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

Extracellular acidification occurs in inflamed tissue and the tumor microenvironment; however,a systematic study on how pH sensing contributes to tissue homeostasis is lacking. In the present study,we examine cell type-specific roles of the pH sensor G protein-coupled receptor 65 (GPR65) and its inflammatory disease-associated Ile231Leu-coding variant in inflammation control. GPR65 Ile231Leu knock-in mice are highly susceptible to both bacterial infection-induced and T cell-driven colitis. Mechanistically,GPR65 Ile231Leu elicits a cytokine imbalance through impaired helper type 17 T cell (TH17 cell) and TH22 cell differentiation and interleukin (IL)-22 production in association with altered cellular metabolism controlled through the cAMP-CREB-DGAT1 axis. In dendritic cells,GPR65 Ile231Leu elevates IL-12 and IL-23 release at acidic pH and alters endo-lysosomal fusion and degradation capacity,resulting in enhanced antigen presentation. The present study highlights GPR65 Ile231Leu as a multistep risk factor in intestinal inflammation and illuminates a mechanism by which pH sensing controls inflammatory circuits and tissue homeostasis. View Publication

Plasmacytoid dendritic cells (pDCs) have been implicated as having a role in antifungal immunity,but mechanisms of their interaction with fungi and the resulting cellular responses are not well understood. In this study,we identify the direct and indirect biological response of human pDCs to the fungal pathogen Aspergillus fumigatus and characterize the expression and regulation of antifungal receptors on the pDC surface. Results indicate pDCs do not phagocytose Aspergillus conidia,but instead bind hyphal surfaces and undergo activation and maturation via the upregulation of costimulatory and maturation markers. Measuring the expression of C-type lectin receptors dectin-1,dectin-2,dectin-3,and mannose receptor on human pDCs revealed intermediate expression of each receptor compared with monocytes. The specific dectin-1 agonist curdlan induced pDC activation and maturation in a cell-intrinsic and cell-extrinsic manner. The indirect activation of pDCs by curdlan was much stronger than direct stimulation and was mediated through cytokine production by other PBMCs. Overall,our data indicate pDCs express various C-type lectin receptors,recognize and respond to Aspergillus hyphal Ag,and serve as immune enhancers or modulators in the overarching fungal immune response. View Publication

BACKGROUND MiR-199a-3p (miR-199a) can enhance the chemosensitivity of hepatocellular carcinoma (HCC). Because of the easy degradation of miRNA by direct infusion,effective vehicle-mediated delivery of miR-199a may represent a new strategy for improving HCC chemotherapy. Considering mesenchymal stem cell (MSC)-derived exosomes as promising natural nanovectors for drug and molecule delivery,we aimed to determine whether exosomes from adipose tissue-derived MSCs (AMSCs) could be used to deliver miR-199a and improve HCC chemosensitivity. METHODS MiR-199a-modified AMSCs (AMSC-199a) were constructed by miR-199a lentivirus infection and puromycin selection. MiR-199-modified exosomes (AMSC-Exo-199a) were isolated from the supernatant of AMSC-199a and were assessed by transmission electron microscopy,nanoparticle tracking analysis,and flow cytometry analysis. The expression levels of miR-199a in HCC samples,AMSCs,exosomes,and HCC cells were quantified by real-time PCR. The effects of AMSC-Exo-199a on HCC chemosensitivity were determined by cell proliferation and apoptosis assays and by i.v. injection into orthotopic HCC mouse models with doxorubicin treatment. MTOR,p-4EBP1 and p-70S6K levels in HCC cells and tissues were quantified by Western blot. RESULTS AMSC-Exo-199a had the classic characteristics of exosomes and could effectively mediate miR-199a delivery to HCC cells. Additionally,AMSC-Exo-199a significantly sensitized HCC cells to doxorubicin by targeting mTOR and subsequently inhibiting the mTOR pathway. Moreover,i.v.-injected AMSC-Exo-199a could distribute to tumor tissue and markedly increased the effect of Dox against HCC in vivo. CONCLUSIONS AMSC-Exo-199a can be an effective vehicle for miR-199a delivery,and they effectively sensitized HCC to chemotherapeutic agents by targeting mTOR pathway. AMSC-Exo-199a administration may provide a new strategy for improving HCC chemosensitivity. View Publication

The maturation of brain microvascular endothelial cells leads to the formation of a tightly sealed monolayer,known as the blood–brain barrier (BBB). The BBB damage is associated with the pathogenesis of age-related neurodegenerative diseases including vascular cognitive impairment and Alzheimer’s disease. Growing knowledge in the field of epigenetics can enhance the understanding of molecular profile of the BBB and has great potential for the development of novel therapeutic strategies or targets to repair a disrupted BBB. Histone deacetylases (HDACs) inhibitors are epigenetic regulators that can induce acetylation of histones and induce open chromatin conformation,promoting gene expression by enhancing the binding of DNA with transcription factors. We investigated how HDAC inhibition influences the barrier integrity using immortalized human endothelial cells (HCMEC/D3) and the human induced pluripotent stem cell (iPSC)-derived brain vascular endothelial cells. The endothelial cells were treated with or without a novel compound named W2A-16. W2A-16 not only activates Wnt/?-catenin signaling but also functions as a class I HDAC inhibitor. We demonstrated that the administration with W2A-16 sustained barrier properties of the monolayer of endothelial cells,as evidenced by increased trans-endothelial electrical resistance (TEER). The BBB-related genes and protein expression were also increased compared with non-treated controls. Analysis of transcript profiles through RNA-sequencing in hCMEC/D3 cells indicated that W2A-16 potentially enhances BBB integrity by influencing genes associated with the regulation of the extracellular microenvironment. These findings collectively propose that the HDAC inhibition by W2A-16 plays a facilitating role in the formation of the BBB. Pharmacological approaches to inhibit HDAC may be a potential therapeutic strategy to boost and/or restore BBB integrity. View Publication

Spinal cord injury (SCI) elicits a hostile microenvironment characterized by inflammation,gliosis,and disrupted signaling pathways that collectively impede neural repair. Neural progenitor cells (NPCs) represent a promising regenerative approach,yet their survival and differentiation are often compromised in this setting. Here,we investigated whether engineering NPCs to overexpress the Notch pathway modulator Delta-like non-canonical Notch ligand 1 (DLK1) could overcome these limitations and improve functional outcomes after cervical SCI in rats. NPCs were engineered to express DLK1 under a Pax6 promoter-driven expression system,ensuring elevated DLK1 levels during the progenitor state. Following transplantation of DLK1-overexpressing NPCs or control NPCs,we assessed graft survival,lineage differentiation,behavioral performance,and electrophysiological integration over 12 weeks. DLK1-expressing NPCs exhibited significantly greater retention in the injured spinal cord and showed enhanced neuronal differentiation alongside reduced astrocytic commitment compared to controls. Behavioral tests—including forelimb grip strength and CatWalk gait assessments—demonstrated that DLK1-modified NPCs conferred robust improvements in forelimb motor coordination and overall locomotion. Concordantly,electrophysiological recordings revealed increased motor-evoked potential amplitudes and area-under-the-curve values in animals receiving DLK1-transduced NPC grafts,indicative of strengthened synaptic integration within the host motor circuitry. View Publication

HNF4A and HNF1A encode transcription factors that are important for the development and function of the pancreas and liver. Mutations in both genes have been directly linked to Maturity Onset Diabetes of the Young (MODY) and type 2 diabetes (T2D) risk. To better define the pleiotropic gene regulatory roles of HNF4A and HNF1A,we generated a comprehensive genome-wide map of their binding targets in pancreatic and hepatic cells using ChIP-Seq. HNF4A was found to bind and regulate known (ACY3,HAAO,HNF1A,MAP3K11) and previously unidentified (ABCD3,CDKN2AIP,USH1C,VIL1) loci in a tissue-dependent manner. Functional follow-up highlighted a potential role for HAAO and USH1C as regulators of beta cell function. Unlike the loss-of-function HNF4A/MODY1 variant I271fs,the T2D-associated HNF4A variant (rs1800961) was found to activate AKAP1,GAD2 and HOPX gene expression,potentially due to changes in DNA-binding affinity. We also found HNF1A to bind to and regulate GPR39 expression in beta cells. Overall,our studies provide a rich resource for uncovering downstream molecular targets of HNF4A and HNF1A that may contribute to beta cell or hepatic cell (dys)function,and set up a framework for gene discovery and functional validation. Here,the authors generated a genome-wide map of the global targets bound by HNF4A and HNF1A in beta cells and hepatic cells,and highlighted notable downstream pathways and target genes that may influence beta cell function. This approach also shed light on a potentially activating effect of a HNF4A type 2 diabetes risk variant. View Publication

SummaryA tissue resident-like phenotype in tumor infiltrating T cells can limit systemic anti-tumor immunity. Enhanced systemic anti-tumor immunity is observed in head and neck cancer patients after neoadjuvant PD-L1 immune checkpoint blockade (ICB) and transforming growth factor β (TGF-β) neutralization. Using T cell receptor (TCR) sequencing and functional immunity assays in a syngeneic model of oral cancer,we dissect the relative contribution of these treatments to enhanced systemic immunity. The addition of TGF-β neutralization to ICB resulted in the egress of expanded and exhausted CD8+ tumor infiltrating lymphocytes (TILs) into circulation and greater systemic anti-tumor immunity. This enhanced egress associated with reduced expression of Itgae (CD103) and its upstream regulator Znf683. Circulating CD8+ T cells expressed higher Cxcr3 after treatment,an observation also made in samples from patients treated with dual TGF-β neutralization and ICB. These findings provide the scientific rationale for the use of PD-L1 ICB and TGF-β neutralization in newly diagnosed patients with carcinomas prior to definitive treatment of locoregional disease. Graphical abstract Highlights•TGF-β blockade reduces Znf683 and CD103 in αPDL1-activated TILs•Reduced TIL CD103 expression associates with egress into circulation•The addition of TGF-β blockade to αPDL1 enhances systemic anti-tumor immunity•Circulating CD8+ T cells express greater CXCR3 after dual TGF-β and PDL1 blockade Natural sciences; Biological sciences; Immunology ; Immune response; Systems biology; Cancer systems biology; Cancer View Publication

Although FOXP3+ regulatory T cells (Treg) depend on IL-2 produced by other cells for their survival and function,the levels of IL-2 in inflamed tissue are low,making it unclear how Treg access this critical resource. Here,we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo,including in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely,endowing monoclonal antibody-directed chimeric antigen receptor (mAbCAR) Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their ability to suppress neuroinflammation in vivo. Together,these data identify a role for HPSE and the ECM in immune tolerance,providing new avenues for improving Treg-based therapy of autoimmunity. Regulatory T cell (Treg) maintenance and function require IL-2,yet this cytokine is only present in low levels in vivo. In this study,the authors demonstrate that that Treg use heparanase to access IL-2 bound to heparan sulfate proteoglycans in the extracellular matrix of inflamed brain tissue in mice. View Publication

SUMMARYRegulatory T cells (Tregs) are promising cellular therapies to induce immune tolerance in organ transplantation and autoimmune disease. The success of chimeric antigen receptor (CAR) T-cell therapy for cancer has sparked interest in using CARs to generate antigen-specific Tregs. Here,we compared CAR with endogenous T cell receptor (TCR)/CD28 activation in human Tregs. Strikingly,CAR Tregs displayed increased cytotoxicity and diminished suppression of antigen-presenting cells and effector T (Teff) cells compared with TCR/CD28 activated Tregs. RNA sequencing revealed that CAR Tregs activate Teff cell gene programs. Indeed,CAR Tregs secreted high levels of inflammatory cytokines,with a subset of FOXP3+ CAR Tregs uniquely acquiring CD40L surface expression and producing IFNγ. Interestingly,decreasing CAR antigen affinity reduced Teff cell gene expression and inflammatory cytokine production by CAR Tregs. Our findings showcase the impact of engineered receptor activation on Treg biology and support tailoring CAR constructs to Tregs for maximal therapeutic efficacy. Graphical Abstract View Publication

Acute myeloid leukaemia (AML) is a haematological malignancy characterised by the accumulation of transformed myeloid progenitors in the bone marrow. Piplartine (PL),also known as piperlongumine,is a pro-oxidant small molecule extracted from peppers that has demonstrated antineoplastic potential in solid tumours and other haematological malignancies. In this work,we explored the potential of PL to treat AML through the use of a combination of cellular and molecular analyses of primary and cultured leukaemia cells in vitro and in vivo. We showed that PL exhibits in vitro cytotoxicity against AML cells,including CD34 + leukaemia-propagating cells,but not healthy haematopoietic progenitors,suggesting anti-leukaemia selectivity. Mechanistically,PL treatment increased reactive oxygen species (ROS) levels and induced ROS-mediated apoptosis in AML cells,which could be prevented by treatment with the antioxidant scavenger N -acetyl-cysteine and the pancaspase inhibitor Z-VAD(OMe)-FMK. PL treatment reduced NFKB1 gene transcription and the level of NF-κB p65 (pS536),which was depleted from the nucleus of AML cells,indicating suppression of NF-κB p65 signalling. Significantly,PL suppressed AML development in a mouse xenograft model,and its combination with current AML treatments (cytarabine,daunorubicin and azacytidine) had synergistic effects,indicating translational therapeutic potential. Taken together,these data position PL as a novel anti-AML candidate drug that can target leukaemia stem/progenitors and is amenable to combinatorial therapeutic strategies. Subject terms: Acute myeloid leukaemia,Cancer stem cells,Pharmacology View Publication

Of all gynecologic cancers,epithelial-ovarian cancer (OCa) stands out with the highest mortality rates. Despite all efforts,90% of individuals who receive standard surgical and cytotoxic therapy experience disease recurrence. The precise mechanism by which leukemia inhibitory factor (LIF) and its receptor (LIFR) contribute to the progression of OCa remains unknown. Analysis of cancer databases revealed that elevated expression of LIF or LIFR was associated with poor progression-free survival of OCa patients and a predictor of poor response to chemotherapy. Using multiple primary and established OCa cell lines or tissues that represent five subtypes of epithelial-OCa,we demonstrated that LIF/LIFR autocrine signaling is active in OCa. Moreover,treatment with LIFR inhibitor,EC359 significantly reduced OCa cell viability and cell survival with an IC 50 ranging from 5-50 nM. Furthermore,EC359 diminished the stemness of OCa cells. Mechanistic studies using RNA-seq and rescue experiments unveiled that EC359 primarily induced ferroptosis by suppressing the glutathione antioxidant defense system. Using multiple in vitro,ex vivo and in vivo models including cell-based xenografts,patient-derived explants,organoids,and xenograft tumors,we demonstrated that EC359 dramatically reduced the growth and progression of OCa. Additionally,EC359 therapy considerably improved tumor immunogenicity by robust CD45 + leukocyte tumor infiltration and polarizing tumor-associated macrophages (TAMs) toward M1 phenotype while showing no impact on normal T-,B-,and other immune cells. Collectively,our findings indicate that the LIF/LIFR autocrine loop plays an essential role in OCa progression and that EC359 could be a promising therapeutic agent for OCa. Subject terms: Molecular medicine,Ovarian cancer View Publication