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We assessed viable Pax7(-/-) mice in 129Sv/J background and observed reduced growth and marked muscle wasting together with a complete absence of functional satellite cells. Acute injury resulted in an extreme deficit in muscle regeneration. However,a small number of regenerated myofibers were detected,suggesting the presence of residual myogenic cells in Pax7-deficient muscle. Rare Pax3(+)/MyoD+ myoblasts were recovered from Pax7(-/-) muscle homogenates and cultures of myofiber bundles but not from single myofibers free of interstitial tissues. Finally,we identified Pax3+ cells in the muscle interstitial environment and demonstrated that they coexpressed MyoD during regeneration. Sublaminar satellite cells in hind limb muscle did not express detectable levels of Pax3 protein or messenger RNA. Therefore,we conclude that interstitial Pax3+ cells represent a novel myogenic population that is distinct from the sublaminar satellite cell lineage and that Pax7 is essential for the formation of functional myogenic progenitors from sublaminar satellite cells. View Publication

Homing of hematopoietic stem cells (HSCs) into the bone marrow (BM) is a prerequisite for establishment of hematopoiesis during development and following transplantation. However,the molecular interactions that control homing of HSCs,in particular,of fetal HSCs,are not well understood. Herein,we studied the role of the alpha6 and alpha4 integrin receptors for homing and engraftment of fetal liver (FL) HSCs and hematopoietic progenitor cells (HPCs) to adult BM by using integrin alpha6 gene-deleted mice and function-blocking antibodies. Both integrins were ubiquitously expressed in FL Lin(-)Sca-1(+)Kit(+) (LSK) cells. Deletion of integrin alpha6 receptor or inhibition by a function-blocking antibody inhibited FL LSK cell adhesion to its extracellular ligands,laminins-411 and -511 in vitro,and significantly reduced homing of HPCs to BM. In contrast,the anti-integrin alpha6 antibody did not inhibit BM homing of HSCs. In agreement with this,integrin alpha6 gene-deleted FL HSCs did not display any homing or engraftment defect compared with wild-type littermates. In contrast,inhibition of integrin alpha4 receptor by a function-blocking antibody virtually abrogated homing of both FL HSCs and HPCs to BM,indicating distinct functions for integrin alpha6 and alpha4 receptors during homing of fetal HSCs and HPCs. View Publication

The molecular and cellular requirements for the development of different populations of human dendritic cells (DC) were studied. Conditions were defined that support DC production from lymphoid progenitors but that fail to induce DC formation from peripheral monocytes. The production of these lymphoid-related DC was severely blocked when hematopoietic progenitors overexpressed Ik7,a mutant dominant-negative Ikaros protein. In contrast,Ik7 did not block the formation of DC in conditions supporting the development of monocyte-derived DC. Furthermore,Ik7 did not block the formation of monocyte/macrophages and enhanced granulopoiesis. One of the molecular mechanisms mediated by Ik7 appears to be down-regulation of the flt3-receptor mRNA. Thus,distinct signals control the formation of DC demonstrating that some aspects of DC diversity are determined in part by distinct molecular cues at the hematopoietic level. (Blood. 2000;95:128-137) View Publication

The hypothesis that bystander inflammatory signals promote memory B cell (B(MEM)) self-renewal and differentiation in an antigen-independent manner is critically evaluated herein. To comprehensively address this hypothesis,a detailed analysis is presented examining the response profiles of B-2 lineage B220(+)IgG(+) B(MEM) toward cognate protein antigen in comparison to bystander inflammatory signals. After in vivo antigen encounter,quiescent B(MEM) clonally expand. Surprisingly,proliferating B(MEM) do not acquire germinal center (GC) B cell markers before generating daughter B(MEM) and differentiating into plasma cells or form structurally identifiable GCs. In striking contrast to cognate antigen,inflammatory stimuli,including Toll-like receptor agonists or bystander T cell activation,fail to induce even low levels of B(MEM) proliferation or differentiation in vivo. Under the extreme conditions of adjuvanted protein vaccination or acute viral infection,no detectable bystander proliferation or differentiation of B(MEM) occurred. The absence of a B(MEM) response to nonspecific inflammatory signals clearly shows that B(MEM) proliferation and differentiation is a process tightly controlled by the availability of cognate antigen. View Publication

Wnt signaling is a key regulator of vertebrate heart development; however,specific roles for human cardiomyocyte development remain uncertain. Here we use human embryonic stem cells (hESCs) to analyze systematically in human cardiomyocyte development the expression of endogenous Wnt signaling components,monitor pathway activity,and dissect stage-specific requirements for canonical and noncanonical Wnt signaling mechanisms using small-molecule inhibitors. Our analysis suggests that WNT3 and WNT8A,via FZD7 and canonical signaling,regulate BRACHYURY expression and mesoderm induction; that WNT5A/5B,via ROR2 and noncanonical signaling,regulate MESP1 expression and cardiovascular development; and that later in development WNT2,WNT5A/5B,and WNT11,via FZD4 and FZD6,regulate functional cardiomyocyte differentiation via noncanonical Wnt signaling. Our findings confirm in human development previously proposed roles for canonical Wnt signaling in sequential stages of vertebrate cardiomyogenesis,and identify more precise roles for noncanonical signaling and for individual Wnt signal and Wnt receptor genes in human cardiomyocyte development. View Publication

The termination of activation signals is a critical step in the control of the immune response; perturbation of inhibitory feedback pathways results in profound immune defects culminating in autoimmunity and overwhelming inflammation. FcgammaRIIB receptor is a well described inhibitory receptor. The ligation of B-cell receptor (BCR) and FcgammaRIIB leads to the inhibition of B-cell activation. Numerous studies have demonstrated that the SH2-domain-containing inositol 5-phosphatase SHIP (referred hereto as SHIP-1) is essential in this process. The cDNA encoding a second SH2-domain-containing inositol 5-phosphatase,SHIP-2,has been cloned [Pesesse,Deleu,De Smedt,Drayer and Erneux (1997) Biochem. Biophys. Res. Commun. 239,697-700]. Here we report the distribution of SHIP-2 in mouse tissues: a Western blot analysis of mouse tissues reveals that SHIP-2 is expressed in both haemopoietic and non-haemopoietic cells. In addition to T-cell and B-cell lines,spleen,thymus and lung are shown to coexpress SHIP-1 and SHIP-2. Moreover,SHIP-2 is detected in fibroblasts,heart and different brain areas. SHIP-2 shows a maximal tyrosine phosphorylation and association to Shc after ligation of BCR to FcgammaRIIB but not after stimulation of BCR alone. Our results therefore suggest a possible role for SHIP-2 in the negative regulation of immunocompetent cells. View Publication

OBJECTIVE The aim of this study was to evaluate the distribution,concentration and toxicity of cinnamaldehyde in electronic cigarette (e-cigarette) refill fluids and aerosols. METHODS The distribution and concentration of cinnamaldehyde were determined in 39 e-cigarette refill fluids plus 6 duplicates using gas chromatography and mass spectrometry (GC/MS). A cinnamaldehyde toxicity profile was established for embryonic and adult cells using a live cell imaging assay,immunocytochemistry,the comet assay and a recovery assay. RESULTS Twenty of the 39 refill fluids contained cinnamaldehyde at concentrations that are cytotoxic to human embryonic and lung cells in the MTT assay. Cinnamon Ceylon aerosol produced in a cartomizer-style e-cigarette was cytotoxic. Cinnamon Ceylon aerosols and refill fluid aerosols (80% propylene glycol or cinnamaldehyde/propylene glycol) made using a tank/boxmod e-cigarette were more cytotoxic at 5 V than 3 V. Using GC/MS,aerosols produced at 5 V contained 10 additional peaks not present in aerosol generated at 3 V. One of these,2,3-butandione (diacetyl),was confirmed with an authentic standard. Cinnamaldehyde depolymerised microtubules in human pulmonary fibroblasts. At concentrations that produced no effect in the MTT assay,cinnamaldehyde decreased growth,attachment and spreading; altered cell morphology and motility; increased DNA strand breaks; and increased cell death. At the MTT IC50 concentration,lung cells were unable to recover from cinnamaldehyde after 2 hours of treatment,whereas embryonic cells recovered after 8 hours. CONCLUSIONS Cinnamaldehyde-containing refill fluids and aerosols are cytotoxic,genotoxic and low concentrations adversely affect cell processes and survival. These data indicate that cinnamaldehyde in e-cigarette refill fluids/aerosols may impair homeostasis in the respiratory system. View Publication

Many breast cancer deaths result from tumors acquiring resistance to available therapies. Thus,new therapeutic agents are needed for targeting drug-resistant breast cancers. Drug-refractory breast cancers include HER2+ tumors that have acquired resistance to HER2-targeted antibodies and kinase inhibitors,and Triple-Negative" Breast Cancers (TNBCs) that lack the therapeutic targets Estrogen Receptor� View Publication

BACKGROUND Triple-negative breast cancer (TNBC) has significantly worse prognosis. Acquired chemoresistance remains the major cause of therapeutic failure of TNBC. In clinic,the relapsed TNBC is commonly pan-resistant to various drugs with completely different resistant mechanisms. Investigation of the mechanisms and development of new drugs to target pan-chemoresistance will potentially improve the therapeutic outcomes of TNBC patients. METHODS In this study,1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT),combination index (CI)-isobologram,western blot,ALDEFLUOR analysis,clonogenic assay and immunocytochemistry were used. RESULTS The chemoresistant MDA-MB-231PAC10 cells are highly cross-resistant to paclitaxel (PAC),cisplatin (CDDP),docetaxel and doxorubicin. The MDA-MB-231PAC10 cells are quiescent with significantly longer doubling time (64.9 vs 31.7 h). This may be caused by high expression of p21(Waf1). The MDA-MB-231PAC10 cells express high aldehyde dehydrogenase (ALDH) activity and a panel of embryonic stem cell-related proteins,for example,Oct4,Sox2,Nanog and nuclealisation of HIF2$$ and NF-$$Bp65. We have previously reported that disulfiram (DS),an antialcoholism drug,targets cancer stem cells (CSCs) and enhances cytotoxicity of anticancer drugs. Disulfiram abolished CSC characters and completely reversed PAC and CDDP resistance in MDA-MB-231PAC10 cells. CONCLUSION Cancer stem cells may be responsible for acquired pan-chemoresistance. As a drug used in clinic,DS may be repurposed as a CSC inhibitor to reverse the acquired pan-chemoresistance. View Publication

In Alzheimer’s disease,amyloid beta (Aβ) and tau pathology are thought to drive synapse loss. However,there is limited information on how endogenous levels of tau,Aβ and other biomarkers relate to patient characteristics,or how manipulating physiological levels of Aβ impacts synapses in living adult human brain. Using live human brain slice cultures,we report that Aβ 1-40 and tau release levels vary with donor age and brain region,respectively. Release of other biomarkers such as KLK-6,NCAM-1,and Neurogranin vary between brain region,while TDP-43 and NCAM-1 release is impacted by sex. Pharmacological manipulation of Aβ in either direction results in a loss of synaptophysin puncta,with increased physiological Aβ triggering potentially compensatory synaptic transcript changes. In contrast,treatment with Aβ-containing Alzheimer’s disease brain extract results in post-synaptic Aβ uptake and pre-synaptic puncta loss without affecting synaptic transcripts. These data reveal distinct effects of physiological and pathological Aβ on synapses in human brain tissue. Subject terms: Alzheimer's disease,Alzheimer's disease View Publication

B cell-activating factor belonging to the TNF family (BAFF) plays a critical role in B cell maturation,yet its precise role in B cell differentiation into Ig-secreting cells (ISCs) remains unclear. In this study,we find that upon isolation human naive and memory B (MB) cells have prebound BAFF on their surface,whereas germinal center (GC) B cells lack detectable levels of prebound BAFF. We attribute their lack of prebound BAFF to cell activation,because we demonstrate that stimulation of naive and MB cells results in the loss of prebound BAFF. Furthermore,the absence of prebound BAFF on GC B cells is not related to a lack of BAFF-binding receptors or an inability to bind exogenous BAFF. Instead,our data suggest that accessibility to soluble BAFF is limited within GCs,perhaps to prevent skewing of the conventional B cell differentiation program. In support of this concept,whereas BAFF significantly enhances ISC differentiation in response to T cell-dependent activation,we report for the first time the ability of BAFF to considerably attenuate ISC differentiation of MB cells in response to CpG stimulation,a form of T cell-independent activation. Our data suggest that BAFF may be providing regulatory signals during specific T cell-independent events,which protect the balance between MB cells and ISCs outside GCs. Taken together,these data define a complex role for BAFF in humoral immune responses and show for the first time that BAFF can also play an inhibitory role in B cell differentiation. View Publication

PPAR gamma is activated by diverse ligands and regulates the differentiation of many cell types. Based on evidence that activation of PPAR gamma 2 by rosiglitazone stimulates adipogenesis and inhibits osteoblastogenesis in U-33/gamma 2 cells,a model mesenchymal progenitor of adipocytes and osteoblasts,we postulated that the increase in marrow fat and the decrease in osteoblast number that occur during aging are due to increased PPAR gamma 2 activation. Here,we show that the naturally occurring PPAR gamma ligands 9,10-dihydroxyoctadecenoic acid,and 15-deoxy-Delta(12,14)-PGJ(2),also stimulate adipocytes and inhibit osteoblast differentiation of U-33/gamma 2 cells. Strikingly,9,10-epoxyoctadecenoic acid and the thiazolidine acetamide ligand GW0072 [(+/-)-(2S,5S)-4-(4-(4-carboxyphenyl)butyl)-2-heptyl-4-oxo-5-thaizolidineN,N-dibenzyl-acetamide] prevent osteoblast differentiation,but do not stimulate adipogenesis,whereas 9-hydroxyoctadecadienoic acid stimulates adipogenesis but does not affect osteoblast differentiation. The divergent effects of PPAR gamma 2 ligands on osteoblast and adipocyte differentiation were confirmed in primary murine bone marrow cultures using rosiglitazone and GW0072. These findings indicate that the proadipogenic and antiosteoblastogenic effects of PPAR gamma 2 are mediated by distinct regulatory pathways that can be differentially modulated depending on the nature of the ligand,and they support the idea that increased fatty acid oxidation during aging may inhibit osteoblast differentiation. Moreover,there may be selective PPAR gamma 2 modulators that block the adverse effects of fatty acid oxidation products while retaining beneficial activities such as insulin sensitization. View Publication