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

Pulmonary metastasis of breast cancer requires recruitment and expansion of T-regulatory cells (Treg) that promote escape from host protective immune cells. However,it remains unclear precisely how tumors recruit Tregs to support metastatic growth. Here we report the mechanistic involvement of a unique and previously undescribed subset of regulatory B cells. These cells,designated tumor-evoked Bregs (tBreg),phenotypically resemble activated but poorly proliferative mature B2 cells (CD19(+) CD25(High) CD69(High)) that express constitutively active Stat3 and B7-H1(High) CD81(High) CD86(High) CD62L(Low) IgM(Int). Our studies with the mouse 4T1 model of breast cancer indicate that the primary role of tBregs in lung metastases is to induce TGF-$\beta$-dependent conversion of FoxP3(+) Tregs from resting CD4(+) T cells. In the absence of tBregs,4T1 tumors cannot metastasize into the lungs efficiently due to poor Treg conversion. Our findings have important clinical implications,as they suggest that tBregs must be controlled to interrupt the initiation of a key cancer-induced immunosuppressive event that is critical to support cancer metastasis. View Publication

NK cells from individuals with X-linked lymphoproliferative (XLP) disease exhibit functional defects when stimulated through the NK receptor,2B4 (CD244). These defects are likely a consequence of aberrant intracellular signaling initiated by mutations of the adaptor molecule SLAM-associated protein. In this report,we show that NK cells from individuals with XLP but not healthy individuals fail to phosphorylate and thereby inactivate glycogen synthase kinase-3 (GSK-3) following 2B4 stimulation. Lack of GSK-3 phosphorylation prevented the accumulation of the transcriptional coactivator beta-catenin in the cytoplasm and its subsequent translocation to the nucleus. Potential signaling pathways leading from 2B4 stimulation to GSK-3 phosphorylation were also investigated. Ligation of 2B4 resulted in the phosphorylation of the guanine nucleotide exchange factor,Vav-1,and subsequent activation of the GTP-binding protein Rac-1 (but not Ras) and the serine-threonine kinase Raf-1 in healthy but not XLP-derived NK cells. In addition,the activity of MEK-2 (but not MEK-1) was up-regulated,and Erk1/2 was phosphorylated in normal NK cells but not those from an individual with XLP suggesting that these proteins relay SLAM-associated protein-dependent signals from 2B4. Finally,inactivation of GSK-3 using a specific inhibitor of GSK-3beta increased the cytotoxicity and cytokine secretion of both healthy and XLP NK cells. These data indicate that the signaling of 2B4 in NK cells is mediated by GSK-3 and beta-catenin,possibly through a signal transduction pathway that involves Vav-1,Rac-1,Raf-1,MEK-2,and Erk1/2 and that this pathway is aberrant in individuals with XLP. View Publication

Current strategies for the production of therapeutic mAbs include the use of mammalian cell systems to recombinantly produce Abs derived from mice bearing human Ig transgenes,humanization of rodent Abs,or phage libraries. Generation of hybridomas secreting human mAbs has been previously reported; however,this approach has not been fully exploited for immunotherapy development. We previously reported the use of transient regulation of cellular DNA mismatch repair processes to enhance traits (e.g.,affinity and titers) of mAb-producing cell lines,including hybridomas. We reasoned that this process,named morphogenics,could be used to improve suboptimal hybridoma cells generated by means of ex vivo immunization and immortalization of antigen-specific human B cells for therapeutic Ab development. Here we present a platform process that combines hybridoma and morphogenics technologies for the generation of fully human mAbs specific for disease-associated human antigens. We were able to generate hybridoma lines secreting mAbs with high binding specificity and biological activity. One mAb with strong neutralizing activity against human granulocyte-macrophage colony-stimulating factor was identified that is now considered for preclinical development for autoimmune disease indications. Moreover,these hybridoma cells have proven suitable for genetic optimization using the morphogenics process and have shown potential for large-scale manufacturing. View Publication

DNA methylation is one of the essential epigenetic processes that play a role in regulating gene expression. Aberrant methylation of CpG-rich promoter regions has been associated with many forms of human cancers. The current method for determining the methylation status relies mainly on bisulfite treatment of genomic DNA,followed by methylation-specific PCR (MSP). The difficulty in acquiring a methylation profiling often is limited by the amount of genomic DNA that can be recovered from a given sample,whereas complex procedures of bisulfite treatment further compromise the effective template for PCR analysis. To circumvent these obstacles,we developed degenerated oligonucleotide primer (DOP)-PCR to enable amplification of bisulfite-modified genomic DNA at a genome-wide scale. A DOP pair was specially designed as follows: first 3' DOP,CTCGAGCTGHHHHHAACTAC,where H is a mixture of base consisting of 50% A,25% T,and 25% C; and second 5' DOP,CTCGAGCTGDDDDDGTTTAG,where D is a mixture of base consisting of 50% T,25% G,and 25% A. Our results showed that bisulfite-modified DNAs from a cell line,cord blood cells,or cells obtained by laser capture microdissection were amplified by up to 1000-fold using this method. Subsequent MSP analysis using these amplified DNAs on nine randomly selected cancer-related genes revealed that the methylation status of these genes remained identical to that derived from the original unamplified template. View Publication

Chimeric antigen receptor (CAR) therapy targeting CD19 has yielded remarkable outcomes in patients with acute lymphoblastic leukemia. To identify potential CAR targets in acute myeloid leukemia (AML),we probed the AML surfaceome for overexpressed molecules with tolerable systemic expression. We integrated large transcriptomics and proteomics datasets from malignant and normal tissues,and developed an algorithm to identify potential targets expressed in leukemia stem cells,but not in normal CD34+CD38- hematopoietic cells,T cells,or vital tissues. As these investigations did not uncover candidate targets with a profile as favorable as CD19,we developed a generalizable combinatorial targeting strategy fulfilling stringent efficacy and safety criteria. Our findings indicate that several target pairings hold great promise for CAR therapy of AML. View Publication

Store-operated calcium entry (SOCE) is a Calcium (Ca2+) influx pathway activated by depletion of intracellular stores that occurs in eukaryotic cells. In neurons,the presence and functions of SOCE are still in question. Here,we show evidences for the existence of SOCE in primary mouse cortical neurons. Endoplasmic reticulum (ER)-Ca2+ depletion using thapsigargin (Tg) triggered a maintained cytosolic Ca2+ increase,which rapidly returned to basal level in the presence of the SOCE blockers 2-Aminoethoxydiphenyl borate (2-APB) and YM-58483. Neural SOCE is also engaged by activation of metabotropic glutamate receptors (mGluRs) with (S)-3,5-dihydroxyphenylglycine (DHPG) (agonist of group I mGluRs),being an essential mechanism to maintain the mGluR-driven Ca2+ signal. Activation of group I of mGluRs triggers long-term depression (LTD) in many brain regions,but the underlying mechanism and,specifically,the necessity of Ca2+ increase in the postsynaptic neuron is controversial. In primary cortical neurons,we now show that the inhibition of Ca2+ influx through SOCE impaired DHPG-LTD,pointing out a key function of calcium and SOCE in synaptic plasticity. View Publication

INTRODUCTION The Comprehensive in vitro Proarrhythmia Assay (CiPA) is a nonclinical Safety Pharmacology paradigm for discovering electrophysiological mechanisms that are likely to confer proarrhythmic liability to drug candidates intended for human use. TOPICS COVERED Key talks delivered at the 'CiPA on my mind' session,held during the 2015 Annual Meeting of the Safety Pharmacology Society (SPS),are summarized. Issues and potential solutions relating to crucial constituents [e.g.,biological materials (ion channels and pluripotent stem cell-derived cardiomyocytes),study platforms,drug solutions,and data analysis] of CiPA core assays are critically examined. DISCUSSION In order to advance the CiPA paradigm from the current testing and validation stages to a research and regulatory drug development strategy,systematic guidance by CiPA stakeholders is necessary to expedite solutions to pending and newly arising issues. Once a study protocol is proved to yield robust and reproducible results within and across laboratories,it can be implemented as qualified regulatory procedure. View Publication

OBJECTIVE: The molecular mechanisms that maintain human pluripotent stem (PS) cells are not completely understood. Here we sought to identify new candidate PS cell regulators to facilitate future improvements in their generation,expansion,and differentiation. MATERIALS AND METHODS: We used bioinformatic analyses of multiple serial-analysis-of-gene-expression libraries (generated from human PS cells and their differentiated derivatives),together with small interfering RNA (siRNA) screening to identify candidate pluripotency regulators. Validation of candidate regulators involved promoter analyses,Affymetrix profiling,real-time PCR,and immunoprecipitation. RESULTS: Promoter analysis of genes differentially expressed across multiple serial-analysis-of-gene-expression libraries identified E2F motifs in the promoters of many PS cell-specific genes (e.g.,POU5F1,NANOG,SOX2,FOXD3). siRNA analyses identified two retinoblastoma binding proteins (RBBP4,RBBP9) as required for maintenance of multiple human PS cell types. Both RBBPs were bound to RB in human PS cells,and E2F motifs were present in the promoters of genes whose expression was altered by decreasing RBBP4 and RBBP9 expression. Affymetrix and real-time PCR studies of siRNA-treated human PS cells showed that reduced RBBP4 or RBBP9 expression concomitantly decreased expression of POU5F1,NANOG,SOX2,and/or FOXD3 plus certain cell cycle genes (e.g.,CCNA2,CCNB1),while increasing expression of genes involved in organogenesis (particularly neurogenesis). CONCLUSIONS: These results reveal new candidate positive regulators of human PS cells,providing evidence of their ability to regulate expression of pluripotency,cell cycle,and differentiation genes in human PS cells. These data provide valuable new leads for further elucidating mechanisms of human pluripotency. View Publication

Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene initiate aggressive forms of leukaemia,which are often refractory to conventional therapies. Many MLL-fusion partners are members of the super elongation complex (SEC),a critical regulator of transcriptional elongation,suggesting that aberrant control of this process has an important role in leukaemia induction. Here we use a global proteomic strategy to demonstrate that MLL fusions,as part of SEC and the polymerase-associated factor complex (PAFc),are associated with the BET family of acetyl-lysine recognizing,chromatin 'adaptor' proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukaemia,via the displacement of the BET family of proteins from chromatin. We show that a novel small molecule inhibitor of the BET family,GSK1210151A (I-BET151),has profound efficacy against human and murine MLL-fusion leukaemic cell lines,through the induction of early cell cycle arrest and apoptosis. I-BET151 treatment in two human leukaemia cell lines with different MLL fusions alters the expression of a common set of genes whose function may account for these phenotypic changes. The mode of action of I-BET151 is,at least in part,due to the inhibition of transcription at key genes (BCL2,C-MYC and CDK6) through the displacement of BRD3/4,PAFc and SEC components from chromatin. In vivo studies indicate that I-BET151 has significant therapeutic value,providing survival benefit in two distinct mouse models of murine MLL-AF9 and human MLL-AF4 leukaemia. Finally,the efficacy of I-BET151 against human leukaemia stem cells is demonstrated,providing further evidence of its potent therapeutic potential. These findings establish the displacement of BET proteins from chromatin as a promising epigenetic therapy for these aggressive leukaemias. View Publication

Cockayne syndrome (CS) is a human premature aging disorder associated with neurological and developmental abnormalities,caused by mutations mainly in the CS group B gene (ERCC6). At the molecular level,CS is characterized by a deficiency in the transcription-couple DNA repair pathway. To understand the role of this molecular pathway in a pluripotent cell and the impact of CSB mutation during human cellular development,we generated induced pluripotent stem cells (iPSCs) from CSB skin fibroblasts (CSB-iPSC). Here,we showed that the lack of functional CSB does not represent a barrier to genetic reprogramming. However,iPSCs derived from CSB patient's fibroblasts exhibited elevated cell death rate and higher reactive oxygen species (ROS) production. Moreover,these cellular phenotypes were accompanied by an up-regulation of TXNIP and TP53 transcriptional expression. Our findings suggest that CSB modulates cell viability in pluripotent stem cells,regulating the expression of TP53 and TXNIP and ROS production. View Publication

We have tracked the fate of immature human B cells at a critical stage in their development when the mature B cell repertoire is shaped. We show that a major subset of bone marrow emigrant immature human B cells,the transitional 2 (T2) B cells,homes to gut-associated lymphoid tissue (GALT) and that most T2 B cells isolated from human GALT are activated. Activation in GALT is a previously unknown potential fate for immature human B cells. The process of maturation from immature transitional B cell through to mature naive B cell includes the removal of autoreactive cells from the developing repertoire,a process which is known to fail in systemic lupus erythematosus (SLE). We observe that immature B cells in SLE are poorly equipped to access the gut and that gut immune compartments are depleted in SLE. Thus,activation of immature B cells in GALT may function as a checkpoint that protects against autoimmunity. In healthy individuals,this pathway may be involved in generating the vast population of IgA plasma cells and also the enigmatic marginal zone B cell subset that is poorly understood in humans. View Publication

MicroRNAs (miRNAs/miRs) are small,endogenous noncoding RNAs that are important post-transcriptional regulators with clear roles in the development of the immune system and immune responses. Using miRNA microarray profiling,we characterized the expression profile of naive and in vivo generated murine effector antiviral CD8+ T cells. We observed that out of 362 measurable mature miRNAs,120 were differentially expressed by at least 2-fold in influenza-specific effector CD8+ CTLs compared with naive CD8+ T cells. One miRNA found to be highly downregulated on both strands in effector CTLs was miR-139. Because previous studies have indicated a role for miR-139-mediated regulation of CTL effector responses,we hypothesized that deletion of miR-139 would enhance antiviral CTL responses during influenza virus infection. We generated miR-139-/- mice or overexpressed miR-139 in T cells to assess the functional contribution of miR-139 expression in CD8+ T cell responses. Our study demonstrates that the development of naive T cells and generation or differentiation of effector or memory CD8+ T cell responses to influenza virus infection are not impacted by miR-139 deficiency or overexpression; yet,miR-139-/- CD8+ T cells are outcompeted by wild-type CD8+ T cells in a competition setting and demonstrate reduced responses to Listeria monocytogenes Using an in vitro model of T cell exhaustion,we confirmed that miR-139 expression similarly does not impact the development of T cell exhaustion. We conclude that despite significant downregulation of miR-139 following in vivo and in vitro activation,miR-139 expression is dispensable for influenza-specific CTL responses. View Publication