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

An attractive target for therapeutic intervention is constitutively activated,mutant FLT3,which is expressed in a subpopulation of patients with acute myelocyic leukemia (AML) and is generally a poor prognostic indicator in patients under the age of 65 years. PKC412 is one of several mutant FLT3 inhibitors that is undergoing clinical testing,and which is currently in late-stage clinical trials. However,the discovery of drug-resistant leukemic blast cells in PKC412-treated patients with AML has prompted the search for novel,structurally diverse FLT3 inhibitors that could be alternatively used to override drug resistance. Here,we report the potent and selective antiproliferative effects of the novel mutant FLT3 inhibitor NVP-AST487 on primary patient cells and cell lines expressing FLT3-ITD or FLT3 kinase domain point mutants. NVP-AST487,which selectively targets mutant FLT3 protein kinase activity,is also shown to override PKC412 resistance in vitro,and has significant antileukemic activity in an in vivo model of FLT3-ITD(+) leukemia. Finally,the combination of NVP-AST487 with standard chemotherapeutic agents leads to enhanced inhibition of proliferation of mutant FLT3-expressing cells. Thus,we present a novel class of FLT3 inhibitors that displays high selectivity and potency toward FLT3 as a molecular target,and which could potentially be used to override drug resistance in AML. View Publication

During persistent murine cytomegalovirus (MCMV) infection,the T cell response is maintained at extremely high intensity for the life of the host. These cells closely resemble human CMV-specific cells,which compose a major component of the peripheral T cell compartment in most people. Despite a phenotype that suggests extensive antigen-driven differentiation,MCMV-specific T cells remain functional and respond vigorously to viral challenge. We hypothesized that a low rate of antigen-driven proliferation would account for the maintenance of this population. Instead,we found that most of these cells divided only sporadically in chronically infected hosts and had a short half-life in circulation. The overall population was supported,at least in part,by memory T cells primed early in infection,as well as by recruitment of naive T cells at late times. Thus,these data show that memory inflation is maintained by a continuous replacement of short-lived,functional cells during chronic MCMV infection. View Publication

BACKGROUND Valproic acid (VPA),a commonly used mood stabilizer that promotes neuronal differentiation,regulates multiple signaling pathways involving extracellular signal-regulated kinase (ERK) and glycogen synthase kinase3beta (GSK3beta). However,the mechanism by which VPA promotes differentiation is not understood. RESULTS We report here that 1 mM VPA simultaneously induces differentiation and reduces proliferation of basic fibroblast growth factor (bFGF)-treated embryonic day 14 (E14) rat cerebral cortex neural progenitor cells (NPCs). The effects of VPA on the regulation of differentiation and inhibition of proliferation occur via the ERK-p21Cip/WAF1 pathway. These effects,however,are not mediated by the pathway involving the epidermal growth factor receptor (EGFR) but via the pathway which stabilizes Ras through beta-catenin signaling. Stimulation of differentiation and inhibition of proliferation in NPCs by VPA occur independently and the beta-catenin-Ras-ERK-p21Cip/WAF1 pathway is involved in both processes. The independent regulation of differentiation and proliferation in NPCs by VPA was also demonstrated in vivo in the cerebral cortex of developing rat embryos. CONCLUSION We propose that this mechanism of VPA action may contribute to an explanation of its anti-tumor and neuroprotective effects,as well as elucidate its role in the independent regulation of differentiation and inhibition of proliferation in the cerebral cortex of developing rat embryos. View Publication

Neonatal hyperoxia impairs vascular and alveolar growth in mice and decreases endothelial progenitor cells. To determine the role of bone marrow-derived cells in restoration of neonatal lung structure after injury,we studied a novel bone marrow myeloid progenitor cell population from Tie2-green fluorescent protein (GFP) transgenic mice (bone marrow-derived angiogenic cells; BMDAC). We hypothesized that treatment with BMDAC would restore normal lung structure in infant mice during recovery from neonatal hyperoxia. Neonatal mice (1-day-old) were exposed to 80% oxygen for 10 days. BMDACs (1 x 10(5)),embryonic endothelial progenitor cells,mouse embryonic fibroblasts (control),or saline were then injected into the pulmonary circulation. At 21 days of age,saline-treated mice had enlarged alveoli,reduced septation,and a reduction in vascular density. In contrast,mice treated with BMDAC had complete restoration of lung structure that was indistinguishable from room air controls. BMDAC comprised 12% of distal lung cells localized to pulmonary vessels or alveolar type II (AT2) cells and persist (8.8%) for 8 wk postinjection. Coculture of AT2 cells or lung endothelial cells (luEC) with BMDAC augmented AT2 and luEC cell growth in vitro. We conclude that treatment with BMDAC after neonatal hyperoxia restores lung structure in this model of bronchopulmonary dysplasia. View Publication

Although promising new radiation therapy techniques such as hadrontherapy are currently being evaluated in the treatment of head and neck malignancies,local control of head and neck squamous cell carcinoma (HNSCC) remains low. Here,we investigated the involvement of cancer stem-like cells (CSCs) in a radioresistant HNSCC cell line (SQ20B). Stem-like cells SQ20B/SidePopulation(SP)/CD44(+)/ALDH(high) were more resistant to both photon and carbon ion irradiation compared with non-CSCs. This was confirmed by a BrdU labeling experiment,which suggests that CSCs were able to proliferate and to induce tumorigenicity after irradiation. SQ20B/SP/CD44(+)/ALDH(high) were capable of an extended G2/M arrest phase in response to photon or carbon ion irradiation compared with non-CSCs. Moreover,our data strongly suggest that resistance of CSCs may result from an imbalance between exacerbated self-renewal and proliferative capacities and the decrease in apoptotic cell death triggering. In order to modulate these processes,two targeted pharmacological strategies were tested. Firstly,UCN-01,a checkpoint kinase (Chk1) inhibitor,induced the relapse of G2/M arrest and radiosensitization of SQ20B-CSCs. Secondly,all-trans retinoic acid (ATRA) resulted in an inhibition of ALDH activity,and induction of the differentiation and radiosensitization of SQ20B/SP/CD44(+)/ALDH(high) cells. The combination of ATRA and UCN-01 treatments with irradiation drastically decreased the surviving fraction at 2Gy of SQ20B-CSCs from 0.85 to 0.38 after photon irradiation,and from 0.45 to 0.21 in response to carbon ions. Taken together,our results suggest that the combination of UCN-01 and ATRA represent a promising pharmacological-targeted strategy that significantly sensitizes CSCs to photon or carbon ion radiation. View Publication

Giant cell tumor of bone (GCTB) is a very rare tumor entity,which is little examined owing to the lack of established cell lines and mouse models and the restriction of available primary cell lines. The stromal cells of GCTB have been made responsible for the aggressive growth and metastasis,emphasizing the presence of a cancer stem cell population. To identify and target such tumor-initiating cells,stromal cells were isolated from eight freshly resected GCTB tissues. Tumorigenic properties were examined by colony and spheroid formation,differentiation,migration,MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay,immunohistochemistry,antibody protein array,Alu in situ hybridization,FACS analysis and xenotransplantation into fertilized chicken eggs and mice. A sub-population of the neoplastic stromal cells formed spheroids and colonies,differentiated to osteoblasts,migrated to wounded regions and expressed the metastasis marker CXC-chemokine receptor type 4,indicating self-renewal,invasion and differentiation potential. Compared with adherent-growing cells,markers for pluripotency,stemness and cancer progression,including the CSC surface marker c-Met,were enhanced in spheroidal cells. This c-Met-enriched sub-population formed xenograft tumors in fertilized chicken eggs and mice. Cabozantinib,an inhibitor of c-Met in phase II trials,eliminated CSC features with a higher therapeutic effect than standard chemotherapy. This study identifies a c-Met(+) tumorigenic sub-population within stromal GCTB cells and suggests the c-Met inhibitor cabozantinib as a new therapeutic option for targeted elimination of unresectable or recurrent GCTB. View Publication

There is little insight into or agreement about the signals that control differentiation of memory B cells (MBCs) and long-lived plasma cells (LLPCs). By performing BrdU pulse-labeling studies,we found that MBC formation preceded the formation of LLPCs in an adoptive transfer immunization system,which allowed for a synchronized Ag-specific response with homogeneous Ag-receptor,yet at natural precursor frequencies. We confirmed these observations in wild-type (WT) mice and extended them with germinal center (GC) disruption experiments and variable region gene sequencing. We thus show that the GC response undergoes a temporal switch in its output as it matures,revealing that the reaction engenders both MBC subsets with different immune effector function and,ultimately,LLPCs at largely separate points in time. These data demonstrate the kinetics of the formation of the cells that provide stable humoral immunity and therefore have implications for autoimmunity,for vaccine development,and for understanding long-term pathogen resistance. View Publication

The myeloma surface proteome (surfaceome) determines tumor interaction with the microenvironment and serves as an emerging arena for therapeutic development. Here,we use glycoprotein capture proteomics to define the myeloma surfaceome at baseline,in drug resistance,and in response to acute drug treatment. We provide a scoring system for surface antigens and identify CCR10 as a promising target in this disease expressed widely on malignant plasma cells. We engineer proof-of-principle chimeric antigen receptor (CAR) T-cells targeting CCR10 using its natural ligand CCL27. In myeloma models we identify proteins that could serve as markers of resistance to bortezomib and lenalidomide,including CD53,CD10,EVI2B,and CD33. We find that acute lenalidomide treatment increases activity of MUC1-targeting CAR-T cells through antigen upregulation. Finally,we develop a miniaturized surface proteomic protocol for profiling primary plasma cell samples with low inputs. These approaches and datasets may contribute to the biological,therapeutic,and diagnostic understanding of myeloma. View Publication

PURPOSE We investigated the effect of the racemic $\beta$-hydroxybutyrate precursor,R,S-1,3-butanediol (BD),on T-cell-related cytokine gene expression within stimulated peripheral blood mononuclear cells (PBMC) following prolonged,strenuous exercise. METHODS A repeated-measures,randomised,crossover study was conducted in nine healthy,trained male cyclists (age,26.7 ± 5.2 years; VO2peak,63.9 ± 2.5 mL kg-1 min-1). Participants ingested 0.35 g kg-1 of BD or placebo 30 min before and 60 min during 85 min of steady-state (SS) exercise,which preceded a {\~{}} 30 min time-trial (TT) (7 kJ kg-1). Blood samples were collected at pre-supplement,pre-exercise,post-SS,post-TT and 1-h post-TT. Whole blood cultures were stimulated with Staphylococcal enterotoxin B (SEB) for 24 h to determine T-cell-related interleukin (IL)-4,IL-10 and interferon (IFN)-$\gamma$ mRNA expression within isolated PBMCs in vitro. RESULTS Serum cortisol,total circulating leukocyte and lymphocyte,and T-cell subset concentrations were similar between trials during exercise and recovery (all p {\textgreater} 0.05). BD ingestion increased T-cell-related IFN-$\gamma$ mRNA expression compared with placebo throughout exercise and recovery (p = 0.011); however,IL-4 and IL-10 mRNA expression and the IFN-$\gamma$/IL-4 mRNA expression ratio were unaltered (all p {\textgreater} 0.05). CONCLUSION Acute hyperketonaemia appears to transiently amplify the initiation of the pro-inflammatory T-cell-related IFN-$\gamma$ response to an immune challenge in vitro during and following prolonged,strenuous exercise; suggesting enhanced type-1 T-cell immunity at the gene level. View Publication

Copy number variations of the human CNTN6 gene,resulting from megabase-scale microdeletions or microduplications in the 3p26.3 region,are frequently implicated in neurodevelopmental disorders such as intellectual disability and developmental delay. However,duplication of the full-length human CNTN6 gene presents with variable penetrance,resulting in phenotypes that range from neurodevelopmental disorders to no visible pathologies,even within the same family. Previously,we obtained a set of induced pluripotent stem cell lines derived from a patient with a CNTN6 gene duplication and from two healthy donors. Our findings demonstrated that CNTN6 expression in neurons carrying the duplication was significantly reduced. Additionally,the expression from the CNTN6 duplicated allele was markedly lower compared to the wild-type allele. Here,we first introduce a system for correcting megabase-scale duplications in induced pluripotent stem cells and secondly analyze the impact of this correction on CNTN6 gene expression. We showed that the deletion of one copy of the CNTN6 duplication did not affect the expression levels of the remaining allele in the neuronal cells. View Publication

Although chimeric antigen receptor (CAR) T cells are effective against B-lineage malignancies,post-CAR relapse is common,and efficacy in other tumors is limited. These challenges may be addressed through rational manipulations to control CAR T cell function. Here we examine the impact of cognate T cell antigen experience on subsequent CD8+ CAR T cell activity. Prior antigen encounter resulted in superior effector function against leukemia expressing low target antigen density at the expense of reduced proliferative capacity and susceptibility to dysfunction at limiting CAR doses. Distinctive temporal transcriptomic and epigenetic profiles in naive-derived and memory-derived CAR T cells identified RUNX family transcription factors as potential targets to augment the function of naive-derived CD8+ CAR T cells. RUNX2 overexpression enhanced antitumor efficacy of mouse CAR T cells,dependent on prior cell state,and heightened human CAR T cell functions. Our data demonstrate that prior antigen experience of CAR T cells determines functional attributes and amenability to transcription factor-mediated functional enhancement. Here,Fry and colleagues examine the impact of antigen experience on subsequent CD8+ CAR T cell activity during the antileukemia response and show that RUNX2 overexpression enhances antitumor activity of these cells. View Publication

PURPOSE: B-cell receptor signaling plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). However,blocking B-cell receptor signaling with dasatinib,an inhibitor of SRC kinase,produced variable results in preclinical and clinical studies. We aim to define the molecular mechanisms underlying the differential dasatinib sensitivity and to uncover more effective therapeutic targets in CLL. EXPERIMENTAL DESIGN: Fresh CLL B cells were treated with dasatinib,and cell viability was followed. The CLL cases were then divided into good and poor responders. The cellular response was correlated with the activities of B-cell receptor signaling molecules,as well as with molecular and cytogenetic prognostic factors. RESULTS: Among 50 CLL cases,dasatinib treatment reduced cell viability by 2% to 90%,with an average reduction of 47% on day 4 of culture. The drug induced CLL cell death through the intrinsic apoptotic pathway mediated by reactive oxygen species. Unexpectedly,phosphorylation of SRC family kinases was inhibited by dasatinib in good,as well as poor,responders. As opposed to SRC family kinases,activities of two downstream molecules,SYK and phospholipase Cgamma2,correlate well with the apoptotic response of CLL cells to dasatinib. CONCLUSIONS: Thus,SYK inhibition predicts cellular response to dasatinib. SYK,together with phospholipase Cgamma2,may serve as potential biomarkers to predict dasatinib therapeutic response in patients. From the pathogenic perspective,our study suggests the existence of alternative mechanisms or pathways that activate SYK,independent of SRC kinase activities. The study further implicates that SYK might serve as a more effective therapeutic target in CLL treatment. View Publication