activated the dendritic cells efficiently. However, lipocalin allergens were not able to modify the lipopolysaccharide‐induced responses. We conclude that an important group of mammal‐derived respiratory allergens, lipocalins, appear not to be able to activate dendritic cells, a major component involved in the allergenicity of some proteins. It is conceivable that this incapacity of lipocalin allergens to arouse innate immunity may be associated with their poor capacity to induce a strong T cell response, verified in several studies.     

Characterization of a Large Panel of Lactic Acid Bacteria Derived from the Human Gut for their Capacity to Polarize Dendritic Cell

Dendritic cells (DCs) are the principal stimulators of naïve T helper (Th) cells and play a pivotal regulatory role in the Th1, Th2 and Treg cell balance. DCs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. Here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate DC. A large panel of human gut-derived Lactobacillus and Bifidobacterium spp. was screened for DC-polarizing capacity by exposing bone marrow-derived murine DC to lethally irradiated bacteria. Cytokines in culture supernatants and DC-surface maturation markers were analysed. Substantial differences were found among strains in the capacity to induce interleukin-12 (IL-12) and tumour necrosis factor (TNF)-α, while the differences for IL-10 and IL-6 were less pronounced. Bifidobacteria tended to be weak IL-12 and TNF-α inducers, while both strong and weak IL-12 inducers were found among the strains of Lactobacillus . Remarkably, strains weak in IL-12 induction inhibited IL-12 and TNF-α production induced by an otherwise strong cytokine-inducing strain of Lactobacillus casei , while IL-10 production remained unaltered. Selected strains were tested for induction of DC maturation markers. Those lactobacilli with greatest capacity to induce IL-12 were most effective in upregulating surface MHC class II and CD86. Moreover, L. casei -induced upregulation of CD86 was reduced in the presence of a weak IL-12-inducing L. reuteri . In conclusion, human Lactobacillus and Bifidobacterium spp. polarize differentially DC maturation. Thus, the potential exists for Th1/Th2/Treg-driving capacities of the gut DC to be modulated according to composition of gut flora including ingested probiotics.     

Adenovirus-Mediated Wnt5a Expression Inhibits the Telogen-To-Anagen Transition of Hair Follicles in Mice

The canonical Wnt/β-catenin pathway plays an important role in hair cycle induction. Wnt5a is a non-canonical Wnt family member that generally antagonizes canonical Wnt signaling in other systems. In hair follicles, Wnt5a and canonical Wnt are both expressed in cells in the telogen stage. Wnt5a has been shown to be critical for controlling hair cell fate. However, the role that Wnt5a plays in the transition from the telogen to anagen stage is unknown. In this study, using whole-mount in situ hybridization, we show that Wnt5a is produced by several other cell types, excluding dermal papilla cells, throughout the hair cycle. For example, Wnt5a is expressed in bulge and secondary hair germ cells in the telogen stage. Our studies focused on the depilated 8-week-old mouse as a synchronized model of hair growth. Interestingly, overexpression of adenovirus Wnt5a in the dorsal skin of mice led to the elongation of the telogen stage and inhibition of the initiation of the anagen stage. However, following an extended period of time, four pelage hair types grew from hairless skin that was induced by Wnt5a, and the structure of these new hair shafts was normal. Using microarray analysis and quantitative arrays, we showed that the expression of β-catenin and some target genes of canonical Wnt signaling decreased after Wnt5a treatment. These data demonstrate that Wnt5a may inhibit the telogen stage to maintain a quiescent state of the hair follicle.     

ADV-miR-184体外转染对人晶状体上皮细胞移行的影响

Objective Investigate the effects of the recombinant adenoviral vector containing microRNA-184 (ADV-miR-184) on the migration of human lens epithelial cells in vitro.Methods ADV-miR-184 WaS amplified,purified,and titmted in 293 cell line.Human lens epithelial cells (HLE-B3) were then transfected with ADV-miR-184.The) effect of miR-184 on the migration of these transfected cells was evaluated by the scratch assay.Results The original titer of ADV-miR-184 was 1.6×109 puf/mL.HLE-B3 cells were transfected with ADV-miR-184 at different multiplicity of infection (MOI),including MOI10,MOI50,MOI100,MOI200 and MOI500,for a period of 72 h.The migrating distances of HLE-B3 cells were inbitied by MOI100 significantly higher titers of ADV-miR-184 transfection.Transfection with inhibited the migration of HLE-B3 cells compared with groups of control,MOI10,or MOI50 (P＜0.05).In contrast,no differences were showed between MOI100 and MOI200 or MOI500 groups.The migrating distances of HLE-B3 ceils transfected with MOI100 were also significantly inhibited at 24h,48h and 96h tremments compared with controls (P＜0.05).Conclusion ADV-miR-184 could be successfully used to tnmsfect human lens epithelial cells.The miR-184 inhibited the migration of human lens epithelial cells,which suggests that the miR virus may play a role in the development of the posterior capsular opacification.     

ADV-miR-184体外转染对人晶状体上皮细胞移行的影响

目的 探讨携带miR-184的重组腺病毒(ADV-miR-184)体外转染对人晶状体上皮细胞移行的影响.方法 ADV-miR-184在293细胞中扩增、纯化并滴定病毒滴度;ADV-miR-184体外感染人晶状体上皮细胞(HLE-B3),采用细胞划痕法测定ADV-miR-184对HLE-B3移行距离的影响.结果 测定ADV-miR-184的病毒滴度为1.6×109 puf/mL;分别将ADV-miR-184以MO110、MO150、MOI100、MOI200、MOI500转染HLE-B3 72 h后,细胞移行距离随着ADV-miR-184的浓度增加而减少,MOI100组与MOI50组、MOI10组相比有明显差异(P＜0.05).但与MOI200、MOI500实验组相比变化不明显.与对照组比较,MOI100感染细胞的移行距离在转染后24 h、48 h、72 h、96 h明显缩短(P＜0.05).结论 ADV-miR-184可成功转染人晶体上皮细胞,并可抑制细胞的移行,提示miR可能参与后发性白内障的形成过程.     

Age‐Matched Dendritic Cell Subpopulations Reference Values in Childhood

Dendritic cells (DCs) are the most potent antigen‐presenting cells and are the key link between the innate and adaptive immune response. Only a few reports with study populations of up to 50 individuals have been published with age‐based reference values for DC subpopulations in healthy children. Therefore, we aimed to establish reference ranges in a larger study population of 100 healthy children, which allowed age‐matched subgroups. Most previous studies were performed using a dual‐platform approach. In this study, a single‐platform approach in a lyse no‐wash procedure was used. DC subpopulations were defined as follows: CD45⁺CD85k⁺HLA‐DR⁺CD14^?CD16^?CD33⁺ cells as myeloid DCs (mDCs) and CD45⁺CD85k⁺HLA‐DR⁺CD14^?CD16^?CD123⁺ cells as plasmacytoid DCs (pDCs). Reference ranges were established using a semi‐parametric regression of age‐matched absolute and relative DC counts. We found a significant decline with increasing age in the medians of mDCs (P = 0.0003) and pDCs per μl peripheral blood (PB) (P = 0.004) and in the 50%, 90% and 95% reference ranges. We also identified significantly lower absolute cell counts of mDCs per μl PB in girls than in boys for all age groups (P = 0.0015). Due to the larger paediatric study population and single‐platform approach, this study may give a more precise overview of the normal age‐matched development of DC subpopulations and may provide a basis for analyzing abnormal DC counts in different illnesses or therapies such as post stem cell transplantation.     

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。