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

Bat immunity has received increasing attention because some bat species are being decimated by the fungal disease,White Nose Syndrome,while other species are potential reservoirs of zoonotic viruses. Identifying specific immune processes requires new specific tools and reagents. In this study,we describe a new mouse monoclonal antibody (mAb) reactive with Eptesicus fuscus immunoglobulins. The epitope recognized by mAb BT1-4F10 was localized to immunoglobulin light (lambda) chains; hence,the mAb recognized serum immunoglobulins and B lymphocytes. The BT1-4F10 epitope appeared to be restricted to Microchiropteran immunoglobulins and absent from Megachiropteran immunoglobulins. Analyses of sera and other E. fuscus fluids showed that most,if not all,secreted immunoglobulins utilized lambda light chains. Finally,mAb BT1-4F10 permitted the identification of B cell follicles in splenic white pulp. This Microchiropteran-specific mAb has potential utility in seroassays; hence,this reagent may have both basic and practical applications for studying immune process. View Publication

Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in organ transplant recipients. The use of granulocyte-colony stimulating factor (G-CSF)-mobilized stem cells from HCMV seropositive donors is suggested to double the risk of late-onset HCMV disease and chronic graft-versus-host disease in recipients when compared to conventional bone marrow transplantation with HCMV seropositive donors,although the etiology of the increased risk is unknown. To understand mechanisms of HCMV transmission in patients receiving G-CSF-mobilized blood products,we generated a NOD-scid IL2Rγ(c)(null)-humanized mouse model in which HCMV establishes latent infection in human hematopoietic cells. In this model,G-CSF induces the reactivation of latent HCMV in monocytes/macrophages that have migrated into organ tissues. In addition to establishing a humanized mouse model for systemic and latent HCMV infection,these results suggest that the use of G-CSF mobilized blood products from seropositive donors pose an elevated risk for HCMV transmission to recipients. View Publication

The synthetic retinoid 6-[3-(adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437),which can bind to and activate the nuclear retinoic acid receptors beta and gamma (RARbeta/gamma),is a potent inducer of apoptosis in various cancer cell lines. However,this effect was reported to be independent of RARs. In this study,we compared and contrasted the potencies and mechanisms of action of CD437 and several other receptor-selective retinoids in induction of apoptosis and modulation of squamous differentiation in UMSCC22B human head and neck squamous cell carcinoma cell line. CD437 and the structurally related retinoid CD2325 exhibited almost equal potency in inducing apoptosis,whereas several other retinoids failed to induce apoptosis. The RAR-specific pan antagonist AGN193109 failed to suppress CD437-induced apoptosis,indicating that the induction of apoptosis by CD437 was RAR-independent. c-Fos expression was induced by CD437 and CD2325 that induced apoptosis in the cell line but not by other retinoids that failed to induce apoptosis,suggesting a role for c-Fos in CD437-induced apoptosis. At low concentration (0.01 microM),CD437 shared with several other receptor-selective retinoids the ability to suppress the mRNA levels of the squamous differentiation markers Spr1,involucrin,and cytokeratin 1. This effect of CD437 could be blocked by AGN193109. We conclude that CD437 can exert its effects in UMSCC22B human human head and neck squamous cell carcinoma cells by at least two mechanisms: RAR-mediated suppression of squamous differentiation and RAR-independent induction of apoptosis. View Publication

MicroRNAs (miRNAs) are pivotal for regulation of hematopoiesis but their critical targets remain largely unknown. Here,we show that ectopic expression of miR-17,-20,-93 and -106,all AAAGUGC seed-containing miRNAs,increases proliferation,colony outgrowth and replating capacity of myeloid progenitors and results in enhanced P-ERK levels. We found that these miRNAs are endogenously and abundantly expressed in myeloid progenitors and down-regulated in mature neutrophils. Quantitative proteomics identified sequestosome 1 (SQSTM1),an ubiquitin-binding protein and regulator of autophagy-mediated protein degradation,as a major target for these miRNAs in myeloid progenitors. In addition,we found increased expression of Sqstm1 transcripts during CSF3-induced neutrophil differentiation of 32D-CSF3R cells and an inverse correlation of SQSTM1 protein levels and miR-106 expression in AML samples. ShRNA-mediated silencing of Sqstm1 phenocopied the effects of ectopic miR-17/20/93/106 expression in hematopoietic progenitors in vitro and in mice. Further,SQSTM1 binds to the ligand-activated colony-stimulating factor 3 receptor (CSF3R) mainly in the late endosomal compartment,but not in LC3 positive autophagosomes. SQSTM1 regulates CSF3R stability and ligand-induced mitogen-activated protein kinase signaling. We demonstrate that AAAGUGC seed-containing miRNAs promote cell expansion,replating capacity and signaling in hematopoietic cells by interference with SQSTM1-regulated pathways. View Publication

During hematopoietic differentiation of human embryonic stem cells (hESCs),early hematopoietic progenitors arise along with endothelial cells within the CD34(+) population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays,their phenotype has not been defined. Here,using hESC differentiation in coculture with OP9 stromal cells,we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45,persisted on differentiating hematopoietic cells,and reliably separated the hematopoietic CD34(+) population from CD34(+)CD43(-)CD31(+)KDR(+) endothelial and CD34(+)CD43(-)CD31(-)KDR(-) mesenchymal cells. Furthermore,we demonstrated that the first-appearing CD34(+)CD43(+)CD235a(+)CD41a(+/-)CD45(-) cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34(+)CD43(+)CD41a(-)CD235a(-)CD45(-) cells. These cells were negative for lineage-specific markers (Lin(-)),expressed KDR,VE-cadherin,and CD105 endothelial proteins,and expressed GATA-2,GATA-3,RUNX1,C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34(+)CD43(+)CD45(-)Lin(-) cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34(+)CD43(+)CD45(+)Lin(-) cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3(high)GATA3(low)RUNX1(low)PU1(high)MPO(high)IL7RA(high) gene expression profile. View Publication

BACKGROUND/AIMS [corrected] Embryonic stem cells (ES cells) have the capacity to propagate indefinitely,maintain pluripotency,and differentiate into any cell type under defined conditions. As a result,they are considered to be the best model system for research into early embryonic development. AICA ribonucleotide (AICAR) is an activator of AMP-activated protein kinase (AMPK) that is thought to affect ES cell function,but its role in ES cell fate decision is unclear. METHODS In this study,we performed microarray analysis to investigate AICAR downstream targets and further understand its effect on ES cells. RESULTS Our microarray data demonstrated that AICAR can significantly up-regulate pluripotency-associated genes and down-regulate differentiation-associated transcription factors. Although AICAR cannot maintain ES cell identity without LIF,it can antagonize the action of RA-induced differentiation. Using those differentially expressed genes identified,we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with the Database for Annotation,Visualization and Integrated Discovery (DAVID) online system. AICAR was not only shown to influence the AMPK pathway,but also act on other signaling pathways such as BMP,MAPK and TGF-β,to maintain the stemness of J1 ES cells. Furthermore,AICAR modulated ES cell epigenetic modification by altering the expression of epigenetic-associated proteins,including Dnmt3a,Dnmt3b,Smarca2,Mbd3,and Arid1a,or through regulating the transcription of long intervening non-coding RNA (lincRNA). CONCLUSION Taken together,our work suggests that AICAR is capable of maintaining ES cell self-renewal and pluripotency,which could be useful in future medical treatment. View Publication

Concomitant inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. The difficulty of discovering kinase inhibitors with an appropriate multitarget profile has,however,necessitated the application of combination therapies,which can pose major clinical development challenges. Epigenetic reader domains of the bromodomain family have recently emerged as new targets for cancer therapy. Here we report that several clinical kinase inhibitors also inhibit bromodomains with therapeutically relevant potencies and are best classified as dual kinase-bromodomain inhibitors. Nanomolar activity on BRD4 by BI-2536 and TG-101348,which are clinical PLK1 and JAK2-FLT3 kinase inhibitors,respectively,is particularly noteworthy as these combinations of activities on independent oncogenic pathways exemplify a new strategy for rational single-agent polypharmacological targeting. Furthermore,structure-activity relationships and co-crystal structures identify design features that enable a general platform for the rational design of dual kinase-bromodomain inhibitors. View Publication

Vascular endothelial growth factor (VEGF) and stem cell factor (SCF) act as growth factors for the hemangioblast,an embryonic progenitor of the hematopoietic and endothelial lineages. Because thrombopoietin (TPO) and its receptor,c-Mpl,regulate primitive hematopoietic populations,including bone marrow hematopoietic stem cells,we investigated whether TPO acts on the hemangioblasts that derive from differentiation of embryonic stem cells in vitro. Reverse transcriptase polymerase chain reaction analysis detected expression of c-Mpl beginning on day 3 of embryoid body differentiation when the hemangioblast first arises. In assays of the hemangioblast colony-forming cell (BL-CFC),TPO alone supported BL-CFC formation and nearly doubled the number of BL-CFC when added together with VEGF and SCF. When replated under the appropriate conditions,TPO-stimulated BL-CFC gave rise to secondary hematopoietic colonies,as well as endothelial cells,confirming their nature as hemangioblasts. Addition of a neutralizing anti-VEGF antibody did not block TPO enhancement of BL-CFC formation,suggesting that TPO acts independently of VEGF. These results establish that Mpl signaling plays a role in the earliest stages of hematopoietic development and that TPO represents a third growth factor influencing hemangioblast formation. View Publication

Previous studies in the mouse indicated that ARID3A plays a critical role in the first cell fate decision required for generation of trophectoderm (TE). Here,we demonstrate that ARID3A is widely expressed during mouse and human placentation and essential for early embryonic viability. ARID3A localizes to trophoblast giant cells and other trophoblast-derived cell subtypes in the junctional and labyrinth zones of the placenta. Conventional Arid3a knockout embryos suffer restricted intrauterine growth with severe defects in placental structural organization. Arid3a null placentas show aberrant expression of subtype-specific markers as well as significant alteration in cytokines,chemokines and inflammatory response-related genes,including previously established markers of human placentation disorders. BMP4-mediated induction of trophoblast stem (TS)-like cells from human induced pluripotent stem cells results in ARID3A up-regulation and cytoplasmic to nuclear translocation. Overexpression of ARID3A in BMP4-mediated TS-like cells up-regulates TE markers,whereas pluripotency markers are down-regulated. Our results reveal an essential,conserved function for ARID3A in mammalian placental development through regulation of both intrinsic and extrinsic developmental programs. View Publication

OBJECTIVE: The role of semaphorins in tumor progression is still poorly understood. In this study,we aimed at elucidating the regulatory role of semaphorin 3A (SEMA3A) in primary tumor growth and metastatic dissemination. METHODS AND RESULTS: We used 3 different experimental approaches in mouse tumor models: (1) overexpression of SEMA3A in tumor cells,(2) systemic expression of SEMA3A following liver gene transfer in mice,and (3) tumor-targeted release of SEMA3A using gene modified Tie2-expressing monocytes as delivery vehicles. In each of these experimental settings,SEMA3A efficiently inhibited tumor growth by inhibiting vessel function and increasing tumor hypoxia and necrosis,without promoting metastasis. We further show that the expression of the receptor neuropilin-1 in tumor cells is required for SEMA3A-dependent inhibition of tumor cell migration in vitro and metastatic spreading in vivo. CONCLUSIONS: In sum,both systemic and tumor-targeted delivery of SEMA3A inhibits tumor angiogenesis and tumor growth in multiple mouse models; moreover,SEMA3A inhibits the metastatic spreading from primary tumors. These data support the rationale for further investigation of SEMA3A as an anticancer molecule. View Publication

The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds,UM171 being the prototype,is independent of suppression of the aryl hydrocarbon receptor,which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy. View Publication

BACKGROUND AIMS microRNAs (miRNAs) are small non-coding RNAs that bind to 3'UTR regions of mRNAs to promote their degradation or block their translation. Mice with disruption of the trefoil factor 1 gene (Tff1) develop gastric neoplasms. We studied these mice to identify conserved miRNA networks involved in gastric carcinogenesis. METHODS We performed next-generation miRNA sequencing analysis of normal gastric tissues (based on histology) from subjects without evidence of gastric neoplasm from patients (n=64) and TFF1-knockout mice (n=22). We validated our findings using 270 normal gastric tissues (including 61 samples from patients without evidence of neoplastic lesions) and 234 gastric tumor tissues from 3 separate cohorts of patients and from mice. We performed molecular and functional assays using cell lines (MKN28,MKN45,STKM2,and AGS cells),gastric organoids,and mice with xenograft tumors. RESULTS We identified 117 miRNAs that were significantly deregulated in mouse and human gastric tumor tissues,compared with non-tumor tissues. We validated changes in levels of 6 miRNAs by quantitative real-time PCR analyses of neoplastic gastric tissues from mice (n=39) and 3 independent cohorts patients (332 patients total). We found levels of MIR135B-5p,MIR196B-5p,and MIR92A-5p to be increased in tumor tissues whereas levels of MIR143-3p,MIR204-5p,and MIR133-3p were decreased in tumor tissues. Levels of MIR143-3p were reduced not only in gastric cancer tissues,but also in normal tissues adjacent to tumors in humans and low-grade dysplasia in mice. Transgenic expression of MIR143-3p in gastric cancer cell lines reduced their proliferation and restored their sensitivity to cisplatin. AGS cells with stable transgenic expression of MIR143-3p grew more slowly as xenograft tumors in mice than control AGS cells; tumor growth from AGS cells that expressed MIR143-3p,but not control cells,was sensitive to cisplatin. We identified and validated bromodomain containing 2 (BRD2) as a direct target of MIR143-3p; increased levels of BRD2 in gastric tumors associated with shorter survival times of patients. CONCLUSIONS In an analysis of miRNA profiles of gastric tumors from mice and human patients,we identified a conserved signature associated with early stages of gastric tumorigenesis. Strategies to restore MIR143-3p or inhibit BRD2 might be developed for treatment of gastric cancer. View Publication