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

Germ line mutations in the Adenomatous polyposis coli tumor suppressor gene cause a hereditary form of intestinal tumorigenesis in both mice and man. Here we show that in Apc(Min/+) mice,which carry a heterozygous germ line mutation at codon 850 of Apc,there is progressive loss of immature and mature thymocytes from approximately 80 days of age with complete regression of the thymus by 120 days. In addition,Apc(Min/+) mice show parallel depletion of splenic natural killer (NK) cells,immature B cells,and B progenitor cells in bone marrow due to complete loss of interleukin 7 (IL-7)-dependent B-cell progenitors. Using bone marrow transplantation experiments into wild-type recipients,we have shown that the capacity of transplanted Apc(Min/+) bone marrow cells for T- and B-cell development appears normal. In contrast,although the Apc(Min/+) bone marrow microenvironment supported short-term reconstitution with wild-type bone marrow,Apc(Min/+) animals that received transplants subsequently underwent lymphodepletion. Fibroblast colony-forming unit (CFU-F) colony assays revealed a significant reduction in colony-forming mesenchymal progenitor cells in the bone marrow of Apc(Min/+) mice compared with wild-type animals prior to the onset of lymphodepletion. This suggests that an altered bone marrow microenvironment may account for the selective lymphocyte depletion observed in this model of familial adenomatous polyposis. 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

The multifunctional signaling protein p75 neurotrophin receptor (p75(NTR)) is a central regulator and major contributor to the highly invasive nature of malignant gliomas. Here,we show that neurotrophin-dependent regulated intramembrane proteolysis (RIP) of p75(NTR) is required for p75(NTR)-mediated glioma invasion,and identify a previously unnamed process for targeted glioma therapy. Expression of cleavage-resistant chimeras of p75(NTR) or treatment of animals bearing p75(NTR)-positive intracranial tumors with clinically applicable gamma-secretase inhibitors resulted in dramatically decreased glioma invasion and prolonged survival. Importantly,proteolytic processing of p75(NTR) was observed in p75(NTR)-positive patient tumor specimens and brain tumor initiating cells. This work highlights the importance of p75(NTR) as a therapeutic target,suggesting that gamma-secretase inhibitors may have direct clinical application for the treatment of malignant glioma. View Publication

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder,with no effective treatment currently available. Recently,non-pharmacological therapy,especially gamma frequency stimulation has shown promising therapeutic effects in Alzheimer’s disease (AD) mouse models. Electric field (EF) is a non-invasive biophysical approach for neuronal protection. However,whether EF is beneficial in AD neuropathology remains unknown. In this study,we exposed the P301S tauopathy mouse model to EF at gamma frequency on the head. We demonstrated that EF treatment significantly improved the cognitive impairments in the P301S mice. This was accompanied by reduced tau pathologies,suppressed microglial activation,neuroinflammation and oxidative stress in the tauopathy mouse brain. Moreover,EF treatment induced cell-specific responses in neural cells,with neurons being more susceptible,followed by microglia and oligodendrocytes. EF also had favorable effects on synaptic protein in neurons,inflammatory response and complement signaling in microglia,and myelination in oligodendrocytes. This study provides strong evidence that EF at gamma frequency may have great potential to be a novel therapeutic intervention for P301S by attenuating neuropathology and offering neuroprotection.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13195-025-01859-8. View Publication

Cell-based therapies against HIV/AIDS have been gaining increased interest. Natural killer (NK) cells are a key component of the innate immune system with the ability to kill diverse tumor cells and virus-infected cells. While NK cells have been shown to play an important role in the control of HIV-1 replication,their functional activities are often compromised in HIV-1-infected individuals. We have previously demonstrated the derivation of NK cells from human embryonic stem cells (hESCs) with the ability to potently kill multiple types of tumor cells both in vitro and in vivo. We now demonstrate the derivation of functional NK cells from human induced pluripotent stem cells (iPSCs). More importantly,both hESC- and iPSC-derived NK cells are able to inhibit HIV-1 NL4-3 infection of CEM-GFP cells. Additional studies using HIV-1-infected human primary CD4(+) T cells illustrated that hESC- and iPSC-derived NK cells suppress HIV-1 infection by at least three distinct cellular mechanisms: killing of infected targets through direct lysis,antibody-dependent cellular cytotoxicity,and production of chemokines and cytokines. Our results establish the potential to utilize hESC- and iPSC-derived NK cells to better understand anti-HIV-1 immunity and provide a novel cellular immunotherapeutic approach to treat HIV/AIDS. View Publication

Resting CD4+ T cells are primary targets of early HIV infection events in vivo,but do not readily support HIV replication in vitro. This barrier to infection can be overcome by exposing resting CD4+ T cells to endothelial cells (ECs). ECs line blood vessels and direct T cell trafficking into inflamed tissues. Cell trafficking pathways have been shown to have overlapping roles in facilitating HIV replication,but their relevance to EC-mediated enhancement of HIV susceptibility in resting CD4+ T cells has not previously been examined. We characterized the phenotype of primary human resting CD4+ T cells that became productively infected with HIV when cocultured with primary human blood and lymphatic ECs. The infected CD4+ T cells were primarily central memory cells enriched for high expression of the integrins LFA-1 and VLA-4. ICAM-1 and VCAM-1,the cognate ligands for LFA-1 and VLA-4,respectively,were expressed by the ECs in the coculture. Blocking LFA-1 and VLA-4 on resting CD4+ T cells inhibited infection by 65.4%-96.9%,indicating that engagement of these integrins facilitates EC-mediated enhancement of productive HIV infection in resting CD4+ T cells. The demonstration that ECs influence cellular HIV susceptibility of resting memory CD4+ T cells through cell trafficking pathways engaged during the transmigration of T cells into tissues highlights the physiological relevance of these findings for HIV acquisition and opportunities for intervention. View Publication