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

We describe the generation of a fusion cytokine consisting of GM-CSF in tandem with N-terminal-truncated MCP-1 (6-76),hereafter GMME1. Treatment of activated T cells with recombinant GMME1 protein leads to proinflammatory cytokine reduction and apoptosis via a CCR2-restricted pathway. Similarly,cell death is triggered in macrophages cultured with GMME1,while an inhibition of Ab production from plasma cells is observed. Treatment of CD4 T cells derived from experimental autoimmune encephalomyelitis mice with GMME1 leads to p38 hyperphosphorylation,inhibition of p44/42,AKT and STAT3 phosphorylation,and caspase-3 activation. GMME1 administration to experimental autoimmune encephalomyelitis mice suppresses symptomatic disease and correlates with decreased levels of inflammatory cytokines including IL-17,MOG-specific Ab titers,and blockade of CD4 and CD8 T cell infiltration in spinal cords. We propose that GMME1 defines a new class of agents for the treatment of autoimmune ailments by selectively targeting lymphomyeloid cells expressing CCR2. View Publication

The concept of cancer stem cells (CSC) embodies two aspects: the stem cell as the initial target of the oncogenic process and the existence of two populations of cells in cancers: the CSC and derived cells. The second is discussed in this review. CSC are defined as cells having three properties: a selectively endowed tumorigenic capacity,an ability to recreate the full repertoire of cancer cells of the parent tumor and the expression of a distinctive repertoire of surface biomarkers. In operational terms,the CSC are among all cancer cells those able to initiate a xenotransplant. Other explicit or implicit assumptions exist,including the concept of CSC as a single unique infrequent population of cells. To avoid such assumptions,we propose to use the operational term tumor-propagating cells (TPC); indeed,the cells that initiate transplants did not initiate the cancer. The experimental evidence supporting the explicit definition is analyzed. Cancers indeed contain a fraction of cells mainly responsible for the tumor development. However,there is evidence that these cells do not represent one homogenous population. Moreover,there is no evidence that the derived cells result from an asymmetric,qualitative and irreversible process. A more general model is proposed of which the CSC model could be one extreme case. We propose that the TPC are multiple evolutionary selected cancer cells with the most competitive properties [maintained by (epi-)genetic mechanisms],at least partially reversible,quantitative rather than qualitative and resulting from a stochastic rather than deterministic process. View Publication

It is unknown how dendritic cells (DCs) become specialized as mucosal DCs and maintain intestinal homeostasis. We report that a subset of bone marrow cells freshly isolated from C57BL/6 mice express the retinoic acid (RA)-synthesizing enzyme aldehyde dehydrogenase family 1,subfamily A2 (ALDH1a2) and are capable of providing RA to DC precursors in the bone marrow microenvironment. RA induced bone marrow-derived DCs to express CCR9 and ALDH1a2 and conferred upon them mucosal DC functions,including induction of Foxp3(+) regulatory T cells,IgA-secreting B cells,and gut-homing molecules. This response of DCs to RA was dependent on a narrow time window and stringent dose effect. RA promoted bone marrow-derived DC production of bioactive TGF-β by inhibiting suppressor of cytokine signaling 3 expression and thereby enhancing STAT3 activation. These RA effects were evident in vivo,in that mucosal DCs from vitamin A-deficient mice had reduced mucosal DC function,namely failure to induce Foxp3(+) regulatory T cells. Furthermore,MyD88 signaling enhanced RA-educated DC ALDH1a2 expression and was required for optimal TGF-β production. These data indicate that RA plays a critical role in the generation of mucosal DCs from bone marrow and in their functional activity. View Publication

Genetic Parkinson disease (PD) has been associated with mutations in PINK1,a gene encoding a mitochondrial kinase implicated in the regulation of mitochondrial degradation. While the studies so far examined PINK1 function in non-neuronal systems or through PINK1 knockdown approaches,there is an imperative to examine the role of endogenous PINK1 in appropriate human-derived and biologically relevant cell models. Here we report the generation of induced pluripotent stem (iPS) cells from skin fibroblasts taken from three PD patients with nonsense (c.1366CtextgreaterT; p.Q456X) or missense (c.509TtextgreaterG; p.V170G) mutations in the PINK1 gene. These cells were differentiated into dopaminergic neurons that upon mitochondrial depolarization showed impaired recruitment of lentivirally expressed Parkin to mitochondria,increased mitochondrial copy number,and upregulation of PGC-1α,an important regulator of mitochondrial biogenesis. Importantly,these alterations were corrected by lentiviral expression of wild-type PINK1 in mutant iPS cell-derived PINK1 neurons. In conclusion,our studies suggest that fibroblasts from genetic PD can be reprogrammed and differentiated into neurons. These neurons exhibit distinct phenotypes that should be amenable to further mechanistic studies in this relevant biological context. View Publication

Although many previous investigations have studied how mercury compounds cause cell death,sub-cytotoxic levels may affect mechanisms essential for the proper development of the nervous system. The present study investigates whether low doses of methylmercury (MeHg) and mercury chloride (HgCl2) can modulate the activity of JAK/STAT signaling,a pathway that promotes gliogenesis. We report that sub-cytotoxic doses of MeHg enhance ciliary neurotrophic factor (CNTF) evoked STAT3 phosphorylation in human SH-SY5Y neuroblastoma and mouse cortical neural progenitor cells (NPCs). This effect is specific for MeHg,since HgCl2 fails to enhance JAK/STAT signaling. Exposing NPCs to these low doses of MeHg (30-300nM) enhances CNTF-induced expression of STAT3-target genes such as glial fibrillary acidic protein (GFAP) and suppressors of cytokine signaling 3 (SOCS3),and increases the proportion of cells expressing GFAP following 2 days of differentiation. Higher,near-cytotoxic concentrations of MeHg and HgCl2 inhibit STAT3 phosphorylation and lead to increased production of superoxide. Lower concentrations of MeHg effective in enhancing JAK/STAT signaling (30nM) do not result in a detectable increase in superoxide nor increased expression of the oxidant-responsive genes,heme oxygenase 1,heat shock protein A5 and sirtuin 1. These findings suggest that low concentrations of MeHg inappropriately enhance STAT3 phosphorylation and glial differentiation,and that the mechanism causing this enhancement is distinct from the reactive oxygen species-associated cell death observed at higher concentrations of MeHg and HgCl2. View Publication

Cellular senescence and the senescence-associated secretory phenotype (SASP) are implicated in aging and age-related disease,and SASP-related inflammation is thought to contribute to tissue dysfunction in aging and diseased animals. However,whether and how SASP factors influence the regenerative capacity of tissues remains unclear. Here,using intestinal organoids as a model of tissue regeneration,we show that SASP factors released by senescent fibroblasts deregulate stem cell activity and differentiation and ultimately impair crypt formation. We identify the secreted N-terminal domain of Ptk7 as a key component of the SASP that activates non-canonical Wnt / Ca2+ signaling through FZD7 in intestinal stem cells (ISCs). Changes in cytosolic [Ca2+] elicited by Ptk7 promote nuclear translocation of YAP and induce expression of YAP/TEAD target genes,impairing symmetry breaking and stem cell differentiation. Our study discovers secreted Ptk7 as a factor released by senescent cells and provides insight into the mechanism by which cellular senescence contributes to tissue dysfunction in aging and disease. View Publication

Juvenile dermatomyositis (JDM) is a pediatric autoimmune disease associated with characteristic rash and proximal muscle weakness. To gain insight into differential lymphocyte gene expression in JDM,peripheral blood mononuclear cells from 4 new-onset JDM patients and 4 healthy controls were sorted into highly enriched lymphocyte populations for RNAseq analysis. NK cells from JDM patients had substantially greater differentially expressed genes (273) than T (57) and B (33) cells. Upregulated genes were associated with the innate immune response and cell cycle,while downregulated genes were associated with decreased ribosomal RNA. Suppressed ribosomal RNA in JDM NK cells was validated by measuring transcription and phosphorylation levels. We confirmed a population of low ribosome expressing NK cells in healthy adults and children. This population of low ribosome NK cells was substantially expanded in 6 treatment-na{\{i}}ve JDM patients and was associated with decreased NK cell degranulation. The enrichment of this NK low ribosome population was completely abrogated in JDM patients with quiescent disease. Together these data suggest NK cells are highly activated in new-onset JDM patients with an increased population of low ribosome expressing NK cells which correlates with decreased NK cell function and resolved with control of active disease." View Publication

ABSTRACTBackgroundIdiopathic pulmonary fibrosis (IPF) is a lethal disease with an unknown etiology and complex pathophysiology that are not fully understood. The disease involves intricate cellular interplay,particularly among various immune cells. Currently,there is no treatment capable of reversing the fibrotic process or aiding lung regeneration. Hepatocyte growth factor (HGF) has demonstrated antifibrotic properties,whereas the adoptive transfer of modified T cells is a well‐established treatment for various malignancies. We aimed to understand the dynamics of T cells in the progression of lung fibrosis and to study the therapeutic benefit of adoptive T cell transfer in a bleomycin‐injured mouse lung (BLM) model.MethodsT cells were isolated from the spleen of naïve mice and transfected in vitro with mouse HGF plasmid and were administered intratracheally to the mice lungs 7 days post‐bleomycin injury to the lung. Lung tissue and bronchoalveolar lavage were collected and analyzed using flow cytometry,histology,qRT‐PCR,ELISA,and hydroxyproline assay.ResultsOur findings demonstrate the successful T cell therapy of bleomycin‐induced lung injury through the adoptive transfer of HGF‐transfected T cells in mice. This treatment resulted in decreased collagen deposition and a balancing of immune cell exhaustion and cytokine homeostasis compared with untreated controls. In vitro testing showed enhanced apoptosis in myofibroblasts induced by HGF‐overexpressing T cells.ConclusionsTaken together,our data highlight the great potential of adoptive T cell transfer as an emerging therapy to counteract lung fibrosis. This study explores the potential of T cells as a therapeutic strategy against idiopathic pulmonary fibrosis (IPF),a progressive lung disease for which there is currently no treatment to reverse fibrosis or restore normal lung function. To investigate an innovative approach using adoptive T cell transfer,T cells isolated from healthy mice were genetically modified to carry a plasmid containing hepatocyte growth factor (HGF). The modified cells were delivered directly into the airways of mice with bleomycin‐induced lung fibrosis. The results showed a significant reduction in fibrotic scarring,improved immune regulation,and increased apoptosis of pathogenic myofibroblasts. These results highlight the potential of HGF‐engineered T cells as a promising therapeutic approach to combat IPF. 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

Immune rejection is one of the most serious challenges in allogeneic transplantation,including allogeneic induced pluripotent stem cell (allo-iPSC)-derived cell therapy. Beta-2-Microglobulin gene-knockout,human leukocyte antigen (HLA) class I-deficient iPSCs can evade immune rejection by host T cells,which occurs due to HLA mismatches. However,natural killer (NK) cells recognize HLA class Ⅰ-deficient cells and reject them,which is known as the missing-self response. Introducing chimeric HLA-E protein to HLA class Ⅰ-deficient iPSCs suppresses the missing-self response of NK cells expressing the inhibitory receptor NKG2A; however,technology to suppress NKG2A-negative NK cells is still required. Here,we developed novel agonists for the other inhibitory receptor,killer immunoglobulin receptor (KIR),on NK cells. We found that antibodies that bind to activating KIR enhance NK cell activation and developed selective agonists for inhibitory KIRs (KIR2DL1,KIR2DL2/3,and KIR3DL1). Introducing these selective inhibitory KIR agonists on T cells and HLA class Ⅰ-deficient iPSCs allowed them to evade immune rejection by NK cells. Additionally,we identified an NKG2A-selective agonist as an alternative to chimeric HLA-E,which stimulates the activating receptor NKG2C. This technology enhances immune tolerance in allo-iPSCs and facilitates the development of various iPSC-derived regenerative medicines. The online version contains supplementary material available at 10.1038/s41598-025-18394-z. Subject terms: Allotransplantation,NK cells View Publication

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy characterized by desmoplastic stroma,immunosuppressive tumor microenvironment (TME),and resistance to standard therapies. Natural killer (NK) cell-based immunotherapies have shown limited efficacy due to impaired persistence,infiltration,and function in PDAC. Methods: We established a direct reprogramming strategy to generate cytotoxic NK cells (1 F-NKs) by targeting BCL11B,a transcription factor essential for T cell lineage commitment,using shRNA or CRISPR/Cas9 in peripheral blood mononuclear cells (PBMCs). A genome-wide CRISPR/Cas9 screen identified tumor-intrinsic modulators of NK resistance. Functional and in vivo studies assesses the efficacy of 1 F-NKs alone and in combination with mesothelin (MSLN)-CAR engineering and PKMYT1 inhibition. Results: BCL11B depletion enabled the generation of CD56brightCD16bright 1 F-NKs with potent cytotoxicity and elevated NKG2D and CX3CR1 expression. Site-specific integration of a mesothelin (MSLN)-CAR into BCL11B locus generated MSLN-1 F-NKs with stable antigen specific activity. A genome-wide screen identified PKMYT1 as a modulator of tumor resistance to NK cell-mediated killing; its inhibition by RP6306 upregulated NKG2D ligands (MICA/B) and CX3CL1,sensitizing PDACs to 1 F-NK cytotoxicity. In PDAC xenograft models,1 F-NKs alone or combined with CAR engineering and RP6306 significantly reduced tumor growth and prolonged survival. Notably,this triple combination elicited a synergistic antitumor effect,outperforming each monotherapy or dual combination. Conclusions: This study presents a synergistic immunotherapy platform that integrates NK reprogramming,CAR engineering,and tumor sensitization. The combinatorial approach significantly enhances antitumor efficacy in PDAC and offers a promising strategy for overcoming immune resistance in solid tumors. View Publication

Innate lymphoid cells (ILCs) are a new family of immune cells that play important roles in innate immunity in mucosal tissues,and in the maintenance of tissue and metabolic homeostasis. Recently,group 2 ILCs (ILC2s) were found to promote the development and effector functions of Th2-type CD4(+) T cells by interacting directly with T cells or by activating dendritic cells,suggesting a role for ILC2s in regulating adaptive immunity. However,our current knowledge on the role of ILCs in humoral immunity is limited. In this study,we found that ILC2s isolated from the lungs of naive BALB/c mice enhanced the proliferation of B1- as well as B2-type B cells and promoted the production of IgM,IgG1,IgA,and IgE by these cells in vitro. Soluble factors secreted by ILC2s were sufficient to enhance B cell Ig production. By using blocking Abs and ILC2s isolated from IL-5-deficient mice,we found that ILC2-derived IL-5 is critically involved in the enhanced production of IgM. Furthermore,when adoptively transferred to Il7r(-/-) mice,which lack ILC2s and mature T cells,lung ILC2s promoted the production of IgM Abs to a polysaccharide Ag,4-hydroxy-3-nitrophenylacetyl Ficoll,within 7 d of airway exposure in vivo. These findings add to the growing body of literature regarding the regulatory functions of ILCs in adaptive immunity,and suggest that lung ILC2s promote B cell production of early Abs to a respiratory Ag even in the absence of T cells. View Publication