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

Glioblastoma multiforme (GBM) is a grade IV malignant brain tumor with high mortality and has been well known to involve many molecular pathways,including G-protein coupled receptor (GPCR)-mediated signaling (such as epithelial growth factor receptor [EGFR] and platelet derived growth factor receptor [PDGFR]). G protein-coupled receptor kinases (GRK) directly regulate GPCR activity by phosphorylating activated agonist-bound receptors to desensitize signaling and internalize receptors through beta-arrestins. Recent studies in various cancers,including prostate and breast cancer,have highlighted the role of change in GRK expression to oncogenesis and tumor proliferation. In this study,we evaluated the expression of GRK5 in grade II to grade IV glioma specimens using immunohistochemistry and found that GRK5 expression levels are highly correlated with aggressiveness of glioma. We used culture conditions to selectively promote the growth of either glioblastoma cells with stem cell markers (GSC) or differentiated glioblastoma cells (DGC) from fresh GBM specimens. GSC are known to be highly invasive and mobile,and have the capacity to self-renew and are more resistant to chemotherapy and radiation compared to differentiated populations of GBM. We examined the expression of GRK5 in these two sets of culturing conditions for GBM cells and found that GRK5 expression is upregulated in GSC compared to differentiated GBM cells. To better understand the role of GRK5 in GBM-derived stem cells,we created stable GRK5 knockdown and evaluated the proliferation rate. Using an ATP chemiluminescence assay,we show,for the first time,that knocking down the expression of GRK5 decreased the proliferation rate of GSC in contrast to control. View Publication

BACKGROUND Neural stem cells (NSCs) display tissue trophic and immune modulatory therapeutic activities after transplantation in central nervous system disorders. The intercellular interplay between stem cells and target immune cells is increased in NSCs exposed to inflammatory cues. Here,we hypothesize that inflammatory cytokine signalling leads to metabolic reprogramming of NSCs regulating some of their immune modulatory effects. METHODS NSC lines were prepared from the subventricular zone (SVZ) of 7-12-week-old mice. Whole secretome-based screening and analysis of intracellular small metabolites was performed in NSCs exposed to cocktails of either Th1-like (IFN-γ,500 U/ml; TNF-α,200 U/ml; IL-1β,100 U/ml) or Th2-like (IL-4,IL-5 and IL-13; 10 ng/ml) inflammatory cytokines for 16 h in vitro. Isotopologues distribution of arginine and downstream metabolites was assessed by liquid chromatography/mass spectrometry in NSCs incubated with U-(13)C6 L-arginine in the presence or absence of Th1 or Th2 cocktails (Th1 NSCs or Th2 NSCs). The expression of arginase I and II was investigated in vitro in Th1 NSCs and Th2 NSCs and in vivo in the SVZ of mice with experimental autoimmune encephalomyelitis,as prototypical model of Th1 cell-driven brain inflammatory disease. The effects of the inflammatory cytokine signalling were studied in NSC-lymph node cells (LNC) co-cultures by flow cytometry-based analysis of cell proliferation following pan-arginase inhibition with N(ω)-hydroxy-nor-arginine (nor-NOHA). RESULTS Cytokine-primed NSCs showed significantly higher anti-proliferative effect in co-cultures vs. control NSCs. Metabolomic analysis of intracellular metabolites revealed alteration of arginine metabolism and increased extracellular arginase I activity in cytokine-primed NSCs. Arginase inhibition by nor-NOHA partly rescued the anti-proliferative effects of cytokine-primed NSCs. CONCLUSIONS Our work underlines the use of metabolic profiling as hypothesis-generating tools that helps unravelling how stem cell-mediated mechanisms of tissue restoration become affected by local inflammatory responses. Among different therapeutic candidates,we identify arginase signalling as novel metabolic determinant of the NSC-to-immune system communication. View Publication

BACKGROUND AND AIMS Hypoxia is one of the central players in shaping the immune context of the tumor microenvironment (TME). However,the complex interplay between immune cell infiltrates within the hypoxic TME of HCC remains to be elucidated. APPROACH AND RESULTS We analyzed the immune landscapes of hypoxia-low and hypoxia-high tumor regions using cytometry by time of light,immunohistochemistry,and transcriptomic analyses. The mechanisms of immunosuppression in immune subsets of interest were further explored using in vitro hypoxia assays. Regulatory T cells (Tregs) and a number of immunosuppressive myeloid subsets,including M2 macrophages and human leukocyte antigen-DR isotype (HLA-DRlo ) type 2 conventional dendritic cell (cDC2),were found to be significantly enriched in hypoxia-high tumor regions. On the other hand,the abundance of active granzyme Bhi PD-1lo CD8+ T cells in hypoxia-low tumor regions implied a relatively active immune landscape compared with hypoxia-high regions. The up-regulation of cancer-associated genes in the tumor tissues and immunosuppressive genes in the tumor-infiltrating leukocytes supported a highly pro-tumorigenic network in hypoxic HCC. Chemokine genes such as CCL20 (C-C motif chemokine ligand 20) and CXCL5 (C-X-C motif chemokine ligand 5) were associated with recruitment of both Tregs and HLA-DRlo cDC2 to hypoxia-high microenvironments. The interaction between Tregs and cDC2 under a hypoxic TME resulted in a loss of antigen-presenting HLA-DR on cDC2. CONCLUSIONS We uncovered the unique immunosuppressive landscapes and identified key immune subsets enriched in hypoxic HCC. In particular,we identified a potential Treg-mediated immunosuppression through interaction with a cDC2 subset in HCC that could be exploited for immunotherapies. View Publication

Chronic decreases in the second messenger cyclic AMP (cAMP) occur in numerous settings,but how cells compensate for such decreases is unknown. We have used a unique system-murine dendritic cells (DCs) with a DC-selective depletion of the heterotrimeric GTP binding protein G$\alpha$s-to address this issue. These mice spontaneously develop Th2-allergic asthma and their DCs have persistently lower cAMP levels. We found that phosphodiesterase 4B (PDE4B) is the primary phosphodiesterase expressed in DCs and that its expression is preferentially decreased in G$\alpha$s-depleted DCs. PDE4B expression is dynamic,falling and rising in a protein kinase A-dependent manner with decreased and increased cAMP concentrations,respectively. Treatment of DCs that drive enhanced Th2 immunity with a PDE4B inhibitor ameliorated DC-induced helper T cell response. We conclude that PDE4B is a homeostatic regulator of cellular cAMP concentrations in DCs and may be a target for treating Th2-allergic asthma and other settings with low cellular cAMP concentrations. View Publication

Type 1 diabetes is an autoimmune disease with no cure,where clinical translation of promising therapeutics has been hampered by the reproducibility crisis. Here,short-term administration of an antagonist to the receptor for advanced glycation end products (sRAGE) protected against murine diabetes at two independent research centers. Treatment with sRAGE increased regulatory T cells (Tregs) within the islets,pancreatic lymph nodes,and spleen,increasing islet insulin expression and function. Diabetes protection was abrogated by Treg depletion and shown to be dependent on antagonizing RAGE with use of knockout mice. Human Tregs treated with a RAGE ligand downregulated genes for suppression,migration,and Treg homeostasis (FOXP3,IL7R,TIGIT,JAK1,STAT3,STAT5b,CCR4). Loss of suppressive function was reversed by sRAGE,where Tregs increased proliferation and suppressed conventional T-cell division,confirming that sRAGE expands functional human Tregs. These results highlight sRAGE as an attractive treatment to prevent diabetes,showing efficacy and reproducibility at multiple research centers and in human T cells. View Publication

The role of NK cells against HIV-1 infections remains to be elucidated in vivo. While humanized mouse models potentially could be used to directly evaluate human NK cell responses during HIV-1 infection,improved functional development of human NK cells in these hosts is needed. Here,we report the humanized MISTRG-6-15 mouse model,in which NK cells were quick to expand and exhibit degranulation,cytotoxicity,and proinflammatory cytokine production in nonlymphoid organs upon HIV-1 infection but had reduced functionality in lymphoid organs. Although HIV-1 infection induced functional impairment of NK cells,antiretroviral therapy reinvigorated NK cells in response to HIV-1 rebound after analytic treatment interruption. Moreover,a broadly neutralizing antibody,PGT121,enhanced NK cell function in vivo,consistent with antibody-dependent cellular cytotoxicity. Monoclonal antibody depletion of NK cells resulted in higher viral loads in multiple nonlymphoid organs. Overall,our results in humanized MISTRG-6-15 mice demonstrated that NK cells provided direct anti-HIV-1 responses in vivo but were limited in their responses in lymphoid organs. View Publication

Krabbe disease (Kd) is a lysosomal storage disorder (LSD) caused by the deficiency of the lysosomal galactosylceramidase (GALC) which cleaves the myelin enriched lipid galactosylceramide (GalCer). Accumulated GalCer is catabolized into the cytotoxic lipid psychosine that causes myelinating cells death and demyelination which recruits microglia/macrophages that fail to digest myelin debris and become globoid cells. Here,to understand the pathological mechanisms of Kd,we used induced pluripotent stem cells (iPSCs) from Kd patients to produce myelinating organoids and microglia. We show that Kd organoids have no obvious defects in neurogenesis,astrogenesis,and oligodendrogenesis but manifest early myelination defects. Specifically,Kd organoids showed shorter but a similar number of myelin internodes than Controls at the peak of myelination and a reduced number and shorter internodes at a later time point. Interestingly,myelin is affected in the absence of autophagy and mTOR pathway dysregulation,suggesting lack of lysosomal dysfunction which makes this organoid model a very valuable tool to study the early events that drive demyelination in Kd. Kd iPSC-derived microglia show a marginal rate of globoid cell formation under normal culture conditions that is drastically increased upon GalCer feeding. Under normal culture conditions,Kd microglia show a minor LAMP1 content decrease and a slight increase in the autophagy protein LC3B. Upon GalCer feeding,Kd cells show accumulation of autophagy proteins and strong LAMP1 reduction that at a later time point are reverted showing the compensatory capabilities of globoid cells. Altogether,this supports the value of our cultures as tools to study the mechanisms that drive globoid cell formation and the compensatory mechanism in play to overcome GalCer accumulation in Kd. View Publication

Neutrophils are essential innate immune cells with unusual anti-microbial properties while dysfunctions of neutrophils lead to severe health problems such as lethal infections. Generation of neutrophils from human induced pluripotent stem cells (hiPSCs) is highly promising to produce off-the-shelf neutrophils for transfusion therapies. However,the anti-microbial potencies of hiPSCs derived neutrophils (iNEUs) remain less documented. Here,we develop a scalable approach to generate iNEUs in a chemical defined condition. iNEUs display typical neutrophil characters in terms of phagocytosis,migration,formation of neutrophil extracellular traps (NETs),etc. Importantly,iNEUs display a strong killing potency against various bacteria such as K.pneumoniae,P.aeruginosa,E.coli and S.aureus. Moreover,transfusions of iNEUs in mice with neutrophil dysfunction largely enhance their survival in lethal infection of different bacteria. Together,our data show that hiPSCs derived neutrophils hold strong anti-microbial potencies to protect severe infections under neutrophil dysfunction conditions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13619-025-00227-z. View Publication

Ageing-dependent low-grade inflammation is a hallmark of central nervous system (CNS) diseases. Vascular and immune abnormalities are implicated in the progression of gliomas and occur in the early stages of Alzheimer's disease (AD); however,the mechanisms by which these alterations manifest in the brain parenchyma remain unclear. Using RNAseq,scRNAseq,bioinformatics tools and a cohort of patients with glioma and Alzheimer's disease for validation of results,we have established an analysis of blood–brain barrier (BBB) dysfunction and neuron loss. A mouse model for glioblastoma pathology was also used that reversed BBB disruption and neuron loss,with the incorporation of the IDH mutation. Finally,we established a characterization of the relevant immune populations with an IHC analysis and transcriptional profile. In this study,molecular analyses of the brain ecosystem revealed that blood–brain barrier dysfunction and neuronal synapse integrity exhibit significant threshold-dependent changes that correlate directly and inversely,respectively,with brain ageing (significant changes at 57 years) and the progression of AD and gliomas (survival of 1525 vs 4084 days for patients with High vs Low BBB dysfunction). Using human samples and mouse models,we identified immunoageing processes characterized by an imbalance between pro-inflammatory and anti-inflammatory signals. This dysregulation promotes the extravasation of monocyte-derived macrophages (85% increase of cells),particularly those with a suppressive phenotype,alongside an increase in inflammatory cytokine levels. Notably,our data show that vascular normalization in a glioma model can reverse neuronal loss and attenuate the aggressiveness of the tumours. Finally,tumour development can be prevented by reactivating the ageing immune system. We propose that the ageing brain represents a common,BBB dysfunction-associated process driving chronic inflammation. This inflammation is regulated by TREM2+/TIM3+ suppressive myeloid cells,which play a central role in disease progression. Our findings suggest that targeting these pathways could offer therapeutic strategies to mitigate CNS pathologies linked to ageing,characterized by toxic neuroinflammation and myeloid dysfunction. This study was funded by ISCIII and co-funded by the European Union. View Publication