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

We analyzed 112 patients with high-risk acute myeloid leukemia (61 in complete remission [CR]; 51 in relapse),who received human leukocyte-antigen (HLA)-haploidentical transplants from natural killer (NK) alloreactive (n = 51) or non-NK alloreactive donors (n = 61). NK alloreactive donors possessed HLA class I,killer-cell immunoglobulin-like receptor (KIR) ligand(s) which were missing in the recipients,KIR gene(s) for missing self recognition on recipient targets,and alloreactive NK clones against recipient targets. Transplantation from NK-alloreactive donors was associated with a significantly lower relapse rate in patients transplanted in CR (3% versus 47%) (P textgreater .003),better event-free survival in patients transplanted in relapse (34% versus 6%,P = .04) and in remission (67% versus 18%,P = .02),and reduced risk of relapse or death (relative risk versus non-NK-alloreactive donor,0.48; 95% CI,0.29-0.78; P textgreater .001). In all patients we tested the missing ligand" model which pools KIR ligand mismatched transplants and KIR ligand-matched transplants from donors possessing KIR(s) for which neither donor nor recipient have HLA ligand(s). Only transplantation from NK-alloreactive donors is associated with a survival advantage." View Publication

Interleukin-17A (IL-17A) and IL-17F are 2 of several cytokines produced by T helper 17 cells (Th17),which are able to indirectly induce the recruitment of neutrophils. Recently,human Th17 cells have been phenotypically characterized and shown to express discrete chemokine receptors,including CCR2 and CCR6. Herein,we show that highly purified neutrophils cultured with interferon-gamma plus lipopolysaccharide produce the CCL2 and CCL20 chemokines,the known ligands of CCR2 and CCR6,respectively. Accordingly,supernatants from activated neutrophils induced chemotaxis of Th17 cells,which was greatly suppressed by anti-CCL20 and anti-CCL2 antibodies. We also discovered that activated Th17 cells could directly chemoattract neutrophils via the release of biologically active CXCL8. Consistent with this reciprocal recruitment,neutrophils and Th17 cells were found in gut tissue from Crohn disease and synovial fluid from rheumatoid arthritis patients. Finally,we report that,although human Th17 cells can directly interact with freshly isolated or preactivated neutrophils via granulocyte-macrophage colony-stimulating factor,tumor necrosis factor-alpha,and interferon-gamma release,these latter cells cannot be activated by IL-17A and IL-17F,because of their lack of IL-17RC expression. Collectively,our results reveal a novel chemokine-dependent reciprocal cross-talk between neutrophils and Th17 cells,which may represent a useful target for the treatment of chronic inflammatory diseases. View Publication

Primitive hematopoietic cells from several species are known to efflux both Hoechst 33342 and Rhodamine-123. We now show that murine hematopoietic stem cells (HSCs) defined by long-term multilineage repopulation assays efflux both dyes variably according to their developmental or activation status. In day 14.5 murine fetal liver,very few HSCs efflux Hoechst 33342 efficiently,and they are thus not detected as side population" (SP) cells. HSCs in mouse fetal liver also fail to efflux Rhodamine-123. Both of these features are retained by most of the HSCs present until 4 weeks after birth but are reversed by 8 weeks of age or after a new HSC population is regenerated in adult mice that receive transplants with murine fetal liver cells. Activation of adult HSCs in vivo following 5-fluorouracil treatment� View Publication

Targeted nucleases,including zinc-finger nucleases (ZFNs),transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9),have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However,realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids,the direct delivery of nuclease proteins to cells provides rapid action and fast turnover,leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression,purification and delivery of ZFN proteins,which are intrinsically cell-permeable; TALEN proteins,which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein,whose nucleofection into cells facilitates rapid induction of multiplexed modifications,are described,along with procedures for evaluating nuclease protein activity. Once they are constructed,nuclease proteins can be expressed and purified within 6 d,and they can be used to induce genomic modifications in human cells within 2 d. View Publication

Circulating Tumor Cells (CTCs) represent an easy,repeatable and representative access to information regarding solid tumors. However,their detection remains difficult because of their paucity,their short half-life,and the lack of reliable surface biomarkers. Flow cytometry (FC) is a fast,sensitive and affordable technique,ideal for rare cells detection. Adapted to CTCs detection (i.e. extremely rare cells),most FC-based techniques require a time-consuming pre-enrichment step,followed by a 2-hours staining procedure,impeding on the efficiency of CTCs detection. We overcame these caveats and reduced the procedure to less than one hour,with minimal manipulation. First,cells were simultaneously fixed,permeabilized,then stained. Second,using low-speed FC acquisition conditions and two discriminators (cell size and pan-cytokeratin expression),we suppressed the pre-enrichment step. Applied to blood from donors with or without known malignant diseases,this protocol ensures a high recovery of the cells of interest independently of their epithelial-mesenchymal plasticity and can predict which samples are derived from cancer donors. This proof-of-concept study lays the bases of a sensitive tool to detect CTCs from a small amount of blood upstream of in-depth analyses. View Publication

BACKGROUND Patients with gene expression profiling-defined high-risk myeloma in relapse have poor outcomes with current therapies. We tested whether natural killer cells expanded by co-culture with K562 cells transfected with 41BBL and membrane-bound interleukin-15 could kill myeloma cells with a high-risk gene expression profile in vitro and in a unique model which recapitulates human myeloma. DESIGN AND METHODS OPM2 and high-risk primary myeloma tumors were grown in human fetal bone implanted into non-obese diabetic severe combined immunodeficiency mice with a deficient interleukin-2 receptor gamma chain. These mice are devoid of endogenous natural killer and T-cell activity and were used to determine whether adoptively transferred expanded natural killer cells could inhibit myeloma growth and myeloma-associated bone destruction. RESULTS Natural killer cells from healthy donors and myeloma patients expanded a median of 804- and 351-fold,respectively,without significant T-cell expansion. Expanded natural killer cells killed both allogeneic and autologous primary myeloma cells avidly via a perforin-mediated mechanism in which the activating receptor NKG2D,natural cytotoxicity receptors,and DNAX-accessory molecule-1 played a central role. Adoptive transfer of expanded natural killer cells inhibited the growth of established OPM2 and high-risk primary myeloma tumors grown in the murine model. The transferred,expanded natural killer cells proliferated in vivo in an interleukin-2 dose-dependent fashion,persisted up to 4 weeks,were readily detectable in the human bone,inhibited myeloma growth and protected bone from myeloma-induced osteolysis. CONCLUSIONS These studies provide the rationale for testing expanded natural killer cells in humans. View Publication

Mast cells are tissue-resident immune cells that play a key role in inflammation and allergy. Here we show that interaction of mast cells with antibody-targeted cells induces the polarized exocytosis of their granules resulting in a sustained exposure of effector enzymes,such as tryptase and chymase,at the cell-cell contact site. This previously unidentified mast cell effector mechanism,which we name the antibody-dependent degranulatory synapse (ADDS),is triggered by both IgE- and IgG-targeted cells. ADDSs take place within an area of cortical actin cytoskeleton clearance in the absence of microtubule organizing centre and Golgi apparatus repositioning towards the stimulating cell. Remarkably,IgG-mediated degranulatory synapses also occur upon contact with opsonized Toxoplasma gondii tachyzoites resulting in tryptase-dependent parasite death. Our results broaden current views of mast cell degranulation by revealing that human mast cells form degranulatory synapses with antibody-targeted cells and pathogens for dedicated secretion and defence. View Publication