[Construction of recombinant human nerve growth factor (rh-ß-NGF) eukaryotic vector and its expression in HEK293 cells].

Human nerve growth factor (NGF) is a nerve cell growth regulation factor, which can provide nutrition for the neurons and promote the neurites outgrowth. In order to produce large-scale recombinant human nerve growth factor (rh-beta-NGF), we constructed a plasmid vector, which can stably express the rh-beta-NGF in the HEK293 cell lines. First, the plasmid of pCMV-beta-NGF-IRES-dhfr was constructed and transformed into HEK293 cells. Then MTX pressurized filter and limiting dilution methods were used to obtain monoclonal HEK293 cell lines. After stepwise reducing serum in culture media, the cells eventually adapted to serum-free medium and secreted rh-beta-NGF. SDS-PAGE analysis revealed that the expression product owned a molecular weight of about 13 kDa and a purity of more than 50%. The peptide mapping sequencing analysis demonstrated the sequences of rh-beta-NGF matched with the theoretical ones. Later we purified this protein by ion exchange and molecular sieve chromatograph. Finally, our experimental results exhibited that the recombinant cell lines can stably express rh-beta-NGF with a high efficiency of more than 20 pg/cell x day. In addition, this protein could successfully induce differentiation of PC12 cells. In summary, our recombinant HEK293 cells can express bio-active rh-beta-NGF with great efficiency and stability, which supply a valid basis to large-scale production of rh-beta-NGF.

Novel alginate three-dimensional static and rotating culture systems for effective ex vivo amplification of human cord blood hematopoietic stem cells and in vivo functional analysis of amplified cells in NOD/SCID mice.

BACKGROUND: Autologous and allogeneic hematopoietic stem cell (HSC) transplantations serve as effective therapy for a variety of hematologic and other diseases. Umbilical cord blood (UCB) is an important source of HSCs. However, it is difficult to obtain a sufficient number of HSCs with complete self-renewal capability derived from a single unit of UCB for use in adult transplantation. In this study, we investigated two novel three-dimensional (3D) culture systems (static and rotating) for ex vivo expansion of HSCs from UCB. STUDY DESIGN AND METHODS: We encapsulated the human cord blood mononuclear cells (CBMCs) in alginate 3D static and rotating culture systems, compared the cell number amplification, the proportion of CD34+ cells, and the colony-forming capacity of these systems to those of the conventional two-dimensional (2D) system. The amplified cells were transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice to confirm the hematopoiesis reconstruction capacity of the cells. RESULTS: The increase in the cell number and the proportion of CD34+ cells in the CBMCs was more effective in these 3D alginate culture systems than in the conventional 2D culture system under the same conditions (p < 0.05). The stem cell maintenance capability was confirmed by flow cytometry and colony-forming assay ex vivo and NOD/SCID mice xenogeneic transplantation model in vivo. CONCLUSION: Our results demonstrated that these 3D alginate culture systems are an efficient way to amplify cord blood HSCs for extended periods without having them lose their self-renewal capacity in vivo. These novel 3D alginate culture systems are promising for the amplification of UCB-derived HSCs for clinical application in the future.

Human dendritic cells engineered to secrete interleukin-18 activate MAGE-A3-specific cytotoxic T lymphocytes in vitro.

Adoptive cell transfer (ACT) involves the administration of tumor specific cytotoxic T lymphocytes (CTLs) into a patient to kill cancer cells. Although a promising cancer therapy, limitations on the generation of activated CTLs have restricted ATC's clinical application. Interleukin-18 (IL-18) is an interferon-γ (IFN-γ) inducing factor that plays an important functional role in regulating CTLs. Here, we attempt to use dendritic cells (DCs) modified with a recombinant adenovirus encoding IL-18 (rAd/IL-18) to improve the generation of activated tumor-specific CTLs. These engineered DCs secrete IL-18, increase the expression of co-stimulatory molecules, and enhance the cytotoxic efficacy of melanoma antigen 3 (MAGE-A3)-specific CTLs in vitro. We show that stimulation of CTLs with rAd/IL-18-loaded DCs increases the specific lysis of MAGE-A3-expressing human breast cancer MCF-7 cells, and at the same time increases the production of activated MAGE-A3-specific CTLs. Our results indicate that transducing DCs with rAd/IL-18 increases both the maturation of DCs and the activation level of MAGE-A3-specific CTLs, greatly enhancing the cytotoxic efficacy of CTLs towards tumor cells.

Regulation of gene expression by FSP27 in white and brown adipose tissue.

BACKGROUND: Brown and white adipose tissues (BAT and WAT) play critical roles in controlling energy homeostasis and in the development of obesity and diabetes. The mouse Fat-Specific protein 27 (FSP27), a member of the cell death-inducing DFF45-like effector (CIDE) family, is expressed in both BAT and WAT and is associated with lipid droplets. Over-expression of FSP27 promotes lipid storage, whereas FSP27 deficient mice have improved insulin sensitivity and are resistant to diet-induced obesity. In addition, FSP27-deficient white adipocytes have reduced lipid storage, smaller lipid droplets, increased mitochondrial activity and a higher expression of several BAT-selective genes. To elucidate the molecular mechanism by which FSP27 controls lipid storage and gene expression in WAT and BAT, we systematically analyzed the gene expression profile of FSP27-deficient WAT by microarray analysis and compared the expression levels of a specific set of genes in WAT and BAT by semi-quantitative real-time PCR analysis. RESULTS: BAT-selective genes were significantly up-regulated, whereas WAT-selective genes were down-regulated in the WAT of FSP27-deficient mice. The expression of the BAT-selective genes was also dramatically up-regulated in the WAT of leptin/FSP27 double deficient mice. In addition, the expression levels of genes involved in multiple metabolic pathways, including oxidative phosphorylation, the TCA cycle, fatty acid synthesis and fatty acid oxidation, were increased in the FSP27-deficient WAT. In contrast, the expression levels for genes involved in extracellular matrix remodeling, the classic complement pathway and TGF-beta signaling were down-regulated in the FSP27-deficient WAT. Most importantly, the expression levels of regulatory factors that determine BAT identity, such as CEBP alpha/beta, PRDM16 and major components of the cAMP pathway, were markedly up-regulated in the WAT of FSP27-deficient mice. The expression levels of these regulatory factors were also up-regulated in leptin/FSP27 double deficient mice. Interestingly, distinct gene expression profiles were observed in the BAT of FSP27-deficient mice. Taken together, these data suggest that the WAT of FSP27-deficient mice have a gene expression profile similar to that of BAT. CONCLUSIONS: FSP27 acts as a molecular determinant that controls gene expression for a diversity of metabolic and signaling pathways and, in particular, the expression of regulatory factors, including CEBP alpha/beta, PRDM16 and components of the cAMP signaling pathway, that control the identity of WAT and BAT.

Fucoidin enhances dendritic cell-mediated T-cell cytotoxicity against NY-ESO-1 expressing human cancer cells.

Scavenger receptor A (SR-A) plays a crucial role in affecting the dendritic cell-mediated presentation of cancer testis antigens to T cells against human cancer cells. Here we use a dendritic cell-mediated model to verify that a sulphated polysaccharide, fucoidin, can regulate the adverse regulatory function of SR-A, and lead to the up-regulation of the anti-tumor immunological response. SR-A is a receptor of calreticulin (CRT) existing on the surface of dendritic cells (DCs). CRT is a specific receptor for a NY-ESO-1 cancer testis antigen, and CRT itself is responsible for the cross-presentation of NY-ESO-1 to CD8+ cells and the induction of anti-tumor immunity. Flow cytometrical analysis (FACS) showed that fucoidin was able to significantly enhance the binding ratio of NY-ESO-1 to human DCs in a concentration dependent manner, and that the addition of fucoidin promoted the DC maturation upon stimulation of NY-ESO-1. Results from a cytotoxicity assay indicated that fucoidin-treated DCs stimulated the CD8+ T cells more effectively than non-treated DCs via a cross-presentation pathway. Furthermore, it was found that after stimulated by fucoidin-treated DCs, the CD8+ T cells can release more IFN-gamma than non-fucoidin-treated cells as detected by intracellular IFN-gamma staining. We conclude that fucoidin enhances the cross-presentation of NY-ESO-1 to T cells leading to an increase of T-cell cytotoxicity against NY-ESO-1 expressing human cancer cells.

Up-regulation of mitochondrial activity and acquirement of brown adipose tissue-like property in the white adipose tissue of fsp27 deficient mice.

Fsp27, a member of the Cide family proteins, was shown to localize to lipid droplet and promote lipid storage in adipocytes. We aimed to understand the biological role of Fsp27 in regulating adipose tissue differentiation, insulin sensitivity and energy balance. Fsp27(-/-) mice and Fsp27/lep double deficient mice were generated and we examined the adiposity, whole body metabolism, BAT and WAT morphology, insulin sensitivity, mitochondrial activity, and gene expression changes in these mouse strains. Furthermore, we isolated mouse embryonic fibroblasts (MEFs) from wildtype and Fsp27(-/-) mice, followed by their differentiation into adipocytes in vitro. We found that Fsp27 is expressed in both brown adipose tissue (BAT) and white adipose tissue (WAT) and its levels were significantly elevated in the WAT and liver of leptin-deficient ob/ob mice. Fsp27(-/-) mice had increased energy expenditure, lower levels of plasma triglycerides and free fatty acids. Furthermore, Fsp27(-/-)and Fsp27/lep double-deficient mice are resistant to diet-induced obesity and display increased insulin sensitivity. Moreover, white adipocytes in Fsp27(-/-) mice have reduced triglycerides accumulation and smaller lipid droplets, while levels of mitochondrial proteins, mitochondrial size and activity are dramatically increased. We further demonstrated that BAT-specific genes and key metabolic controlling factors such as FoxC2, PPAR and PGC1alpha were all markedly upregulated. In contrast, factors inhibiting BAT differentiation such as Rb, p107 and RIP140 were down-regulated in the WAT of Fsp27(-/-) mice. Remarkably, Fsp27(-/-) MEFs differentiated in vitro show many brown adipocyte characteristics in the presence of the thyroid hormone triiodothyronine (T3). Our data thus suggest that Fsp27 acts as a novel regulator in vivo to control WAT identity, mitochondrial activity and insulin sensitivity.

Cideb regulates diet-induced obesity, liver steatosis, and insulin sensitivity by controlling lipogenesis and fatty acid oxidation.

OBJECTIVE: Our previous study suggests that Cidea, a member of Cide family proteins that share sequence homology with the DNA fragmentation factor and are expressed at high levels in brown adipose tissue, plays an important role in the development of obesity. Cideb, another member of Cide family protein, is highly expressed in the liver. We would like to understand the physiological role of Cideb in the regulation of energy expenditure and lipid metabolism. RESEARCH DESIGN AND METHODS: We generated Cideb-null mice by homolog recombination and then fed both wild-type and Cideb-null mice with high-fat diet (58% fat). We then characterized the animals' adiposity index, food intake, whole-body metabolic rate, liver morphology, rate of fatty acid synthesis and oxidation, insulin sensitivity, and gene expression profile. RESULTS: Cideb-null mice had lower levels of plasma triglycerides and free fatty acids and were resistant to high-fat diet-induced obesity and live steatosis. In addition, Cideb mutant mice displayed significantly increased insulin sensitivity and enhanced rate of whole-body metabolism and hepatic fatty acid oxidation. More importantly, Cideb-null mice showed decreased lipogenesis and reduced expression levels of acetyl-CoA carboxylase, fatty acid synthase, and stearol-CoA desaturase. We further demonstrated that expression levels of sterol response element binding protein 1c was significantly decreased in Cideb-deficient mice. CONCLUSIONS: Our data demonstrate that Cideb is a novel important regulator in lipid metabolism in the liver. Cideb may represent a new therapeutic target for the treatment of obesity, diabetes, and liver steatosis.