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

Interactions with Ag-specific T cells drive B cell activation and fate choices that ultimately determine the quality of high-affinity Ab responses. As such,these interactions,and especially the long-lived interactions that occur before germinal center formation,may be important checkpoints to regulate undesirable responses. Using mouse model Ag systems,we directly observed interactions between T and B cells responding to the self-antigen myelin oligodendrocyte glycoprotein (MOG) and found that they are of lower quality compared with interactions between cells responding to the model foreign Ag nitrophenyl-haptenated OVA. This was associated with reduced expression of molecules that facilitate these interactions on the B cells,but not on T cells. B cell expression of these molecules was not dictated by the T cell partner,nor could the relative lack of expression on MOG-specific (MOG-sp.) B cells be reversed by a multivalent Ag. Instead,MOG-sp. B cells were inherently less responsive to BCR stimulation than MOG-non-sp. cells. However,the phenotype of MOG-sp. B cells was not consistent with previous descriptions of autoimmune B cells that had been tolerized via regular exposure to systemically expressed self-antigen. This suggests that alternate anergy pathways may exist to limit B cell responses to tissue-restricted self-antigens. 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

Simple SummaryAtypical teratoid rhabdoid tumors (ATRTs) and diffuse intrinsic pontine gliomas (DIPGs) are lethal pediatric brain tumors that can resist chemotherapy and be influenced by their microenvironment. Astrocytes are the key components of the brain tumor microenvironment and can support tumor growth. We investigated the effects of astrocytes on cisplatin sensitivity in pediatric brain cancer cells. The crosstalk between astrocytes and cancer cells activated astrocytes and promoted cancer cell proliferation. Moreover,the tumor cells expressed elevated levels of drug resistance genes in the presence of astrocytes. In conclusion,astrocytes can significantly improve the growth of these tumor cells and modulate their chemosensitivity,highlighting their role in therapeutic resistance. AbstractBackground: ATRTs and DIPGs are deadly pediatric brain tumors with poor prognosis. These tumors can develop resistance to chemotherapies,which may be significantly influenced by their microenvironment. Since astrocytes are the most abundant glial cell type in the brain microenvironment and may support tumor growth and chemoresistance,this study investigated the effects of induced pluripotent stem cell-derived astrocytes (iPSC-astrocytes) on cisplatin sensitivity in CHLA-05-ATRT and SF8628 (DIPG) cells. iPSCs provide an unlimited and standardized source of nascent astrocytes,which enables modeling the interaction between childhood brain tumor cells and iPSC-astrocytes within a controlled coculture system. Methods: To study the effects on tumor growth,the iPSC-astrocytes were cocultured with tumor cells. Additionally,the tumor cells were exposed to various concentrations of cisplatin to evaluate their chemosensitivity in the presence of astrocytes. Results: The paracrine interaction of iPSC-astrocytes with tumor cells upregulated astrocyte activation markers GFAP and STAT3 and promoted tumor cell proliferation. Moreover,the cisplatin treatment significantly decreased the viability of CHLA-05-ATRT and SF8628 cells. However,tumor cells exhibited reduced sensitivity to cisplatin in the coculture with iPSC-astrocytes. During cisplatin treatment,DIPG cells in particular showed upregulation of resistance markers,ERK1,STAT3,and MTDH,which are associated with enhanced proliferation and invasion. They also had increased expression of APEX1,which is involved in the base excision repair pathway following cisplatin-induced DNA damage. Conclusion: These findings underscore the significance of the tumor microenvironment in modulating tumor cell survival and chemosensitivity. 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

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

CD8+ T cells are central to targeting and eliminating cancer cells. Their function is critically supported by type 1 conventional dendritic cells (cDC1s),which both prime antigen‐specific CD8+ T cells in tumour‐draining lymph nodes (tdLNs) and sustain primed CD8+ T cells within tumours. Despite their importance,the spatiotemporal organisation of cDC1s within tumours and their diverse functional roles remain poorly understood. Here,we use scRNAseq and unbiased spatial analysis to construct a detailed map of cDC1 states and distribution within immunogenic mouse tumours during CD8+ T‐cell‐mediated rejection. We reveal two distinct cDC1 activation states characterised by differential expression of genes linked to anti‐tumour immunity,including Cxcl9 and Il12b. Strikingly,Il12b‐expressing cDC1s are CCR7+ and enriched at tumour borders,where they closely associate with stem‐like TCF1+ CD8+ T cells. In contrast,CCR7–Cxcl9‐expressing cDC1s are preferentially found within the tumour parenchyma alongside effector CD8+ T cells. Analysis of a published dataset of human tumours similarly reveals a spatial association between CCR7+ cDC1 and stem‐like TCF1+ CD8+ T cells. These findings uncover a highly spatially coordinated interaction between cDC1s and CD8+ T cells within tumours,shedding light on the intricate cellular dynamics that underpin effective anti‐tumour immunity. Using scRNAseq and spatial analysis,we analyse cDC1 states and spatial distribution in tumours during immune‐mediated rejection. We identify two cDC1 activation states,each occupying different regions and associated with distinct CD8+ T cell populations. This reveals the spatial organisation of cDC1 states that may be key to anti‐tumour immunity. 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

Induced pluripotent stem cell (iPSC)-derived mesenchymal stromal cells (iMSCs) offer a promising alternative to primary mesenchymal stromal cells (MSCs) and their derivatives,particularly extracellular vesicles (EVs),for use in advanced therapy medicinal products. In this study we evaluated the immunomodulatory and regenerative potential of iMSCs as well as iMSC-EVs,alongside primary human umbilical cord-derived mesenchymal stromal cells (hUCMSCs). Our findings demonstrate that iMSCs exhibit comparable abilities to hUCMSCs in regulating lymphocyte proliferation and inducing an anti-inflammatory phenotype in monocytes. We also observed decreased TNFα levels and increased IL-10 induction,indicating a potential mechanism for their immunomodulatory effects. Furthermore,iMSC-EVs also showed effective immunomodulation by inhibiting T cell proliferation and inducing macrophage polarization similar to their parental cells. Additionally,iMSC-EVs exhibited pro-regenerative potential akin to hUCMSC-EVs in in vitro scratch assays. Notably,priming iMSCs with pro-inflammatory cytokines significantly enhanced the immunomodulatory potential of iMSC-EVs. These results underscore the considerable promise of iMSCs and iMSCs-EVs as an alternate source for MSC-derived therapeutics,given their potent immunomodulatory and regenerative properties. The online version contains supplementary material available at 10.1038/s41598-024-75956-3. View Publication

Natural killer (NK) cells are an important innate defense against malignancies,and exogenous sources of NK cells have been developed as anti-cancer agents. Nevertheless,the apparent limitations of NK cells in clearing cancers have suggested that their efficacy might be augmented by combination with other treatments. We have developed cell-penetrating peptides that target the transcription factors ATF5,CEBPB,and CEBPD and that promote apoptotic cancer cell death both in vitro and in vivo without apparent toxicity to non-transformed cells. We report here that one such peptide,Dpep,significantly sensitizes a variety of tumor cell types to the cytotoxic activity of the NK cell line,NK-92MI. Such sensitization requires pre-exposure of tumor cells to Dpep and does not appear due to effects of Dpep on NK cells themselves. Our findings suggest that Dpep acts in this context to lower the apoptotic threshold of tumor cells to NK cell toxicity. Additionally,while Dpep pre-treatment does not prevent tumor cells from causing NK cell “inactivation”,it sensitizes cancer cells to repeated rounds of exposure to fresh NK cells. These findings thus indicate that Dpep pre-treatment is an effective strategy to sensitize cancer cells to the cytotoxic actions of NK cells. View Publication

BACKGROUND Asthma is a heterogenous disease characterized by chronic inflammation and airway remodeling. An increase in the severity of airway remodeling is associated with a more severe form of asthma. There is increasing interest in the epithelial to mesenchymal transition process and mechanisms involved in the differentiation and repair of the airway epithelium,especially as they apply to severe asthma. Growing evidence suggests that Epithelial-Mesenchymal transition (EMT) could contribute to airway remodeling and fibrosis in asthma. Severe asthmatic patients with remodeled airways have a neutrophil driven inflammation. Neutrophils are an important source of TGF-$\beta$1,which plays a role in recruitment and activation of inflammatory cells,extracellular matrix (ECM) production and fibrosis development,and is a potent inducer of EMT. OBJECTIVE As there is little data examining the contribution of neutrophils and/or their mediators to the induction of EMT in airway epithelial cells,the objective of this study was to better understand the potential role of neutrophils in severe asthma in regards to EMT. METHODS We used an in vitro system to investigate the neutrophil-epithelial cell interaction. We obtained peripheral blood neutrophils from severe asthmatic patients and control subjects and examined for their ability to induce EMT in primary airway epithelial cells. RESULTS Our data indicate that neutrophils from severe asthmatic patients induce changes in morphology and EMT marker expression in bronchial epithelial cells consistent with the EMT process when co-cultured. TGF-$\beta$1 levels in the culture medium of severe asthmatic patients were increased compared to that from co-cultures of non-asthmatic neutrophils and epithelial cells. CONCLUSIONS AND CLINICAL RELEVANCE As an inducer of EMT and an important source of TGF-$\beta$1,neutrophils may play a significant role in the development of airway remodeling and fibrosis in severe asthmatic airways. View Publication

The persistence of latent HIV proviruses in long-lived CD4(+) T cells despite antiretroviral therapy (ART) is a major obstacle to viral eradication. Because current candidate latency-reversing agents (LRAs) induce HIV transcription,but fail to clear these cellular reservoirs,new approaches for killing these reactivated latent HIV reservoir cells are urgently needed. HIV latency depends upon the transcriptional quiescence of the integrated provirus and the circumvention of immune defense mechanisms. These defenses include cell-intrinsic innate responses that use pattern-recognition receptors (PRRs) to detect viral pathogens,and that subsequently induce apoptosis of the infected cell. Retinoic acid (RA)-inducible gene I (RIG-I,encoded by DDX58) forms one class of PRRs that mediates apoptosis and the elimination of infected cells after recognition of viral RNA. Here we show that acitretin,an RA derivative approved by the US Food and Drug Administration (FDA),enhances RIG-I signaling ex vivo,increases HIV transcription,and induces preferential apoptosis of HIV-infected cells. These effects are abrogated by DDX58 knockdown. Acitretin also decreases proviral DNA levels in CD4(+) T cells from HIV-positive subjects on suppressive ART,an effect that is amplified when combined with suberoylanilide hydroxamic acid (SAHA),a histone deacetylase inhibitor. Pharmacological enhancement of an innate cellular-defense network could provide a means by which to eliminate reactivated cells in the latent HIV reservoir. View Publication

Monitoring pluripotent stem cell behaviors (self-renewal and differentiation to specific lineages/phenotypes) is critical for a fundamental understanding of stem cell biology and their translational applications. In this study,a multi-modal stem cell monitoring system was developed to quantitatively characterize physico-electrochemical changes of the cells in real time,in relation to cellular activities during self-renewal or lineage-specific differentiation,in a non-destructive,label-free manner. The system was validated by measuring physical (mass) and electrochemical (impedance) changes in human induced pluripotent stem cells undergoing self-renewal,or subjected to mesendodermal or ectodermal differentiation,and correlating them to morphological (size,shape) and biochemical changes (gene/protein expression). An equivalent circuit model was used to further dissect the electrochemical (resistive and capacitive) contributions of distinctive cellular features. Overall,the combination of the physico-electrochemical measurements and electrical circuit modeling collectively offers a means to longitudinally quantify the states of stem cell self-renewal and differentiation. View Publication