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

OBJECTIVES: Our objective was to develop and assess a novel endogenous progenitor cell (EPC) assay based on aldehyde dehydrogenase (ALDH) activity,and to define the relationship of ALDH-bright (ALDH(br)) cells with previously defined EPCs,patient age,and extent of coronary artery disease. BACKGROUND: Accurate assessment of circulating EPCs is of significant interest,yet current assays have limitations. Progenitor cells display high levels of ALDH activity. An assay based on ALDH activity may offer a simple means for enumerating EPCs. METHODS: We simultaneously determined the numbers of EPCs based on ALDH activity and cell surface expression of CD133,CD34,and vascular endothelial growth factor receptor-2 in 110 patients undergoing cardiac catheterization. We assessed the reproducibility of these estimates,correlation among EPC assays,and the association of ALDH(br) numbers with age and disease severity. RESULTS: Aldehyde dehydrogenase-bright cells were easily identified in nonmobilized peripheral blood with median and mean frequencies of 0.041% and 0.074%,respectively. Aldehyde dehydrogenase-bright cells expressed CD34 or CD133 cell surface markers (57.0% and 27.1%,respectively),correlated closely with CD133+CD34+ cells (r = 0.72; p textless 0.001),and differentiated into endothelial cells with greater efficiency than CD133+CD34+ cells. Aldehyde dehydrogenase-bright cell numbers were inversely associated with patient age and coronary disease severity. CONCLUSIONS: Aldehyde dehydrogenase activity represents a novel simplified method for quantifying EPCs. The correlation of ALDH(br) cells with clinical factors and outcomes warrants further study. View Publication

We examined the signaling pathways and cell type-specific responses of IFN regulatory factor (IRF) 5,an immune-regulatory transcription factor. We show that the protein kinases IKK$\alpha$,IKK$\beta$,IKK$\epsilon$,and TANK-binding kinase 1 each confer IRF5 phosphorylation/dimerization,thus extending the family of IRF5 activator kinases. Among primary human immune cell subsets,we found that IRF5 is most abundant in plasmacytoid dendritic cells (pDCs). Flow cytometric cell imaging revealed that IRF5 is specifically activated by endosomal TLR signaling. Comparative analyses revealed that IRF3 is activated in pDCs uniquely through RIG-I-like receptor (RLR) signaling. Transcriptomic analyses of pDCs show that the partitioning of TLR7/IRF5 and RLR/IRF3 pathways confers differential gene expression and immune cytokine production in pDCs,linking IRF5 with immune regulatory and proinflammatory gene expression. Thus,TLR7/IRF5 and RLR-IRF3 partitioning serves to polarize pDC response outcome. Strategies to differentially engage IRF signaling pathways should be considered in the design of immunotherapeutic approaches to modulate or polarize the immune response for specific outcome. View Publication

In vitro differentiation of embryonic stem cells is tightly regulated by the same key signaling pathways that control pattern formation during embryogenesis. Small molecules that selectively target these developmental pathways,including Wnt,and BMP signaling may be valuable for directing differentiation of pluripotent stem cells toward many desired tissue types,but to date only few such compounds have been shown to promote cardiac differentiation. Here,we show that XAV939,a recently discovered small molecule inhibitor of Wnt/β-catenin signaling,can robustly induce cardiomyogenesis in mouse ES cells. Our results suggest that a timely administration of XAV939 immediately following the formation of mesoderm progenitor cells promotes cardiomyogenic development at the expense of other mesoderm derived lineages,including the endothelial,smooth muscle,and hematopoietic lineages. Given the critical role that Wnt/β-catenin signaling plays in many aspects of embryogenesis and tissue regeneration,XAV939 is a valuable chemical probe to dissect in vitro differentiation of stem cells and to explore their regenerative potential in a variety of contexts. View Publication

Mast cells (MCs) play an important role in the regulation of protective adaptive immune responses against pathogens. However,it is still unclear whether MCs promote such host defense responses via direct effects on T cells or rather by modifying the functions of antigen-presenting cells. To identify the underlying mechanisms of the immunoregulatory capacity of MCs,we investigated the impact of MCs on dendritic cell (DC) maturation and function. We found that murine peritoneal MCs underwent direct crosstalk with immature DCs that induced DC maturation as evidenced by enhanced expression of costimulatory molecules. Furthermore,the MC/DC interaction resulted in the release of the T-cell modulating cytokines IFN-γ,IL-2,IL-6 and TGF-β into coculture supernatants and increased the IL-12p70,IFN-γ,IL-6 and TGF-β secretion of LPS-matured DCs. Such MC-primed" DCs subsequently induced efficient CD4+ T-cell proliferation. Surprisingly� View Publication

Canonical Wnt/β-catenin signaling has been suggested to promote self-renewal of pluripotent mouse and human embryonic stem cells. Here,we show that SB-216763,a glycogen synthase kinase-3 (GSK3) inhibitor,can maintain mouse embryonic stem cells (mESCs) in a pluripotent state in the absence of exogenous leukemia inhibitory factor (LIF) when cultured on mouse embryonic fibroblasts (MEFs). MESCs maintained with SB-216763 for one month were morphologically indistinguishable from LIF-treated mESCs and expressed pluripotent-specific genes Oct4,Sox2,and Nanog. Furthermore,Nanog immunostaining was more homogenous in SB-216763-treated colonies compared to LIF. Embryoid bodies (EBs) prepared from these mESCs expressed early-stage markers for all three germ layers,and could efficiently differentiate into cardiac-like cells and MAP2-immunoreactive neurons. To our knowledge,SB-216763 is the first GSK3 inhibitor that can promote self-renewal of mESC co-cultured with MEFs for more than two months. View Publication

Due to viral envelope glycoprotein D binding to cellular membrane HVEM receptor,HSV-1 can infect certain dendritic cells,which becomes an event in the viral strategy to interfere with the host's immune system. We previously generated the HSV-1 mutant strain M6,which produced an attenuated phenotype in mice and rhesus monkeys. The attenuated M6 strain was used to investigate how HSV-1 infection of dendritic cells interferes with both innate and adaptive immunity. Our study showed that dendritic cells membrane HVEM receptors could mediate infection of the wild-type strain and attenuated M6 strain and that dendritic cells infected by both viruses in local tissues of animals exhibited changes in transcriptional profiles associated with innate immune and inflammatory responses. The infection of pDCs and cDCs by the two strains promoted cell differentiation to the CD103+ phenotype,but varied transcriptional profiles were observed,implying a strategy that the HSV-1 wild-type strain interferes with antiviral immunity,probably due to viral modification of the immunological phenotype of dendritic cells during processing and presentation of antigen to T cells,leading to a series of deviations in immune responses,ultimately generating the deficient immune phenotype observed in infected individuals in the clinical. View Publication

BACKGROUND Two opposing B cell subsets have been defined based on their cytokine profile: IL-6 producing effector B cells (B-effs) versus IL-10 producing regulatory B cells (B-regs) that respectively positively or negatively regulate immune responses. B-regs are decreased and/or impaired in many autoimmune diseases and inflammatory conditions. Since there is increasing evidence that links B cells and B cell-rich lymphoid follicles to the pathogenesis of COPD,the aim of this study was to investigate the presence and function of B-regs in COPD. METHODS First,presence of IL-10 producing regulatory B cells in human lung tissue was determined by immunohistochemistry. Secondly,quantification of IL-10??+??B-regs and IL-6??+??B-effs in peripheral blood mononuclear cells (PBMCs) from healthy controls,smokers without airflow limitation,and COPD patients (GOLD stage I-IV) was performed by flow cytometry. Thirdly,we exposed blood-derived B cells from COPD patients in vitro to cigarette smoke extract (CSE) and quantified IL-10??+??B-regs and IL-6??+??B-effs. Furthermore,we aimed at restoring the perturbed IL10 production by blocking BAFF. Fourthly,we determined mRNA expression of transcription factors involved in IL-10 production in FACS sorted memory- and naive B cells upon exposure to medium or CSE. RESULTS The presence of IL-10 producing regulatory B cells in parenchyma and lymphoid follicles in lungs was confirmed by immunohistochemistry. The percentage of IL-10??+??B-regs was significantly decreased in blood-derived memory B cell subsets from smokers without airflow limitation and patients with COPD,compared to never smokers. Furthermore,the capacity of B cells to produce IL-10 was reduced upon in vitro exposure to CSE and this could not be restored by BAFF-blockade. Finally,upon CSE exposure,mRNA levels of the transcription factors IRF4 and HIF-1$\alpha$,were decreased in memory B cells. CONCLUSION Decreased numbers and impaired function of B-regs in smokers and patients with COPD might contribute to the initiation and progression of the disease. View Publication

Cancer stem cells (CSCs) residing in colorectal cancer tissues have tumorigenic capacity and contribute to chemotherapeutic resistance and disease relapse. It is well known that the survival of colorectal CSCs after 5-fluorouracil (5-FU)-based therapy leads to cancer recurrence. Thus CSCs represent a promising drug target. Here,we designed and synthesized novel hybrid molecules linking 5-FU with the plant-derived compound thymoquinone (TQ) and tested the potential of individual compounds and their combination to eliminate colorectal CSCs. Both,Combi and SARB hybrid showed augmented cytotoxicity against colorectal cancer cells,but were non-toxic to organoids prepared from healthy murine small intestine. NanoString analysis revealed a unique signature of deregulated gene expression in response to the combination of TQ and 5-FU (Combi) and SARB treatment. Importantly,two principle stem cell regulatory pathways WNT/{\ss}-Catenin and PI3K/AKT were found to be downregulated after Combi and hybrid treatment. Furthermore,both treatments strikingly eliminated CD133+ CSC population,accompanying the depleted self-renewal capacity by eradicating long-term propagated 3D tumor cell spheres at sub-toxic doses. In vivo xenografts on chicken eggs of SARB-treated HCT116 cells showed a prominent nuclear {\ss}-Catenin and E-cadherin staining. This was in line with the reduced transcriptional activity of {\ss}-Catenin and diminished cell adhesion under SARB exposure. In contrast to 5-FU,both,Combi and SARB treatment effectively reduced the angiogenic capacity of the remaining resistant tumor cells. Taken together,combination or hybridization of single compounds target simultaneously a broader spectrum of oncogenic pathways leading to an effective eradication of colorectal cancer cells. View Publication

An imbalance between suppressor and effector immune responses may preclude cure in chronic parasitic diseases. In the case of Trypanosoma cruzi infection,specialized regulatory Foxp3+ T (Treg) cells suppress protective type-1 effector responses. Herein,we investigated the kinetics and underlying mechanisms behind the regulation of protective parasite-specific CD8+ T cell immunity during acute T. cruzi infection. Using the DEREG mouse model,we found that Treg cells play a role during the initial stages after T. cruzi infection,restraining the magnitude of CD8+ T cell responses and parasite control. Early Treg cell depletion increased the frequencies of polyfunctional short-lived,effector T cell subsets,without affecting memory precursor cell formation or the expression of activation,exhaustion and functional markers. In addition,Treg cell depletion during early infection minimally affected the antigen-presenting cell response but it boosted CD4+ T cell responses before the development of anti-parasite effector CD8+ T cell immunity. Crucially,the absence of CD39 expression on Treg cells significantly bolstered effector parasite-specific CD8+ T cell responses,preventing increased parasite replication in T. cruzi infected mice adoptively transferred with Treg cells. Our work underscores the crucial role of Treg cells in regulating protective anti-parasite immunity and provides evidence that CD39 expression by Treg cells represents a key immunomodulatory mechanism in this infection model. Author summaryChagas disease,caused by Trypanosoma cruzi,can result in severe health complications. While the exact mechanisms underlying the disease’s pathogenesis remain incompletely understood,the host’s inflammatory immune response is believed to play a critical role. To shed light on disease mechanisms and potential treatments,we investigated the impact of regulatory T (Treg) cells on the development of effector immune responses against T. cruzi. Our findings reveal that Treg cells dampen parasite-specific CD8+ T cells,a crucial arm of the immune response in counteracting the parasite. Notably,this regulatory influence occurs primarily during the early stages of T. cruzi infection. Furthermore,we observed that while Treg cells have minimal effects on antigen-presenting cells,they modulate the magnitude and phenotype of conventional CD4+ T cells. Importantly,we identified CD39,a molecule involved in the purinergic pathway,as essential for the suppressive functions of Treg cells during T. cruzi infection. Our findings enhance the understanding of the regulatory response during the acute phase of T. cruzi infection and may have implications for the development of novel therapeutic strategies. View Publication

Toll-like receptor 9 (TLR9) recognizes bacterial,viral and self DNA and play an important role in immunity and inflammation. However,the role of TLR9 in obesity is less well-studied. Here,we generate B-cell-specific Tlr9-deficient (Tlr9fl/fl/Cd19Cre+/-,KO) B6 mice and model obesity using a high-fat diet. Compared with control mice,B-cell-specific-Tlr9-deficient mice exhibited increased fat tissue inflammation,weight gain,and impaired glucose and insulin tolerance. Furthermore,the frequencies of IL-10-producing-B cells and marginal zone B cells were reduced,and those of follicular and germinal center B cells were increased. This was associated with increased frequencies of IFNγ-producing-T cells and increased follicular helper cells. In addition,gut microbiota from the KO mice induced a pro-inflammatory state leading to immunological and metabolic dysregulation when transferred to germ-free mice. Using 16 S rRNA gene sequencing,we identify altered gut microbial communities including reduced Lachnospiraceae,which may play a role in altered metabolism in KO mice. We identify an important network involving Tlr9,Irf4 and Il-10 interconnecting metabolic homeostasis,with the function of B and T cells,and gut microbiota in obesity. Although the function of Toll-like receptor 9 (TLR9) in immunity and inflammation is well-established,its role in obesity is less well-studied. In this study,the authors demonstrate that TLR9 deficiency in B cells is associated with obesity in mice and results in altered frequencies of T and B lymphocyte subsets and gut microbiome dysbiosis. View Publication

PRDX2 is significantly expressed in various cancers and is associated with the proliferation of tumor cells. Nonetheless,the precise mechanism of PRDX2 in tumor immunity remains incompletely understood. This study aims to investigate the impact of PRDX2,which is highly expressed in lung adenocarcinoma,on T cells in the tumor immune microenvironment,and its immune action target to promote the immune escape of lung cancer cells,to provide a theoretical basis for lung adenocarcinoma treatment with PRDX2 as the target. Mouse animal models to verify the effect of Conoidin A treatment on tumor growth and T cell infiltration. Flow cytometry and Western blot verified tumor cell apoptosis in the in vitro co-culture system as well as granzyme B and perforin expression in T cells. RNA-Seq was used to obtain the downstream immune molecule. si-RNA knockdown of Galectin-9 was co-cultured with T cells in vitro. Immunofluorescence and Western blot verified that PRDX2 regulates Galectin-9 expression through HDAC3. PRDX2 expression was negatively correlated with CD8 + T cell expression in LUAD patients. Inhibition of PRDX2 significantly enhanced T-cell killing of LUAD cells and reduced tumor load in both in vitro and in vivo models. Mechanistically,Conoidin A or shRNA_PRDX2 decreased Galectin-9 expression by down-regulating the phosphorylation of HDAC3,consequently enhancing the infiltration and function of CD8 + T cells. This study reveals the role of the PRDX2/HDAC3/Galectin-9 axis in LUAD immune escape and indicates Galectin-9 as a promising target for immunotherapy. The online version contains supplementary material available at 10.1186/s12967-024-05888-z. View Publication