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

ObjectiveThis study investigated the impact of innate lymphoid cell type 2 (ILC2s) on the function of regulatory T cells (Treg) and CD8+ T cells in chronic lymphocytic leukemia (CLL) through IL-9.MethodsPeripheral blood samples were collected from CLL patients (n = 52) and healthy controls (n = 30). ILC2 proportions and IL-9 levels were assessed using flow cytometry and ELISA. Immunofluorescence staining was performed to stain GATA3,CRTH2,and IL-9 in cervical lymph nodes from CLL patients (n = 10) and control subjects with reactive lymphadenitis (n = 10). Correlation analysis between ILC2s and IL-9 was conducted using the Spearman test. ILC2s were sorted and cultured from CLL patients,followed by co-culture experiments with PBMCs of healthy controls and MEC-1 cells,with or without anti-IL-9 antibody intervention. Flow cytometry was used to measure the proportions of ILC2s,Treg cells,PD-1+/TIGIT+/CTLA-4+ Treg subsets,and granzyme B+/perforin+ CD8+ T cells,along with MEC-1 cell apoptosis.ResultsThe proportions of ILC2s and Treg,along with serum IL-9 levels,were significantly elevated in CLL patients (P < 0.05). Peripheral blood ILC2s were positively correlated with IL-9 (r = 0.609,P < 0.001). The average fluorescence intensity of GATA3,CRTH2,and IL-9 in the cervical lymph nodes of CLL patients increased significantly (P < 0.001),and IL-9 showed colocalization with GATA3 and CRTH2. In vitro,IL-9 levels in the supernatant of sorted ILC2s from CLL patients increased. Treatment with anti-IL-9 antibody significantly reduced the PD-1+ Treg and TIGIT+ Treg cells while increasing granzyme B+ CD8+ T cells (P < 0.05). However,there was no significant effect on Treg,CTLA-4+ Treg,and perforin+ CD8+ T cells (P > 0.05). Additionally,anti-IL-9 antibody significantly increased early apoptosis (P < 0.05).ConclusionILC2s affect CD8+ T cells and Treg cells through IL-9,weakening the anti-tumor effects of CD8+ T cells and enhancing the immunosuppressive effects of Treg cells,thereby contributing to CLL pathogenesis.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-025-04082-4. View Publication

Stem cells from bone marrow,skeletal muscle and possibly other tissues can be identified by the 'side-population' (SP) phenotype. Although it has been assumed that expression of ABC transporters is responsible for this phenotype,the specific molecules involved have not been defined. Here we show that expression of the Bcrp1 (also known as Abcg2 murine/ABCG2 human) gene is a conserved feature of stem cells from a wide variety of sources. Bcrp1 mRNA was expressed at high levels in primitive murine hematopoietic stem cells,and was sharply downregulated with differentiation. Enforced expression of the ABCG2 cDNA directly conferred the SP phenotype to bone-marrow cells and caused a reduction in maturing progeny both in vitro and in transplantation-based assays. These results show that expression of the Bcrp1/ABCG2 gene is an important determinant of the SP phenotype,and that it might serve as a marker for stem cells from various sources. View Publication

Systemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MCs) and their progenitors. In most cases,neoplastic cells display the D816V-mutated variant of KIT. KIT D816V exhibits constitutive tyrosine kinase (TK) activity and has been implicated in increased survival and growth of neoplastic MCs. Recent data suggest that the proapoptotic BH3-only death regulator Bim plays a role as a tumor suppressor in various myeloid neoplasms. We found that KIT D816V suppresses expression of Bim in Ba/F3 cells. The KIT D816-induced down-regulation of Bim was rescued by the KIT-targeting drug PKC412/midostaurin. Both PKC412 and the proteasome-inhibitor bortezomib were found to decrease growth and promote expression of Bim in MC leukemia cell lines HMC-1.1 (D816V negative) and HMC-1.2 (D816V positive). Both drugs were also found to counteract growth of primary neoplastic MCs. Furthermore,midostaurin was found to cooperate with bortezomib and with the BH3-mimetic obatoclax in producing growth inhibition in both HMC-1 subclones. Finally,a Bim-specific siRNA was found to rescue HMC-1 cells from PKC412-induced cell death. Our data show that KIT D816V suppresses expression of proapoptotic Bim in neoplastic MCs. Targeting of Bcl-2 family members by drugs promoting Bim (re)-expression,or by BH3-mimetics such as obatoclax,may be an attractive therapy concept in SM. View Publication

Recent evidence suggests that Notch signaling may play a role in regulation of cancer stem cell (CSC) self-renewal and differentiation hence presenting a promising target for development of novel therapies for aggressive cancers such as triple negative breast cancer (TNBC). We generated Notch1 monoclonal antibodies (mAbs) that specifically bind to the negative regulatory region of human Notch1. Notch1 inhibition in TNBC Sum149 and patient derived xenograft (PDX) 144580 models led to significant TGI particularly in combination with docetaxel. More interestingly,Notch1 mAbs caused a reduction in mammosphere formation and CD44+/CD24-/lo cell population. It also resulted in decreased tumor incidence upon re-implantation and delay in tumor recurrence. Our data demonstrated a potent antitumor efficacy of Notch1 mAbs,with a remarkable activity against CSCs. These findings suggest that anti-Notch1 mAbs may provide novel therapies to improve the efficacy of conventional therapies by directly targeting the CSC niche. They may also delay tumor recurrence and hence have a major impact on cancer patient survival. View Publication

Reduction of adult hippocampal neurogenesis is an early critical event in Alzheimer’s disease (AD),contributing to progressive memory loss and cognitive decline. Reduced levels of the nucleoporin 153 (Nup153),a key epigenetic regulator of NSC stemness,characterize the neural stem cells isolated from a mouse model of AD (3×Tg) (AD-NSCs) and determine their altered plasticity and gene expression. Nup153-regulated mechanisms contributing to NSC function were investigated: (1) in cultured NSCs isolated from AD and wild type (WT) mice by proteomics; (2) in vivo by lentiviral-mediated delivery of Nup153 or GFP in the hippocampus of AD and control mice analyzing neurogenesis and cognitive function; (3) in human iPSC-derived brain organoids obtained from AD patients and control subjects as a model of neurodevelopment. Proteomic approach identified Nup153 interactors in WT- and AD-NSCs potentially implicated in neurogenesis regulation. Gene ontology (GO) analysis showed that Nup153-bound proteins in WT-NSCs were involved in RNA metabolism,nuclear import and epigenetic mechanisms. Nup153-bound proteins in AD-NSCs were involved in pathways of neurodegeneration,mitochondrial dysfunction,proteasomal processing and RNA degradation. Furthermore,recovery of Nup153 levels in AD-NSCs reduced the levels of oxidative stress markers and recovered proteasomal activity. Lentiviral-mediated delivery of Nup153 in the hippocampal niche of AD mice increased the proliferation of early progenitors,marked by BrdU/DCX and BrdU/PSANCAM positivity and,later,the integration of differentiating neurons in the cell granule layer (BrdU/NeuN + cells) compared with GFP-injected AD mice. Consistently,Nup153-injected AD mice showed an improvement of cognitive performance in comparison to AD-GFP mice at 1 month after virus delivery assessed by Morris Water Maze. To validate the role of Nup153 in neurogenesis we took advantage of brain organoids derived from AD-iPSCs characterized by fewer neuroepithelial progenitor loops and reduced differentiation areas. The upregulation of Nup153 in AD organoids recovered the formation of neural-like tubes and differentiation. Our data suggest that the positive effect of Nup153 on neurogenesis is based on a complex regulatory network orchestrated by Nup153 and that this protein is a valuable disease target. The online version contains supplementary material available at 10.1186/s13287-024-03805-1. View Publication

Primary antiphospholipid syndrome (PAPS) is a life-threatening clotting disorder mediated by pathogenic autoantibodies. Here we dissect the origin of self-reactive B cells in human PAPS using peripheral blood and bone marrow of patients with triple-positive PAPS via combined single-cell RNA sequencing,B cell receptors (BCR) repertoire profiling,CITEseq analysis and single cell immortalization. We find that antiphospholipid (aPL)-specific B cells are present in the naive compartment,polyreactive,and derived from the natural repertoire. Furthermore,B cells with aPL specificities are not eliminated in patients with PAPS,persist until the memory and long-lived plasma cell stages,likely after defective germinal center selection,while becoming less polyreactive. Lastly,compared with the non-PAPS cells,PAPS B cells exhibit distinct IFN and APRIL signature as well as dysregulated mTORC1 and MYC pathways. Our findings may thus elucidate the survival mechanisms of these autoreactive B cells and suggest potential therapeutic targets for the treatment of PAPS. Primary antiphospholipid syndrome (PAPS) is a clotting disorder attributed to autoreactive antibodies produced by B cells. Here the authors show,using single cell omics and B cell repertoire data,that autoreactive B cells originate from the natural B cell repertoire and escape germinal center selection to persist in PAPS patient via potential dysregulation of mTORC1 and MYC pathways. View Publication

Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however,the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects,with a median of 14 individual epitopic regions targeted per person (range,2 to 42),and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median,4,245) among all study participants. However,the number of epitopic regions targeted,the protein subunits recognized,and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals,with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid,sensitive,specific,and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response,even if a comprehensive pan-genome screening approach is applied. View Publication

The role of epigenetic regulators in the control of adult neurogenesis is largely undefined. We show that the histone demethylase enzyme Kdm5b (Jarid1b) negatively regulates neurogenesis from adult subventricular zone (SVZ) neural stem cells (NSCs) in culture. shRNA-mediated depletion of Kdm5b in proliferating adult NSCs decreased proliferation rates and reduced neurosphere formation in culture. When transferred to differentiation culture conditions,Kdm5b-depleted adult NSCs migrated from neurospheres with increased velocity. Whole-genome expression screening revealed widespread transcriptional changes with Kdm5b depletion,notably the up-regulation of reelin (Reln),the inhibition of steroid biosynthetic pathway component genes and the activation of genes with intracellular transport functions in cultured adult NSCs. Kdm5b depletion increased extracellular reelin concentration in the culture medium and increased phosphorylation of the downstream reelin signaling target Disabled-1 (Dab1). Sequestration of extracellular reelin with CR-50 reelin-blocking antibodies suppressed the increase in migratory velocity of Kdm5b-depleted adult NSCs. Chromatin immunoprecipitation revealed that Kdm5b is present at the proximal promoter of Reln,and H3K4me3 methylation was increased at this locus with Kdm5b depletion in differentiating adult NSCs. Combined the data suggest Kdm5b negatively regulates neurogenesis and represses Reln in neural stem cells from the adult SVZ. View Publication

BACKGROUND: Age and cardiovascular disease status appear to alter numbers and function of circulating endothelial progenitor cells (EPCs). Despite no universal phenotypic definition,numerous studies have implicated progenitors with apparent endothelial potential in local responses to vascular injury and with cardiovascular disease in general. To further define the role of this lineage in peripheral artery disease (PAD),we developed a multiparameter flow cytometry assay to analyze multiple phenotypic definitions of progenitor cells (PCs),EPCs,and mature endothelial cells (ECs) and evaluate effects of age and PAD on baseline levels of each subset. METHODS: Blood was collected from young healthy subjects (N = 9,mean age 33 +/- 8 years),older healthy subjects (N = 13,mean age 66 +/- 8 years),and older subjects with PAD (N = 15,mean age 69 +/- 8 years). After ammonium chloride lysis,cells were stained and analyzed on a Becton-Dickinson LSR II with a 5-color antibody panel: FITC-anti-CD31,PE-anti-CD146,PE-anti-CD133,PerCP-Cy5.5-anti-CD3,-CD19,-CD33 (lineage panel),PE-Cy7-anti-CD34,and APC-anti-VEGF-R2. Viability was assessed by propidium iodide exclusion,and only viable,low to medium side scatter lineage-negative singlets were analyzed. In some studies,cells were sorted for morphological studies. Subsets were defined as indicated later. RESULTS: Our results,using a comprehensive flow cytometric panel,indicate that CD133+,CD34+,and CD133+/CD34+ PCs are elevated in younger healthy individuals compared to older individuals,both healthy and with PAD. However,the number of EPCs and mature ECs did not significantly differ among the three groups. Assessment of endothelial colony forming units and dual acLDL-lectin staining supported the flow cytometric findings. CONCLUSIONS: We describe a comprehensive flow cytometric method to detect circulating mature and progenitor endothelial populations confirmed by conventional morphological and functional assays. Our findings suggest that aging may influence circulating levels of PCs,but not EPCs or ECs; PAD had no effect on baseline levels of any populations investigated. This study provides the basis for evaluating the potential effects of acute stress and therapeutic intervention on circulating progenitor and endothelial populations as a biomarker for cardiovascular status. View Publication

BACKGROUND: Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However,relatively little is known about the mechanisms responsible for their immunomodulatory effects,which could be influenced by both the cell source and culture conditions. DESIGN AND METHODS: We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells,in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. RESULTS: The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology,immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency,umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition,these cells do not express hTERT and telomerase activity,do express p16(ink4a) protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses,umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation,(ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10,while decreasing interferon-gamma secretion,in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects,a prostaglandin E(2)-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G. CONCLUSIONS: Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency,proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications. View Publication

The active form of vitamin D,1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)),has potent immunomodulatory properties that have promoted its potential use in the prevention and treatment of infectious disease and autoimmune conditions. A variety of immune cells,including macrophages,dendritic cells,and activated T cells express the intracellular vitamin D receptor and are responsive to 1,25(OH)(2)D(3.) Despite this,how 1,25(OH)(2)D(3) regulates adaptive immunity remains unclear and may involve both direct and indirect effects on the proliferation and function of T cells. To further clarify this issue,we have assessed the effects of 1,25(OH)(2)D(3) on human CD4(+)CD25(-) T cells. We observed that stimulation of CD4(+)CD25(-) T cells in the presence of 1,25(OH)(2)D(3) inhibited production of proinflammatory cytokines including IFN- gamma,IL-17,and IL-21 but did not substantially affect T cell division. In contrast to its inhibitory effects on inflammatory cytokines,1,25(OH)(2)D(3) stimulated expression of high levels of CTLA-4 as well as FoxP3,the latter requiring the presence of IL-2. T cells treated with 1,25(OH)(2)D(3) could suppress proliferation of normally responsive T cells,indicating that they possessed characteristics of adaptive regulatory T cells. Our results suggest that 1,25(OH)(2)D(3) and IL-2 have direct synergistic effects on activated T cells,acting as potent anti-inflammatory agents and physiologic inducers of adaptive regulatory T cells. View Publication

There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB),used in the Aldeflour assay,has been considered a specific inhibitor for ALDH1A1 isoform. In this study,we explore the effects of human ALDH isoenzymes,ALDH1A2 and ALDH2,on drug resistance and proliferation,and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay,Western blot,RT-PCR,and Aldefluor assay. Both cell lines,with either ALDH1A2 or ALDH2,exhibited higher cell proliferation rates,higher clonal efficiency,and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors,DEAB and disulfiram,we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65-90%. Furthermore,our TLDA results revealed that ALDH1,ALDH7,ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition,other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells. View Publication