Prospective, randomized, double-blind phase 2B trial of the TLPO and TLPLDC vaccines to prevent recurrence of resected stage III/IV melanoma: a prespecified 36-month analysis.

BACKGROUND: The tumor lysate, particle-loaded, dendritic cell (TLPLDC) vaccine is made by ex vivo priming matured autologous dendritic cells (DCs) with yeast cell wall particles (YCWPs) loaded with autologous tumor lysate (TL). The tumor lysate, particle only (TLPO) vaccine uses autologous TL-loaded YCWPs coated with silicate for in vivo DC loading. Here we report the 36-month prespecified analyses of this prospective, randomized, double-blind trial investigating the ability of the TLPO and TLPLDC (±granulocyte-colony stimulating factor (G-CSF)) vaccines to prevent melanoma recurrence in high-risk patients. METHODS: Patients with clinically disease-free stage III/IV melanoma were randomized 2:1 initially to TLPLDC versus placebo (n=124) and subsequently TLPO versus TLPLDC (n=63). All patients were randomized and blinded; however, the placebo control arm was replaced in the second randomization scheme with another novel vaccine; some analyses in this paper therefore reflect a combination of the two randomization schemes. Patients receiving the TLPLDC vaccine were further divided by their method of DC harvest (with or without G-CSF pretreatment); this was not randomized. The use of standard of care checkpoint inhibitors was not stratified between groups. Safety was assessed and Kaplan-Meier and log-rank analyses compared disease-free (DFS) and overall survival (OS). RESULTS: After combining the two randomization processes, a total of 187 patients were allocated between treatment arms: placebo (n=41), TLPLDC (n=103), or TLPO (n=43). The allocation among arms created by the addition of patients from the two separate randomization schemes does not reflect concurrent randomization among all treatment arms. TLPLDC was further divided by use of G-CSF in DC harvest: no G-CSF (TLPLDC) (n=47) and with G-CSF (TLPLDC+G) (n=56). Median follow-up was 35.8 months. Only two patients experienced a related adverse event ≥grade 3, one each in the TLPLDC+G and placebo arms. DFS was 27.2% (placebo), 55.4% (TLPLDC), 22.9% (TLPLDC+G), and 60.9% (TLPO) (p<0.001). OS was 62.5% (placebo), 93.6% (TLPLDC), 57.7% (TLPLDC+G), and 94.6% (TLPO) (p=0.002). CONCLUSIONS: The TLPO and TLPLDC (without G-CSF) vaccines were associated with improved DFS and OS in this clinical trial. Given production and manufacturing advantages, the efficacy of the TLPO vaccine will be confirmed in a phase 3 trial. TRIAL REGISTRATION NUMBER: NCT02301611.

Divergent clinical outcomes in a phase 2B trial of the TLPLDC vaccine in preventing melanoma recurrence and the impact of dendritic cell collection methodology: a randomized clinical trial.

BACKGROUND: A randomized, double-blind, placebo-controlled phase 2b trial of the tumor lysate, particle-loaded, dendritic cell (TLPLDC) vaccine was conducted in patients with resected stage III/IV melanoma. Dendritic cells (DCs) were harvested with and without granulocyte-colony stimulating factor (G-CSF). This analysis investigates differences in clinical outcomes and RNA gene expression between DC harvest methods. METHODS: The TLPLDC vaccine is created by loading autologous tumor lysate into yeast cell wall particles (YCWPs) and exposing them to phagocytosis by DCs. For DC harvest, patients had a direct blood draw or were pretreated with G-CSF before blood draw. Patients were randomized 2:1 to receive TLPLDC or placebo. Differences in disease-free survival (DFS) and overall survival (OS) were evaluated. RNA-seq analysis was performed on the total RNA of TLPLDC + G and TLPLDC vaccines to compare gene expression between groups. RESULTS: 144 patients were randomized: 103 TLPLDC (47 TLPLDC/56 TLPLDC + G) and 41 placebo (19 placebo/22 placebo + G). Median follow-up was 27.0 months. Both 36-month DFS (55.8% vs. 24.4% vs. 30.0%, p = 0.010) and OS (94.2% vs. 69.8% vs. 70.9%, p = 0.024) were improved in TLPLDC compared to TLPLDC + G or placebo, respectively. When compared to TLPLDC + G vaccine, RNA-seq from TLPLDC vaccine showed upregulation of genes associated with DC maturation and downregulation of genes associated with DC suppression or immaturity. CONCLUSIONS: Patients receiving TLPLDC vaccine without G-CSF had improved OS and DFS. Outcomes remained similar between patients receiving TLPLDC + G and placebo. Direct DC harvest without G-CSF had higher expression of genes linked to DC maturation, likely improving clinical efficacy.

Immunotherapeutic effects of intratumorally injected Zymosan-Adenovirus conjugates encoding constant active IRF3 in a melanoma mouse model.

M2-like tumor-associated macrophages (TAMs) play a significant role in immunosuppressive conditions in the tumor microenvironment (TME). TAM reprogramming, a dual-pronged therapy, reduces immunosuppression and induces immune favorable conditions in the TME. In this study, recombinant adenoviruses encoding active forms of interferon regulatory factor 3 (IRF3) were conjugated to zymosan particles to target phagocytic cells to create a pro-inflammatory immunomodulatory therapy. We determined TAM reprogramming by upregulation and downregulation of M1- and M2-associated genes, respectively, as well as cytokine and transcription factor expression in vitro. The overall shift to immune favorable conditions in the TME was suggested by metabolic, cytokine, and immune cell gene expression. Our data indicated that the zymosan:adenovirus (Zym:Ad) particle itself induced a shift from M2-like to M1-like TAMs, a shift in immune status of the TME, and systemic tumor immunity as determined using a double tumor melanoma mouse model and splenocyte functional assay. Notably, direct intratumoral injection of Zym:Ad IRF3 reduced tumor growth more significantly than Zym:Ad GFP, indicating additional therapeutic benefits due to incorporation of constant active IRF3.

Manipulation of the tumor immuno-microenvironment via TAM-targeted expression of transcription factors.

An immunosuppressive tumor microenvironment (TME) leads to cancer growth, metastasis, and therapeutic resistance. Immunomodulatory immunotherapy aims to skew the immunosuppressive TME back to an immune active state. Tumor-associated macrophages (TAMs) are a critical component of the TME that are actively involved in tumor-specific inflammation and immunosuppression. TAMs exhibit a diverse range of phenotypes and functions, from pro-tumor to anti-tumor. The plasticity of TAMs makes them a promising target for immunotherapy, and TAM-targeted therapies via different strategies have shown great potential. This review discusses current TAM-specific delivery targets and genes of interest for TAM-reprogramming. As phagocytic cells, TAMs have several receptors that have been used to increase TAM-targeted in vivo delivery. Furthermore, a promising approach for reprogramming TAMs is to activate or suppress specific transcription factors in the signal transducers and activators of transcription (STAT) and interferon regulatory factor (IRF) families. Altering TAM transcription factor expression results in a potent shift in cytokine expression and overall TAM function potentially tipping the balance from an immunosuppressive to an immune active TME.

Comprehensive Analysis of miRNAs and Target mRNAs between Immature and Mature Testis Tissue in Chinese Red Steppes Cattle.

This study aims to screen potential regulators and regulate fecundity networks between microRNAs (miRNAs) and target genes. The bovine testes of immature and mature Chinese Red Steppes were performed by genome-wide analysis of mRNAs and miRNAs. Compared with testicular tissues of newborns, 6051 upregulated genes and 7104 downregulated genes in adult cattle were identified as differentially expressed genes (DEGs). The DEGs were significantly enriched in 808 GO terms (p < 0.05) including male gonad development, male genitalia development, spermatogenesis, and sperm motility. Moreover, DEGs were also significantly enriched in 105 KEGG pathways (p < 0.05), including cGMP-PKG signaling pathway and calcium signaling pathway. To explore the expression of miRNA-regulated gene expression, 896 differentially expressed target genes negatively regulated with the expression levels of 31 differentially expressed miRNAs (DERs) were predicted and analyzed, and a network-integrated analysis was constructed. Furthermore, real-time PCR was performed to verify the expression levels of DEGs and DERs. Our results identified novel candidate DEGs and DERs correlated with male reproduction and intricate regulating networks between miRNAs and genes, which will be valuable for future genetic and epigenetic studies of sperm development and maturity, as well as providing valuable insights into the molecular mechanisms of male fertility and spermatogenesis in cattle.

Integrative analysis of miRNAs and mRNAs revealed regulation of lipid metabolism in dairy cattle.

Lipid metabolism in bovine mammary epithelial cells has been the primary focus of the research of milk fat percentage of dairy cattle. Functional microRNAs can affect lipid metabolism by regulating the expression of candidate genes. The purpose of the study was to screen and identify differentially expressed miRNAs, candidate genes, and co-regulatory pathways related to the metabolism of milk fat. To achieve this aim, we used miRNA and transcriptome data from the mammary epithelial cells of dairy cattle with high (H, 4.85%) and low milk fat percentages (L, 3.41%) during mid-lactation. One hundred ninety differentially expressed genes and 33 differentially expressed miRNAs were significantly enriched in related regulatory networks, of which 27 candidate genes regulated by 18 differentially expressed miRNAs significantly enriched in pathways related to lipid metabolism (p < 0.05). Target relationships between PDE4D and bta-miR-148a, PEG10 and bta-miR-877, SOD3 and bta-miR-2382-5p, and ADAMTS1 and bta-miR-2425-5p were verified using luciferase reporter assays and quantitative RT-PCR. The detection of triglyceride production in BMECs showed that bta-miR-21-3p and bta-miR-148a promote triglyceride synthesis, whereas bta-miR-124a, bta-miR-877, bta-miR-2382-5p, and bta-miR-2425-5p inhibit triglyceride synthesis. The conjoint analysis could identify functional miRNAs and regulatory candidate genes involved in lipid metabolism within the co-expression networks of the dairy cattle mammary system, which contributes to the understanding of potential regulatory mechanisms of genetic element and gene signaling networks involved in milk fat metabolism.

miR-2382-5p Regulates Lipid Metabolism by Targeting NDRG2 in Mammary Epithelial Cells of Dairy Cattle.

microRNA is a class of single-stranded RNA molecules of about 22-24 nucleotides in length, which regulate a variety of biological processes, including lipid metabolism and triglyceride synthesis at transcriptional and translational levels by degrading target mRNAs or interfering with the protein production. In this study, the effect of miR-2382-5p on triglyceride levels was examined in bovine mammary epithelial cells (BMECs), and the results showed that miR-2382-5p could decrease the content of triglyceride. Furthermore, miR-2382-5p regulated the expression of lipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma co-activator 1beta (PPARGC1B), hormone-sensitive lipase (HSL), and peroxisome proliferator-activated receptor gamma (PPARγ), which are known to increase triglyceride decomposition in lipid metabolism. Luciferase reporter assay and quantitative real-time PCR (qPCR) validated that miR-2382-5p downregulated the mRNA expression of target gene N-myc downstream-regulated gene 2 (NDRG2) by specifically recognizing and binding to its 3'-untranslated region (UTR). Meanwhile, overexpression of NDRG2 led to increased triglyceride and cholesterol production in BMECs. In summary, this study suggested that miR-2382-5p regulated lipid metabolism by targeting NDRG2, which might be a potential target for molecular manipulation of milk fat composition to produce healthy milk. This study also provided basic data for further understanding lipid metabolism in dairy cattle.

Lentiviral delivery of novel fusion protein IL12/FasTI for cancer immune/gene therapy.

Many of the cytokine-based cancer immunotherapies are hindered by the devastating side effects of systemic delivery of the cytokines. To address this problem, we previously described a novel approach to locally achieve high doses of interleukin-12 (IL-12) in tumors and demonstrated that bi-functional fusion protein mIL-12/FasTI expressed by stable clones of TC-1 cells efficiently suppressed tumor proliferation by activating natural killer (NK) cells and other cytolytic killer cells and sending apoptotic signals into tumor cells. In the present study, we employed a lentiviral vector-based gene delivery system to deliver this fusion construct directly into tumor cells. We show that lentiviral vector efficiently delivers the fusion constructs into Hela cells in vitro as assayed by RT-PCR and immunohistochemistry (IHC). We also confirm that fusion protein mIL-12/FasTI delivered by the viral vector significantly enhanced killer cell activation, increased caspase-3 activity and decreased tumor growth in vitro. This study offers a further step for fusion protein cancer therapy for cancer patients.

Effect of QSOX1 on cattle carcass traits as well as apoptosis and triglyceride production in bovine fetal fibroblasts and mammary epithelial cells.

QSOX1 (quiescin-sulfhydryl oxidase 1) is involved in various processes, including apoptosis and the development of breast diseases. Here, we investigated the effect of QSOX1 on the meat quality of Simmental cattle by analyzing the correlation between QSOX1 single nucleotide polymorphisms (SNPs), I2 204 C>T and I2 378 C>T, and certain meat quality traits. The effects of QSOX1 on triglyceride synthesis and cell apoptosis were further validated by gene silencing or overexpression in bovine fetal fibroblasts and mammary epithelial cells. The results showed that I2 204 C>T and I2 378 C>T had significant correlations with loin thickness, hind hoof weight, fat coverage, liver weight, heart weight, marbling and back fat thickness (P<0.05). QSOX1 overexpression also increased triglyceride production and suppressed apoptosis. In summary, QSOX1 is an important factor for meat quality, lipid metabolism, and cell apoptosis, indicating that QSOX1 could be used as a biomarker to assist in breeding cattle with superior meat.

Initial phase I/IIa trial results of an autologous tumor lysate, particle-loaded, dendritic cell (TLPLDC) vaccine in patients with solid tumors.

INTRODUCTION: Tumor vaccines use various strategies to generate immune responses, commonly targeting generic tumor-associated antigens. The tumor lysate, particle-loaded, dendritic cell (TLPLDC) vaccine is produced from DC loaded with autologous tumor antigens, creating a patient-specific vaccine. Here, we describe initial phase I/IIa trial results. METHODS: This trial includes patients with any stage solid tumors, ECOG ≤1, and >4 months life-expectancy. A personalized vaccine is created using 1 mg of tumor and 120 ml blood (to isolate DC). Primary vaccination series (PVS) is four monthly inoculations. Patients are followed per standard of care (SOC). Endpoints include safety and tumor response (RECIST v1.1). RESULTS: 44 patients were enrolled and vaccinated consisting of 31 late stage patients with residual/measurable disease, and 13 disease-free patients after SOC therapies. While 4 patients progressed before completing the PVS, 12/31 (39%) demonstrated clinical benefit (2 complete responses, 4 partial responses, 6 stable disease). In the adjuvant setting, 46% of late stage patients remain disease free at a median of 22.5 months. CONCLUSIONS: The TLPLDC vaccine is scalable, generates a personalized DC vaccine, and requires little autologous tumor tissue and few DC. The vaccine is safe, with primarily grade 0-2 toxicities, and nearly 40% clinical benefit rate in varied tumors, warranting further study. TRIAL REGISTRATION: ISRCTN81339386, Registered 2/17/2016.

Tumor lysate particle loaded dendritic cell vaccine: preclinical testing of a novel personalized cancer vaccine.

AIM: We developed a novel approach to efficiently deliver autologous tumor antigens to the cytoplasm of dendritic cells (DC) using yeast cell wall particles (YCWP). MATERIALS AND METHODS: Loading of YCWP, leakage of protein from loaded YCWP and cytoplasmic delivery of YCWP content was assessed using fluorescent-tagged experiments. Spectrophotometric analysis compared the epitope-specific T-cell responses following antigen presentation via YCWP versus exogenous loading. The in vivo effectiveness of tumor lysate (TL) particle loaded DC (TLPLDC) vaccine was assessed using murine melanoma models. RESULTS: In fluorescence-tagged experiments, YCWP efficiently delivered antigen to the cytoplasm of DC. TLPLDC loading was more effective than conventional exogenous loading of DC. Finally, in murine melanoma models, TLPLDC outperformed an analogous dendritoma vaccine. CONCLUSION: The TLPLDC vaccine is commercially scalable and holds the potential of producing personalized vaccines.

Comparative genome-wide methylation analysis of longissimus dorsi muscles between Japanese black (Wagyu) and Chinese Red Steppes cattle.

DNA methylation is an important epigenetic mechanism involved in expression of genes in many biological processes including muscle growth and development. Its effects on economically important traits are evinced from reported significant differences in meat quality traits between Japanese black (Wagyu) and Chinese Red Steppes cattle, thus presenting a unique model for analyzing the effects of DNA methylation on these traits. In the present study, we performed whole genome DNA methylation analysis in the two breeds by whole genome bisulfite sequencing (WGBS). Overall, 23150 differentially methylated regions (DMRs) were identified which were located in 8596 genes enriched in 9922 GO terms, of which 1046 GO terms were significantly enriched (p<0.05) including lipid translocation (GO: 0034204) and lipid transport (GO: 0015914). KEGG analysis showed that the DMR related genes were distributed among 276 pathways. Correlation analysis found that 331 DMRs were negatively correlated with the expression levels of differentially expressed genes (DEGs) with 21 DMRs located in promoter regions. Our results identified novel candidate DMRs and DEGs correlated with meat quality traits, which will be valuable for future genomic and epigenomic studies of muscle development and for marker assisted selection of meat quality traits.

MICA/IL-12: A novel bifunctional protein for killer cell activation.

Natural killer (NK) cells have the potential to be effective killers of tumor cells. They are governed by inhibitory and activating receptors such as NKG2D, whose ligands are normally upregulated in cells that are stressed, like cancer cells. Advanced cancer cells, however, have ways to reduce the expression of these ligands, leaving them less detectable by NK cells. Along with these receptors, NK cells also require activating cytokines, such as IL-12. A previous study in our laboratory showed that a fusion protein of the extracellular domain of mouse UL-16 binding protein-like transcript 1 (MULT1E) and mouse interleukin 12 (IL-12) can effectively activate mouse NK cells by in vitro assays and in vivo in animal tumor models. The aim of the present study was to expand the concept of developing a novel bifunctional fusion protein for enhanced NK cell activation to human killer cells. The proposed protein combines the extracellular domain of a human NKG2D ligand, MHC class I polypeptide-related sequence A (MICA) and IL-12. It is hypothesized that when expressed by tumor cells, the protein will activate human NK and other killer cells using the NKG2D receptor, and deliver IL-12 to the NK cells where it can interact with the IL-12R and enhance cytotoxicity. The fusion protein, when expressed by engineered tumor cells, indeed activated NK92 cells as measured by an increase in interferon-γ (IFN-γ) production and an increase in cytotoxicity of tumor cells. The fusion protein was also able to increase the proliferation of human peripheral blood mononuclear cells (PBMCs) and augment their production of IFN-γ. This study along with the data from the previous mouse studies suggest that the MICA/IL-12 bifunctional fusion protein represents an effective activator of killer cells for cancer treatment.

Bovine lipid metabolism related gene GPAM: Molecular characterization, function identification, and association analysis with fat deposition traits.

Glycerol-3-phosphate acyltransferase mitochondrial (GPAM) is the enzyme that catalyzes the initial and committed step of glycerolipid synthesis in animal lipometabolism related pathway. In the present study, the correlation of GPAM expression level with adipogenesis was examined in vitro by RNAi and gene expression to silencing and over-expression of GPAM gene in BEF cells. And then, 2 novel polymorphisms were identified within key functional domain of bovine GPAM gene by sequencing and the relationship between variants of GPAM gene with fat deposition traits of Chinese Simmental-cross steers was analyzed using statistical methods. The result showed that the knockdown of GPAM expression significantly reduced the synthesis of triglycerides and expression of lipid metabolism-related gene in BEF cells (p<0.05), and the over-expression of GPAM could significantly increased the levels of triglyceride and expression of lipid metabolism-related gene (p<0.05). The results of the correlation analysis also showed that cattle with TC genotype of GPAM E20-2823C>T was significantly associated with fatty acid composition of intramuscular fat (had higher content of arachidic acid, eicosenoic acid, and arachidonic acid), individuals with A-allele homozygotes and AG-allele heterozygotes of E20-3386G>A both had higher content of myristic acid, α-linolenic acid, and linoleic acid in intramuscular fat than those with G-allele homozygotes (p<0.05). The individuals with AA genotype of E20-3386G>A was significantly associated with higher marbling score, and the individuals with CC homozygotes of E20-2823C>T had higher GFW and omasum fat weight in beef cattle population. GPAM play a pivotal role in the regulation of cellular triacylglycerol and phospholipid levels, and its mutations could serve as a useful molecular marker used for marker assisted selection in beef cattle breeding process.

miR-29b Targets LPL and TDG Genes and Regulates Apoptosis and Triglyceride Production in MECs.

microRNAs are involved in various biological processes by regulating the degradation or repressing the translation of target genes. In this study, the target genes of miR-29b were analyzed and predicted by bioinformatics. And lipoprotein lipase (LPL) and thymine DNA glycosylase (TDG) were selected for further validation by dual luciferase reporter assay. In addition, we investigated the effects of miR-29b on triglyceride synthesis and mammary epithelial cell (MEC) apoptosis. The result showed that luciferase activity was significantly lower in cells that miR-29b cotransfected with LPL and TDG gene reporter vectors (pmiR-RB-REPORT-LPL-WT, pmiR-RB-REPORT-TDG-WT) than in cells of miR-29b cotransfected with gene reporter vectors (pmiR-RB-REPORT-LPL-mut and pmiR-RB-REPORT-LPL-si; pmiR-RB-REPORT-TDG-mut and pmiR-RB-REPORT-TDG-si) (p < 0.05), indicating that target sites existed in 3'UTR of LPL and TDG. Furthermore, the expressions of miR-29b were negatively correlated with levels of mRNA and protein of LPL and TDG gene using quantitative real-time polymerase chain reaction and western blot analysis, suggesting that miR-29b might play an important role in regulation of LPL and TDG gene expression. Finally, miR-29b promoted triglyceride production and suppressed apoptosis in MECs, which might be attributed to the expressions of target genes LPL and TDG.

Mouse interleukin-12/FasTI: A novel bi-functional fusion protein for cancer immuno/gene therapy.

Whereas cancer immunotherapy with cytokines in recent research was demonstrated effective in activating immune response against tumor cells, one major obstacle with the use of these cytokines is their severe side effects when delivered systemically at high doses. Another challenge is that advanced tumor cells often evade immunosurveillance of the immune system as well as of the Fas-mediated apoptosis by various mechanisms. We report the design and preliminary evaluation of the antitumor activity of a novel fusion protein-mIL-12/FasTI, consisting of mouse interleukin-12 and the transmembrane and intracellular domains of mouse Fas. The fusion construct (pmIL-12/FasTI) was transfected into mouse lung carcinoma cell line TC-1. Stable cell clones expressing the fusion protein were established as assayed by RT-PCR and immunohistochemistry. ELISA and cell proliferation analyses demonstrated that NK cells were effectively activated by the fusion protein with increased IFN-γ production and cytotoxicity. Enhanced caspase-3 activity of the clones when co-cultured with NK cells indicated that apoptosis was induced through Fas/FasL signaling pathway. The preliminary results suggest a synergized anticancer activity of the fusion protein. It may represent a promising therapeutic agent for cancer treatment.

A phase I/IIa clinical trial in stage IV melanoma of an autologous tumor-dendritic cell fusion (dendritoma) vaccine with low dose interleukin-2.

BACKGROUND: Stage IV melanoma has high mortality, largely unaffected by traditional therapies. Immunotherapy including cytokine therapies and checkpoint inhibitors improves outcomes, but has significant toxicities. In this phase I/IIa trial, we investigated safety and efficacy of a dendritoma vaccine, an active, specific immunotherapy, in stage IV melanoma patients. METHODS: Autologous tumor lysate and dendritic cells were fused creating dendritoma vaccines for each patient. Phase I patients were vaccinated every 3 months with IL-2 given for 5 days after initial inoculation. Phase IIa patients were vaccinated every 6 weeks with IL-2 given on days 1, 3 and 5 after initial inoculation. Toxicity and clinical outcomes were assessed. RESULTS: Twenty-five patients were enrolled and inoculated. All dendritoma and IL-2 toxicities were

Novel dendritic cell-based vaccination in late stage melanoma.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play an important role in stimulating an immune response of both CD4(+) T helper cells and CD8(+) cytotoxic T lymphocytes (CTLs). As such, DCs have been studied extensively in cancer immunotherapy for their capability to induce a specific anti-tumor response when loaded with tumor antigens. However, when the most relevant antigens of a tumor remain to be identified, alternative approaches are required. Formation of a dentritoma, a fused DC and tumor cells hybrid, is one strategy. Although initial studies of these hybrid cells are promising, several limitations interfere with its clinical and commercial application. Here we present early experience in clinical trials and an alternative approach to manufacturing this DC/tumor cell hybrid for use in the treatment of late stage and metastatic melanoma.

A biotin-streptavidin-biotin bridge dramatically enhances cell fusion.

Although the generation of hybrid cells by cell fusion plays a significant role in biotechnology and biomedicine, the low cell-fusion rates and the limitation of large-scale cell fusion for clinical applications of the two widely used approaches, polyethylene-glycol (PEG)-mediated cell fusion and electrofusion, hinder the application of this critical technology in certain key areas, including cancer immunotherapy. In the present study, a simple procedure that can not only significantly increase the heterologous cell fusion but is also capable of producing fused cells on a large scale is reported. A biotin-streptavidin-biotin (BSB) bridge was created by coating one to-be-fused cell with biotin and the other with biotin-streptavidin. The BSB bridge enhances cell-fusion rates induced with PEG fusion or electrofusion by 10-30% depending on the cell types when compared with cell fusions without the bridge. The procedure described increases heterologous cell pairing and eliminates the alignment step required for the majority of electrofusions. Notably, it can be used to make large-scale cell fusions for clinical applications.

The effect of leader peptide mutations on the biological function of bovine myostatin gene.

The growth of muscle fibers can be negatively regulated by bovine myostatin. The first two exons of myostatin gene code for the N-propeptide and its third exon codes for the C-polypeptide. Myostatin is secreted as a latent configuration formed by dimerization of two matured C peptides non-covalently linked with the N terminal pro-peptide. Pro-peptide has two distinct functions in guiding protein folding and regulating biological activity of myostatin. When the structure of the leader peptide is altered via mutations resulting in more tight binding with the mature peptide, myostatin function is inhibited, resulting in the changes of P21 and CDK2 expression levels which are related to the regulation of cell cycle. In the present study, the coding region of bMSTN (bovine myostatin) gene was amplified and mutated (A224C and G938A) through fusion PCR, and the mutated bMSTN gene (bMSTN-mut) was inserted in frame into the pEF1a-IRES-DsRed-Express2 vector and transfected into bovine fibroblast cells. The expression levels of bMSTN-mut, P21 and CDK2 (cyclin dependent kinase 2) were examined with qPCR and Western-blotting. Changes in cell cycle after transfection were also analyzed with flow cytometry. The results indicated that leader peptide mutation resulted in down-regulation of P21 expression levels and up-regulation of CDK2 expression levels. The flow cytometry results showed that the proportion of cells in the G0/G1-phase was lower and that of cells in the S-phase was higher in bMSTN-mut transfected group than that in the control group. The proliferation rate of bMSTN-mut transfected cells was also significantly higher than that of the control cells. In conclusion, the studies have shown that the pEF1a-IRES-DsRed-Express2-bMSTN-mut recombinant plasmid could effectively promote the proliferation of bovine fibroblast cells. The site-directed mutagenesis of bMSTN gene leader peptide and in vitro expression in bovine fibroblast cells could be helpful to further the studies of bMSTN in regulating bovine muscle cell growth and development.