参考文献

Tandem pleckstrin homology domain proteins (TAPPs) are recruited to the plasma membrane via binding to phosphoinositides produced by phosphoinositide 3-kinases (PI3Ks). Whereas PI3Ks are critical for B-cell activation,the functions of TAPP proteins in B cells are unknown. We have identified 40 potential interaction partners of TAPP2 in B cells,including proteins involved in cytoskeletal rearrangement,signal transduction and endocytic trafficking. The association of TAPP2 with the cytoskeletal proteins utrophin and syntrophin was confirmed by Western blotting. We found that TAPP2,syntrophin,and utrophin are coexpressed in normal human B cells and B-chronic lymphocytic leukemia (B-CLL) cells. TAPP2 and syntrophin expression in B-CLL was variable from patient to patient,with significantly higher expression in the more aggressive disease subset identified by zeta-chain-associated protein kinase of 70 kDa (ZAP70) expression and unmutated immunoglobulin heavy chain (IgH) genes. We examined whether TAPP can regulate cell adhesion,a known function of utrophin/syntrophin in other cell types. Expression of membrane-targeted TAPP2 enhanced B-cell adhesion to fibronectin and laminin,whereas PH domain-mutant TAPP2 inhibited adhesion. siRNA knockdown of TAPP2 or utrophin,or treatment with PI3K inhibitors,significantly inhibited adhesion. These findings identify TAPP2 as a novel link between PI3K signaling and the cytoskeleton with potential relevance for leukemia progression. View Publication

BACKGROUND: MicroRNAs are required for maintenance of pluripotency as well as differentiation,but since more microRNAs have been computationally predicted in genome than have been found,there are likely to be undiscovered microRNAs expressed early in stem cell differentiation. METHODOLOGY/PRINCIPAL FINDINGS: SOLiD ultra-deep sequencing identified textgreater10(7) unique small RNAs from human embryonic stem cells (hESC) and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs,demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs,including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs. CONCLUSIONS/SIGNIFICANCE: Extending the classic definition of microRNAs,this large number of new microRNA genes,the majority of which are less conserved than their canonical counterparts,likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes,the presence of chromatin marks indicative of regulated gene expression,and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation. View Publication

IFN-beta-1a has been used over the past 15 years as a primary therapy for relapsing-remitting multiple sclerosis (MS). However,the immunomodulatory mechanisms that provide a therapeutic effect against this CNS inflammatory disease are not yet completely elucidated. The effect of IFN-beta-1a on Th17 cells,which play a critical role in the development of the autoimmune response,has not been extensively studied in humans. We have investigated the effect of IFN-beta-1a on dendritic cells (DCs) and naive CD4(+)CD45RA(+) T cells derived from untreated MS patients and healthy controls in the context of Th17 cell differentiation. We report that IFN-beta-1a treatment down-regulated the expression of IL-1beta and IL-23p19 in DCs,whereas it induced the gene expression of IL-12p35 and IL-27p28. We propose that IFN-beta-1a-mediated up-regulation of the suppressor of cytokine signaling 3 expression,induced via STAT3 phosphorylation,mediates IL-1beta and IL-23 down-regulation,while IFN-beta-1a-induced STAT1 phosphorylation induces IL-27p28 expression. CD4(+)CD45RA(+) naive T cells cocultured with supernatants from IFN-beta-1a-treated DCs exhibited decreased gene expression of the Th17 cell markers retinoic acid-related orphan nuclear hormone receptor c (RORc),IL-17A,and IL-23R. A direct IFN-beta-1a treatment of CD45RA(+) T cells cultured in Th17-polarizing conditions also down-regulated RORc,IL-17A,and IL-23R,but up-regulated IL-10 gene expression. Studies of the mechanisms involved in the Th17 cell differentiation suggest that IFN-beta-1a inhibits IL-17 and induces IL-10 secretion via activated STAT1 and STAT3,respectively. IFN-beta's suppression of Th17 cell differentiation may represent its most relevant mechanism of selective suppression of the autoimmune response in MS. View Publication

The presentation of proteins on surfaces is fundamental to numerous cell culture and tissue engineering applications. While a number of physisorption and cross-linking methods exist to facilitate this process,few avoid denaturation of proteins or allow control over protein orientation,both of which are critical to the functionality of many signal proteins and ligands. Often recombinant protein sequences include a poly-histidine tag to facilitate purification. We utilize this sequence to anchor proteins to biosurfaces via a peptide bonded to the surface which conjugates with the poly-histidine tag in the presence of zinc rather than nickel,which is more traditionally used to conjugate poly-histidine tags to surfaces. We demonstrate that this strategy enables the display of proteins on 2D and 3D surfaces without compromising protein function through direct cross-linking or physisorption. View Publication

BACKGROUND: Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in multiple intracellular signaling pathways promoting tumor growth. mTOR is aberrantly activated in a significant portion of breast cancers and is a promising target for treatment. Rapamycin and its analogues are in clinical trials for breast cancer treatment. Patterns of gene expression (metagenes) may also be used to simulate a biologic process or effects of a drug treatment. In this study,we tested the hypothesis that the gene-expression signature regulated by rapamycin could predict disease outcome for patients with breast cancer. RESULTS: Colony formation and sulforhodamine B (IC50 textless 1 nM) assays,and xenograft animals showed that MDA-MB-468 cells were sensitive to treatment with rapamycin. The comparison of in vitro and in vivo gene expression data identified a signature,termed rapamycin metagene index (RMI),of 31 genes upregulated by rapamycin treatment in vitro as well as in vivo (false discovery rate of 10%). In the Miller dataset,RMI did not correlate with tumor size or lymph node status. High (textgreater75th percentile) RMI was significantly associated with longer survival (P = 0.015). On multivariate analysis,RMI (P = 0.029),tumor size (P = 0.015) and lymph node status (P = 0.001) were prognostic. In van 't Veer study,RMI was not associated with the time to develop distant metastasis (P = 0.41). In the Wang dataset,RMI predicted time to disease relapse (P = 0.009). CONCLUSION: Rapamycin-regulated gene expression signature predicts clinical outcome in breast cancer. This supports the central role of mTOR signaling in breast cancer biology and provides further impetus to pursue mTOR-targeted therapies for breast cancer treatment. View Publication

Experimentation with the progenitor/stem cells in adult prostate epithelium can be inconvenient due to a tight time line from tissue acquisition to cell isolation and to downstream experiments. To circumvent this inconvenience,we developed a simple technical procedure for culturing epithelial cells derived from human prostate tissue. In this study,benign prostate tissue was enzymatically digested and fractionated into epithelium and stroma,which were then cultured in the medium designed for prostate epithelial and stromal cells,respectively. The cultured cells were analyzed by immunocytochemical staining and flow cytometry. Prostate tissue-regenerating capacity of cultured cells in vitro was determined by co-culturing epithelial and stromal cells in dihydrotestosterone-containing RPMI. Cell lineages in formed acini-like structures were determined by immunohistochemistry. The culture of epithelial cells mainly consisted of basal cells. A minor population was negative for known lineage markers and positive for CD133. The culture also contained cells with high activity of aldehyde dehydrogenase. After co-culturing with stromal cells,the epithelial cells were able to form acini-like structures containing multiple cell lineages. Thus,the established culture of prostate epithelial cells provides an alternative source for studying progenitor/stem cells of prostate epithelium. View Publication

Herpes simplex virus (HSV) tegument proteins are released into the cytoplasm during viral entry and hence are among the first viral proteins encountered by an infected cell. Despite the implied importance of these proteins in the evasion of host defenses,the function of some,like virion protein 11/12 (VP11/12),have not been clearly defined. Previously,we reported that VP11/12 is strongly tyrosine phosphorylated during the infection of lymphocytes but not in fibroblasts or an epithelial cell line (G. Zahariadis,M. J. Wagner,R. C. Doepker,J. M. Maciejko,C. M. Crider,K. R. Jerome,and J. R. Smiley,J. Virol. 82:6098-6108,2008). We also showed that tyrosine phosphorylation depends in part on the activity of the lymphocyte-specific Src family kinase (SFK) Lck in Jurkat T cells. These data suggested that VP11/12 is a substrate of Lck and that Lck is activated during HSV infection. Here,we show that HSV infection markedly increases the fraction of Lck phosphorylated on its activation loop tyrosine (Y394),a feature characteristic of activated Lck. A previous report implicated the immediate-early protein ICP0 and the viral serine/threonine kinases US3 and UL13 in the induction of a similar activated phenotype of SFKs other than Lck in fibroblasts and suggested that ICP0 interacts directly with SFKs through their SH3 domain. However,we were unable to detect an interaction between ICP0 and Lck in T lymphocytes,and we show that ICP0,US3,and UL13 are not strictly required for Lck activation. In contrast,VP11/12 interacted with Lck or Lck signaling complexes and was strictly required for Lck activation during HSV infection. Thus,VP11/12 likely modulates host cell signaling pathways for the benefit of the virus. View Publication

Mtb influences DC activity and T cell-mediated immune responses. We show that the treatment of immature monocyte-derived DC with Mtb elicited the formation of mature DC,producing TNF-alpha,IL-1beta,IL-6,and IL-23 and instructing CD4(+) cells to secrete IFN-gamma and IL-17. Mtb-induced cytokine release by DC depended on dectin-1 receptor engagement,whereas MR or DC-SIGN stimulation inhibited this process. A selective dectin-1 binding by the receptor agonist glucan was sufficient to enable DC to generate Th1/Th17 lymphocytes,showing features comparable with those induced by Mtb-treated DC. Interestingly,DC-SIGN or MR engagement inhibited Th17 and increased Th1 generation by glucan- or Mtb-treated DC. Our results indicate that Mtb modulates the lymphocyte response by affecting DC maturation and cytokine release. Dectin-1 engagement by Mtb enables DC to promote a Th1/Th17 response,whereas DC-SIGN and MR costimulation limits dectin-1-dependent Th17 generation and favors a Th1 response,probably by interfering with release of cytokines. View Publication

BACKGROUND: Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However,relatively little is known about the mechanisms responsible for their immunomodulatory effects,which could be influenced by both the cell source and culture conditions. DESIGN AND METHODS: We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells,in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. RESULTS: The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology,immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency,umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition,these cells do not express hTERT and telomerase activity,do express p16(ink4a) protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses,umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation,(ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10,while decreasing interferon-gamma secretion,in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects,a prostaglandin E(2)-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G. CONCLUSIONS: Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency,proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications. View Publication

Although activation of the B-cell receptor (BCR) signaling pathway is implicated in the pathogenesis of chronic lymphocytic leukemia (CLL),its clinical impact and the molecular correlates of such response are not clearly defined. T-cell leukemia 1 (TCL1),the AKT modulator and proto-oncogene,is differentially expressed in CLL and linked to its pathogenesis based on CD5(+) B-cell expansions arising in TCL1-transgenic mice. We studied here the association of TCL1 levels and its intracellular dynamics with the in vitro responses to BCR stimulation in 70 CLL cases. The growth kinetics after BCR engagement correlated strongly with the degree and timing of induced AKT phospho-activation. This signaling intensity was best predicted by TCL1 levels and the kinetics of TCL1-AKT corecruitment to BCR membrane activation complexes,which further included the kinases LYN,SYK,ZAP70,and PKC. High TCL1 levels were also strongly associated with aggressive disease features,such as advanced clinical stage,higher white blood cell counts,and shorter lymphocyte doubling time. Higher TCL1 levels independently predicted an inferior clinical outcome (ie,shorter progression-free survival,P textless .001),regardless of therapy regimen,especially for ZAP70(+) tumors. We propose TCL1 as a marker of the BCR-responsive CLL subset identifying poor prognostic cases where targeting BCR-associated kinases may be therapeutically useful. View Publication

Genetic reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells or iPSCs) by over-expression of specific genes has been accomplished using mouse and human cells. However,it is still unclear how similar human iPSCs are to human Embryonic Stem Cells (hESCs). Here,we describe the transcriptional profile of human iPSCs generated without viral vectors or genomic insertions,revealing that these cells are in general similar to hESCs but with significant differences. For the generation of human iPSCs without viral vectors or genomic insertions,pluripotent factors Oct4 and Nanog were cloned in episomal vectors and transfected into human fetal neural progenitor cells. The transient expression of these two factors,or from Oct4 alone,resulted in efficient generation of human iPSCs. The reprogramming strategy described here revealed a potential transcriptional signature for human iPSCs yet retaining the gene expression of donor cells in human reprogrammed cells free of viral and transgene interference. Moreover,the episomal reprogramming strategy represents a safe way to generate human iPSCs for clinical purposes and basic research. View Publication

The dose response curve is the gold standard for measuring the effect of a drug treatment,but is rarely used in genomic scale transcriptional profiling due to perceived obstacles of cost and analysis. One barrier to examining transcriptional dose responses is that existing methods for microarray data analysis can identify patterns,but provide no quantitative pharmacological information. We developed analytical methods that identify transcripts responsive to dose,calculate classical pharmacological parameters such as the EC50,and enable an in-depth analysis of coordinated dose-dependent treatment effects. The approach was applied to a transcriptional profiling study that evaluated four kinase inhibitors (imatinib,nilotinib,dasatinib and PD0325901) across a six-logarithm dose range,using 12 arrays per compound. The transcript responses proved a powerful means to characterize and compare the compounds: the distribution of EC50 values for the transcriptome was linked to specific targets,dose-dependent effects on cellular processes were identified using automated pathway analysis,and a connection was seen between EC50s in standard cellular assays and transcriptional EC50s. Our approach greatly enriches the information that can be obtained from standard transcriptional profiling technology. Moreover,these methods are automated,robust to non-optimized assays,and could be applied to other sources of quantitative data. View Publication