参考文献

Aldehyde dehydrogenase 1A1 (ALDH) activity is one hallmark of human bone marrow (BM),umbilical cord blood (UCB),and peripheral blood (PB) primitive progenitors presenting high reconstitution capacities in vivo. In this study,we have identified ALDH(+) cells within human skeletal muscles,and have analyzed their phenotypical and functional characteristics. Immunohistofluorescence analysis of human muscle tissue sections revealed rare endomysial cells. Flow cytometry analysis using the fluorescent substrate of ALDH,Aldefluor,identified brightly stained (ALDH(br)) cells with low side scatter (SSC(lo)),in enzymatically dissociated muscle biopsies,thereafter abbreviated as SMALD(+) (for skeletal muscle ALDH(+)) cells. Phenotypical analysis discriminated two sub-populations according to CD34 expression: SMALD(+)/CD34(-) and SMALD(+)/CD34(+) cells. These sub-populations did not initially express endothelial (CD31),hematopoietic (CD45),and myogenic (CD56) markers. Upon sorting,however,whereas SMALD(+)/CD34(+) cells developed in vitro as a heterogeneous population of CD56(-) cells able to differentiate in adipoblasts,the SMALD(+)/CD34(-) fraction developed in vitro as a highly enriched population of CD56(+) myoblasts able to form myotubes. Moreover,only the SMALD(+)/CD34(-) population maintained a strong myogenic potential in vivo upon intramuscular transplantation. Our results suggest that ALDH activity is a novel marker for a population of new human skeletal muscle progenitors presenting a potential for cell biology and cell therapy. View Publication

Microtubules are among the most strategic subcellular targets of anticancer chemotherapeutics. Despite this fact,new antimicrotubule agents that possess unique mechanisms of cytotoxic action and have broader antineoplastic spectra than the vinca alkaloids have not been introduced over the last several decades--until the recent development of taxol. Unlike classical antimicrotubule agents like colchicine and the vinca alkaloids,which induce depolymerization of microtubules,taxol induces tubulin polymerization and forms extremely stable and nonfunctional microtubules. Taxol has demonstrated broad activity in preclinical screening studies,and antineoplastic activity has been observed in several classically refractory tumors. These tumors include cisplatin-resistant ovarian carcinoma in phase II trials and malignant melanoma and non-small cell lung carcinoma in phase I studies. Taxol's structural complexity has hampered the development of feasible processes for synthesis,and its extreme scarcity has limited the use of a conventional,broad-scale screening approach for evaluation of clinical antitumor activity. However,taxol's unique mechanism of action,its spectrum of preclinical antitumor activity,and tumor responses in early clinical trials have generated renewed interest in pursuing its development. View Publication

Since the derivation of human embryonic stem (hES) cells,their translation to clinical therapies has been met with several challenges,including the need for large-scale expansion and controlled differentiation processes. Suspension bioreactors are an effective alternative to static culture flasks as they enable the generation of clinically relevant cell numbers with greater efficacy in a controlled culture system. We,along with other groups,have developed bioreactor protocols for the expansion of pluripotent murine ES cells. Here we present a novel bioreactor protocol that yields a 25-fold expansion of hES cells over 6 days. Using immunofluorescence,flow cytometry,and teratoma formation assays,we demonstrated that these bioreactor cultures retained high levels of pluripotency and a normal karyotype. Importantly,the use of bioreactors enables the expansion of hES cells in the absence of feeder layers or matrices,which will facilitate the adaptation of good manufacturing process (GMP) standards to the development of hES cell therapies. View Publication

The origin and function of human double negative (DN) TCR-alphabeta+ T cells is unknown. They are thought to contribute to the pathogenesis of systemic lupus erythematosus because they expand and accumulate in inflamed organs. In this study,we provide evidence that human TCR-alphabeta+ CD4- CD8- DN T cells can derive from activated CD8+ T cells. Freshly isolated TCR-alphabeta+ DN T cells display a distinct gene expression and cytokine production profile. DN cells isolated from peripheral blood as well as DN cells derived in vitro from CD8+ T cells produce a defined array of proinflammatory mediators that includes IL-1beta,IL-17,IFN-gamma,CXCL3,and CXCL2. These results indicate that,upon activation,CD8+ T cells have the capacity to acquire a distinct phenotype that grants them inflammatory capacity. View Publication

Cancer stem cells are known for their inherent resistance to therapy. Here we investigated whether normal stem cells with acquired resistance to stress can be used to identify novel markers of cancer stem cells. For this,we generated a human embryonic stem cell line resistant to Trichostatin A and analyzed changes in its gene expression. The resistant cells over-expressed various genes associated with tumor aggressiveness,many of which are also expressed in the CD133+ glioma cancer stem cells. These findings suggest that stress-resistant stem cells generated in vitro may be useful for the discovery of novel markers of cancer stem cells. View Publication

Hematopoietic stem cell (HSC) engraftment is a multistep process involving HSC homing to bone marrow,self-renewal,proliferation,and differentiation to mature blood cells. Here,we show that loss of p190-B RhoGTPase activating protein,a negative regulator of Rho GTPases,results in enhanced long-term engraftment during serial transplantation. This effect is associated with maintenance of functional HSC-enriched cells. Furthermore,loss of p190-B led to marked improvement of HSC in vivo repopulation capacity during ex vivo culture without altering proliferation and multilineage differentiation of HSC and progeny. Transcriptional analysis revealed that p190-B deficiency represses the up-regulation of p16(Ink4a) in HSCs in primary and secondary transplantation recipients,providing a possible mechanism of p190-B-mediated HSC functions. Our study defines p190-B as a critical transducer element of HSC self-renewal activity and long-term engraftment,thus suggesting that p190-B is a target for HSC-based therapies requiring maintenance of engraftment phenotype. View Publication

Antiangiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane. Bortezomib's inhibitory effects on chorioallantoic membrane vascularization were abrogated in the presence of distinct tumor xenografts,thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy,we identified GRP-78,a chaperone protein of the unfolded protein response,as being responsible for bortezomib resistance. Indeed,a variety of bortezomib-resistant solid tumor cell lines (PC-3,HRT-18),but not myeloma cell lines (U266,OPM-2),were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP-78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib but induced prosurvival signals by phosphorylation of extracellular signal-related kinase and inhibited p53-mediated expression of proapoptotic Bok and Noxa proteins in endothelial cells. From our data,we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment,thus demonstrating a hitherto unknown mechanism of resistance to bortezomib. View Publication

The RAS-RAF-mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway provides numerous opportunities for targeted oncology therapeutics. In particular,the MEK enzyme is attractive due to high selectivity for its target ERK and the central role that activated ERK plays in driving cell proliferation. The structural,pharmacologic,and pharmacokinetic properties of RDEA119/BAY 869766,an allosteric MEK inhibitor,are presented. RDEA119/BAY 869766 is selectively bound directly to an allosteric pocket in the MEK1/2 enzymes. This compound is highly efficacious at inhibiting cell proliferation in several tumor cell lines in vitro. In vivo,RDEA119/BAY 869766 exhibits potent activity in xenograft models of melanoma,colon,and epidermal carcinoma. RDEA119/BAY 869766 exhibits complete suppression of ERK phosphorylation at fully efficacious doses in mice. RDEA119/BAY 869766 shows a tissue selectivity that reduces its potential for central nervous system-related side effects. Using pharmacokinetic and pharmacodynamic data,we show that maintaining adequate MEK inhibition throughout the dosing interval is likely more important than achieving high peak levels because greater efficacy was achieved with more frequent but lower dosing. Based on its longer half-life in humans than in mice,RDEA119/BAY 869766 has the potential for use as a once- or twice-daily oral treatment for cancer. RDEA119/BAY 869766,an exquisitely selective,orally available MEK inhibitor,has been selected for clinical development because of its potency and favorable pharmacokinetic profile. View Publication

Interferon regulatory factor 3 (IRF-3) is essential for innate intracellular immune defenses that limit virus replication,but these defenses fail to suppress human immunodeficiency virus (HIV) infection,which can ultimately associate with opportunistic coinfections and the progression to AIDS. Here,we examined antiviral defenses in CD4+ cells during virus infection and coinfection,revealing that HIV type 1 (HIV-1) directs a global disruption of innate immune signaling and supports a coinfection model through suppression of IRF-3. T cells responded to paramyxovirus infection to activate IRF-3 and interferon-stimulated gene expression,but they failed to mount a response against HIV-1. The lack of response associated with a marked depletion of IRF-3 but not IRF-7 in HIV-1-infected cells,which supported robust viral replication,whereas ectopic expression of active IRF-3 suppressed HIV-1 infection. IRF-3 depletion was dependent on a productive HIV-1 replication cycle and caused the specific disruption of Toll-like receptor and RIG-I-like receptor innate immune signaling that rendered cells permissive to secondary virus infection. IRF-3 levels were reduced in vivo within CD4+ T cells from patients with acute HIV-1 infection but not from long-term nonprogressors. Our results indicate that viral suppression of IRF-3 promotes HIV-1 infection by disrupting IRF-3-dependent signaling pathways and innate antiviral defenses of the host cell. IRF-3 may direct an innate antiviral response that regulates HIV-1 replication and viral set point while governing susceptibility to opportunistic virus coinfections. View Publication

The anthrax protective antigen (PA) is the receptor-binding subunit common to lethal toxin (LT) and edema toxin (ET),which are responsible for the high mortality rates associated with inhalational Bacillus anthracis infection. Although recombinant PA (rPA) is likely to be an important constituent of any future anthrax vaccine,evaluation of the efficacies of the various candidate rPA vaccines is currently difficult,because the specific B-cell epitopes involved in toxin neutralization have not been completely defined. In this study,we describe the identification and characterization of two murine monoclonal immunoglobulin G1 antibodies (MAbs),1-F1 and 2-B12,which recognize distinct linear neutralizing epitopes on domain 4 of PA. 1-F1 recognized a 12-mer peptide corresponding to residues 692 to 703; this epitope maps to a region of domain 4 known to interact with the anthrax toxin receptor CMG-2 and within a conformation-dependent epitope recognized by the well-characterized neutralizing MAb 14B7. As expected,1-F1 blocked PA's ability to associate with CMG-2 in an in vitro solid-phase binding assay,and it protected murine macrophage cells from intoxication with LT. 2-B12 recognized a 12-mer peptide corresponding to residues 716 to 727,an epitope located immediately adjacent to the core 14B7 binding site and a stretch of amino acids not previously identified as a target of neutralizing antibodies. 2-B12 was as effective as 1-F1 in neutralizing LT in vitro,although it only partially inhibited PA binding to its receptor. Mice passively administered 1-F1 or 2-B12 were partially protected against a lethal challenge with LT. These results advance our fundamental understanding of the mechanisms by which antibodies neutralize anthrax toxin and may have future application in the evaluation of candidate rPA vaccines. View Publication