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

There has been a recent drive to replace in vivo studies with in vitro studies in the field of toxicity testing. Therefore,instead of conventional animal or planar cell culture models,there is an urgent need for in vitro systems whose conditions can be strictly controlled,including cell–cell interactions and sensitivity to low doses of chemicals. Neural organoids generated from human-induced pluripotent stem cells (iPSCs) are a promising in vitro platform for modeling human brain development. In this study,we developed a new tool based on various iPSCs to study and predict chemical-induced toxicity in humans. The model displayed several neurodevelopmental features and showed good reproducibility,comparable to that of previously published models. The results revealed that basic fibroblast growth factor plays a key role in the formation of the embryoid body,as well as complex neural networks and higher-order structures such as layered stacking. Using organoid models,pesticide toxicities were assessed. Cells treated with low concentrations of rotenone underwent apoptosis to a greater extent than those treated with high concentrations of rotenone. Morphological changes associated with the development of neural progenitor cells were observed after exposure to low doses of chlorpyrifos. These findings suggest that the neuronal organoids developed in this study mimic the developmental processes occurring in the brain and nerves and are a useful tool for evaluating drug efficacy,safety,and toxicity. View Publication

The pathogenesis of malarial anemia is multifactorial,and the mechanisms responsible for its high mortality are poorly understood. Studies indicate that host mediators produced during malaria infection may suppress erythroid progenitor development (Miller,K.L.,J.C. Schooley,K.L. Smith,B. Kullgren,L.J. Mahlmann,and P.H. Silverman. 1989. Exp. Hematol. 17:379-385; Yap,G.S.,and M.M. Stevenson. 1991. Ann. NY Acad. Sci. 628:279-281). We describe an intrinsic role for macrophage migration inhibitory factor (MIF) in the development of the anemic complications and bone marrow suppression that are associated with malaria infection. At concentrations found in the circulation of malaria-infected patients,MIF suppressed erythropoietin-dependent erythroid colony formation. MIF synergized with tumor necrosis factor and gamma interferon,which are known antagonists of hematopoiesis,even when these cytokines were present in subinhibitory concentrations. MIF inhibited erythroid differentiation and hemoglobin production,and it antagonized the pattern of mitogen-activated protein kinase phosphorylation that normally occurs during erythroid progenitor differentiation. Infection of MIF knockout mice with Plasmodium chabaudi resulted in less severe anemia,improved erythroid progenitor development,and increased survival compared with wild-type controls. We also found that human mononuclear cells carrying highly expressed MIF alleles produced more MIF when stimulated with the malarial product hemozoin compared with cells carrying low expression MIF alleles. These data suggest that polymorphisms at the MIF locus may influence the levels of MIF produced in the innate response to malaria infection and the likelihood of anemic complications. View Publication

Human rhinovirus (HRV)-induced respiratory infections are associated with elevated levels of IFN-gamma-inducible protein 10 (IP-10),which is an enhancer of T lymphocyte chemotaxis and correlates with symptom severity and T lymphocyte number. Increased IP-10 expression is exhibited by airway epithelial cells following ex vivo HRV challenge and requires intracellular viral replication; however,there are conflicting reports regarding the necessity of type I IFN receptor ligation for IP-10 expression. Furthermore,the involvement of resident airway immune cells,predominantly bronchoalveolar macrophages,in contributing to HRV-stimulated IP-10 elaboration remains unclear. In this regard,our findings demonstrate that ex vivo exposure of human peripheral blood monocytes and bronchoalveolar macrophages (monocytic cells) to native or replication-defective HRV serotype 16 (HRV16) resulted in similarly robust levels of IP-10 release,which occurred in a time- and dose-dependent manner. Furthermore,HRV16 induced a significant increase in type I IFN (IFN-alpha) release and STAT1 phosphorylation in monocytes. Neutralization of the type I IFN receptor and inhibition of JAK or p38 kinase activity strongly attenuated HRV16-stimulated STAT1 phosphorylation and IP-10 release. Thus,this work supports a model,wherein HRV16-induced IP-10 release by monocytic cells is modulated via autocrine/paracrine action of type I IFNs and subsequent JAK/STAT pathway activity. Our findings demonstrating robust activation of monocytic cells in response to native and/or replication-defective HRV16 challenge represent the first evidence indicating a mechanistic disparity in the activation of macrophages when compared with epithelial cells and suggest that macrophages likely contribute to cytokine elaboration following HRV challenge in vivo. View Publication

STAT3 is constitutively activated in many cancer types,including lung cancer,and can induce cancer cell proliferation and cancer stem cell (CSC) maintenance. STAT3 is activated by tyrosine kinases,such as JAK and SRC,but the mechanism by which STAT3 maintains its activated state in cancer cells remains unclear. Here,we show that PRMT5 directly methylates STAT3 and enhances its activated tyrosine phosphorylation in non-small cell lung cancer (NSCLC) cells. PRMT5 expression is also induced by STAT3,suggesting the presence of a positive feedback loop in cancer cells. Furthermore,methylation of STAT3 at arginine 609 by PRMT5 is important for its transcriptional activity and support of tumour growth and CSC maintenance. Indeed,NSCLC cells expressing the STAT3 mutant which R609 was replaced to alanine (R609K) show significantly impaired tumour growth in nude mice. Overall,our study reveals a mechanism by which STAT3 remains activated in NSCLC and provides a new target for cancer therapeutic approaches. Subject terms: Oncogenes,Non-small-cell lung cancer,Growth factor signalling View Publication

TCR/CD28 engagement triggers the initiation of a variety of signal transduction pathways that lead to changes in gene transcription. Although reorganization of the actin cytoskeleton is required for T cell activation,the molecular pathways controlled by the actin cytoskeleton are ill defined. To this end,we analyzed TCR/CD28-stimulated signaling pathways in cytochalasin D-treated T cells to determine the cytoskeletal requirements for T cell activation. Cytochalasin D treatment impaired T cell activation by causing a reduction in TCR/CD28-mediated calcium flux,and blocked activation of two regulatory elements within the IL-2 promoter,NFAT/AP-1 and CD28RE/AP. Treatment had no effect on signaling leading to the activation of either AP-1 or NF-kappaB. Significantly,we found that NFAT1 is required for optimal c-rel up-regulation in response to TCR/CD28 stimulation. In fact,NFAT1 could be detected bound at the c-rel promoter in response to TCR/CD28 stimulation,and targeting of NFAT1 using RNA interference in human CD4(+) T cells abrogated c-rel transcription. Overall,these findings establish that disrupting actin cytoskeletal dynamics impairs TCR/CD28-mediated calcium flux required for NFAT1-mediated c-rel transcription and,thus,activation of the CD28RE/AP. View Publication

HIV up-regulates cell-surface expression of specific ligands for the activating NKG2D receptor,including ULBP-1,-2,and -3,but not MICA or MICB,in infected cells both in vitro and in vivo. However,the viral factor(s) involved in NKG2D ligand expression still remains undefined. HIV-1 Vpr activates the DNA damage/stress-sensing ATR kinase and promotes G(2) cell-cycle arrest,conditions known to up-regulate NKG2D ligands. We report here that HIV-1 selectively induces cell-surface expression of ULBP-2 in primary CD4(+) T lymphocytes by a process that is Vpr dependent. Importantly,Vpr enhanced the susceptibility of HIV-1-infected cells to NK cell-mediated killing. Strikingly,Vpr alone was sufficient to up-regulate expression of all NKG2D ligands and thus promoted efficient NKG2D-dependent NK cell-mediated killing. Delivery of virion-associated Vpr via defective HIV-1 particles induced analogous biologic effects in noninfected target cells,suggesting that Vpr may act similarly beyond infected cells. All these activities relied on Vpr ability to activate the ATR-mediated DNA damage/stress checkpoint. Overall,these results indicate that Vpr is a key determinant responsible for HIV-1-induced up-regulation of NKG2D ligands and further suggest an immunomodulatory role for Vpr that may not only contribute to HIV-1-induced CD4(+) T-lymphocyte depletion but may also take part in HIV-1-induced NK-cell dysfunction. View Publication

Endogenous long double-stranded RNAs (dsRNAs),which are not edited by the RNA editing enzyme ADAR1,may activate the antiviral dsRNA receptor MDA5 to trigger interferon-mediated immune responses. Among the large number of endogenous long dsRNAs,the key substrates that activate MDA5—termed as immunogenic dsRNAs—remain largely unidentified. Here we reveal that human immunogenic dsRNAs constitute a surprisingly small fraction of all cellular dsRNAs. We found that these immunogenic dsRNAs were highly enriched in mRNAs and depleted of introns,consistent with their role as cytosolic MDA5 substrates. We validated the MDA5-dependent immunogenicity of these dsRNAs,which was dampened following ADAR1-mediated RNA editing. Notably,immunogenic dsRNAs were enriched at genetic susceptibility loci associated with common inflammatory diseases,implying their functional importance. We anticipate that a focused analysis of immunogenic dsRNAs will enhance our understanding and treatment of cancer and inflammatory diseases,where the roles of dsRNA editing and sensing are increasingly recognized. The authors show that only a small subset of cytosolic double-stranded RNAs (dsRNAs) requires ADAR1-mediated RNA editing to evade an MDA5-dependent immune response. These immunogenic dsRNAs are enriched in mRNAs and overlap with GWAS signals for common inflammatory diseases. View Publication

PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed,and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs,as determined in the modified Boyden chamber,as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly,in addition to inhibition of Src kinase,PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells,including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR,PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases,the activities of the epidermal growth factor receptor,fibroblast growth factor receptor-1,and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872,whereas PDGF alpha-receptor was susceptible,albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events,creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity,such as vascular remodeling and restenosis. View Publication

The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably,several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP,but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site,revealing a mechanism of filovirus inhibition. View Publication

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for many diverse blood and immune diseases. However,HSCT regimens currently commonly utilize genotoxic chemotherapy and/or total body irradiation (TBI) conditioning which causes significant morbidity and mortality through inducing broad tissue damage triggering infections,graft vs. host disease,infertility,and secondary cancers. We previously demonstrated that targeted monoclonal antibody (mAb)-based HSC depletion with anti(α)-CD117 mAbs could be an effective alternative conditioning approach for HSCT without toxicity in severe combined immunodeficiency (SCID) mouse models,which has prompted parallel clinical αCD117 mAbs to be developed and tested as conditioning agents in clinical trials starting with treatment of patients with SCID. Subsequent efforts have built upon this work to develop various combination approaches,though none are optimal and how any of these mAbs fully function is unknown. To improve efficacy of mAb-based conditioning as a stand-alone conditioning approach for all HSCT settings,it is critical to understand the mechanistic action of αCD117 mAbs on HSCs. Here,we compare the antagonistic properties of αCD117 mAb clones including ACK2,2B8,and 3C11 as well as ACK2 fragments in vitro and in vivo in both SCID and wildtype (WT) mouse models. Further,to augment efficacy,combination regimens were also explored. We confirm that only ACK2 inhibits SCF binding fully and prevents HSC proliferation in vitro. Further,we verify that this corresponds to HSC depletion in vivo and donor engraftment post HSCT in SCID mice. We also show that SCF-blocking αCD117 mAb fragment derivatives retain similar HSC depletion capacity with enhanced engraftment post HSCT in SCID settings,but only full αCD117 mAb ACK2 in combination with αCD47 mAb enables enhanced donor HSC engraftment in WT settings,highlighting that the Fc region is not required for single-agent efficacy in SCID settings but is required in immunocompetent settings. This combination was the only non-genotoxic conditioning approach that enabled robust donor engraftment post HSCT in WT mice. These findings shed new insights into the mechanism of αCD117 mAb-mediated HSC depletion. Further,they highlight multiple approaches for efficacy in SCID settings and optimal combinations for WT settings. This work is likely to aid in the development of clinical non-genotoxic HSCT conditioning approaches that could benefit millions of people world-wide. The online version contains supplementary material available at 10.1186/s13287-024-03981-0. View Publication