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

Despite the fact that glucocorticoids and long acting beta agonists are effective treatments for asthma,their effects on human mast cells (MC) appear to be modest. Although MC are one of the major effector cells in the underlying inflammatory reactions associated with asthma,their regulation by these drugs is not yet fully understood and,in some cases,controversial. Using a human immortalized MC line (LAD2),we studied the effects of fluticasone propionate (FP) and salmeterol (SM),on the release of early and late phase mediators. LAD2 cells were pretreated with FP (100 nM),SM (1 µM),alone and in combination,at various incubation times and subsequently stimulated with agonists substance P,C3a and IgE/anti-IgE. Degranulation was measured by the release of β-hexosaminidase. Cytokine and chemokine expression were measured using quantitative PCR,ELISA and cytometric bead array (CBA) assays. The combination of FP and SM synergistically inhibited degranulation of MC stimulated with substance P (33% inhibition compared to control,n = 3,P>05). Degranulation was inhibited by FP alone,but not SM,when MC were stimulated with C3a (48% inhibition,n = 3,P>05). As previously reported,FP and SM did not inhibit degranulation when MC were stimulated with IgE/anti-IgE. FP and SM in combination inhibited substance P-induced release of tumor necrosis factor (TNF),CCL2,and CXCL8 (98%,99% and 92% inhibition,respectively,n = 4,P>05). Fluticasone and salmeterol synergistically inhibited mediator production by human MC stimulated with the neuropeptide substance P. This synergistic effect on mast cell signaling may be relevant to the therapeutic benefit of combination therapy in asthma. View Publication

Human induced pluripotent stem cells (iPSCs) derived cardiomyocytes (iCMCs) would provide an unlimited cell source for regenerative medicine and drug discoveries. The objective of our study is to generate functional cardiomyocytes from human iPSCs and to develop a novel method of measuring contractility of CMCs. In a series of experiments,adult human skin fibroblasts (HSF) and human umbilical vein endothelial cells (HUVECs) were treated with a combination of pluripotent gene DNA and mRNA under specific conditions. The iPSC colonies were identified and differentiated into various cell lineages,including CMCs. The contractile activity of CMCs was measured by a novel method of frame-by-frame cross correlation (particle image velocimetry-PIV) analysis. Our treatment regimen transformed 4% of HSFs into iPSC colonies at passage 0,a significantly improved efficiency compared with use of either DNA or mRNA alone. The iPSCs were capable of differentiating both in vitro and in vivo into endodermal,ectodermal and mesodermal cells,including CMCs with<88% of cells being positive for troponin T (CTT) and Gata4 by flow cytometry. We report a highly efficient combination of DNA and mRNA to generate iPSCs and functional iCMCs from adult human cells. We also report a novel approach to measure contractility of iCMCs. View Publication

OBJECTIVE: Standard drugs post-myocardial infarction (MI) such as angiotensin converting enzyme (ACE) and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) increase levels of endothelial progenitor cells (EPC). However,potential underlying mechanisms have not yet been investigated. METHODS AND RESULTS: We studied the effects of ACE inhibition or statin treatment on EPC levels and on bone marrow molecular pathways involved in EPC mobilization after MI in rats. Three days post-infarction,acetylated LDL (acLDL)+/Ulex europeus-1 (UEA-1)+/VEGF receptor-2+/eNOS+ EPC levels and formation of endothelial colony forming units (CFU) were reduced to 60+/-12% (p textless 0.05) and 68+/-7% (p textless 0.05). In bone marrow,extracellular signal-regulated kinase (ERK) phosphorylation and matrix metalloproteinase (MMP)-9 activity were repressed. Endothelial nitric oxide synthase (eNOS) activity was unchanged,whereas reactive oxygen species (ROS) were increased two-fold in bone marrow. ACE or HMG-CoA reductase inhibition resulted in significant increases in EPC levels. ACE inhibition increased bone marrow ERK phosphorylation and MMP-9 activity. Statin therapy enhanced bone marrow VEGF protein levels,Akt phosphorylation,eNOS activity and normalized increased ROS levels. Augmented EPC levels in the early post-infarction phase by ACE inhibition or statin treatment were associated with improved cardiac function and increased capillary density in the peri-infarct area 7 days after MI. Moreover,increased EPC levels in response to ACE inhibition or statin treatment were sustained 10 weeks post-infarction. CONCLUSIONS: Increased ROS and impaired MMP-9 activity in bone marrow likely contribute to reduced EPC mobilization in the early post-infarction phase. ACE inhibition or statin treatment increased EPC levels with distinct drug-specific effects on bone marrow molecular alterations. View Publication

Langerhans cells (LC) are a unique subset of dendritic cells (DC),present in the epidermis and serving as the first line of defense against pathogens invading the skin. To investigate the role of human LCs in innate immune responses,we examined TLR expression and function of LC-like DCs derived from CD34+ progenitor cells and compared them to DCs derived from peripheral blood monocytes (monocyte-derived DC; Mo-DC). LC-like DCs and Mo-DCs expressed TLR1-10 mRNAs at comparable levels. Although many of the TLR-induced cytokine patterns were similar between the two cell types,stimulation with the TLR3 agonist poly(I:C) triggered significantly higher amounts of the IFN-inducible chemokines CXCL9 (monokine induced by IFN-gamma) and CXCL11 (IFN-gamma-inducible T cell alpha chemoattractant) in LC-like DCs as compared with Mo-DCs. Supernatants from TLR3-activated LC-like DCs reduced intracellular replication of vesicular stomatitis virus in a type I IFN-dependent manner. Finally,CXCL9 colocalized with LCs in skin biopsy specimens from viral infections. Together,our data suggest that LCs exhibit a direct antiviral activity that is dependent on type I IFN as part of the innate immune system. View Publication

Chronic graft-versus-host disease (cGVHD) is a common late complication of allogeneic stem cell transplantation (allo-SCT). Some cGVHD patients develop skin lesions,and the skin lesions in sclerodermatous cGVHD (s-cGVHD) patients resemble those in progressive systemic sclerosis (PSS),which is characterized by impaired production of circulating endothelial progenitor cells (EPCs). We investigated,retrospectively,whether low EPC production may promote the development of sclerodermatous lesions in cGVHD. Peripheral blood (PB) was obtained from 14 healthy volunteers and 27 allo-SCT patients. Five patients developed s-cGVHD. CD34(+) cells were purified by using the magnetic cell-sorting separation system,and the CD34(+)/CD133(+)/vascular endothelial growth factor (VEGF) receptor-2(+) EPCs were quantified. The endothelial cell colony-formation potential was evaluated. Serum VEGF and basic fibroblast growth factor (b-FGF) concentrations were measured by ELISA. The s-cGVHD patients had significantly lower median circulating EPCs frequencies than non-s-cGVHD patients or control (145 of 20 mL [interquartial range-IQR 107-193] versus 1083.5 [IQR 669.3-2151]; P = .0023,and versus 1530.5 [IQR 961.3-2158]; P = .0012,respectively). They also had impaired median endothelial-forming ability compared to non-s-cGVHD patients or controls (3.8 [IQR 1.0-4.3] versus 12.8 [IQR 8.8-28.8],and versus 26.4 [IQR 23.6-30.6],respectively; P = .0012). Their VEGF and b-FGF serum levels were also higher than in controls. In conclusion,s-cGVHD patients show findings consistent with those seen in PSS with impaired vasculogenesis that may limit blood perfusion and may contribute to the development of sclerodermatous lesions. View Publication

Bromodomain-containing protein 4 (BRD4) is an important epigenetic reader implicated in the pathogenesis of a number of different cancers and other diseases. Brd4-null mouse embryos die shortly after implantation and are compromised in their ability to maintain the inner cell mass,which gives rise to embryonic stem cells (ESCs). Here we report that BRD4 regulates expression of the pluripotency factor Nanog in mouse ESCs and preimplantation embryos,as well as in human ESCs and embryonic cancer stem cells. Inhibition of BRD4 function using a chemical inhibitor,small interfering RNAs,or a dominant-negative approach suppresses Nanog expression,and abolishes the self-renewal ability of ESCs. We also find that BRD4 associates with BRG1 (brahma-related gene 1,aka Smarca4 (SWI/SNF-related,matrix-associated,actin-dependent regulator of chromatin,subfamily a,member 4)),a key regulator of ESC self-renewal and pluripotency,in the Nanog regulatory regions to regulate Nanog expression. Our study identifies Nanog as a novel BRD4 target gene,providing new insights for the biological function of BRD4 in stem cells and mouse embryos. Knowledge gained from these non-cancerous systems will facilitate future investigations of how Brd4 dysfunction leads to cancers.Cell Death and Differentiation advance online publication,22 August 2014; doi:10.1038/cdd.2014.124. View Publication

BACKGROUND: Embryonic stem (ES) cells self-renew as coherent colonies in which cells maintain tight cell-cell contact. Although intercellular communications are essential to establish the basis of cell-specific identity,molecular mechanisms underlying intrinsic cell-cell interactions in ES cells at the signaling level remain underexplored.backslashnbackslashnMETHODOLOGY/PRINCIPAL FINDINGS: Here we show that endogenous Rho signaling is required for the maintenance of cell-cell contacts in ES cells. siRNA-mediated loss of function experiments demonstrated that Rock,a major effector kinase downstream of Rho,played a key role in the formation of cell-cell junctional assemblies through regulation of myosin II by controlling a myosin light chain phosphatase. Chemical engineering of this signaling axis by a Rock-specific inhibitor revealed that cell-cell adhesion was reversibly controllable and dispensable for self-renewal of mouse ES cells as confirmed by chimera assay. Furthermore,a novel culture system combining a single synthetic matrix,defined medium,and the Rock inhibitor fully warranted human ES cell self-renewal independent of animal-derived matrices,tight cell contacts,or fibroblastic niche-forming cells as determined by teratoma formation assay.backslashnbackslashnCONCLUSIONS/SIGNIFICANCE: These findings demonstrate an essential role of the Rho-Rock-Myosin signaling axis for the regulation of basic cell-cell communications in both mouse and human ES cells,and would contribute to advance in medically compatible xeno-free environments for human pluripotent stem cells. View Publication

While the determination of the trophoblast lineage and the facilitation of placental morphogenesis by trophoblast interactions with other cells of the placenta are crucial components for the establishment of pregnancy,these processes are not tractable at the time of human implantation. Embryonic stem cells (ESCs) provide an embryonic surrogate to derive insights into these processes. In this review,we will summarize current paradigms which promote trophoblast differentiation from ESCs,and potential opportunities for their use to further define signals directing morphogenesis of the placenta following implantation of the embryo into the endometrium. View Publication

The aim of the present study was to determine the effect of AZD8055 on proliferation,apoptosis and glycolysis in the human cervical cancer cell line HeLa and to investigate the underlying mechanism(s) of action. HeLa human cervical cancer cells were treated with 10 nM AZD8055 for 24,48 or 72 h. MTT was used to determine cell proliferation. Annexin V/propidium iodide staining was used to determine cell apoptosis analyzed by fluorescence-activated cell sorting (FACS). Glycolytic activity was determined by measuring the activity of the key enzyme lactate dehydrogenase (LDH) and lactate production. RNA and protein expression were examined by qRT-PCR and western blotting,respectively. Treatment with AZD8055 inhibited proliferation and glycolysis,and induced apoptosis in HeLa cells in a time-dependent manner. During the prolonged treatment with AZD8055,the phosphorylation of mammalian target of rapamycin (mTOR) C1 substrates p70S6K and phosphorylation of the mTORC2 substrate Akt were deregulated,suggesting that the activity of mTOR was downregulated. Furthermore,our study showed that the expression of miR-143 was upregulated in a time-dependent manner in HeLa cells treated with AZD8055. In summary,the present study reveals a novel antitumor mechanism of AZD8055 in HeLa human cervical cancer cells. View Publication

Patient-derived induced pluripotent stem cells (iPSCs) provide a novel tool to investigate the pathophysiology of poorly known diseases,in particular those affecting the nervous system,which has been difficult to study for its lack of accessibility. In this emerging and promising field,recent iPSCs studies are mostly used as proof-of-principle" experiments that are confirmatory of previous findings obtained from animal models and postmortem human studies; its promise as a discovery tool is just beginning to be realized. A recent number of studies point to the functional similarities between in vitro neurogenesis and in vivo neuronal development� View Publication

The relevance of blood-based assays to monitor minimal residual disease (MRD) in non-metastatic prostate cancer (PCa) remains unclear. Proving that clinically relevant circulating tumor cells (CTCs) can be detected with available technologies could address this. This study aimed to improve CTC detection in non-metastatic PCa patients by combining three independent CTC assays: the CellSearch system,an in vivo CellCollector and the EPISPOT. Peripheral blood samples from high-risk PCa patients were screened for CTCs before and three months after radical prostatectomy (RP). Combining the results of both time points,CTCs were detected in 37{\%},54.9{\%} and 58.7{\%} of patients using CellSearch,CellCollector and EPISPOT,respectively. The cumulative positivity rate of the three CTC assays was 81.3{\%} (87/107) with 21.5{\%} (23/107) of patients harboring ≥5 CTCs/7.5 ml blood. Matched pair analysis of 30 blood samples taken before and after surgery indicated a significant decrease in CTCs captured by the CellCollector from 66{\%} before RP to 34{\%} after therapy (p = 0.031). CTC detection by EPISPOT before RP significantly correlated with PSA serum values (p {\textless} 0.0001) and clinical tumor stage (p = 0.04),while the other assays showed no significant correlations. In conclusion,CTC-based liquid biopsies have the potential to monitor MRD in patients with non-metastatic prostate cancer. View Publication