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

Alzheimer's disease (AD) is emerging as a synaptopathology driven by metaplasticity. Indeed,reminiscent of metaplasticity,oligomeric forms of the amyloid-beta$ peptide (oAbeta$) prevent induction of long-term potentiation (LTP) via the prior activation of GluN2B-containing NMDA receptors (NMDARs). However,the downstream Ca2+-dependent signaling molecules that mediate aberrant metaplasticity are unknown. In this study,we show that oAbeta$ promotes the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) via GluN2B-containing NMDARs. Importantly,we find that CaMKII inhibition rescues both the LTP impairment and the dendritic spine loss mediated by oAbeta$. Mechanistically resembling metaplasticity,oAbeta$ prevents subsequent rounds of plasticity from inducing CaMKII T286 autophosphorylation,as well as the associated anchoring and accumulation of synaptic AMPA receptors (AMPARs). Finally,prolonged oAbeta$ treatment-induced CaMKII misactivation leads to dendritic spine loss via the destabilization of surface AMPARs. Thus,our study demonstrates that oAbeta$ engages synaptic metaplasticity via aberrant CaMKII activation. View Publication

Natural killer (NK) cell recognition of tumor cells is mediated through activating receptors such as CD226,with suppression of effector functions often controlled by negative regulatory transcription factors such as FOXO1. Here we show that CD226 regulation of NK cell cytotoxicity is facilitated through inactivation of FOXO1. Gene-expression analysis of NK cells isolated from syngeneic tumors grown in wild-type or CD226-deficient mice revealed dysregulated expression of FOXO1-regulated genes in the absence of CD226. In vitro cytotoxicity and stimulation assays demonstrated that CD226 is required for optimal killing of tumor target cells,with engagement of its ligand CD155 resulting in phosphorylation of FOXO1. CD226 deficiency or anti-CD226 antibody blockade impaired cytotoxicity with concomitant compromised inactivation of FOXO1. Furthermore,inhibitors of FOXO1 phosphorylation abrogated CD226-mediated signaling and effector responses. These results define a pathway by which CD226 exerts control of NK cell responses against tumors. View Publication

Immune activation may underlie the pathogenesis of irritable bowel syndrome (IBS),but the evidence is conflicting. We examined whether peripheral CD4+ T-cells from IBS patients demonstrated immune activation and changes in cytokine production. To gain mechanistic insight,we examined whether immune activation correlated with psychological stress and changing symptoms over time. IBS patients (n = 29) and healthy volunteers (HV; n = 29) completed symptom and psychological questionnaires. IBS patients had a significant increase in CD4+ T-cells expressing the gut homing marker integrin beta7 (p = 0.023) and lymphoid marker CD62L (p = 0.026) compared to HV. Furthermore,phytohaemagglutinin stimulated CD4+ T-cells from IBS-D patients demonstrated increased TNFalpha secretion when compared to HV (p = 0.044). Increased psychological scores in IBS did not correlate with TNFalpha production,while stress hormones inhibited cytokine secretion from CD4+ T-cells of HV in vitro. IBS symptoms,but not markers of immune activation,decreased over time. CD4+ T-cells from IBS-D patients exhibit immune activation,but this did not appear to correlate with psychological stress measurements or changing symptoms over time. This could suggest that immune activation is a surrogate of an initial trigger and/or ongoing parallel peripheral mechanisms. View Publication

BACKGROUND Thrombocytopenia leading to life-threatening excessive bleeding can be treated by platelet transfusion. Currently,such treatments are totally dependent on donor-derived platelets. To support future applications in the use of in vitro-derived platelets,we sought to identify genes whose manipulation might improve the efficiency of megakaryocyte production and resulting hemostatic effectiveness. Disruption of Lyn kinase has previously been shown to improve cell survival,megakaryocyte ploidy and TPO-mediated activation in mice,but its role in human megakaryocytes and platelets has not been examined. METHODS To analyze the role of Lyn at defined differentiation stages during human megakaryocyte differentiation,conditional Lyn-deficient cells were generated using CRISPR/Cas9 technology in iPS cells. The efficiency of Lyn-deficient megakaryocytes to differentiate and become activated in response to a range of platelet agonists was analyzed in iPSC-derived megakaryocytes. RESULTS Temporally controlled deletion of Lyn improved the in vitro differentiation of hematopoietic progenitor cells into mature megakaryocytes,as measured by the rate and extent of appearance of CD41+ CD42+ cells. Lyn-deficient megakaryocytes also demonstrated improved hemostatic effectiveness,as reported by their ability to mediate clot formation in rotational thromboelastometry. Finally,Lyn-deficient megakaryocytes produced increased numbers of platelet-like particles (PLP) in vitro. CONCLUSIONS Conditional deletion of Lyn kinase increases the hemostatic effectiveness of megakaryocytes and their progeny as well as improving their yield. Adoption of this system during generation of in vitro-derived platelets may contribute to both their efficiency of production and their ability to support hemostasis. View Publication

Immunogenicity following inactivated SARS-CoV-2 vaccination among solid organ transplant recipients has not been assessed. Seventy-five patients (37 kidney transplant [KT] recipients and 38 healthy controls) received two doses,at 4-week intervals,of an inactivated whole-virus SARS-CoV-2 vaccine. SARS-CoV-2-specific humoral (HMI) and cell-mediated immunity (CMI) were measured before,4 weeks post-first dose,and 2 weeks post-second dose. The median (IQR) age of KT recipients was 50 (42-54) years and 89% were receiving calcineurin inhibitors/mycophenolate/corticosteroid regimens. The median (IQR) time since transplant was 4.5 (2-9.5) years. Among 35 KT patients,the median (IQR) of anti-RBD IgG level measured by CLIA after vaccination was not different from baseline,but was significantly lower than in controls (2.4 [1.1-3.7] vs. 1742.0 [747.7-3783.0] AU/ml,p < .01) as well as percentages of neutralizing antibody inhibition measured by surrogate viral neutralization test (0 [0-0] vs. 71.2 [56.8-92.2]%,p < .01). However,the median (IQR) of SARS-CoV-2 mixed peptides-specific T cell responses measured by ELISpot was significantly increased compared with baseline (30 [4-120] vs. 12 [0-56] T cells/106  PBMCs,p = .02) and not different from the controls. Our findings revealed weak HMI but comparable CMI responses in fully vaccinated KT recipients receiving inactivated SARS-CoV-2 vaccination compared to immunocompetent individuals (Thai Clinical Trials Registry,TCTR20210226002). View Publication

BACKGROUND Monocytes are thought to be involved in venous thrombosis but the role of individual monocyte subpopulations on thrombus formation,clot inflammation,and degradation is an important unresolved issue. We investigate the role of inflammatory Ly6Chi monocytes in deep vein thrombosis and their potential therapeutic impact. METHODS Frequencies and compositions of blood monocytes were analyzed by flow cytometry in CCR2-/- (C-C chemokine receptor type 2) and wild-type mice of different ages and after treatment with the NR4A1 (nuclear receptor group 4 family A member 1,Nur77) agonist CnsB (cytosporone B). TF (tissue factor) sufficient and deficient Ly6Chi monocytes were adoptively transferred into aged CCR2-/- mice. Thrombus formation and size were followed by ultrasound over a 3-week period after surgical reduction of blood flow (stenosis) in the inferior vena cava. RESULTS Reduced numbers of peripheral monocytes in aged (>30 w) CCR2-/- mice are accompanied by reduced thrombus formation after inferior vena cava ligation. Reducing the number of inflammatory Ly6Chi monocytes in wild-type mice by CsnB treatment before ligation,similarly suspends clotting,while later treatment (d1 or d4) reduces thrombus growth and accelerates resolution. We describe how changes in inflammatory monocyte numbers affect the gradual differentiation of monocytes in thrombi and show that only tissue factor-competent Ly6Chi monocytes restore thrombosis in aged CCR2-/- mice. CONCLUSIONS We conclude that the number of inflammatory Ly6Chi monocytes controls deep vein thrombosis formation,growth,and resolution and can be therapeutically manipulated with a NR4A1 agonist at all disease stages. View Publication

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS-CoV-2 infection is critical for developing treatments for severe COVID-19. Here,we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID-19 patients early after symptom onset,correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFN? and of systemic inflammatory cytokines CXCL10 and IL-6. Using an in vitro stem cell-based human pDC model,we further demonstrate that pDCs,while not supporting SARS-CoV-2 replication,directly sense the virus and in response produce multiple antiviral (interferons: IFN? and IFN?1) and inflammatory (IL-6,IL-8,CXCL10) cytokines that protect epithelial cells from de novo SARS-CoV-2 infection. Via targeted deletion of virus-recognition innate immune pathways,we identify TLR7-MyD88 signaling as crucial for production of antiviral interferons (IFNs),whereas Toll-like receptor (TLR)2 is responsible for the inflammatory IL-6 response. We further show that SARS-CoV-2 engages the receptor neuropilin-1 on pDCs to selectively mitigate the antiviral interferon response,but not the IL-6 response,suggesting neuropilin-1 as potential therapeutic target for stimulation of TLR7-mediated antiviral protection. View Publication

BACKGROUND Bispecific T-cell engager (BiTE) molecules induce redirected lysis of cancer cells by T cells and are an emerging modality for solid tumor immunotherapy. While signs of clinical activity have been demonstrated,efficacy of T-cell engagers (TCEs) in solid tumors settings,molecular determinants of response,and underlying mechanisms of resistance to BiTE therapy require more investigation. METHODS To uncover cancer cell-intrinsic genetic modifiers of TCE-mediated cytotoxicity,we performed genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loss-of-function and CRISPRa (CRISPR activation) gain-of-function screens using TCEs against two distinct tumor-associated antigens (TAAs). By using in vitro T-cell cytotoxicity assays and in vivo efficacy studies,we validated the roles of two common pathways identified in our screen,T-cell costimulation pathway and apoptosis pathway,as key modifiers of BiTE activity. RESULTS Our genetic screens uncovered TAAs-independent cancer cell-intrinsic genes with functions in autophagy,T-cell costimulation,the apoptosis pathway,chromatin remodeling,and cytokine signaling that altered responsiveness to BiTE-mediated killing. Notably,loss of CD58 (the ligand of the CD2 T-cell costimulatory receptor),a gene frequently altered in cancer,led to decreased TCE-mediated cytotoxicity,T-cell activation and antitumor efficacy in vitro and in vivo. Moreover,the effects of CD58 loss were synergistically compounded by concurrent loss of CD80/CD86 (ligands for the CD28 T-cell costimulatory receptor),whereas joint CD2 and CD28 costimulation additively enhanced TCE-mediated killing,indicating non-redundant costimulatory mechanisms between the two pathways. Additionally,loss of CFLAR (Caspase-8 and FADD Like Apoptosis Regulator),BCL2L1,and BID (BH3 Interacting Domain Death Agonist) induced profound changes in sensitivity to TCEs,indicating that key regulators of apoptosis,which are frequently altered in cancer,impact tumor responsiveness to BiTE therapy. CONCLUSIONS This study demonstrates that genetic alterations central to carcinogenesis and commonly detected in cancer samples lead to significant modulation of BiTE antitumor activity in vitro and in vivo,findings with relevance for a better understanding of patient responses to BiTE therapy and novel combinations that enhance TCE efficacy. View Publication

Allogeneic T cells are key immune therapeutic cells to fight cancer and other clinical indications. High T cell dose per patient and increasing patient numbers result in clinical demand for a large number of allogeneic T cells. This necessitates a manufacturing platform that can be scaled up while retaining cell quality. Here we present a closed and scalable platform for T cell manufacturing to meet clinical demand. Upstream manufacturing steps of T cell activation and expansion are done in-vessel,in a stirred-tank bioreactor. T cell selection,which is necessary for CAR-T-based therapy,is done in the bioreactor itself,thus maintaining optimal culture conditions through the selection step. Platform's attributes of automation and performing the steps of T cell activation,expansion,and selection in-vessel,greatly contribute to enhancing process control,cell quality,and to the reduction of manual labor and contamination risk. In addition,the viability of integrating a closed,automated,downstream process of cell concentration,is demonstrated. The presented T cell manufacturing platform has scale-up capabilities while preserving key factors of cell quality and process control. View Publication

BACKGROUND The use of intralesional Mycobacterium bovis BCG (intralesional live BCG) for the treatment of metastatic melanoma resulted in regression of directly injected,and occasionally of distal lesions. However,intralesional-BCG is less effective in patients with visceral metastases and did not significantly improve overall survival. METHODS We generated a novel BCG lysate and developed it into a thermosensitive PLGA-PEG-PLGA hydrogel (BCG hydrogel),which was injected adjacent to the tumor to assess its antitumor effect in syngeneic tumor models (B16F10,MC38). The effect of BCG hydrogel treatment on contralateral tumors,lung metastases,and survival was assessed to evaluate systemic long-term efficacy. Gene expression profiles of tumor-infiltrating immune cells and of tumor-draining lymph nodes from BCG hydrogel-treated mice were analyzed by single-cell RNA sequencing (scRNA-seq) and CD8+ T cell receptor (TCR) repertoire diversity was assessed by TCR-sequencing. To confirm the mechanistic findings,RNA-seq data of biopsies obtained from in-transit cutaneous metastases of patients with melanoma who had received intralesional-BCG therapy were analyzed. RESULTS Here,we show that BCG lysate exhibits enhanced antitumor efficacy compared to live mycobacteria and promotes a proinflammatory tumor microenvironment and M1 macrophage (M$\Phi$) polarization in vivo. The underlying mechanisms of BCG lysate-mediated tumor immunity are dependent on M$\Phi$ and dendritic cells (DCs). BCG hydrogel treatment induced systemic immunity in melanoma-bearing mice with suppression of lung metastases and improved survival. Furthermore,BCG hydrogel promoted cathepsin S (CTSS) activity in M$\Phi$ and DCs,resulting in enhanced antigen processing and presentation of tumor-associated antigens. Finally,BCG hydrogel treatment was associated with increased frequencies of melanoma-reactive CD8+ T cells. In human patients with melanoma,intralesional-BCG treatment was associated with enhanced M1 M$\Phi$,mature DC,antigen processing and presentation,as well as with increased CTSS expression which positively correlated with patient survival. CONCLUSIONS These findings provide mechanistic insights as well as rationale for the clinical translation of BCG hydrogel as cancer immunotherapy to overcome the current limitations of immunotherapies for the treatment of patients with melanoma. View Publication

Exosomes derived from mesenchymal stem cells (MSCs) could protect against myocardial infarction (MI). TLR4 is reported to play an important role in MI,while microRNA-182-5p (miR-182-5p) negatively regulates TLR4 expression. Therefore,we hypothesize that MSCs-derived exosomes overexpressing miR-182-5p may have beneficial effects on MI. We generated bone marrow mesenchymal stem cells (BM-MSCs) and overexpressed miR-182-5p in these cells for exosome isolation. H2O2-stimulated neonatal mouse ventricle myocytes (NMVMs) and MI mouse model were employed,which were subjected to exosome treatment. The expression of inflammatory factors,heart function,and TLR4 signaling pathway activation were monitored. It was found that miR-182-5p decreased TLR4 expression in BM-MSCs and NMVMs. Administration of exosomes overexpressing miR-182-5p to H2O2-stimulated NMVMs enhanced cell viability and suppressed the expression of inflammatory cytokines. In addition,they promoted heart function,suppressed inflammatory responses,and de-activated TLR4/NF-$\kappa$B signaling pathway in MI mice. In conclusion,miR-182-5p transferred by the exosomes derived from BM-MSCs protected against MI-induced impairments by targeting TLR4. View Publication