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

Charge-coupled device detectors are vulnerable to cosmic rays that can contaminate Raman spectra with positive going spikes. Because spikes can adversely affect spectral processing and data analyses,they must be removed. Although both hardware-based and software-based spike removal methods exist,they typically require parameter and threshold specification dependent on well-considered user input. Here,we present a fully automated spike removal algorithm that proceeds without requiring user input. It is minimally dependent on sample attributes,and those that are required (e.g.,standard deviation of spectral noise) can be determined with other fully automated procedures. At the core of the method is the identification and location of spikes with coincident second derivatives along both the spectral and spatiotemporal dimensions of two-dimensional datasets. The method can be applied to spectra that are relatively inhomogeneous because it provides fairly effective and selective targeting of spikes resulting in minimal distortion of spectra. Relatively effective spike removal obtained with full automation could provide substantial benefits to users where large numbers of spectra must be processed. View Publication

The increased application of in vitro systems in pharmacology and toxicology requires cell culture systems that facilitate the cultivation process and ensure stable,reproducible and controllable cultivation conditions. Up to now,some devices have been developed for the cultivation of cells under submersed conditions. However,systems meeting the requirements of an air-liquid interface (ALI) cultivation for the special needs of bronchial epithelial cells for example are still lacking. In order to obtain in vivo like organization and differentiation of these cells they need to be cultivated under ALI conditions on microporous membranes in direct contact with the environmental atmosphere. For this purpose,a Long-Term-Cultivation system was developed (CULTEX(®) LTC-C system) for the computer-controlled cultivation of such cells. The transwell inserts are placed in an incubator module (24 inserts),which can be adjusted for the medium level (ultrasonic pulse-echosensor),time and volume-dependent medium exchange,and frequency for mixing the medium with a rotating disc for homogeneous distribution of medium and secretion components. Normal primary freshly isolated bronchial epithelial cells were cultivated for up to 38 days to show the efficiency of such a cultivation procedure for generating 3D cultures exhibiting in vivo-like pseudostratified organization of the cells as well as differentiation characteristics like mucus-producing and cilia-forming cells. View Publication

Systemic lupus erythematosus (SLE) is an autoimmune/inflammatory disease characterized by autoantibody production and abnormal T cells that infiltrate tissues through not well-known mechanisms. We report that SLE T lymphocytes display increased levels of CD44,ezrin,radixin,and moesin (ERM) phosphorylation,stronger actin polymerization,higher polar cap formation,and enhanced adhesion and chemotactic migration compared with T cells from patients with rheumatoid arthritis and normal individuals. Silencing of CD44 by CD44 small interfering RNA in SLE T cells inhibited significantly their ability to adhere and migrate as did treatment with Rho kinase and actin polymerization inhibitors. Forced expression of T567D-ezrin,a phosphorylation-mimic form,enhanced remarkably the adhesion and migration rate of normal T cells. Anti-CD3/TCR autoantibodies present in SLE sera caused increased ERM phosphorylation,adhesion,and migration in normal T cells. pERM and CD44 are highly expressed in T cells infiltrating in the kidneys of patients with lupus nephritis. These data prove that increased ERM phosphorylation represents a key molecular abnormality that guides T cell adhesion and migration in SLE patients. View Publication

Toll-like receptors (TLR) recognize pathogen-associated molecular patterns and play important roles in the innate immune system. While single-stranded viral RNA is the natural ligand of TLR7/TLR8,the imidazoquinoline resiquimod (R-848) is recognized as a potent synthetic agonist of TLR7/TLR8. We investigated the effects of TLR7/8 activation on lipid mediator production in polymorphonuclear leukocytes exposed to R-848. Although R-848 had minimal effects by itself,it strongly enhanced leukotriene B4 formation on subsequent stimulation by fMLP,platelet-activating factor,and the ionophore A23187. R-848 acted via TLR8 but not TLR7 as shown by the lack of effect of the TLR7-specific ligand imiquimod. Priming with R-848 also resulted in enhanced arachidonic acid release and platelet-activating factor formation following fMLP stimulation,as well as enhanced prostaglandin E2 synthesis following the addition of arachidonic acid. Western blot analysis demonstrated that R-848 induced the phosphorylation of the cytosolic phospholipase A2alpha,promoted 5-lipoxygenase translocation and potently stimulated the expression of the type 2 cyclooxygenase. Bafilomycin A1,an inhibitor of endosomal acidification,efficiently inhibited all R-848-induced effects. These studies demonstrate that TLR8 signaling strongly promotes inflammatory lipid mediator biosynthesis and provide novel insights on innate immune response to viral infections. View Publication

Electrospun poly-l-lactic acid (PLLA) nanofiber mats carrying surface amine groups,previously introduced by nitrogen atmospheric pressure nonequilibrium plasma,are embedded into aqueous solutions of oligomeric acrylamide-end capped AGMA1,a biocompatible polyamidoamine with arg-gly-asp (RGD)-reminiscent repeating units. The resultant mixture is finally cured giving PLLA-AGMA1 hydrogel composites that absorb large amounts of water and,in the swollen state,are translucent,soft,and pliable,yet as strong as the parent PLLA mat. They do not split apart from each other when swollen in water and remain highly flexible and resistant,since the hydrogel portion is covalently grafted onto the PLLA nanofibers via the addition reaction of the surface amine groups to a part of the terminal acrylic double bonds of AGMA1 oligomers. Preliminary tested as scaffolds,the composites prove capable of maintaining short-term undifferentiated cultures of human pluripotent stem cells in feeder-free conditions. View Publication

Recurrent/metastatic head and neck cancer remains a devastating disease with insufficient treatment options. We investigated the MET receptor tyrosine kinase as a novel target for the treatment of head and neck squamous cell carcinoma (HNSCC). MET/phosphorylated MET and HGF expression was analyzed in 121 tissues (HNSCC/normal) by immunohistochemistry,and in 20 HNSCC cell lines by immunoblotting. The effects of MET inhibition using small interfering RNA/two small-molecule inhibitors (SU11274/PF-2341066) on signaling,migration,viability,and angiogenesis were determined. The complete MET gene was sequenced in 66 head and neck cancer tissue samples and eight cell lines. MET gene copy number was determined in 14 cell lines and 23 tumor tissues. Drug combinations of SU11274 with cisplatin or erlotinib were tested in SCC35/HN5 cell lines. Eighty-four percent of the HNSCC samples showed MET overexpression,whereas 18 of 20 HNSCC cell lines (90%) expressed MET. HGF overexpression was present in 45% of HNSCC. MET inhibition with SU11274/PF-2341066 abrogated MET signaling,cell viability,motility/migration in vitro,and tumor angiogenesis in vivo. Mutational analysis of 66 tumor tissues and 8 cell lines identified novel mutations in the semaphorin (T230M/E168D/N375S),juxtamembrane (T1010I/R988C),and tyrosine kinase (T1275I/V1333I) domains (incidence: 13.5%). Increased MET gene copy number was present with textgreater10 copies in 3 of 23 (13%) tumor tissues. A greater-than-additive inhibition of cell growth was observed when combining a MET inhibitor with cisplatin or erlotinib and synergy may be mediated via erbB3/AKT signaling. MET is functionally important in HNSCC with prominent overexpression,increased gene copy number,and mutations. MET inhibition abrogated MET functions,including proliferation,migration/motility,and angiogenesis. MET is a promising,novel target for HNSCC and combination approaches with cisplatin or EGFR inhibitors should be explored. View Publication

We demonstrate an essential role for the proteasome complex in two proteolytic processes required for activation of the transcription factor NF-kappa B. The p105 precursor of the p50 subunit of NF-kappa B is processed in vitro by an ATP-dependent process that requires proteasomes and ubiquitin conjugation. The C-terminal region of p105 is rapidly degraded,leaving the N-terminal p50 domain. p105 processing can be blocked in intact cells with inhibitors of the proteasome or in yeast with proteasome mutants. These inhibitors also block the activation of NF-kappa B and the rapid degradation of I kappa B alpha induced by tumor necrosis factor alpha. Thus,the ubiquitin-proteasome pathway functions not only in the complete degradation of polypeptides,but also in the regulated processing of precursors into active proteins. View Publication

Astrocyte phenotypes change in a process called reactive gliosis after traumatic central nervous system (CNS) injury. Astrogliosis is characterized by expansion of the glial fibrillary acidic protein (GFAP) cytoskeleton,adoption of stellate morphologies,and differential expression of some extracellular matrix molecules. The astrocytic response immediately after injury is beneficial,but in the chronic injury phase,reactive astrocytes produce inhibitory factors (i.e.,chondroitin sulfate proteoglycans [CSPGs]) that limit the regrowth of injured axons. There are no drugs that promote axon regeneration or functional recovery after CNS trauma in humans. To develop novel therapeutics for the injured CNS,we screened various libraries in a phenotypic assay to identify compounds that promote neurite outgrowth. However,the effects these compounds have on astrocytes are unknown. Specifically,we were interested in whether compounds could alter astrocytes in a manner that mimics the glial reaction to injury. To test this hypothesis,we developed cell-based phenotypic bioassays to measure changes in (1) GFAP morphology/localization and (2) CSPG expression/immunoreactivity from primary astrocyte cultures. These assays were optimized for six-point dose-response experiments in 96-well plates. The GFAP morphology assay is suitable for counter-screening with a Z-factor of 0.44±0.03 (mean±standard error of the mean; N=3 biological replicates). The CSPG assay is reproducible and informative,but does not satisfy common metrics for a screenable" assay. As proof of principle we tested a small set of hit compounds from our neurite outgrowth bioassay and identified one that can enhance axon growth without exacerbating the deleterious characteristics of reactive gliosis. View Publication

Metabolic imaging of brain tumors using (13)C Magnetic Resonance Spectroscopy (MRS) of hyperpolarized [1-(13)C] pyruvate is a promising neuroimaging strategy which,after a decade of preclinical success in glioblastoma (GBM) models,is now entering clinical trials in multiple centers. Typically,the presence of GBM has been associated with elevated hyperpolarized [1-(13)C] lactate produced from [1-(13)C] pyruvate,and response to therapy has been associated with a drop in hyperpolarized [1-(13)C] lactate. However,to date,lower grade gliomas had not been investigated using this approach. The most prevalent mutation in lower grade gliomas is the isocitrate dehydrogenase 1 (IDH1) mutation,which,in addition to initiating tumor development,also induces metabolic reprogramming. In particular,mutant IDH1 gliomas are associated with low levels of lactate dehydrogenase A (LDHA) and monocarboxylate transporters 1 and 4 (MCT1,MCT4),three proteins involved in pyruvate metabolism to lactate. We therefore investigated the potential of (13)C MRS of hyperpolarized [1-(13)C] pyruvate for detection of mutant IDH1 gliomas and for monitoring of their therapeutic response. We studied patient-derived mutant IDH1 glioma cells that underexpress LDHA,MCT1 and MCT4,and wild-type IDH1 GBM cells that express high levels of these proteins. Mutant IDH1 cells and tumors produced significantly less hyperpolarized [1-(13)C] lactate compared to GBM,consistent with their metabolic reprogramming. Furthermore,hyperpolarized [1-(13)C] lactate production was not affected by chemotherapeutic treatment with temozolomide (TMZ) in mutant IDH1 tumors,in contrast to previous reports in GBM. Our results demonstrate the unusual metabolic imaging profile of mutant IDH1 gliomas,which,when combined with other clinically available imaging methods,could be used to detect the presence of the IDH1 mutation in vivo. View Publication

The lack of natural killer (NK) cell-specific markers,as well as the overlap among several common surface antigens and functional properties,has obscured the delineation between NK cells and dendritic cells. Here,novel subsets of peripheral blood CD3/14/19(neg) NK cells and monocyte/dendritic cell (DC)-like cells were identified on the basis of CD7 and CD4 expression. Coexpression of CD7 and CD56 differentiates NK cells from CD56+ monocyte/DC-like cells,which lack CD7. In contrast to CD7+CD56+ NK cells,CD7(neg)CD56+ cells lack expression of NK cell-associated markers,but share commonalities in their expression of various monocyte/DC-associated markers. Using CD7,we observed approximately 60% of CD4+CD56+ cells were CD7(neg) cells,indicating the actual frequency of activated CD4+ NK cells is much lower in the blood than previously recognized. Functionally,only CD7+ NK cells secrete gamma interferon (IFNgamma) and degranulate after interleukin-12 (IL-12) plus IL-18 or K562 target cell stimulation. Furthermore,using CD7 to separate CD56+ NK cells and CD56+ myeloid cells,we demonstrate that unlike resting CD7+CD56+ NK cells,the CD7(neg)CD56+ myeloid cells stimulate a potent allogeneic response. Our data indicate that CD7 and CD56 coexpression discriminates NK cells from CD7(neg)CD56+ monocyte/DC-like cells,thereby improving our ability to study the intricacies of NK-cell subset phenotypes and functions in vivo. View Publication

In mice there are two families of MHC class I-specific receptors,namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1(b) and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently,how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study,we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers,but not Ly49 molecules,and their cytotoxicity is inhibited by Qa-1(b) on target cells. Using RT-PCR analysis,we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order,with their order of acquisition being first CD94; subsequently NKG2D,NKG2A,and NKG2E; and finally,NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells,and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells,suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic,orderly,and cumulative. View Publication

We used primary peripheral blood T cells,a population that exists in G(0) and can be stimulated to enter the cell cycle synchronously,to define more precisely the effects of nicotine on pathways that control cell cycle entry and progression. Our data show that nicotine decreased the ability of T cells to transit through the G(0)/G(1) boundary (acquire competence) and respond to progression signals. These effects were due to nuclear factor of activated T cells c2 (NFATc2)-dependent repression of cyclin-dependent kinase 4 (CDK4) expression. Growth arrest at the G(0)/G(1) boundary was further enforced by inhibition of cyclin D2 expression and by increased expression and stabilization of p27Kip1. Intriguingly,T cells from habitual users of tobacco products and from NFATc2-deficient mice constitutively expressed CDK4 and were resistant to the antiproliferative effects of nicotine. These results indicate that nicotine impairs T cell cycle entry through NFATc2-dependent mechanisms and suggest that,in the face of chronic nicotine exposure,selection may favor cells that can evade these effects. We postulate that cross talk between nicotinic acetylcholine receptors and growth factor receptor-activated pathways offers a novel mechanism by which nicotine may directly impinge on cell cycle progression. This offers insight into possible reasons that underlie the unique effects of nicotine on distinct cell types and identifies new targets that may be useful control tobacco-related diseases. View Publication