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

Chaetocin,a thiodioxopiperazine natural product previously unreported to have anticancer effects,was found to have potent antimyeloma activity in IL-6-dependent and -independent myeloma cell lines in freshly collected sorted and unsorted patient CD138(+) myeloma cells and in vivo. Chaetocin largely spares matched normal CD138(-) patient bone marrow leukocytes,normal B cells,and neoplastic B-CLL (chronic lymphocytic leukemia) cells,indicating a high degree of selectivity even in closely lineage-related B cells. Furthermore,chaetocin displays superior ex vivo antimyeloma activity and selectivity than doxorubicin and dexamethasone,and dexamethasone- or doxorubicin-resistant myeloma cell lines are largely non-cross-resistant to chaetocin. Mechanistically,chaetocin is dramatically accumulated in cancer cells via a process inhibited by glutathione and requiring intact/unreduced disulfides for uptake. Once inside the cell,its anticancer activity appears mediated primarily through the imposition of oxidative stress and consequent apoptosis induction. Moreover,the selective antimyeloma effects of chaetocin appear not to reflect differential intracellular accumulation of chaetocin but,instead,heightened sensitivity of myeloma cells to the cytotoxic effects of imposed oxidative stress. Considered collectively,chaetocin appears to represent a promising agent for further study as a potential antimyeloma therapeutic. View Publication

The immunodeficiency disorder,X-linked agammaglobulinemia (XLA),results from mutations in the gene encoding Bruton tyrosine kinase (Btk). Btk is required for pre-B cell clonal expansion and B-cell antigen receptor signaling. XLA patients lack mature B cells and immunoglobulin and experience recurrent bacterial infections only partially mitigated by life-long antibody replacement therapy. In pursuit of definitive therapy for XLA,we tested ex vivo gene therapy using a lentiviral vector (LV) containing the immunoglobulin enhancer (Emu) and Igbeta (B29) minimal promoter to drive B lineage-specific human Btk expression in Btk/Tec(-/-) mice,a strain that reproduces the features of human XLA. After transplantation of EmuB29-Btk-LV-transduced stem cells,treated mice showed significant,albeit incomplete,rescue of mature B cells in the bone marrow,peripheral blood,spleen,and peritoneal cavity,and improved responses to T-independent and T-dependent antigens. LV-treated B cells exhibited enhanced B-cell antigen receptor signaling and an in vivo selective advantage in the peripheral versus central B-cell compartment. Secondary transplantation showed sustained Btk expression,viral integration,and partial functional responses,consistent with long-term stem cell marking; and serial transplantation revealed no evidence for cellular or systemic toxicity. These findings strongly support pursuit of B lineage-targeted LV gene therapy in human XLA. View Publication

Class switch DNA recombination (CSR) is central to the maturation of the Ab response because it diversifies Ab effector functions. Like somatic hypermutation,CSR requires activation-induced cytidine deaminase (AID),whose expression is restricted to B cells,as induced by CD40 engagement or dual TLR-BCR engagement (primary CSR-inducing stimuli). By constructing conditional knockout Igh(+/C)γ(1-cre)Rab7(fl/fl) mice,we identified a B cell-intrinsic role for Rab7,a small GTPase involved in intracellular membrane functions,in mediating AID induction and CSR. Igh(+/C)γ(1-cre)Rab7(fl/fl) mice displayed normal B and T cell development and were deficient in Rab7 only in B cells undergoing Igh(C)γ(1-cre) Iγ1-Sγ1-Cγ1-cre transcription,as induced--like Igh germline Iγ1-Sγ1-Cγ1 and Iε-Sε-Cε transcription--by IL-4 in conjunction with a primary CSR-inducing stimulus. These mice could not mount T-independent or T-dependent class-switched IgG1 or IgE responses while maintaining normal IgM levels. Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells showed,in vivo and in vitro,normal proliferation and survival,normal Blimp-1 expression and plasma cell differentiation,as well as intact activation of the noncanonical NF-κB,p38 kinase,and ERK1/2 kinase pathways. They,however,were defective in AID expression and CSR in vivo and in vitro,as induced by CD40 engagement or dual TLR1/2-,TLR4-,TLR7-,or TLR9-BCR engagement. In Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells,CSR was rescued by enforced AID expression. These findings,together with our demonstration that Rab7-mediated canonical NF-κB activation,as critical to AID induction,outline a novel role of Rab7 in signaling pathways that lead to AID expression and CSR,likely by promoting assembly of signaling complexes along intracellular membranes. View Publication

BACKGROUND The goal of antiretroviral therapy (ART) is to suppress HIV-1 replication and reconstitute CD4+ T cells. Here,we report on HIV-infected individuals who had a paradoxical decline in CD4+ T cells despite ART-mediated suppression of plasma HIV-1 load (pVL). We defined such an immunological outcome as extreme immune decline (EXID). METHODS EXID's clinical and immunological characteristics were compared to immunological responders (IRs),immunological nonresponders (INRs),healthy controls (HCs),and idiopathic CD4+ lymphopenia (ICL) patients. T cell immunophenotyping and assembly/activation of inflammasomes were evaluated by flow cytometry. PBMC transcriptome analysis and genetic screening for pathogenic variants were performed. Levels of cytokines/chemokines were measured by electrochemiluminescence. Luciferase immunoprecipitation system and NK-mediated antibody-dependent cellular cytotoxicity (ADCC) assays were used to identify anti-lymphocyte autoantibodies. RESULTS EXIDs were infected with non-B HIV-1 subtypes and after 192 weeks of consistent ART-mediated pVL suppression had a median CD4+ decrease of 157 cells/mul,compared with CD4+ increases of 193 cells/mul and 427 cells/mul in INR and IR,respectively. EXID had reduced naive CD4+ T cells,but similar proportions of cycling CD4+ T cells and HLA-DR+CD38+CD8+ T cells compared with IR and INR. Levels of inflammatory cytokines were also similar in EXID and INR,but the IL-7 axis was profoundly perturbed compared with HC,IR,INR,and ICL. Genes involved in T cell and monocyte/macrophage function,autophagy,and cell migration were differentially expressed in EXID. Two of the 5 EXIDs had autoantibodies causing ADCC,while 2 different EXIDs had an increased inflammasome/caspase-1 activation despite consistently ART-suppressed pVL. CONCLUSIONS EXID is a distinct immunological outcome compared with previously described INR. Anti-CD4+ T cell autoantibodies and aberrant inflammasome/caspase-1 activation despite suppressed HIV-1 viremia are among the mechanisms responsible for EXID. View Publication

Binding of ICAM-1 (intercellular adhesion molecule-1) to the β2-integrin LFA-1 (leukocyte function associated antigen-1) is known to induce crosstalk to the α4β1 integrin. Using different LFA-1 monoclonal antibodies we have been able to study the requirement and mechanism of action for the crosstalk in considerable detail. LFA-1 activating antibodies and those inhibitory antibodies that signal to α4β1 induce phosphorylation of Thr-758 on the β2-chain,which is followed by binding of 14-3-3 proteins and signaling through the G protein exchange factor Tiam1. This results in dephosphorylation of Thr-788/789 on the β1-chain of α4β1 and loss of binding to its ligand VCAM-1 (vascular cell adhesion molecule-1). The results show that with LFA-1 antibodies,we can either 1) activate LFA-1 and inhibit α4β1,2) inhibit both LFA-1 and α4β1,3) inhibit LFA-1 but not α4β1 or 4) not affect LFA-1 or α4β1 These findings are important for the understanding of integrin regulation and for the interpretation of the effect of integrin antibodies and their use in clinical applications. View Publication

NK cells play a critical role in the rejection of xenografts. In this study,we report on an investigation of the effect of complement regulatory protein,a decay accelerating factor (DAF: CD55),in particular,on NK cell-mediated cytolysis. Amelioration of human NK cell-mediated pig endothelial cell (PEC) and pig fibroblast cell lyses by various deletion mutants and point substitutions of DAF was tested,and compared with their complement regulatory function. Although wild-type DAF and the delta-short consensus repeat (SCR) 1-DAF showed clear inhibition of both complement-mediated and NK-mediated PEC lyses,delta-SCR2-DAF and delta-SCR3-DAF failed to suppress either process. However,delta-SCR4-DAF showed a clear complement regulatory effect,but had no effect on NK cells. Conversely,the point substitution of DAF (L147 x F148 to SS and KKK(125-127) to TTT) was half down-regulated in complement inhibitory function,but the inhibition of NK-mediated PEC lysis remained unchanged. Other complement regulatory proteins,such as the cell membrane-bound form factor H,fH-PI,and C1-inactivator,C1-INH-PI,and CD59 were also assessed,but no suppressive effect on NK cell-mediated PEC lysis was found. These data suggest,for DAF to function on NK cells,SCR2-4 is required but no relation to its complement regulatory function exists. View Publication

Oligonucleotides with a CpG" motif trigger a proinflammatory response through activation of Toll-like receptor 9 (TLR9) and are being studied to exploit these properties for use as adjuvants and cancer therapies. However� View Publication

Current therapies for delayed- or nonunion bone fractures are still largely ineffective. Previous studies indicated that the VEGF homolog placental growth factor (PlGF) has a more significant role in disease than in health. Therefore we investigated the role of PlGF in a model of semi-stabilized bone fracture healing. Fracture repair in mice lacking PlGF was impaired and characterized by a massive accumulation of cartilage in the callus,reminiscent of delayed- or nonunion fractures. PlGF was required for the early recruitment of inflammatory cells and the vascularization of the fracture wound. Interestingly,however,PlGF also played a role in the subsequent stages of the repair process. Indeed in vivo and in vitro findings indicated that PlGF induced the proliferation and osteogenic differentiation of mesenchymal progenitors and stimulated cartilage turnover by particular MMPs. Later in the process,PlGF was required for the remodeling of the newly formed bone by stimulating osteoclast differentiation. As PlGF expression was increased throughout the process of bone repair and all the important cell types involved expressed its receptor VEGFR-1,the present data suggest that PlGF is required for mediating and coordinating the key aspects of fracture repair. Therefore PlGF may potentially offer therapeutic advantages for fracture repair. View Publication

V-domain immunoglobulin suppressor of T cell activation (VISTA) is a recently discovered immune checkpoint ligand that functions to suppress T cell activity. The therapeutic potential of activating this immune checkpoint pathway to reduce inflammatory responses remains untapped,largely due to the inability to derive agonists targeting its unknown receptor. A dimeric construct of the IgV domain of VISTA (VISTA-Fc) was shown to suppress the activation of T cells in vitro. However,this effect required its immobilization on a solid surface,suggesting that VISTA-Fc may display limited efficacy as a VISTA-receptor agonist in vivo. Herein,we have designed a stable pentameric VISTA construct (VISTA.COMP) by genetically fusing its IgV domain to the pentamerization domain from the cartilage oligomeric matrix protein (COMP). In contrast to VISTA-Fc,VISTA.COMP does not require immobilization to inhibit the proliferation of CD4+ T cells undergoing polyclonal activation. Furthermore,we show that VISTA.COMP,but not VISTA-Fc,functions as an immunosuppressive agonist in vivo capable of prolonging the survival of skin allografts in a mouse transplant model as well as rescuing mice from acute concanavalin-A-induced hepatitis. Collectively,we believe our data demonstrate that VISTA.COMP is a checkpoint receptor agonist and the first agent to our knowledge targeting the putative VISTA-receptor to suppress T cell-mediated immune responses. View Publication

NLRC5 is a transcriptional regulator of MHC class I (MHCI),which maintains high MHCI expression particularly in T cells. Recent evidence highlights an important NK-T-cell crosstalk,raising the question on whether NLRC5 specifically modulates this interaction. Here we show that NK cells from Nlrc5-deficient mice exhibit moderate alterations in inhibitory receptor expression and responsiveness. Interestingly,NLRC5 expression in T cells is required to protect them from NK-cell-mediated elimination upon inflammation. Using T-cell-specific Nlrc5-deficient mice,we show that NK cells surprisingly break tolerance even towards 'self' Nlrc5-deficient T cells under inflammatory conditions. Furthermore,during chronic LCMV infection,the total CD8(+) T-cell population is severely decreased in these mice,a phenotype reverted by NK-cell depletion. These findings strongly suggest that endogenous T cells with low MHCI expression become NK-cell targets,having thus important implications for T-cell responses in naturally or therapeutically induced inflammatory conditions. View Publication