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

While a third of the world carries the burden of tuberculosis,disease control has been hindered by a lack of tools,including a rapid,point-of-care diagnostic and a protective vaccine. In many infectious diseases,antibodies (Abs) are powerful biomarkers and important immune mediators. However,in Mycobacterium tuberculosis (Mtb) infection,a discriminatory or protective role for humoral immunity remains unclear. Using an unbiased antibody profiling approach,we show that individuals with latent tuberculosis infection (Ltb) and active tuberculosis disease (Atb) have distinct Mtb-specific humoral responses,such that Ltb infection is associated with unique Ab Fc functional profiles,selective binding to FcγRIII,and distinct Ab glycosylation patterns. Moreover,compared to Abs from Atb,Abs from Ltb drove enhanced phagolysosomal maturation,inflammasome activation,and,most importantly,macrophage killing of intracellular Mtb. Combined,these data point to a potential role for Fc-mediated Ab effector functions,tuned via differential glycosylation,in Mtb control. View Publication

Hypoxia-inducible factor-1 (HIF-1) regulates the transcription of genes whose products play critical roles in energy metabolism,erythropoiesis,angiogenesis,and cell survival. Limited information is available concerning its function in mammalian hematopoiesis. Previous studies have demonstrated that homozygosity for a targeted null mutation in the Hif1alpha gene,which encodes the hypoxia-responsive alpha subunit of HIF-1,causes cardiac,vascular,and neural malformations resulting in lethality by embryonic day 10.5 (E10.5). This study revealed reduced myeloid multilineage and committed erythroid progenitors in HIF-1alpha-deficient embryos,as well as decreased hemoglobin content in erythroid colonies from HIF-1alpha-deficient yolk sacs at E9.5. Dysregulation of erythropoietin (Epo) signaling was evident from a significant decrease in mRNA levels of Epo receptor (EpoR) in Hif1alpha-/- yolk sac as well as Epo and EpoR mRNA in Hif1alpha-/- embryos. The erythropoietic defects in HIF-1alpha-deficient erythroid colonies could not be corrected by cytokines,such as vascular endothelial growth factor and Epo,but were ameliorated by Fe-SIH,a compound delivering iron into cells independently of iron transport proteins. Consistent with profound defects in iron homeostasis,Hif1alpha-/- yolk sac and/or embryos demonstrated aberrant mRNA levels of hepcidin,Fpn1,Irp1,and frascati. We conclude that dysregulated expression of genes encoding Epo,EpoR,and iron regulatory proteins contributes to defective erythropoiesis in Hif1alpha-/- yolk sacs. These results identify a novel role for HIF-1 in the regulation of iron homeostasis and reveal unexpected regulatory differences in Epo/EpoR signaling in yolk sac and embryonic erythropoiesis. View Publication

Tools permitting the isolation of live pancreatic cell subsets for culture and/or molecular analysis are limited. To address this,we developed a collection of monoclonal antibodies with selective surface labeling of endocrine and exocrine pancreatic cell types. Cell type labeling specificity and cell surface reactivity were validated on mouse pancreatic sections and by gene expression analysis of cells isolated using FACS. Five antibodies which marked populations of particular interest were used to isolate and study viable populations of purified pancreatic ducts,acinar cells,and subsets of acinar cells from whole pancreatic tissue or of alpha or beta cells from isolated mouse islets. Gene expression analysis showed the presence of known endocrine markers in alpha and beta cell populations and revealed that TTR and DPPIV are primarily expressed in alpha cells whereas DGKB and GPM6A have a beta cell specific expression profile. View Publication

Embryonic stem cell (ESC) identity and self-renewal is maintained by extrinsic signaling pathways and intrinsic gene regulatory networks. Here,we show that three members of the Ccr4-Not complex,Cnot1,Cnot2,and Cnot3,play critical roles in maintaining mouse and human ESC identity as a protein complex and inhibit differentiation into the extraembryonic lineages. Enriched in the inner cell mass of blastocysts,these Cnot genes are highly expressed in ESC and downregulated during differentiation. In mouse ESCs,Cnot1,Cnot2,and Cnot3 are important for maintenance in both normal conditions and the 2i/LIF medium that supports the ground state pluripotency. Genetic analysis indicated that they do not act through known self-renewal pathways or core transcription factors. Instead,they repress the expression of early trophectoderm (TE) transcription factors such as Cdx2. Importantly,these Cnot genes are also necessary for the maintenance of human ESCs,and silencing them mainly lead to TE and primitive endoderm differentiation. Together,our results indicate that Cnot1,Cnot2,and Cnot3 represent a novel component of the core self-renewal and pluripotency circuitry conserved in mouse and human ESCs. View Publication

Down syndrome (DS) is among the most frequent genetic causes of intellectual disability,and ameliorating this deficit is a major goal in support of people with trisomy 21. The Ts65Dn mouse recapitulates some major brain structural and behavioral phenotypes of DS,including reduced size and cellularity of the cerebellum and learning deficits associated with the hippocampus. We show that a single treatment of newborn mice with the Sonic hedgehog pathway agonist SAG 1.1 (SAG) results in normal cerebellar morphology in adults. Further,SAG treatment at birth rescued phenotypes associated with hippocampal deficits that occur in untreated adult Ts65Dn mice. This treatment resulted in behavioral improvements and normalized performance in the Morris water maze task for learning and memory. SAG treatment also produced physiological effects and partially rescued both N-methyl-d-aspartate (NMDA) receptor-dependent synaptic plasticity and NMDA/AMPA receptor ratio,physiological measures associated with memory. These outcomes confirm an important role for the hedgehog pathway in cerebellar development and raise the possibility for its direct influence in hippocampal function. The positive results from this approach suggest a possible direction for therapeutic intervention to improve cognitive function for this population. View Publication

Zika virus is a mosquito-borne flavivirus closely related to dengue virus that can cause severe disease in humans,including microcephaly in newborns and Guillain-Barr{\'{e}} syndrome in adults. Specific treatments and vaccines for Zika virus are not currently available. Here,we isolate and characterize four monoclonal antibodies (mAbs) from an infected patient that target the non-structural protein NS1. We show that while these antibodies are non-neutralizing,NS1-specific mAbs can engage Fc$\gamma$R without inducing antibody dependent enhancement (ADE) of infection in vitro. Moreover,we demonstrate that mAb AA12 has protective efficacy against lethal challenges of African and Asian lineage strains of Zika virus in Stat2-/- mice. Protection is Fc-dependent,as a mutated antibody unable to activate known Fc effector functions or complement is not protective in vivo. This study highlights the importance of the ZIKV NS1 protein as a potential vaccine antigen. View Publication

We have examined a number of reagents for their ability to modulate activity of the Hh signaling pathway during embryonic development of Xenopus. In particular we have focused on regulation of events occurring during tailbud stages and later. Two inducible protein reagents based on the Gli1 and Gli3 transcription factors were generated and the activity of these proteins was compared to the Hh signaling pathway inhibitor,cyclopamine,and the activators,Smoothened agonist (SAG) and purmorphamine (PMA). Effectiveness of reagents was assayed using both molecular biological techniques and biological readouts. We found that the small molecule modulators of the Hh pathway were highly specific and effective and produced results generally superior to the more conventional protein reagents for examination of later stage developmental processes. View Publication

Williams syndrome is a genetic neurodevelopmental disorder characterized by an uncommon hypersociability and a mosaic of retained and compromised linguistic and cognitive abilities. Nearly all clinically diagnosed individuals with Williams syndrome lack precisely the same set of genes,with breakpoints in chromosome band 7q11.23 (refs 1-5). The contribution of specific genes to the neuroanatomical and functional alterations,leading to behavioural pathologies in humans,remains largely unexplored. Here we investigate neural progenitor cells and cortical neurons derived from Williams syndrome and typically developing induced pluripotent stem cells. Neural progenitor cells in Williams syndrome have an increased doubling time and apoptosis compared with typically developing neural progenitor cells. Using an individual with atypical Williams syndrome,we narrowed this cellular phenotype to a single gene candidate,frizzled 9 (FZD9). At the neuronal stage,layer V/VI cortical neurons derived from Williams syndrome were characterized by longer total dendrites,increased numbers of spines and synapses,aberrant calcium oscillation and altered network connectivity. Morphometric alterations observed in neurons from Williams syndrome were validated after Golgi staining of post-mortem layer V/VI cortical neurons. This model of human induced pluripotent stem cells fills the current knowledge gap in the cellular biology of Williams syndrome and could lead to further insights into the molecular mechanism underlying the disorder and the human social brain. View Publication

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations,including their reliance on exogenous materials or electrical fields,which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30–80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material,such as carbon nanotubes,proteins,and siRNA,to 11 cell types,including embryonic stem cells and immune cells. When used for the delivery of transcription factors,the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed,its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications. View Publication

We report on a forward RNAi screen in primary human hematopoietic stem and progenitor cells,using pooled lentiviral shRNA libraries deconvoluted by next generation sequencing. We identify MAPK14/p38α as a modulator of ex vivo stem cell proliferation and show that pharmacologic inhibition of p38 dramatically enhances the stem cell activity of cultured umbilical cord blood derived hematopoietic cells. p38 inhibitors should thus be considered in strategies aiming at expanding stem cells for clinical benefit. View Publication

The immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins,the widely expressed basic helix-loop-helix transcription factors,contribute to HSC and MPP activity,but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches,we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. E47-deficient progenitors show competent myeloid production in short-term assays in vitro and in vivo. However,long-term myeloid and lymphoid differentiation is compromised because of a progressive loss of HSC self-renewal that is associated with diminished p21 expression and hyperproliferation. The activity of E47 is shown to be cell-intrinsic. Moreover,E47-deficient HSCs and MPPs have altered expression of genes associated with cellular energy metabolism,and the size of the MPP pool but not downstream lymphoid precursors in bone marrow or thymus is rescued in vivo by antioxidant. Together,these observations suggest a role for E47 in the tight control of HSC proliferation and energy metabolism,and demonstrate that E47 is not required for short-term myeloid differentiation. View Publication

The stem cell factor (SCF)/Kit system has served as a classic model in deciphering molecular signaling events in the hematopoietic compartment,and Kit expression is a most critical marker for hematopoietic stem cells (HSCs) and progenitors. However,it remains to be elucidated how Kit expression is regulated in HSCs. Herein we report that a cytoplasmic tyrosine phosphatase Shp2,acting downstream of Kit and other RTKs,promotes Kit gene expression,constituting a Kit-Shp2-Kit signaling axis. Inducible ablation of PTPN11/Shp2 resulted in severe cytopenia in BM,spleen,and peripheral blood in mice. Shp2 removal suppressed the functional pool of HSCs/progenitors,and Shp2-deficient HSCs failed to reconstitute lethally irradiated recipients because of defects in homing,self-renewal,and survival. We show that Shp2 regulates coordinately multiple signals involving up-regulation of Kit expression via Gata2. Therefore,this study reveals a critical role of Shp2 in maintenance of a functional HSC/progenitor pool in adult mammals,at least in part through a kinase-phosphatase-kinase cascade. View Publication