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

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems have evolved as an adaptive surveillance and defense mechanism in bacteria and archaea that uses short RNAs to direct degradation of foreign genetic elements. Here,we present our protocol for utilizing the S. pyogenes type II bacterial CRISPR system to achieve sequence-specific genome alterations in human cells. In principle,any genomic sequence of the form N(19)NGG can be targeted with the generation of custom guide RNA (gRNA) which functions to direct the Cas9 protein to genomic targets and induce DNA cleavage. Here,we describe our methods for designing and generating gRNA expression constructs either singly or in a multiplexed manner,as well as optimized protocols for the delivery of Cas9-gRNA components into human cells. Genomic alterations at the target site are then introduced either through nonhomologous end joining (NHEJ) or through homologous recombination (HR) in the presence of an appropriate donor sequence. This RNA-guided editing tool offers greater ease of customization and synthesis in comparison to existing sequence-specific endonucleases and promises to become a highly versatile and multiplexable human genome engineering platform. View Publication

Aim Reactive oxygen species (ROS) produced by enzymes of the NADPH oxidase family serve as second messengers for cellular signaling. Processes such as differentiation and proliferation are regulated by NADPH oxidases. In the intestine,due to the exceedingly fast and constant renewal of the epithelium both processes have to be highly controlled and balanced. Nox1 is the major NADPH oxidase expressed in the gut,and its function is regulated by cytosolic subunits such as NoxO1. We hypothesize that the NoxO1-controlled activity of Nox1 contributes to a proper epithelial homeostasis and renewal in the gut. Results NoxO1 is highly expressed in the colon. Knockout of NoxO1 reduces the production of superoxide in colon crypts and is not subsidized by an elevated expression of its homolog p47phox. Knockout of NoxO1 increases the proliferative capacity and prevents apoptosis of colon epithelial cells. In mouse models of dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS induced colon cancer,NoxO1 has a protective role and may influence the population of natural killer cells. Conclusion NoxO1 affects colon epithelium homeostasis and prevents inflammation. View Publication

Durable response to chimeric antigen receptor T (CART) cell therapy remains limited in part due to CART cell exhaustion. Here,we investigate the regulation of CART cell exhaustion with three independent approaches including: a genome-wide CRISPR knockout screen using an in vitro model for exhaustion,RNA and ATAC sequencing on baseline and exhausted CART cells,and RNA and ATAC sequencing on pre-infusion CART cell products from responders and non-responders in the ZUMA-1 clinical trial. Each of these approaches identify interleukin (IL)-4 as a regulator of CART cell dysfunction. Further,IL-4-treated CD8+ CART cells develop signs of exhaustion independently of the presence of CD4+ CART cells. Conversely,IL-4 pathway editing or the combination of CART cells with an IL-4 monoclonal antibody improves antitumor efficacy and reduces signs of CART cell exhaustion in mantle cell lymphoma xenograft mouse models. Therefore,we identify both a role for IL-4 in inducing CART exhaustion and translatable approaches to improve CART cell therapy. The application and therapeutic success of CAR-T cell approaches are limited by the development of T cell exhaustion. Here,Stewart et al discover a role for IL-4 in driving CD8+ CAR-T cell exhaustion and demonstrate the improvement of CAR-T cell effectivity with interruption of IL-4 signalling. View Publication

Heme oxygenase-1 (HO-1,HMOX1 ) degrades heme protecting cells from heme-induced oxidative damage. Beyond its well-established cellular functions,heme has emerged as a stabilizer of G-quadruplexes. These secondary DNA structures interfere with DNA replication. We recently revealed that nuclear HO-1 colocalizes with DNA G-quadruplexes and promotes their removal. Here,we investigate whether HO-1 safeguards cells against replication stress. Experiments were conducted in control and HMOX1 -deficient HEK293T cell lines. Immunostaining unveiled that DNA G-quadruplexes accumulated in the absence of HO-1,the effect that was further enhanced in response to δ-aminolevulinic acid (ALA),a substrate in heme synthesis. This was associated with replication stress,as evidenced by an elevated proportion of stalled forks analyzed by fiber assay. We observed the same effects in hematopoietic stem cells isolated from Hmox1 knockout mice and in a lymphoblastoid cell line from an HMOX1 -deficient patient. Interestingly,in the absence of HO-1,the speed of fork progression was higher,and the response to DNA conformational hindrance less stringent,indicating dysfunction of the PARP1-p53-p21 axis. PARP1 activity was not decreased in the absence of HO-1. Instead,we observed that HO-1 deficiency impairs the nuclear import and accumulation of p53,an effect dependent on the removal of excess heme. We also demonstrated that administering ALA is a more specific method for increasing intracellular free heme compared to treatment with hemin,which in turn induces strong lipid peroxidation. Our results indicate that protection against replication stress is a universal feature of HO-1,presumably contributing to its widely recognized cytoprotective activity. View Publication

The ability of NK cells to directly recognize pathogens and be activated via Toll-like receptors (TLR) is increasingly recognized. Nevertheless,controversial results on the NK cell ability to be directly activated by lipopolysaccharide (LPS),the ligand of TLR4,have been recently reported. To start elucidating the reasons explaining the contrasting observations of the literature,we focused on the potential role of currently used NK cell purification procedures to condition putative NK cell responsiveness to LPS. To do so,human NK cells were isolated by negative selection,using three different commercial kits,to be comparatively evaluated for the production of IFNgamma in response to ultra-pure LPS and/or IL-2. Despite the lack of surface TLR4,we found that two out of the three NK cell-enriched populations released IFNgamma (and one of the two,IL-12p70 as well) in response to the LPS plus IL-2 combination,whereas the last one did not. However,the two LPS plus IL-2-responsive NK cell populations were found variably contaminated with 6-sulfo LacNAc(+) dendritic cells (slanDC),demonstrated responsible for triggering,via the production of IL-12p70 in response to LPS,the release of IFNgamma by IL-2-stimulated NK cells. Accordingly,slanDC depletion completely abrogated the capacity to produce both IL-12p70 and IFNgamma in response to LPS plus IL-2 by slanDC-containing NK cells. Taken together,our data uncover that two commercially available kits,specifically designed to isolate NK cells by negative selection,also co-purify variable amounts of slanDC. The latter cells may dramatically affect the outcome of experiments carried on to evaluate NK cell responsiveness to TLR agonists such as LPS. View Publication

The activating receptor NKG2D,expressed by natural killer (NK) cells and CD8(+) T cells,has a role in the specific killing of transformed cells. We examined NKG2D expression in patients with glioblastoma multiforme and found that NKG2D was downregulated on NK cells and CD8(+) T cells. Expression of NKG2D on lymphocytes significantly increased following tumor resection and correlated with an increased ability to kill NKG2D ligand-positive tumor targets. Despite the presence of soluble NKG2D ligands in the sera of glioblastoma patients,NKG2D downregulation was primarily caused by tumor-derived tumor growth factor-beta,suggesting that blocking of this cytokine may have therapeutic benefit. View Publication

Cells demonstrate plasticity following injury,but the extent of this phenomenon and the cellular mechanisms involved remain underexplored. Using single-cell RNA sequencing (scRNA-seq) and lineage tracing,we uncover that myoepithelial cells (MECs) of the submucosal glands (SMGs) proliferate and migrate to repopulate the airway surface epithelium (SE) in multiple injury models. Specifically,SMG-derived cells display multipotency and contribute to basal and luminal cell types of the SMGs and SE. Ex vivo expanded MECs have the potential to repopulate and differentiate into SE cells when grafted onto denuded airway scaffolds. Significantly,we find that SMG-like cells appear on the SE of both extra- and intra-lobular airways of large animal lungs following severe injury. We find that the transcription factor SOX9 is necessary for MEC plasticity in airway regeneration. Because SMGs are abundant and present deep within airways,they may serve as a reserve cell source for enhancing human airway regeneration. View Publication

Retinoid-related molecules (RRM) are novel agents with tumor-selective cytotoxic/antiproliferative activity,a different mechanism of action from classic retinoids and no cross-resistance with other chemotherapeutics. ST1926 and CD437 are prototypic RRMs,with the former currently undergoing phase I clinical trials. We show here that ST1926,CD437,and active congeners cause DNA damage. Cellular and subcellular COMET assays,H2AX phosphorylation (gamma-H2AX),and scoring of chromosome aberrations indicate that active RRMs produce DNA double-strand breaks (DSB) and chromosomal lesions in NB4,an acute myeloid leukemia (AML) cell line characterized by high sensitivity to RRMs. There is a direct quantitative correlation between the levels of DSBs and the cytotoxic/antiproliferative effects induced by RRMs. NB4.437r blasts,which are selectively resistant to RRMs,do not show any sign of DNA damage after treatment with ST1926,CD437,and analogues. DNA damage is the major mechanism underlying the antileukemic activity of RRMs in NB4 and other AML cell lines. In accordance with the S-phase specificity of the cytotoxic and antiproliferative responses of AML cells to RRMs,increases in DSBs are maximal during the S phase of the cell cycle. Induction of DSBs precedes inhibition of DNA replication and is associated with rapid activation of ataxia telangectasia mutated,ataxia telangectasia RAD3-related,and DNA-dependent protein kinases with subsequent stimulation of the p38 mitogen-activated protein kinase. Inhibition of ataxia telangectasia mutated and DNA-dependent protein kinases reduces phosphorylation of H2AX. Cells defective for homologous recombination are particularly sensitive to ST1926,indicating that this process is important for the protection of cells from the RRM-dependent DNA damage and cytotoxicity. View Publication

The mTOR protein is a master regulator of cell growth and proliferation,and inhibitors of its kinase activity have the potential to become new class of anticancer drugs. Starting from quinoline 1,which was identified in a biochemical mTOR assay,we developed a tricyclic benzonaphthyridinone inhibitor 37 (Torin1),which inhibited phosphorylation of mTORC1 and mTORC2 substrates in cells at concentrations of 2 and 10 nM,respectively. Moreover,Torin1 exhibits 1000-fold selectivity for mTOR over PI3K (EC(50) = 1800 nM) and exhibits 100-fold binding selectivity relative to 450 other protein kinases. Torin1 was efficacious at a dose of 20 mg/kg in a U87MG xenograft model and demonstrated good pharmacodynamic inhibition of downstream effectors of mTOR in tumor and peripheral tissues. These results demonstrate that Torin1 is a useful probe of mTOR-dependent phenomena and that benzonaphthridinones represent a promising scaffold for the further development of mTOR-specific inhibitors with the potential for clinical utility. View Publication

BACKGROUND There is an unmet need for developing faithful animal models for preclinical evaluation of immunotherapy. The current approach to generate preclinical models for immunotherapy evaluation has been to transplant CD34+ cells from umbilical cord blood into immune-deficient mice followed by implantation of patient derived tumor cells. However,current models are associated with high tumor rejection rate secondary to the allograft vs. tumor response from human leukocyte antigen (HLA) mismatches. We herein report the first development of a novel,humanized patient-derived xenograft (PDX) model using autologous CD34+ cells from bone marrow aspirate obtained from a patient with metastatic clear cell renal cell carcinoma (mRCC) from whom a PDX had been developed. CASE DESCRIPTION This is a 68-year-old Caucasian man diagnosed with mRCC with metastasis to the liver in 2014. He was treated with sunitinib +/- AGS-003 and underwent a cytoreductive right nephrectomy,left adrenalectomy and partial liver resection. PDX was generated using resected nephrectomy specimen. After surgery,patient received multiple lines of standard of care therapy including sunitinib,axitinib,bevacizumab,everolimus and cabozantinib. While progressing on cabozantinib,he was treated with nivolumab. Seven years after initiation of nivolumab,and 4 years after stopping systemic therapy,he remains in complete remission. To generate autologous PDX model,bone marrow aspirate was performed and CD34+ hematopoietic stem/progenitor cells (HSPCs) were isolated and injected into 150 rad irradiated non-obese diabetic scid gamma null (NSG) mice. At 11 weeks post-transplant,the matched patient PDX was injected subcutaneously into the humanized mice and the mice were treated with nivolumab. CONCLUSIONS Our case represents successful therapy of nivolumab in mRCC. Furthermore,HPSCs obtained from a single bone marrow aspirate were able to reconstitute an immune system in the mice that allowed nivolumab to inhibit the tumor growth of PDX and recapitulated the durable remission observed in the patient with nivolumab. We observed the reconstitution of human T cells,B cells and natural killer (NK) cells and unlike the humanized mouse model using cord blood,our model system eliminates the tumor rejection from mis-matched HLA. Our autologous humanized renal cell carcinoma (RCC) PDX model provides an effective tool to study immunotherapy in a preclinical setting. View Publication

Microfold (M) cells residing in the follicle-associated epithelium (FAE) of the gut-associated lymphoid tissue are specialized for antigen uptake to initiate mucosal immune responses. The molecular machinery and biological significance of M cell differentiation,however,remain to be fully elucidated. Here,we demonstrate that Sox8,a member of the SRY-related HMG box transcription factor family,is specifically expressed by M cells in the intestinal epithelium. The expression of Sox8 requires activation of RANKL-RelB signaling. Chromatin immunoprecipitation and luciferase assays revealed that Sox8 directly binds the promoter region of Gp2 to increase Gp2 expression,which is the hallmark of functionally mature M cells. Furthermore,genetic deletion of Sox8 causes a marked decrease in the number of mature M cells,resulting in reduced antigen uptake in Peyer's patches. Consequently,juvenile Sox8-deficient mice showed attenuated germinal center reactions and antigen-specific IgA responses. These findings indicate that Sox8 plays an essential role in the development of M cells to establish mucosal immune responses. View Publication

AbstractIntroduction
Preeclampsia and other placental pathologies are characterized by a lack of spiral artery remodeling associated with insufficient invasion by extravillous trophoblast cells (EVT). Because trophoblast invasion occurs in early pregnancy when access to human placental tissue is limited,there is a need for model systems for the study of trophoblast differentiation and invasion. Human embryonic stem cells (hESC) treated with BMP4- differentiate to trophoblast,and express HLA-G,a marker of EVT. The goals of the present study were to further characterize the HLA-G+ cells derived from BMP4-treated hESC,and determine their suitability as a model.
Methods
HESC were treated with BMP4 under 4% or 20% oxygen and tested in Matrigel invasion chambers. Both BMP4-treated hESC and primary human placental cells were separated into HLA-G+ and HLA-G−/TACSTD2+ populations with immunomagnetic beads and expression profiles analyzed by microarray.
Results
There was a 10-fold increase in invasion when hESC were BMP4-treated. There was also an independent,stimulatory effect of oxygen on this process. Invasive cells expressed trophoblast marker KRT7,and the majority were also HLA-G+. Gene expression profiles revealed that HLA-G+,BMP4-treated hESC were similar to,but distinct from,HLA-G+ cells isolated from first trimester placentas. Whereas HLA-G+ and HLA-G− cells from first trimester placentas had highly divergent gene expression profiles,HLA-G+ and HLA-G− cells from BMP4-treated hESC had somewhat similar profiles,and both expressed genes characteristic of early trophoblast development.
Conclusions
We conclude that hESC treated with BMP4 provide a model for studying transition to the EVT lineage. View Publication