技术资料

Caveolin 1 (Cav-1) is a major protein of a specific membrane lipid raft known as caveolae. Cav-1 interacts with the gp41 of the human immunodeficiency virus (HIV) envelope,but the role of Cav-1 in HIV replication and pathogenesis is not known. In this report,we demonstrate that HIV infection in primary human monocyte-derived macrophages (MDMs),THP-1 macrophages,and U87-CD4 cells results in a dramatic upregulation of Cav-1 expression mediated by HIV Tat. The activity of p53 is essential for Tat-induced Cav-1 expression,as our findings show enhanced phosphorylation of serine residues at amino acid positions 15 and 46 in the presence of Tat with a resulting Cav-1 upregulation. Furthermore,inhibition of p38 mitogen-activated protein kinase (MAPK) blocked phosphorylation of p53 in the presence of Tat. Infection studies of Cav-1-overexpressing cells reveal a significant reduction of HIV production. Taken together,these results suggest that HIV infection enhances the expression of Cav-1,which subsequently causes virus reduction,suggesting that Cav-1 may contribute to persistent infection in macrophages. View Publication

OBJECTIVE: To test the graft-promoting effects of mesenchymal stem cells (MSCs) in a cynomolgus monkey model of islet/bone marrow transplantation. RESEARCH DESIGN AND METHODS: Cynomolgus MSCs were obtained from iliac crest aspirate and characterized through passage 11 for phenotype,gene expression,differentiation potential,and karyotype. Allogeneic donor MSCs were cotransplanted intraportally with islets on postoperative day (POD) 0 and intravenously with donor marrow on PODs 5 and 11. Recipients were followed for stabilization of blood glucose levels,reduction of exogenous insulin requirement (EIR),C-peptide levels,changes in peripheral blood T regulatory cells,and chimerism. Destabilization of glycemia and increases in EIR were used as signs of rejection; additional intravenous MSCs were administered to test the effect on reversal of rejection. RESULTS: MSC phenotype and a normal karyotype were observed through passage 11. IL-6,IL-10,vascular endothelial growth factor,TGF-β,hepatocyte growth factor,and galectin-1 gene expression levels varied among donors. MSC treatment significantly enhanced islet engraftment and function at 1 month posttransplant (n = 8),as compared with animals that received islets without MSCs (n = 3). Additional infusions of donor or third-party MSCs resulted in reversal of rejection episodes and prolongation of islet function in two animals. Stable islet allograft function was associated with increased numbers of regulatory T-cells in peripheral blood. CONCLUSIONS: MSCs may provide an important approach for enhancement of islet engraftment,thereby decreasing the numbers of islets needed to achieve insulin independence. Furthermore,MSCs may serve as a new,safe,and effective antirejection therapy. View Publication

AIMS: Macrophage scavenger receptor A (SR-A) and class B scavenger receptor CD36 (CD36) contribute to foam cell formation and atherogenesis via uptake of modified lipoproteins. So far,the role of these scavenger receptors has been studied mainly using knockout models totally lacking these receptors. We studied the role of SR-A and CD36 in foam cell formation and atherogenesis by RNA interference (RNAi)-mediated silencing,which is a clinically feasible method to down-regulate the expression of these receptors. METHODS AND RESULTS: We constructed lentivirus vectors encoding short hairpin RNAs (shRNAs) against mouse SR-A and CD36. Decreased SR-A but not CD36 expression led to reduced foam cell formation caused by acetylated low-density lipoprotein (LDL) in mouse macrophages,whereas the uptake of oxidized LDL was not altered. More importantly,silencing of SR-A upregulates CD36 and vice versa. In LDL receptor-deficient apolipoprotein B100 (LDLR(-/-)ApoB(100/100)) mice kept on a western diet,silencing of either SR-A or CD36 in bone marrow cells led to a marked decrease (37.4 and 34.2%,respectively) in cross-sectional lesion area,whereas simultaneous silencing of both receptors was not effective. CONCLUSION: Our results suggest that silencing of either SR-A or CD36 alone reduces atherogenesis in mice. However,due to reciprocal upregulation,silencing of both SR-A and CD36 is not effective. View Publication

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state,and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics,these two reprogramming methods reset genomic methylation,an epigenetic modification of DNA that influences gene expression,leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin,which favours their differentiation along lineages related to the donor cell,while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming,or by treatment of iPSCs with chromatin-modifying drugs. In contrast,the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming,which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment. View Publication

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy with one of the worst outcomes among all cancers. PDA often recurs after initial treatment to result in patient death despite the use of chemotherapy or radiation therapy. PDA contains a subset of tumor-initiating cells capable of extensive self-renewal known as cancer stem cells (CSC),which may contribute to therapeutic resistance and metastasis. At present,conventional chemotherapy and radiotherapy are largely ineffective in depleting CSC pool,suggesting the need for novel therapies that specifically target the cancer-sustaining stem cells for tumor eradication and to improve the poor prognosis of PDA patients. In this study,we report that death receptor 5 (DR5) is enriched in pancreatic CSCs compared with the bulk of the tumor cells. Treating a collection of freshly generated patient-derived PDA xenografts with gemcitabine,the first-line chemotherapeutic agent for PDA,is initially effective in reducing tumor size,but largely ineffective in diminishing the CSC populations,and eventually culminated in tumor relapse. However,a combination of tigatuzumab,a fully humanized DR5 agonist monoclonal antibody,with gemcitabine proved to be more efficacious by providing a double hit to kill both CSCs and bulk tumor cells. The combination therapy produced remarkable reduction in pancreatic CSCs,tumor remissions,and significant improvements in time to tumor progression in a model that is considered more difficult to treat. These data provide the rationale to explore the DR5-directed therapies in combination with chemotherapy as a therapeutic option to improve the current standard of care for pancreatic cancer patients. View Publication

BACKGROUND AND AIMS: Atherosclerosis is known to be an inflammatory disease and there is increasing evidence that chylomicron remnants (CMR),the lipoproteins which carry dietary fats in the blood,cause macrophage foam cell formation and inflammation. In early atherosclerosis the frequency of activated monocytes in the peripheral circulation is increased,and clearance of CMR from blood may be delayed,however,whether CMR contribute directly to monocyte activation and subsequent egress into the arterial wall has not been established. Here,the contribution of CMR to activation of monocyte pro-inflammatory pathways was assessed using an in vitro model. METHODS AND RESULTS: Primary human monocytes and CMR-like particles (CRLP) were used to measure several endpoints of monocyte activation. Treatment with CRLP caused rapid and prolonged generation of reactive oxygen species by monocytes. The pro-inflammatory chemokines MCP-1 and IL-8 were secreted in nanogram quantities by the cells in the absence of CRLP. IL-8 secretion was transiently increased after CRLP treatment,and CRLP maintained secretion in the presence of pharmacological inhibitors of IL-8 production. In contrast,exposure to CRLP significantly reduced MCP-1 secretion. Chemotaxis towards MCP-1 was increased in monocytes pre-exposed to CRLP and was reversed by addition of exogenous MCP-1. CONCLUSION: Our findings indicate that CRLP activate human monocytes and augment their migration in vitro by reducing cellular MCP-1 expression. Our data support the current hypothesis that CMR contribute to the inflammatory milieu of the arterial wall in early atherosclerosis,and suggest that this may reflect direct interaction with circulating blood monocytes. View Publication

Human embryonic stem cells (hESCs) offer an inexhaustible supply of human somatic cell types through their ability to self-renew while retaining pluripotency. As such,hESC-derived cell types are important for applications ranging from in vitro modeling to therapeutic use. However,for their full potential to be realized,both the growth of the undifferentiated cells and their derivatives must be performed in defined culture conditions. Many research groups maintain hESCs using mouse embryonic fibroblasts (MEF) and MEF conditioned medium (CM). The use of murine systems to support hESCs has been imperative in developing hESC technology; however,they suffer from some major limitations including lack of definition,xenobiotic nature,batch-to-batch variation,and labor-intensive production. Therefore,hESC culture definition is essential if hESC lines,and their derivatives are to be quality assured and manufactured to GMP. We have initiated the process of standardizing hESC tissue culture and have employed two serum-free media: mTeSR (MT) and Stem Pro (SP). hESCs were maintained in a pluripotent state,for over 30 passages using MT and SP. Additionally,we present evidence that hESCs maintained in MT and SP generate equivalent levels of human hepatic endoderm as observed with CM. This data suggests that MT and SP are effective replacements for MEF-CM in hESC culture,contributing to the standardization of hESC in vitro models and ultimately their application. View Publication

Human embryonic stem cells (hESCs),unlike mouse ones (mESCs),are vulnerable to apoptosis upon dissociation. Here,we show that the apoptosis,which is of a nonanoikis type,is caused by ROCK-dependent hyperactivation of actomyosin and efficiently suppressed by the myosin inhibitor Blebbistatin. The actomyosin hyperactivation is triggered by the loss of E-cadherin-dependent intercellular contact and also observed in dissociated mouse epiblast-derived pluripotent cells but not in mESCs. We reveal that Abr,a unique Rho-GEF family factor containing a functional Rac-GAP domain,is an indispensable upstream regulator of the apoptosis and ROCK/myosin hyperactivation. Rho activation coupled with Rac inhibition is induced in hESCs upon dissociation,but not in Abr-depleted hESCs or mESCs. Furthermore,artificial Rho or ROCK activation with Rac inhibition restores the vulnerability of Abr-depleted hESCs to dissociation-induced apoptosis. Thus,the Abr-dependent Rho-high/Rac-low" state plays a decisive role in initiating the dissociation-induced actomyosin hyperactivation and apoptosis in hESCs." View Publication

BACKGROUND As one of the least studied bone morphogenetic proteins (BMPs),BMP9 is one of the most osteogenic BMPs. Retinoic acid (RA) signaling is known to play an important role in development,differentiation and bone metabolism. In this study,we investigate the effect of RA signaling on BMP9-induced osteogenic differentiation of mesenchymal progenitor cells (MPCs). METHODOLOGY/PRINCIPAL FINDINGS Both primary MPCs and MPC line are used for BMP9 and RA stimulation. Recombinant adenoviruses are used to deliver BMP9,RARalpha and RXRalpha into MPCs. The in vitro osteogenic differentiation is monitored by determining the early and late osteogenic markers and matrix mineralization. Mouse perinatal limb explants and in vivo MPC implantation experiments are carried out to assess bone formation. We find that both 9CRA and ATRA effectively induce early osteogenic marker,such as alkaline phosphatase (ALP),and late osteogenic markers,such as osteopontin (OPN) and osteocalcin (OC). BMP9-induced osteogenic differentiation and mineralization is synergistically enhanced by 9CRA and ATRA in vitro. 9CRA and ATRA are shown to induce BMP9 expression and activate BMPR Smad-mediated transcription activity. Using mouse perinatal limb explants,we find that BMP9 and RAs act together to promote the expansion of hypertrophic chondrocyte zone at growth plate. Progenitor cell implantation studies reveal that co-expression of BMP9 and RXRalpha or RARalpha significantly increases trabecular bone and osteoid matrix formation. CONCLUSION/SIGNIFICANCE Our results strongly suggest that retinoid signaling may synergize with BMP9 activity in promoting osteogenic differentiation of MPCs. This knowledge should expand our understanding about how BMP9 cross-talks with other signaling pathways. Furthermore,a combination of BMP9 and retinoic acid (or its agonists) may be explored as effective bone regeneration therapeutics to treat large segmental bony defects,non-union fracture,and/or osteoporotic fracture. View Publication

This study evaluated the hypothesis that smoke from harm reduction cigarettes impedes attachment and proliferation of H9 human embryonic stem cells (hESCs). Smoke from three harm reduction brands was compared with smoke from a conventional brand. Doses of smoke were measured in puff equivalents (PE) (1 PE = the amount of smoke in one puff that dissolves in 1 ml of medium). Cytotoxic doses were determined using morphological criteria and trypan blue staining,and apoptosis was confirmed using Magic Red staining. Attachment and proliferation of hESC were followed at a noncytotoxic dose in time-lapse videos collected using BioStation technology. Data were mined from videos either manually or using video bioinformatics subroutines developed with CL-Quant software. Mainstream (MS) and sidestream (SS) smoke from conventional and harm reduction cigarettes induced apoptosis in hESC colonies at 1 PE. At 0.1 PE (noncytotoxic),SS smoke from all brands inhibited attachment of hESC colonies to Matrigel with the strongest inhibition occurring in harm reduction brands. At 0.1 PE,SS smoke,but not MS smoke,from all brands inhibited hESC growth,and two harm reduction brands were more potent than the conventional brand. In general,hESC appeared more sensitive to smoke than their mouse ESC counterparts. Although harm reduction cigarettes are often marketed as safer than conventional brands,our assays show that SS smoke from harm reduction cigarettes was at least as potent or in some cases more potent than smoke from a conventional brand and that SS smoke was more inhibitory than MS smoke in all assays. View Publication

Extracorporeal photochemotherapy (ECP) is widely used to treat cutaneous T-cell lymphoma,graft-versus-host disease,and allografted organ rejection. Its clinical and experimental efficacy in cancer immunotherapy and autoreactive disorders suggests a novel mechanism. This study reveals that ECP induces a high percentage of processed monocytes to enter the antigen-presenting dendritic cell (DC) differentiation pathway,within a single day,without added cytokines,as determined by enhanced expression of relevant genes. The resulting DCs are capable of processing and presentation of exogenous and endogenous antigen and are largely maturationally synchronized,as assessed by the level of expression of costimulatory surface molecules. Principal component analysis of the ECP-induced monocyte transcriptome reveals that activation or suppression of more than 1100 genes produces a reproducible distinctive molecular signature,common to ECP-processed monocytes from normal subjects,and those from patients. Because ECP induces normal monocytes to enter the DC differentiation pathway,this phenomenon is independent of disease state. The efficiency with which ECP stimulates new functional DCs supports the possibility that these cells participate prominently in the clinical successes of the treatment. Appropriately modified by future advances,ECP may potentially offer a general source of therapeutic DCs. View Publication

Current methodology for pluripotent human embryonic stem cells (hESCs) expansion relies on murine sarcoma basement membrane substrates (Matrigel™),which precludes the use of these cells in regenerative medicine. To realize the clinical efficacy of hESCs and their derivatives,expansion of these cells in a defined system that is free of animal components is required. This study reports the successful propagation of hESCs (HES-3 and H1) for textgreater 20 passages on tissue culture-treated polystyrene plates,coated from 5 μg/mL of human plasma-purified vitronectin (VN) solution. Cells maintain expression of pluripotent markers Tra1-60 and OCT-4 and are karyotypically normal after 20 passages of continuous culture. In vitro and in vivo differentiation of hESC by embryoid body formation and teratoma yielded cells from the ecto-,endo-,and mesoderm lineages. VN immobilized on tissue culture polystyrene was characterized using a combination of X-ray photoemission spectroscopy,atomic force microscopy,and quantification of the VN surface density with a Bradford protein assay. Ponceau S staining was used to measure VN adsorption and desorption kinetics. Tuning the VN surface density,via the concentration of depositing solution,revealed a threshold surface density of 250 ng/cm²,which is required for hESCs attachment,proliferation,and differentiation. Cell attachment and proliferation assays on VN surface densities above this threshold show the substrate properties to be equally viable. View Publication