Wnt5a Inhibits Human Monocyte‐Derived Myeloid Dendritic Cell Generation

Wnt5a is a non‐canonical Wnt protein that is expressed at elevated levels in inflammatory conditions. Its role in inflammation remains unclear, although it is known that Wnt5a is expressed at a higher level in monocyte‐derived myeloid dendritic cells (Mo‐mDCs) than in monocytes and macrophages. The function of Wnt5a in dendritic cells (DCs) remains relatively unexplored. Here, we found that under Mo‐mDC culture conditions, Wnt5a inhibited the generation of CD14^+/low Mo‐mDCs while promoting the generation of CD14^+/++ CD16⁺ monocytes. We could further show that stimulation of monocytes with rWnt5a induced a rapid IL‐6 production and that the rWnt5a treated Mo‐mDC differentiation was restored upon blocking of IL‐6. Also, conditioned media from Wnt5a stimulated human breast cancer cells producing IL‐6, specifically inhibited Mo‐mDC differentiation. These observations are strengthened by our finding that patients with sepsis, a disease involving elevated Wnt5a and IL‐6 levels, also showed a significant increase in the CD14⁺ CD16⁺⁺/CD14^+/++ CD16⁺ monocyte populations, which was accompanied by a significant decrease in circulating mDCs. We finally show that under typical Mo‐mDC culture conditions, monocytes isolated from patients with sepsis as compared to healthy controls, preferentially differentiated into CD14^+/++ HLA‐DR⁺⁺ cells. We suggest that Wnt5a is a possible candidate mediator for the CD14^+/++ CD16⁺ monocyte accumulation seen in patients with infectious disease and cancer.     

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.     

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.     

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.     

Clathrin‐Mediated Endocytosis of Gold Nanoparticles In Vitro

Gold nanoparticles (AuNPs) have potential biomedical and scientific applications. In this study, we evaluated the uptake and internalization of FBS‐coated 20 nm AuNPs into lung fibroblasts and liver cells by different microscopy techniques. AuNP aggregates were observed inside MRC5 lung fibroblasts and Chang liver cells under light microscopy, especially after enhancement with automegallography. Clusters of AuNPs were observed to be adsorbed on the cell surface by scanning electron microscopy. Ultrathin sections showed that AuNPs were mainly enclosed within cytoplasmic vesicles when viewed under transmission electron microscopy. We also investigated the mechanism of uptake for AuNPs, using endocytosis inhibitors and quantification of Au with inductively coupled plasma mass spectrometry. Cells treated with concanavalin A and chlorpromazine showed significant decrease of Au uptake in MRC5 lung fibroblasts and Chang liver cells, respectively, implying that the uptake of AuNPs was facilitated by clathrin‐mediated endocytosis. It would therefore appear that uptake of 20 nm AuNPs in both cell types with different tissues of origin, was dependent upon clathrin‐mediated endocytosis. Anat Rec, 298:418–427, 2015. © 2014 Wiley Periodicals, Inc.     

Dendritic Cells Enhance the Differentiation of Naïve B Cells into Plasma Cells In Vitro

We have shown previously that in vitro- generated human dendritic cells have an effect on the response of B cells at various stages of their differentiation. In a culture system described for the in vitro induction of plasma-cell differentiation, it was reported that naïve B cells have a poor propensity to differentiate into plasma cells. In such a culture system, 12% of naïve B cells differentiated into plasma cells in the presence of IL-2 and IL-10, despite the interruption of CD40 signalling which is necessary for plasma-cell differentiation. However, as reported herein, naïve B cells differentiated fully into plasma cells in response to dendritic cells. Addition of dendritic cells enhanced this differentiation strikingly by recruiting 57% of B cells as plasma cells producing IgM, but also IgG and IgA. In this model, dendritic cells act in synergy with IL-2 at an early stage of CD40-dependent B-cell differentiation, while IL-2 and IL-10 act together, at a later stage, in the generation of plasma cells in a CD40-independent manner. Thus, in addition to the key role played by dendritic cells in the initiation of T-cell responses, our results suggest that dendritic cells regulate humoral responses.     

Interaction of Human Dendritic Cells with Graphene Oxide Nanoparticles In Vitro

Bulletin of Experimental Biology and Medicine - We studied the effect of graphene oxide nanoparticles on the differentiation of human dendritic cells and uptake of nanoparticles by these cells in...     

用GM-CSF和 IL-4在体外诱导高纯度CD14+树突状细胞

本研究改进传统的树突状细胞（DC）诱导方法，用GM－CSF（150ng/ml）和IL－4（80U／ml），在体外经7d从健康人外周血中诱导出了大量高纯度DC，其高表达HLA－I、HLA－II类分子，共刺激分子和黏附分子，同时还高表达其前体单核细胞的特异性标志CD14分子，显示出成熟 DC的特征。这些CD14^ DC能强烈诱导同种异体淋巴细胞的增殖，其内吞能力在第3天达最高，之后明显下降。此结果丰富了DC的类型，并为CD14^ DC的深入研究和应用奠定了基础。     

Murine Dendritic Cells Pulsed In Vitro with Toxoplasma gondii Antigens Induce Protective Immunity In Vivo

The activation of a predominant T-helper-cell subset plays a critical role in disease resolution. In the case of Toxoplasma gondii, the available evidence indicates that CD4⁺ protective cells belong to the Th1 subset. The aim of this study was to determine whether T. gondii antigens (in T. gondii sonicate [TSo]) presented by splenic dendritic cells (DC) were able to induce a specific immune response in vivo and to protect CBA/J mice orally challenged with T. gondii cysts. CBA/J mice immunized with TSo-pulsed DC exhibited significantly fewer cysts in their brains after oral infection with T. gondii 76K than control mice did. Protected mice developed a strong humoral response in vivo, with especially high levels of anti-TSo immunoglobulin G2a antibodies in serum. T. gondii antigens such as SAG1 (surface protein), SAG2 (surface protein), MIC1 (microneme protein), ROP2 through ROP4 (rhoptry proteins), and MIC2 (microneme protein) were recognized predominantly. Furthermore, DC loaded with TSo, which synthesized high levels of interleukin-12 (IL-12), triggered a strong cellular response in vivo, as assessed by the proliferation of lymph node cells in response to TSo restimulation in vitro. Cellular proliferation was associated with gamma interferon and IL-2 production. Taken together, these results indicate that immunization of CBA/J mice with TSo-pulsed DC can induce both humoral and Th1-like cellular immune responses and affords partial resistance against the establishment of chronic toxoplasmosis.     

E-Selectin Involvement in In Vitro Adhesion of Blood Dendritic Cells to Human Umbilical Cord Endothelial Cells

Peripheral blood dendritic cells (BDC) are potent antigen-presenting lymphoid cells. In the present study, we have examined the in vitro adhesion of BDC to human umbilical cord venous endothelial cells (HUVEC) and studied the expression of CD molecules and oligosaccharide haptens on BDC and endothelial cells. Immunohistochemistry showed that BDC were strongly positive for antibodies against HLA-DR, CD 11c, CD 18, CD44 and CD54, and moderately positive for anti-CD 11a, CD31,CD43 and CD58. In addition, BDC were moderately positive for anti-Sialyl Lewis a and strongly positive for anti-Sialyl Lewis x and CD77 (Galα l 4Galβl- 4Glc) Non-stimulated HUVEC were positive for anti-CD29, CD31 and CD77. An in vitro adhesion assay showed that only a small percentage of radiolabelled BDC bound to non-stimulated HUVEC (16.9 ± 5.9%, mean±SD). Stimulation of the HUVEC with IL-1 for 4 h produced a significant increase (P<0.002) in the percentage of radiolabelled BDC that bound to HUVEC (42.3 ± 7.1%). Preincubation of HUVEC with antibodies against E-selectin (10μg/ml) significantly inhibited (P<0.02) the binding of radiolabelled BDC to activated HUVEC (32.2±1.3%) whereas preincubution of BDC with antibodies against CD54, CD18, CD11b, CD11c and Sialyl Lewis x did not produce any significant inhibition. Preincubation of BDC with Sialyl Lewis a antibody and with isotype-matched control antibodies did not affect the increased binding of BDC to IL-l-activated HUVEC. Thus, E-selectin seems to be involved in adhesion of BDC to IL-1-stimulated HUVEC.     

Smooth-Muscle-Like Cells Derived from Human Embryonic Stem Cells Support and Augment Cord-Like Structures In Vitro

Engineering vascularized tissue is crucial for its successful implantation, survival, and integration with the host tissue. Vascular smooth muscle cells (v-SMCs) provide physical support to the vasculature and aid in maintaining endothelial viability. In this study, we show an efficient derivation of v-SMCs from human embryonic stem cells (hESCs), and demonstrate their functionality and ability to support the vasculature in vitro. Human ESCs were differentiated in monolayers and supplemented with platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta 1 (TGF-β1). Human ESC-derived smooth-muscle-like cells (SMLCs) were found to highly express specific smooth muscle cell (SMC) markers—including α-smooth muscle actin, calponin, SM22, and smooth muscle myosin heavy chain—to produce and secrete fibronectin and collagen, and to contract in response to carbachol. In vitro tubulogenesis assays revealed that these hESC-derived SMLCs interacted with human endothelial progenitor cell (EPCs) to form longer and thicker cord-like structures in vitro. We have demonstrated a simple protocol for the efficient derivation of highly purified SMLCs from hESCs. These in vitro functional SMLCs interacted with EPCs to support and augment capillary-like structures (CLSs), demonstrating the potential of hESCs as a cell source for therapeutic vascular tissue engineering.     

人外周血树状突细胞在体外对LAK杀伤活性的影响

本文采用三因素不同水平的析因设计，研究了人外周血树状突细胞在体外对ＬＡＫ抗肿瘤作用的影响。结果显示，３种浓度的树状突细胞均能增强ＬＡＫ的杀伤活性（Ｐ＜０．００１），以中等浓度的树状突细胞作用最为明显。加入ＩＬ－２后，树状突细胞对ＬＡＫ抗肿瘤活性的增强程度进一步提高。     

A Clinical Study of HBsAg‐Activated Dendritic Cells and Cytokine‐Induced Killer Cells During the Treatment for Chronic Hepatitis B

We aim to study the therapeutic effects of HBsAg‐activated DCs and cytokine‐induced killer (CIK) cells as adoptive immunotherapy in patients with Chronic Hepatitis B (CHB). Autologous HBsAg‐activated DC–CIK cells were infused into patients with CHB to evaluate their effect on HBV‐DNA, HBsAg, ALT, etc. The viral load in the treatment group decreased significantly (P < 0.001), while that in the control group did not decrease (P > 0.05). Twenty‐one patients (63.6% efficiency) in the treatment group had a viral response (≥2 log decrease in viral load), while four patients (14.8% efficiency) from the control group had a viral response. There were significant differences in the viral responses of the two groups (the control group 63.6% versus the control group 14.8%, P < 0.001). We concluded that the immunity was enhanced after HBsAg activation in DCs and CIK cells. Reinfusion of autologous HBsAg‐activated DC–CIK cells inhibited HBV proliferation in 21 of 33 (63.6%) patients.     

Generation of Full-Length Functional Antibody against PreS2 of Hepatitis B Virus in Hepatic Cells In Vitro from Bicistrons Mediated by Gutless Adenovirus

Background

Monoclonal antibodies (mAbs) have been developed as effective therapeutics for a wide variety of diseases. Delivery of mAbs by gene transfer provides an option for overcoming the difficulties in mAb production and manufacturing processes. However, for the polymeric structure of full-length mAbs, it is important to design an optimal gene transfer system for mAb generation.

Methods

Gutless adenovirus and liver-specific promoter transthyretin (TTR) were combined to deliver bicistronic mAb genes in human hepatic cell lines. In order to optimize the bicistrons for mAb generation, four bicistrons were designed and compared, and the most efficient one was selected. ELISA and Western blot were conducted to evaluate mAb products in the supernatants.

Results

Our data showed that all of four gutless adenoviruses elicited liver-specific mAb production in HepG2 and Hep3B hepatic cell lines. It was observed that the L2AH bicistron construct (comprising an immunoglobulin light-chain cDNA situated 50 of a heavy-chain cDNA, with a foot-and-mouth disease virus 2A cleavage site in the middle, subcloned into the helper-dependent adenovirus plasmid pGL) could induce the highest level expression of mAb (about 5.0 μg/mL in Hep3B) among these four constructs. Importantly, the mAb products by gene transfer methods retained specific antigen-binding activity.

Conclusion

Our studies gave further evidence that it was feasible to produce active full-length mAb in human hepatic cell lines in vitro by a special gene delivery system. Moreover, we developed an optimized bicistron gene transfer system for future gene therapy research, which may also be of use in industrial mAb production.     

人类胚胎生殖细胞体外分化为心肌细胞的研究

胚胎生殖（EG）细胞是来源于胚胎原始生殖细胞（PGCs）的多潜能干细胞。采用无饲养层细胞、无细胞因子等的基础培养液（DMEM＋20％NBS＋0．1mM 2Me）培养EG细胞，部分在基础培养液添加10μmol／LRA＋0．75％DMSO或10μmol／L 5-氮胞苷（5-AZA）诱导人类EG细胞向心肌细胞分化，以检测其是否具有向心肌细胞自发分化的能力。9例（8．49％，9／106）胎儿EG细胞在体外分化得到20个节律性心脏跳动样细胞团，其搏动节律为20～120次／min，体外维持节律性搏动最短2d，最长至15d，呈PAS，Myoglobin，α-actin阳性；对K^＋、Ca^＋、肾上腺素等具有与在体心脏相似的反应性；透射电镜观察具有心肌细胞样结构。添加DMSO和RA或5-AZA诱导未得到跳动样心肌细胞，但可提高心肌α-actin免疫组织化学染色阳性率；表明人类胚胎生殖细胞具有向心肌细胞分化的潜能。     

Lipopolysaccharide‐Induced In Vivo Activation of Follicular Dendritic Cells is Tumor Necrosis Factor Receptor‐1 Independent

It is well recognized that tumor necrosis factor receptor‐1 (TNFR1) signaling pathway (with lymphotoxin‐β receptor) is of critical importance for the development, activation, and clustering of follicular dendritic cells (FDCs) within the lymphoid follicles. However, further information on the molecular control of these processes is very sparse. Here, we show that intravenous application of lipopolysaccharide induces the clear and prominent morphological signs of FDC development and activation in vivo, which is independent of TNFR1 pathway. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.     

Polystyrene Brushes on Fully Deuterated Organic Nanoparticles by Surface‐Initiated Nitroxide‐Mediated Radical Polymerization

The synthesis of polystyrene (PS) brushes on fully deuterated PS nanoparticles by surface‐initiated nitroxide‐mediated radical polymerization (SI‐NMRP) is reported. Due to the high demand of deuterated monomers, an efficient deuteration procedure of suitable and readily available precursors is developed. SI‐NMRP of styrene is improved regarding reaction control, grafting density, and conversion. Insights into the scaling behavior and conformational features of surface‐attached PS chains on deuterated particles are investigated by using dynamic light scattering measurements, proving that polymer brushes are formed. The particles with surface‐attached initiator are shown to be uniform spherical core‐shell particles by small‐angle neutron scattering measurements.