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

The macrophages from Crohn's Disease (CD) patients are defective to control the replication of CD-associated adherent-invasive E. coli (AIEC). We aimed to identify the host factors associated with AIEC replication focusing on polymorphisms related to autophagy. Peripheral blood monocyte-derived macrophages (MDM),obtained from 95 CD patient,30 ulcerative colitis (UC) patients and 15 healthy subjects,were genotyped for several CD-associated polymorphisms. AIEC bacteria survival increased within MDM from CD patients compared to UC (p = 0.0019). AIEC bacteria survival increased in patients with CD-associated polymorphism IRGM (p = 0.05) and reduced in those with CD-associated polymorphisms XBP-1 (p = 0.026) and ULK-1 (p = 0.033). AIEC infection led to an increase of pro-inflammatory cytokines IL-1$\beta$ (p {\textless} 0.0001) and TNF-$\alpha$ (p {\textless} 0.0001) in CD macrophages. ULK-1 expression increased in AIEC-infected MDM from CD patients compared to MDM from UC patients or healthy subjects (p = 0.0056) and correlated with AIEC survival (p = 0.0013). Moreover,the expression of ULK-1 phosphorylation on Serine 757 decreased following to AIEC infection (p {\textless} 0.0001). Short-term silencing of ULK-1 and IRGM genes restricted and promote,respectively,AIEC survival within MDM (p = 0.0018 and p = 0.0291). In conclusion,the macrophage defect to mediate AIEC clearance in CD patients is linked to polymorphisms related to autophagy such as IRGM and ULK-1. View Publication

Filoviruses of the genus Ebolavirus include five species with marked differences in their ability to cause disease in humans. From the highly virulent Ebola virus to the seemingly nonpathogenic Reston virus,case-fatality rates can range between 0-90{\%}. In order to understand the molecular basis of these differences it is imperative to establish disease models that recapitulate human disease as faithfully as possible. Non-human primates are the gold-standard models for filovirus pathogenesis,but comparative studies are skewed by the fact that Reston virus infection can be lethal for NHP. Here we have used HLA-A2 transgenic,NOD-scid-interleukin 2$\gamma$ receptor knockout (NSG-A2) mice reconstituted with human hematopoiesis to compare Ebola virus and Reston virus pathogenesis in a human-like environment. While significantly less pathogenic than Ebola virus,Reston virus killed 20{\%} of infected mice,a finding that was linked to exacerbated inflammation and viral replication in the liver. In addition,'humanized' mice recapitulated the case-fatality ratios of different Ebolavirus species in humans. Our findings point out at humanized mice as a putative model to test the pathogenicity of newly discovered filoviruses,and warrants further investigations on Reston virus pathogenesis in humans. View Publication

Sustained,indolent immune injury of the vasculature of a heart transplant limits long-term graft and recipient survival. This injury is mitigated by a poorly characterized,maladaptive repair response. Vascular endothelial cells respond to proangiogenic cues in the embryo by differentiation to specialized phenotypes,associated with expression of apelin. In the adult,the role of developmental proangiogenic cues in repair of the established vasculature is largely unknown. We found that human and minor histocompatibility-mismatched donor mouse heart allografts with alloimmune-mediated vasculopathy upregulated expression of apelin in arteries and myocardial microvessels. In vivo,loss of donor heart expression of apelin facilitated graft immune cell infiltration,blunted vascular repair,and worsened occlusive vasculopathy in mice. In vitro,an apelin receptor agonist analog elicited endothelial nitric oxide synthase activation to promote endothelial monolayer wound repair and reduce immune cell adhesion. Thus,apelin acted as an autocrine growth cue to sustain vascular repair and mitigate the effects of immune injury. Treatment with an apelin receptor agonist after vasculopathy was established markedly reduced progression of arterial occlusion in mice. Together,these initial data identify proangiogenic apelin as a key mediator of coronary vascular repair and a pharmacotherapeutic target for immune-mediated injury of the coronary vasculature. View Publication

Products derived from bacterial members of the gut microbiota evoke immune signalling pathways of the host that promote immunity and barrier function in the intestine. How immune reactions to enteric viruses support intestinal homeostasis is unknown. We recently demonstrated that infection by murine norovirus (MNV) reverses intestinal abnormalities following depletion of bacteria,indicating that an intestinal animal virus can provide cues to the host that are typically attributed to the microbiota. Here,we elucidate mechanisms by which MNV evokes protective responses from the host. We identify an important role for the viral protein NS1/2 in establishing local replication and a type I interferon (IFN-I) response in the colon. We further show that IFN-I acts on intestinal epithelial cells to increase the proportion of CCR2-dependent macrophages and interleukin (IL)-22-producing innate lymphoid cells,which in turn promote pSTAT3 signalling in intestinal epithelial cells and protection from intestinal injury. In addition,we demonstrate that MNV provides a striking IL-22-dependent protection against early-life lethal infection by Citrobacter rodentium. These findings demonstrate novel ways in which a viral member of the microbiota fortifies the intestinal barrier during chemical injury and infectious challenges. View Publication

Neutrophils are the first immune cells to kill invading microbes at sites of infection using a variety of processes,including the release of proteases,phagocytosis and the production of neutrophil extracellular traps (NETs). NET formation,or NETosis,is a specific and highly efficient process,which is induced by a variety of stimuli leading to expulsion of DNA,proteases and antimicrobial peptides to the extracellular space. However,uncontrolled NETosis may lead to adverse effects and exert tissue damage in pathological conditions. Here,we show that the ATP channel pannexin1 (Panx1) is functionally expressed by bone marrow-derived neutrophils (BMDNs) of wild-type (WT) mice and that ATP contributes to NETosis induced in vitro by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate (PMA). Interestingly,neutrophils isolated from Panx1-/- mice showed reduced and/or delayed induction of NETosis. Brilliant blue FCF dye (BB-FCF),a Panx1 channel inhibitor,decreased NETosis in wild-type neutrophils to the extent observed in Panx1-/- neutrophils. Thus,we demonstrate that ATP and Panx1 channels contribute to NETosis and may represent a therapeutic target. View Publication

Static gene expression programs have been extensively characterized in stem cells and mature human cells. However,the dynamics of RNA isoform changes upon cell-state-transitions during cell differentiation,the determinants and functional consequences have largely remained unclear. Here,we established an improved model for human neurogenesis in vitro that is amenable for systems-wide analyses of gene expression. Our multi-omics analysis reveals that the pronounced alterations in cell morphology correlate strongly with widespread changes in RNA isoform expression. Our approach identifies thousands of new RNA isoforms that are expressed at distinct differentiation stages. RNA isoforms mainly arise from exon skipping and the alternative usage of transcription start and polyadenylation sites during human neurogenesis. The transcript isoform changes can remodel the identity and functions of protein isoforms. Finally,our study identifies a set of RNA binding proteins as a potential determinant of differentiation stage-specific global isoform changes. This work supports the view of regulated isoform changes that underlie state-transitions during neurogenesis. Synopsis Multi-omics analysis of a newly established human neuronal cell differentiation model reveals widespread dynamic changes in RNA isoform expression,their functional consequences and potential determinants,providing evidence that they underlie cell-state-transitions during neurogenesis. Dynamic changes in RNA and protein levels are strongly correlated during all stages of neuronal differentiation.Nanopore sequencing (ONT-seq) during human neurogenesis reveals 12,019 non-annotated RNA isoforms,a large number of which are differentially expressed during differentiation.70% of new RNA isoforms result from the use of alternative transcription start sites (TSSs) or polyadenylation (pA) sites and exon skipping.RNA isoform changes underlie protein isoform changes during human neurogenesis as revealed by integrating ONT-seq,RNA-seq and proteomics time course data.RNA motif enrichment,RNA-seq and available CLIP-seq data uncover a set of RNA binding proteins (RBPs) as potential determinants of differentiation stage-specific global isoform changes. Multi-omics analysis of a newly established human neuronal cell differentiation model reveals widespread dynamic changes in RNA isoform expression,their functional consequences and potential determinants,providing evidence that they underlie cell-state-transitions during neurogenesis. View Publication

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disorder characterized by sodium channel dysfunction. However,the clinical management of ARVC remains challenging. Identifying novel compounds for the treatment of ARVC is crucial for advancing drug development.PurposeIn this study,we aim to identify novel compounds for treating ARVC.MethodsMachine learning (ML) models were constructed using proteins analyzed from the scRNA-seq data of ARVC rats and their corresponding protein-protein interaction (PPI) network to predict binding affinity (BA). To validate these predictions,a series of experiments in cardiac organoids were conducted,including Western blotting,ELISA,MEA,and Masson staining to assess the effects of these compounds.ResultsWe first discovered and identified SCN5A as the most significantly affected sodium channel protein in ARVC. ML models predicted that Kaempferol binds to SCN5A with high affinity. In vitro experiments further confirmed that Kaempferol exerted therapeutic effects in ARVC.ConclusionThis study presents a novel approach for identifying potential compounds to treat ARVC. By integrating ML modeling with organoid validation,our platform provides valuable support in addressing the public health challenges posed by ARVC,with broad application prospects. Kaempferol shows promise as a lead compound for ARVC treatment. View Publication

Energetic stress compels cells to evolve adaptive mechanisms to adjust their metabolism. Inhibition of mTOR kinase complex 1 (mTORC1) is essential for cell survival during glucose starvation. How mTORC1 controls cell viability during glucose starvation is not well understood. Here we show that the mTORC1 effectors eukaryotic initiation factor 4E binding proteins 1/2 (4EBP1/2) confer protection to mammalian cells and budding yeast under glucose starvation. Mechanistically,4EBP1/2 promote NADPH homeostasis by preventing NADPH-consuming fatty acid synthesis via translational repression of Acetyl-CoA Carboxylase 1 (ACC1),thereby mitigating oxidative stress. This has important relevance for cancer,as oncogene-transformed cells and glioma cells exploit the 4EBP1/2 regulation of ACC1 expression and redox balance to combat energetic stress,thereby supporting transformation and tumorigenicity in vitro and in vivo. Clinically,high EIF4EBP1 expression is associated with poor outcomes in several cancer types. Our data reveal that the mTORC1-4EBP1/2 axis provokes a metabolic switch essential for survival during glucose starvation which is exploited by transformed and tumor cells. How cells adapt to glucose starvation is still elusive. Here,Levy et al. show that the mTOR substrate 4EBP1 protects human,mouse,and yeast cells from glucose starvation and is exploited by cancer cells to promote tumorigenesis. View Publication

Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by loss-of-function mutations in the MECP2 gene,resulting in diverse cellular dysfunctions. Here,we investigated the role of the long noncoding RNA (lncRNA) NEAT1 in the context of MeCP2 deficiency using human neural cells and RTT patient samples. Through single-cell RNA sequencing and molecular analyses,we found that NEAT1 is markedly downregulated in MECP2 knockout (KO) cells at various stages of neural differentiation. NEAT1 downregulation correlated with aberrant activation of the mTOR pathway,abnormal protein metabolism,and dysregulated autophagy,contributing to the accumulation of protein aggregates and impaired mitochondrial function. Reactivation of NEAT1 in MECP2-KO cells rescued these phenotypes,indicating its critical role downstream of MECP2. Furthermore,direct RNA–RNA interaction was revealed as the key process for NEAT1 influence on autophagy genes,leading to altered subcellular localization of specific autophagy-related messenger RNAs and impaired biogenesis of autophagic complexes. Importantly,NEAT1 restoration rescued the morphological defects observed in MECP2-KO neurons,highlighting its crucial role in neuronal maturation. Overall,our findings elucidate lncRNA NEAT1 as a key mediator of MeCP2 function,regulating essential pathways involved in protein metabolism,autophagy,and neuronal morphology. View Publication

BackgroundTrichloroethylene (TCE) is a ubiquitous pollutant with potential capacity to induce congenital heart disease (CHD). However,the mechanisms underlying TCE-induced CHD are largely unraveled.MethodsWe exposed zebrafish embryos to TCE to investigate its cardiac development toxicity and related response factor through bulk RNA sequencing. We constructed transgenic fluorescent fish and employed the CRISPR/dCas9 system along with single-cell RNA sequencing to identify the genetic cause of TCE-induced CHD.ResultsWe found that early-stage exposure to TCE induced significant cardiac defects characterized by elongated SV-BA distance,thinned myocardium,and attenuated contractility. Gremlin1 encoding gene,grem1a,a putative target showing high expression at the beginning of cardiac development,was sharply down-regulated by TCE. Consistently,grem1a knockdown in zebrafish induced cardiac phenotypes generally like those of the TCE-treated group,accompanying the disarrangement of myofibril structure. Single-cell RNA-seq depicted that mitochondrial respiration in grem1a-repressed cardiomyocytes was greatly enhanced,ultimately leading to a branch from the normal trajectory of myocardial development. Accordingly,in vitro results demonstrated that GREM1 repression increased mitochondrial content,ATP production,mitochondrial reactive oxygen species,mitochondrial membrane potential,and disrupted myofibril expansion in hPSC-CMs.ConclusionsThese results suggested that TCE-induced gremlin1 repression could result in mitochondrial hyperfunction,thereby hampering cardiomyocyte development and causing cardiac defects in zebrafish embryos. This study not only provided a novel insight into the etiology for environmental stressor-caused cardiac development defects,but also offered a potential therapeutic and preventive target for TCE-induced CHD.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-025-02314-9. View Publication

BackgroundAt present,hepatic ischemia-reperfusion injury (IRI) is an important complication of partial hepatectomy and liver transplantation,and it is an important cause of poor prognosis. Spleen tyrosine kinase(SYK) plays an important role in a variety of signaling pathways in the liver,but its role in hepatic IRI is still unclear. This study aims to investigate the role and mechanism of SYK in hepatic IRI and tumor recurrence.MethodsWe first observed the activation of SYK in the liver of mice in response to hepatic IRI. Subsequently,Pharmacological inhibitions of SYK were used to evaluated the effect of SYK on neutrophil recruitment and NETosis,and further explored the effect of SYK on IRI and tumor recurrence.ResultsOur study shows that SYK is activated in response to hepatic IRI and aggravates liver injury. On the one hand,neutrophils SYK during the early stage of liver reperfusion increases neutrophil extracellular traps (NETs) production by promoting Pyruvate kinase M2(PKM2) nuclear translocation leading to upregulation of phosphorylated STAT3,thereby exacerbating liver inflammation and tumor recurrence. On the other hand,macrophages SYK can promote the recruitment of neutrophils and increase the activation of NLRP3 inflammasome and IL1β,which further promotes the formation of NETs.ConclusionsOur study demonstrates that neutrophil and macrophage SYK synergistically promote hepatic IRI and tumor recurrence,and SYK may be a potential target to improve postoperative hepatic IRI and tumor recurrence.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10020-024-00907-7. View Publication

AbstractBackgroundAcute graft‐versus‐host disease (aGVHD) arises from the imbalance of host T cells. Galectin‐9 negatively regulates CD4 effector T cell (Th1 and Th17) function by binding to Tim‐3. However,the relationship between Galectin‐9/Tim‐3 and CD4+ T subsets in patients with aGVHD after Haplo‐HSCT (haploidentical peripheral blood hematopoietic stem cell transplantation) has not been fully elucidated. Here,we investigated the role of Galectin‐9 and CD4+T subsets in aGVHD after haplo‐HSCT.MethodsForty‐two patients underwent Haplo‐HSCT (26 without aGVHD and 16 with aGVHD),and 20 healthy controls were included. The concentrations of Galectin‐9,interferon‐gamma (IFN‐γ),interleukin (IL)‐4,transforming growth factor (TGF)‐β,and IL‐17 in the serum and culture supernatant were measured using enzyme‐linked immunosorbent assay or cytometric bead array. The expression levels of Galectin‐9,PI3K,p‐PI3K,and p‐mTOR protein were detected by western blot analysis. Flow cytometry was used to analyze the proportions of CD4+ T cell subsets. Bioinformatics analysis was performed.ResultsIn patients with aGVHD,regulatory T (Treg) cells and Galectin‐9 decreased,and the Th1,Th17,and Treg cells were significantly imbalanced. Moreover,Treg and Galectin‐9 were rapidly reconstituted in the early stage of patients without aGVHD after Haplo‐HSCT,but Th17 cells were reconstituted slowly. Furthermore,Tim‐3 upregulation on Th17 and Th1 cells was associated with excessive activation of the PI3K/AKT pathway in patients with aGVHD. Specifically,in vitro treatment with Galectin‐9 reduced IFN‐γ and IL‐17 production while augmenting TGF‐β secretion. Bioinformatics analysis suggested the potential involvement of the PI3K/AKT/mTOR pathway in aGVHD. Mechanistically,exogenous Galectin‐9 was found to mitigate aGVHD by restoring the Treg/Teffs (effector T cells) balance and suppressing PI3K.ConclusionGalectin‐9 may ameliorate aGVHD after haplo‐HSCT by modulating Treg/Teffs balance and regulating the PI3K/AKT/mTOR pathway. Targeting Galectin‐9 may hold potential value for the treatment of aGVHD. In patients with acute graft‐versus‐host disease (aGVHD),the expression of Tim‐3 is significantly increased. Galectin‐9 binding to Tim‐3 may inhibit the activation of the PI3K/AKT pathway and enhance the function of Treg cells. On the other hand,transforming growth factor (TGF)‐β promotes the differentiation of Treg cells through autocrine secretion,while TGF‐β induces the expression of Galectin‐9 in a paracrine manner. The increased Treg cells can inhibit the activation of Th1 and Th17 cells by secreting TGF‐β,thus alleviating aGVHD and inducing immune tolerance View Publication