技术资料

Viruses have evolved an assortment of mechanisms for regulating the Akt signaling pathway to establish a cellular environment more favorable for viral replication. Myxoma virus (MYXV) is a rabbit-specific poxvirus that encodes many immunomodulatory factors,including an ankyrin repeat-containing host range protein termed M-T5 that functions to regulate tropism of MYXV for rabbit lymphocytes and certain human cancer cells. MYXV permissiveness in these human cancer cells is dependent upon the direct interaction between M-T5 and Akt,which has been shown to induce the kinase activity of Akt. In this study,an array of compounds that selectively manipulate Akt signaling was screened and we show that only a subset of Akt inhibitors significantly decreased the ability of MYXV to replicate in previously permissive human cancer cells. Furthermore,reduced viral replication efficiency was correlated with lower levels of phosphorylated Akt. In contrast,the PP2A-specific phosphatase inhibitor okadaic acid promoted increased Akt kinase activation and rescued MYXV replication in human cancer cells that did not previously support viral replication. Finally,phosphorylation of Akt at residue Thr308 was shown to dictate the physical interaction between Akt and M-T5,which then leads to phosphorylation of Ser473 and permits productive MYXV replication in these human cancer cells. The results of this study further characterize the mechanism by which M-T5 exploits the Akt signaling cascade and affirms this interaction as a major tropism determinant that regulates the replication efficiency of MYXV in human cancer cells. View Publication

Human embryonic stem cells (hESC) are capable to give rise to all cell types in the human body during the normal course of development. Therefore,these cells hold a great promise in regenerative cell replacement based therapeutical approaches. However,some controversy exists in literature concerning the ultimate fate of hESC after exposure to genotoxic agents,in particular,regarding the effect of DNA damaging insults on pluripotency of hESC. To comprehensively address this issue,we performed an analysis of the expression of marker genes,associated with pluripotent state of hESC,such as Oct-4,Nanog,Sox-2,SSEA-4,TERT,TRA-1-60 and TRA-1-81 up to 65h after exposure to ionizing radiation (IR) using flow cytometry,immunocytochemistry and quantitative real-time polymerase chain reaction techniques. We show that irradiation with relatively low doses of gamma-radiation (0.2Gy and 1Gy) does not lead to loss of expression of the pluripotency-associated markers in the surviving hESC. While changes in the levels of expression of some of the pluripotency markers were observed at different time points after IR exposure,these alterations were not persistent,and,in most cases,the expression of the pluripotency-associated markers remained significantly higher than that observed in fully differentiated human fibroblasts,and in hESCs differentiated into definitive endodermal lineage. Our data suggest that exposure of hESC to relatively low doses of IR as a model genotoxic agent does not significantly affect pluripotency of the surviving fraction of hESC. View Publication

Growth factor independence-1B (Gfi-1B) is a transcriptional repressor essential for erythropoiesis and megakaryopoiesis. Targeted gene disruption of GFI1B in mice leads to embryonic lethality resulting from failure to produce definitive erythrocytes,hindering the study of Gfi-1B function in adult hematopoiesis. We here show that,in humans,Gfi-1B controls the development of erythrocytes and megakaryocytes by regulating the proliferation and differentiation of bipotent erythro-megakaryocytic progenitors. We further identify in this cell population the type III transforming growth factor-beta receptor gene,TGFBR3,as a direct target of Gfi-1B. Knockdown of Gfi-1B results in altered transforming growth factor-beta (TGF-beta) signaling as shown by the increase in Smad2 phosphorylation and its inability to associate to the transcription intermediary factor 1-gamma (TIF1-gamma). Because the Smad2/TIF1-gamma complex is known to specifically regulate erythroid differentiation,we propose that,by repressing TGF-beta type III receptor (TbetaRIotaII) expression,Gfi-1B favors the Smad2/TIF1-gamma interaction downstream of TGF-beta signaling,allowing immature progenitors to differentiate toward the erythroid lineage. View Publication

OBJECTIVES: In this study,we hypothesized that an antihuman-CD34 antibody immobilized on the surface of commercially available sirolimus-eluting stents (SES) could enhance re-endothelialization compared with SES alone. BACKGROUND: Previous experience with antihuman-CD34 antibody surface modified Genous stents (GS) (OrbusNeich Medical,Fort Lauderdale,Florida) has shown enhanced stent endothelialization in vivo. METHODS: In the phase 1 study,stents were deployed in 21 pig coronary arteries for single stenting (9 vessels: 3 GS,3 SES,and 3 bare-metal stents) and overlapping stenting with various combinations (12 vessels: 4 GS+GS,4 SES+SES,and 4 GS+SES) and harvested at 14 days for scanning electron and confocal microscopy. In phase 2,immobilized anti-CD34 antibody coating was applied on commercially available SES (SES-anti-CD34,n = 7) and compared with GS (n = 8) and SES (n = 7) and examined at 3 and 14 days by scanning electron/confocal microscopy analysis. RESULTS: In phase 1,single stent implantation showed greatest endothelialization in GS (99%) and in bare-metal stent (99%) compared with SES (55%,p = 0.048). In overlapping stents,endothelialization at the overlapping zone was significantly greater in GS+GS (95 +/- 6%) and GS+SES (79 +/- 5%) compared with the SES+SES (36 +/- 14%) group (p = 0.007). In phase 2,SES-anti-CD34 resulted in increased endothelialization compared with SES alone at 3 days (SES-anti-CD34 36 +/- 26%; SES 7 +/- 3%; and GS 76 +/- 8%; p = 0.01),and 14 days (SES-anti-CD34 82 +/- 8%; SES 53 +/- 20%; and GS 98 +/- 2%; p = 0.009). CONCLUSIONS: Immobilization of anti-CD34 antibody on SES enhances endothelialization and may potentially be an effective therapeutic alternative to improve currently available drug-eluting stents. View Publication

The interaction between the linker for activation of T cells (LAT) with PLC-gamma1 is important for TCR-mediated Ca(2+) signaling and MAPK activation. Knock-in mice harboring a mutation at the PLC-gamma1 binding site (Y136) of LAT develop a severe lymphoproliferative syndrome. These mice have defective thymic development and selection and lack natural regulatory T cells,implicating a breakdown of both central and peripheral tolerance. To bypass this developmental defect,we developed a conditional knock-in line in which only LATY136F is expressed in mature T cells after deletion of the wild type LAT allele. Analysis of LATY136F T cells indicated that the interaction between LAT and PLC-gamma1 plays an important role in TCR-mediated signaling,proliferation,and IL-2 production. Furthermore,the deletion of LAT induced development of the lymphoproliferative syndrome in these mice. Although Foxp3(+) natural Treg cells were present in these mice after deletion,they were unable to suppress the proliferation of conventional T cells. Our data indicate that the binding of LAT to PLC-gamma1 is essential for the suppressive function of CD4(+)CD25(+) regulatory T cells. View Publication

CD8(+) T lymphocytes play a key role in host defense,in particular against important persistent viruses,although the critical functional properties of such cells in tissue are not fully defined. We have previously observed that CD8(+) T cells specific for tissue-localized viruses such as hepatitis C virus express high levels of the C-type lectin CD161. To explore the significance of this,we examined CD8(+)CD161(+) T cells in healthy donors and those with hepatitis C virus and defined a population of CD8(+) T cells with distinct homing and functional properties. These cells express high levels of CD161 and a pattern of molecules consistent with type 17 differentiation,including cytokines (e.g.,IL-17,IL-22),transcription factors (e.g.,retinoic acid-related orphan receptor gamma-t,P = 6 x 10(-9); RUNX2,P = 0.004),cytokine receptors (e.g.,IL-23R,P = 2 x 10(-7); IL-18 receptor,P = 4 x 10(-6)),and chemokine receptors (e.g.,CCR6,P = 3 x 10(-8); CXCR6,P = 3 x 10(-7); CCR2,P = 4 x 10(-7)). CD161(+)CD8(+) T cells were markedly enriched in tissue samples and coexpressed IL-17 with high levels of IFN-gamma and/or IL-22. The levels of polyfunctional cells in tissue was most marked in those with mild disease (P = 0.0006). These data define a T cell lineage that is present already in cord blood and represents as many as one in six circulating CD8(+) T cells in normal humans and a substantial fraction of tissue-infiltrating CD8(+) T cells in chronic inflammation. Such cells play a role in the pathogenesis of chronic hepatitis and arthritis and potentially in other infectious and inflammatory diseases of man. View Publication

The activating receptor NKG2D,expressed by natural killer (NK) cells and CD8(+) T cells,has a role in the specific killing of transformed cells. We examined NKG2D expression in patients with glioblastoma multiforme and found that NKG2D was downregulated on NK cells and CD8(+) T cells. Expression of NKG2D on lymphocytes significantly increased following tumor resection and correlated with an increased ability to kill NKG2D ligand-positive tumor targets. Despite the presence of soluble NKG2D ligands in the sera of glioblastoma patients,NKG2D downregulation was primarily caused by tumor-derived tumor growth factor-beta,suggesting that blocking of this cytokine may have therapeutic benefit. View Publication

Neuroblastoma is the most common extracranial solid tumor of childhood. Focal adhesion kinase (FAK) is an intracellular kinase that regulates both cellular adhesion and apoptosis. FAK is overexpressed in a number of human tumors including neuroblastoma. Previously,we have shown that the MYCN oncogene,the primary adverse prognostic indicator in neuroblastoma,regulates the expression of FAK in neuroblastoma. In this study,we have examined the effects of FAK inhibition upon neuroblastoma using a small molecule [1,2,4,5-benzenetetraamine tetrahydrochloride (Y15)] to inhibit FAK expression and the phosphorylation of FAK at the Y397 site. Utilizing both non-isogenic and isogenic MYCN(+)/MYCN(-) neuroblastoma cell lines,we found that Y15 effectively diminished phosphorylation of the Y397 site of FAK. Treatment with Y15 resulted in increased detachment,decreased cell viability and increased apoptosis in the neuroblastoma cell lines. We also found that the cell lines with higher MYCN are more sensitive to Y15 treatment than their MYCN negative counterparts. In addition,we have shown that treatment with Y15 in vivo leads to less tumor growth in nude mouse xenograft models,again with the greatest effects seen in MYCN(+) tumor xenografts. The results of the current study suggest that FAK and phosphorylation at the Y397 site plays a role in neuroblastoma cell survival,and that the FAK Y397 phosphorylation site is a potential therapeutic target for this childhood tumor. View Publication

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor (EPOR). The presence of EPO and its receptor in the CNS suggests a different function for EPO other than erythropoiesis. The purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation of neonatal spinal cord-derived neural progenitor cells. The effect of EPO on cell cycle progression was also examined,as well as the signaling cascades involved in this process. Our results showed that EPOR was present in the neural progenitor cells and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated neural progenitor cells indicated a reduced percentage of cells in G0/G1 phase,whereas the cell proliferation index (S phase plus G2/M phase) was increased. EPO also increased the proportion of 5-bromo-2-deoxyuridine (BrdU)-positive cells. With respect to the cell cycle signaling,we examined the cyclin-dependent kinases D1,D2 and E,and cyclin-dependent kinase inhibitors,p21cip1,p27kip1 and p57kip2. No significant differences were observed in the expression of these transcripts after EPO administration. Interestingly,the anti-apoptotic factors,mcl-1 and bcl-2 were significantly increased twofold. Moreover,these specific effects of EPO were eliminated by incubation of the progenitor cells with anti-EPO neutralizing antibody. Those observations suggested that EPO may play a role in normal spinal cord development by regulating cell proliferation and apoptosis. View Publication

Comprehensive in vitro and in vivo studies comparing EpCAM-based methods with other cytometric CTC enrichment technologies in metastatic colorectal cancer (mCRC) patients are lacking. We compare four manual cytometric methods to detect CTCs in vitro and in mCRC patients. The EpCAM-based technology,MACS HEA MicroBeads((R)),showed a significant better tumor cell recovery rate compared to other cytometric methods (p-valuetextless0.0001). CTCs of 38 mCRC patients were enriched with MACS HEA MicroBeads(R). Progression-free survival did significantly differ between mCRC patients without detectable and with textgreateror= 1 CTCs (p=0.007). CTC enrichment with EpCAM coupled antibodies is superior to other cytometric methods and is a feasible method for CTC detection in mCRC patients. View Publication

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support,but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study,five separate laboratories,each with experience in human embryonic stem cell culture,used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods,with propagation in the presence of Knockout Serum Replacer,FGF-2,and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment,death,and differentiated morphology by phase contrast microscopy,for growth by serial cell counts,and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems,only the control and those based on two commercial media,mTeSR1 and STEMPRO,supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment,cell death,or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study,and the lack of success with other formulations from academic groups compared to previously published results,include: the complex combination of growth factors present in the commercial preparations; improved development,manufacture,and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. View Publication

The pressing demand to elucidate the biology of human embryonic stem (ES) cells and to realize their therapeutic potential has greatly promoted the progresses in the optimization of the culture systems used for this highly promising cell type. These progresses include the characterization of exogenous regulators of pluripotency and differentiation,the development of animal-free,defined,and scalable culture systems,and some pioneering efforts to establish good manufactory practice facilities to derive and expand clinical-grade human ES cells and their derivatives. All of these advancements appear to be also applicable to the derivation and culture of human induced pluripotent stem cells,an ES cell-like cell type derived from somatic cells via reprogramming. This review attempts to summarize these progresses and discuss some of the remaining challenges. View Publication