技术资料

Proteasome inhibition is a new strategy in cancer therapy. We synthesized three new peptide aldehyde inhibitors linked to the benzamide derivative structure to use their cytotoxic activity against malignant melanoma cells. Of these,10 displayed the highest cytotoxicity (0.18 +/- 0.16 microM). A radiosynthesis of the iodine aldehyde was performed. Its drug biodistribution showed that some selectivity of the benzamide group toward malignant melanoma tissue was conserved. View Publication

The potential to differentiate human embryonic stem cells (hESCs) in vitro to provide an unlimited source of human hepatocytes for use in biomedical research,drug discovery,and the treatment of liver diseases holds great promise. Here we describe a three-stage process for the efficient and reproducible differentiation of hESCs to hepatocytes by priming hESCs towards definitive endoderm with activin A and sodium butyrate prior to further differentiation to hepatocytes with dimethyl sulfoxide,followed by maturation with hepatocyte growth factor and oncostatin M. We have demonstrated that differentiation of hESCs in this process recapitulates liver development in vivo: following initial differentiation,hESCs transiently express characteristic markers of the primitive streak mesendoderm before turning to the markers of the definitive endoderm; with further differentiation,expression of hepatocyte progenitor cell markers and mature hepatocyte markers emerged sequentially. Furthermore,we have provided evidence that the hESC-derived hepatocytes are able to carry out a range of hepatocyte functions: storage of glycogen,and generation and secretion of plasma proteins. More importantly,the hESC-derived hepatocytes express several members of cytochrome P450 isozymes,and these P450 isozymes are capable of converting the substrates to metabolites and respond to the chemical stimulation. Our results have provided evidence that hESCs can be differentiated efficiently in vitro to functional hepatocytes,which may be useful as an in vitro system for toxicity screening in drug discovery. View Publication

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients,the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg,which are involved in the activation of Fanconi pathway,in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors,but not that of postmitotic neurons,was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice,we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition,embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis. View Publication

Germinal center (GC) responses to T-dependent Ags require effective collaboration between Th cells,activated B cells,and follicular dendritic cells within a highly organized microenvironment. Studies using gene-targeted mice have highlighted nonredundant molecules that are key for initiating and maintaining the GC niche,including the molecules of the ICOS,CD40,and lymphotoxin (LT) pathways. Signaling through ICOS has multiple consequences,including cytokine production,expression of CD40L on Th cells,and differentiation into CXCR5(+) follicular Th cells,all of which are important in the GC reaction. We have therefore taken advantage of ICOS(-/-) mice to dissect which downstream elements are required to initiate the formation of GC. In the context of a T-dependent immune response,we found that GC B cells from ICOS(-/-) mice express lower levels of LTalphabeta compared with wild-type GC B cells in vivo,and stimulation of ICOS on T cells induces LTalphabeta on B cells in vitro. Administration of agonistic anti-LTbeta receptor Ab was unable to restore the GC response in ICOS(-/-) mice,suggesting that additional input from another pathway is required for optimal GC generation. In contrast,treatment with agonistic anti-CD40 Ab in vivo recovered GC networks and restored LTalphabeta expression on GC B cells in ICOS(-/-) mice,and this effect was dependent on LTbeta receptor signaling. Collectively,these data demonstrate that ICOS activation is a prerequisite for the up-regulation of LTalphabeta on GC B cells in vivo and provide a model for cooperation between ICOS,CD40,and LT pathways in the context of the GC response. View Publication

Human stem cells are powerful tools by which to investigate molecular mechanisms of cell growth and differentiation under normal and pathological conditions. Hedgehog signaling,the dysregulation of which causes several pathologies,such as congenital defects and cancer,is involved in several cell differentiation processes and interferes with adipocyte differentiation of rodent cells. The present study was aimed at investigating the effect of Hedgehog pathway modulation on adipocyte phenotype using different sources of human mesenchymal cells,such as bone marrow stromal cells and human multipotent adipose-derived stem cells. We bring evidence that Hedgehog signaling decreases during human adipocyte differentiation. Inhibition of this pathway is not sufficient to trigger adipogenesis,but activation of Hedgehog pathway alters adipocyte morphology as well as insulin sensitivity. Analysis of glycerol-3-phosphate dehydrogenase activity and expression of adipocyte marker genes indicate that activation of Hedgehog signaling by purmorphamine impairs adipogenesis. In sharp contrast to reports in rodent cells,the maturation process,but not the early steps of human mesenchymal stem cell differentiation,is affected by Hedgehog activation. Hedgehog interferes with adipocyte differentiation by targeting CCAAT enhancer-binding protein alpha and peroxisome proliferator-activated receptor (PPAR) gamma2 expression,whereas PPARgamma1 level remains unaffected. Although Hedgehog pathway stimulation does not modify the total number of adipocytes,adipogenesis appears dramatically impaired,with reduced lipid accumulation,a decrease in adipocyte-specific markers,and acquisition of an insulin-resistant phenotype. This study indicates that a decrease in Hedgehog signaling is necessary but not sufficient to trigger adipocyte differentiation and unveils a striking difference in the adipocyte differentiation process between rodent and human mesenchymal stem cells. View Publication

Infection with human immunodeficiency virus 1 (HIV-1) results in the dissemination of virus to gut-associated lymphoid tissue. Subsequently,HIV-1 mediates massive depletion of gut CD4+ T cells,which contributes to HIV-1-induced immune dysfunction. The migration of lymphocytes to gut-associated lymphoid tissue is mediated by integrin alpha4beta7. We demonstrate here that the HIV-1 envelope protein gp120 bound to an activated form of alpha4beta7. This interaction was mediated by a tripeptide in the V2 loop of gp120,a peptide motif that mimics structures presented by the natural ligands of alpha4beta7. On CD4+ T cells,engagement of alpha4beta7 by gp120 resulted in rapid activation of LFA-1,the central integrin involved in the establishment of virological synapses,which facilitate efficient cell-to-cell spreading of HIV-1. View Publication

BACKGROUND Human embryonic stem cells (hESC) should enable novel insights into early human development and provide a renewable source of cells for regenerative medicine. However,because the three-dimensional hESC aggregates [embryoid bodies (hEB)] typically employed to reveal hESC developmental potential are heterogeneous and exhibit disorganized differentiation,progress in hESC technology development has been hindered. METHODOLOGY/PRINCIPAL FINDINGS Using a centrifugal forced-aggregation strategy in combination with a novel centrifugal-extraction approach as a foundation,we demonstrated that hESC input composition and inductive environment could be manipulated to form large numbers of well-defined aggregates exhibiting multi-lineage differentiation and substantially improved self-organization from single-cell suspensions. These aggregates exhibited coordinated bi-domain structures including contiguous regions of extraembryonic endoderm- and epiblast-like tissue. A silicon wafer-based microfabrication technology was used to generate surfaces that permit the production of hundreds to thousands of hEB per cm(2). CONCLUSIONS/SIGNIFICANCE The mechanisms of early human embryogenesis are poorly understood. We report an ultra high throughput (UHTP) approach for generating spatially and temporally synchronised hEB. Aggregates generated in this manner exhibited aspects of peri-implantation tissue-level morphogenesis. These results should advance fundamental studies into early human developmental processes,enable high-throughput screening strategies to identify conditions that specify hESC-derived cells and tissues,and accelerate the pre-clinical evaluation of hESC-derived cells. View Publication

We have resolved B220+ IgM- B-lineage cells in mouse bone marrow into four fractions based on differential cell surface expression of determinants recognized by S7 (leukosialin,CD43),BP-1,and 30F1 (heat stable antigen). Functional differences among these fractions can be correlated with Ig gene rearrangement status. The largest fraction,lacking S7,consists of pre-B cells whereas the others,expressing S7,include B lineage cells before pre-B. These S7+ fractions,provisionally termed Fr. A,Fr. B,and Fr. C,can differentiate in a stromal layer culture system. Phenotypic alteration during such culture suggests an ordering of these stages from Fr. A to Fr. B to Fr. C and thence to S7- pre-B cells. Using polymerase chain reaction amplification with pairs of oligonucleotide primers for regions 5' of JH1,DFL16.1,and Jk1,we find that the Ig genes of Fr. A are in germline configuration,whereas Fr. B and C are pro-B cell stages with increasing D-J rearrangement,but no V-D-J. Finally,functional analysis demonstrates that the proliferative response to IL-7,an early B lineage growth factor,is restricted to S7+ stages and,furthermore,that an additional,cell contact-mediated signal is essential for survival of Fr. A. View Publication

Transcripts expressed in cytotoxic T lymphocytes (CTL) have mechanistic and diagnostic importance in transplantation. We used microarrays to select CTL-associated transcripts (CATs) expressed in human CD4(+) CTL,CD8(+) CTL and NK cells,excluding transcripts expressed in B cells,monocytes and kidney. This generated three transcript sets: CD4(+)-associated,CD8(+)-associated and NK-associated. Surprisingly,many CATs were expressed in effector memory cells e.g. granzyme B/GZMB,interferon-gamma/IFNG. Transcript expression was very similar between CD4(+) and CD8(+) CTL. There were no transcripts highly selective for CD4(+) CTL or CD8(+) CTL: for example,cytotoxic molecule transcripts (perforin,granzymes,granulysin) were shared between CD8(+) CTL and CD4(+) CTL although expression remained higher in CD8(+) CTL. Transcripts that differentiated between CD8(+) CTL and CD4(+) CTL were primarily those shared between CD8(+) CTL and NK cells (e.g. NK receptors KLRC1,KLRC3,KLRD1,KLRK1). No transcripts could differentiate CD4(+) CTL from CD8(+) CTL but NK cell-associated transcripts could differentiate NK cells from CTL. This study serves as a foundation for the interpretation of CATs in rejecting allografts and highlights the extensive sharing of CATs among CD4(+) CTL,CD8(+) CTL and effector memory T cells. View Publication

In response to inflammatory stimuli,monocytes/macrophages secrete greater quantities of the proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha),interleukin-1beta (IL-1beta) and IL-6. The inflammatory process and the innate immune response are related to the activation of several transcription factors,such as nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1). The proteasome is a multimeric protease complex,which plays a vital role in several cellular functions,including the regulation of transcription factors like NF-kappaB. In this study,we used the human monocyte cell line U937 stimulated with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) as a model to investigate the in vitro effects of MG132,a proteasome inhibitor,on the release of TNF-alpha,IL-1beta and IL-6 and on the expression of their membrane and soluble receptors TNF-R1,IL-1R1 and IL-6R. We also analysed the effects of MG132 on the activation of NF-kappaB and AP-1 and on the IkappaB molecule. MG132 significantly inhibited the secretion of those proinflammatory cytokines. MG132 increased the release of the soluble receptors TNF-R1 and IL-1R1 from U937 cells and decreased their cell-surface expression. MG132 also increased IL-6R cell-surface expression and decreased its release. Proteasome inhibition also led to an increase in LPS+PMA-induced AP-1 activation and the attenuation of LPS+PMA-induced IkappaB degradation,resulting in the abolition of NF-kappaB activation. Our experiments strongly suggest that the proteasome is an important factor in the regulation of proinflammatory cytokines and their receptors. View Publication

Lysophospholipids are signaling molecules that play broad and major roles within the nervous system during both early development and neural injury. We used neural differentiation of human embryonic stem cells (hESC) as an in vitro model to examine the specific effects of lysophosphatidic acid (LPA) at various stages of neural development,from neural induction to mature neurons and glia. We report that LPA inhibits neurosphere formation and the differentiation of neural stem cells (NSC) toward neurons,without modifying NSC proliferation,apoptosis,or astrocytic differentiation. LPA acts through the activation of the Rho/ROCK and the phosphatidylinositol 3-kinase/Akt pathways to inhibit neuronal differentiation. This study is the first demonstration of a role for LPA signaling in neuronal differentiation of hESC. As LPA concentrations increase during inflammation,the inhibition of neuronal differentiation by LPA might contribute to the low level of neurogenesis observed following neurotrauma. View Publication

CD24 is a signal-transducing molecule on the surfaces of most human B cells that can modulate their response to activation signals by antagonizing IL-induced differentiation into antibody-forming cells and inducing proliferation in combination with signals generated by Ag receptors. A cDNA that directs the expression of CD24 on the surfaces of transfected COS cells was cloned by its homology to a cDNA encoding the murine M1/69-J11d heat stable Ag. The CD24 cDNA encodes a mature peptide of only 31 to 35 amino acids that is extensively glycosylated and is attached to the outer surface of the plasma membrane by a glycosyl phosphatidylinositol lipid anchor. Although CD24 is structurally similar to M1/69-J11d,and the two Ag appear to have a common genetic ancestry,the homology of CD24 to the M1/69-J11d Ag is confined to a small cluster of amino acids comprising potential N-linked glycosylation sites. Combined with the differences in expression patterns of the human and murine Ag,this indicates that CD24 and M1/69-J11d may not have equivalent functional roles in lymphoid development. The novel structure of CD24 suggests that signaling could be triggered by the binding of a lectin-like ligand to the carbohydrates projecting from the CD24 peptide,and transduced through the release of second messengers derived from the glycosyl phosphatidylinositol membrane anchor of CD24. View Publication