搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Increasing evidence suggests that the deposition of amyloid plaques,composed primarily of the amyloid-beta protein (Abeta),within the cerebrovasculature is a frequent occurrence in Alzheimer's disease and may play a significant role in disease progression. Accordingly,the pathogenic mechanisms by which Abeta can alter vascular function may have therapeutic implications. Despite observations that Abeta elicits a number of physiological responses in endothelial cells,ranging from alteration of protein expression to cell death,the Abeta species accountable for these responses remains unexplored. In the current study,we show that isolated soluble Abeta aggregation intermediates activate human brain microvascular endothelial cells for both adhesion and subsequent transmigration of monocyte cells in the absence of endothelial cell death and monolayer disruption. In contrast,unaggregated Abeta monomer and mature Abeta fibril fail to induce any change in endothelial adhesion or transmigration. Correlations between average Abeta aggregate size and observed increases in adhesion illustrate that smaller soluble aggregates are more potent activators of endothelium. These results support previous studies demonstrating heightened neuronal activity of soluble Abeta aggregates,including Abeta-derived diffusible ligands,oligomers,and protofibrils,and further show that soluble aggregates also selectively exhibit activity in a vascular cell model. View Publication

Adoptive cell therapy is an emerging treatment strategy for a number of serious diseases. Regulatory T (Treg) cells represent 1 cell type of particular interest for therapy of inflammatory conditions,as they are responsible for controlling unwanted immune responses. Initial clinical trials of adoptive transfer of Treg cells in patients with graft-versus-host disease were shown to be safe. However,obtaining sufficient numbers of highly pure and functional Treg cells with minimal contamination remains a challenge. We developed a novel approach to isolate untouched" human Treg cells from healthy donors on the basis of negative selection using the surface markers CD49d and CD127. This procedure View Publication

Proof of drug-target engagement in physiologically-relevant contexts is a key pillar of successful therapeutic target validation. We developed two orthogonal technologies,the cellular thermal shift assay (CETSA) and a covalent chemical probe reporter approach (harnessing sulfonyl fluoride tyrosine labeling and subsequent click chemistry) to measure the occupancy of the mRNA-decapping scavenger enzyme DcpS by a small molecule inhibitor in live cells. Enzyme affinity determined using isothermal dose response fingerprinting (ITDRFCETSA) and the concentration required to occupy 50% of the enzyme (OC50) using the chemical probe reporter assay were very similar. In this case,the chemical probe method worked well due to the long offset kinetics of the reversible inhibitor (determined using a fluorescent dye-tagged probe). This work suggests that CETSA could become the first choice assay to determine in-cell target engagement due to its simplicity. View Publication

High-grade serous ovarian cancer remains a poorly understood disease with a high mortality rate. Although most patients respond to cytotoxic therapies,a majority will experience recurrence. This may be due to a minority of drug-resistant cancer stem-like cells (CSC) that survive chemotherapy and are capable of repopulating heterogeneous tumors. It remains unclear how CSCs are supported in the tumor microenvironment (TME) particularly during chemotherapy exposure. Tumor-associated macrophages (TAM) make up half of the immune population of the ovarian TME and are known to support CSCs and contribute to cancer progression. TAMs are plastic cells that alter their phenotype in response to environmental stimuli and thus may influence CSC maintenance during chemotherapy. Given the plasticity of TAMs,we studied the effects of carboplatin on macrophage phenotypes using both THP1- and peripheral blood mononuclear cell (PBMC)–derived macrophages and whether this supports CSCs and ovarian cancer progression following treatment. We found that carboplatin exposure induces an M1-like proinflammatory phenotype that promotes SOX2 expression,spheroid formation,and CD117 + ovarian CSCs,and that macrophage-secreted CCL2/MCP-1 is at least partially responsible for this effect. Depletion of TAMs during carboplatin exposure results in fewer CSCs and prolonged survival in a xenograft model of ovarian cancer. This study supports a role for platinum-based chemotherapies in promoting a transient proinflammatory M1-like TAM that enriches for CSCs during treatment. Improving our understanding of TME responses to cytotoxic drugs and identifying novel mechanisms of CSC maintenance will enable the development of better therapeutic strategies for high-grade serous ovarian cancer. Significance: We show that chemotherapy enhances proinflammatory macrophage phenotypes that correlate with ovarian cancer progression. Given that macrophages are the most prominent immune cell within these tumors,this work provides the foundation for future translational studies targeting specific macrophage populations during chemotherapy,a promising approach to prevent relapse in ovarian cancer. View Publication

PURPOSE: The propensity of tumor cells to escape immune elimination could limit,if not defeat,the long-term benefits of effective immunotherapeutic protocols. Immunologic targeting of tumor stroma could significantly reduce the ability of tumors to evade immune elimination. Murine studies have shown that inducing immunity against angiogenesis-associated products engenders potent antitumor immunity without significant pathology. It is,however,not known whether T cells corresponding to stromal products are present in humans. In this study,we describe a method to screen for human stromal products that have not triggered significant tolerance and could therefore serve as candidate antigens for cancer immunotherapy. EXPERIMENTAL DESIGN: To identify candidates for human stromal antigens,we used an in vitro-screening method to determine whether dendritic cells transfected with mRNA encoding products,which are overexpressed in the tumor stroma,are capable of stimulating cytotoxic CD8(+) (CTL) responses from human peripheral blood mononuclear cells. RESULTS: CTL responses could be consistently generated against fibroblast activation protein (FAP) but not against matrix metalloproteinase-9 (MMP-9) or MMP-14. To enhance the immunogenicity of the mRNA-translated FAP product,a lysosomal targeting signal derived from lysosome-associated membrane protein-1 (LAMP-1) was fused to the COOH terminus of FAP to redirect the translated product into the class II presentation pathway. Dendritic cells transfected with mRNA encoding the FAP-LAMP fusion product stimulated enhanced CD4(+) and CD8(+) T-cell responses. CONCLUSION: This study identifies FAP,a protease preferentially expressed in tumor-associated fibroblasts,as a candidate human stromal antigen to target in the setting of cancer immunotherapy,and shows that differential expression of stromal products is not a sufficient criteria to indicate its immunogenicity in a vaccination setting. View Publication

Human induced pluripotent stem cell-derived sensory neuron (iPSC-dSN) models are a valuable resource for the study of neurotoxicity but are affected by poor replicability and reproducibility,often due to a lack of optimization. Here,we identify experimental factors related to culture conditions that substantially impact cellular drug response in vitro and determine optimal conditions for improved replicability and reproducibility. Treatment duration and cell seeding density were both found to be significant factors,while cell line differences also contributed to variation. A replicable dose–response in viability was demonstrated after 48-h exposure to docetaxel or paclitaxel. Additionally,a replicable dose-dependent reduction in neurite outgrowth was demonstrated,demonstrating the applicability of the model for the examination of additional phenotypes. Overall,we have established an optimized iPSC-dSN model for the study of taxane-induced neurotoxicity. View Publication

The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055,an ATP-competitive mTOR inhibitor,by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors,including RMS. Therefore,in the present study,we searched for AZD8055-based combination therapies. Here,we identify a new synergistic lethality of AZD8055 together with ABT-737,a BH3 mimetic that antagonizes Bcl-2,Bcl-xL,and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index textless 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level,as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential,activation of caspases,and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055,the PI3K/mTOR inhibitor NVP-BEZ235,the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis,whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly,molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly,knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737,our findings have important implications for the development of molecular targeted therapies for RMS. View Publication

Booster of pluripotency: RSC133,a new synthetic derivative of indoleacrylic acid/indolepropionic acid,exhibits dual activity by inhibiting histone deacetylase and DNA methyltransferase. Furthermore it potently improves the reprogramming of human somatic cells into a pluripotent state and aids the growth and maintenance of human pluripotent stem cells (hPSCs). View Publication

Previous studies have shown the relevance of bone marrow-derived MSCs (BM-MSCs) in controlling graft-versus-host disease (GVHD) after allogeneic transplantation. Since adipose tissue-derived MSCs (Ad-MSCs) may constitute a good alternative to BM-MSCs,we have expanded MSCs derived from human adipose tissue (hAd-MSCs) and mouse adipose tissue (mAd-MSCs),investigated the immunoregulatory properties of these cells,and evaluated their capacity to control GVHD in mice. The phenotype and immunoregulatory properties of expanded hAd-MSCs were similar to those of human BM-MSCs. Moreover,hAd-MSCs inhibited the proliferation and cytokine secretion of human primary T cells in response to mitogens and allogeneic T cells. Similarly,ex vivo expanded mAd-MSCs had an equivalent immunophenotype and exerted immunoregulatory properties similar to those of hAd-MSCs. Moreover,the infusion of mAd-MSCs in mice transplanted with haploidentical hematopoietic grafts controlled the lethal GVHD that occurred in control recipient mice. These findings constitute the first experimental proof that Ad-MSCs can efficiently control the GVHD associated with allogeneic hematopoietic transplantation,opening new perspectives for the clinical use of Ad-MSCs. View Publication

The FLT3/FLK2 receptor tyrosine kinase is closely related to two receptors,c-Kit and c-Fms,which function with their respective ligands,Kit ligand and macrophage colony-stimulating factor to control differentiation of haematopoietic and non-haematopoietic cells. FLT3/FLK2 is thought to be present on haematopoietic stem cells and found in brain,placenta and testis. We have purified to homogeneity and partially sequenced a soluble form of the FLT3/FLK2 ligand produced by mouse thymic stromal cells. We isolated several mouse and human complementary DNAs that encode polypeptides with identical N termini and different C termini. Some variants contain hydrophobic transmembrane segments,suggesting that processing may be required to release soluble ligand. The purified ligand enhances the response of mouse stem cells and a primitive human progenitor cell population to other growth factors such as interleukins IL-3 and IL-6 and to granulocyte-macrophage colony-stimulating factor,and also stimulates fetal thymocytes. View Publication

Although obesity is known to be critical for cancer development,how obesity negatively impacts antitumor immune responses remains largely unknown. Here,we show that increased fatty acid oxidation (FAO) driven by activated STAT3 in CD8+ T effector cells is critical for obesity-associated breast tumor progression. Ablating T cell Stat3 or treatment with an FAO inhibitor in obese mice spontaneously developing breast tumor reduces FAO,increases glycolysis and CD8+ T effector cell functions,leading to inhibition of breast tumor development. Moreover,PD-1 ligation in CD8+ T cells activates STAT3 to increase FAO,inhibiting CD8+ T effector cell glycolysis and functions. Finally,leptin enriched in mammary adipocytes and fat tissues downregulates CD8+ T cell effector functions through activating STAT3-FAO and inhibiting glycolysis. We identify a critical role of increased oxidation of fatty acids driven by leptin and PD-1 through STAT3 in inhibiting CD8+ T effector cell glycolysis and in promoting obesity-associated breast tumorigenesis. View Publication