技术资料

Microbial products serving as superantigens (SAgs) have been implicated in triggering various T cell-mediated chronic inflammatory disorders,including severe asthma. Given earlier evidence demonstrating that airway smooth muscle (ASM) cells express MHC class II molecules,we investigated whether ASM can present SAg to resting CD4(+) T cells,and further examined whether this action reciprocally elicits proasthmatic changes in ASM responsiveness. Coincubation of CD4(+) T cells with human ASM cells pulsed with the SAg,staphylococcal enterotoxin A (SEA),elicited adherence and clustering of class II and CD3 molecules at the ASM/T cell interface,indicative of immunological synapse formation,in association with T cell activation. This ASM/T cell interaction evoked up-regulated mRNA expression and pronounced release of the Th2-type cytokine,IL-13,into the coculture medium,which was MHC class II dependent. Moreover,when administering the conditioned medium from the SEA-stimulated ASM/T cell cocultures to isolated naive rabbit ASM tissues,the latter exhibited proasthmatic-like changes in their constrictor and relaxation responsiveness that were prevented by pretreating the tissues with an anti-IL-13 neutralizing Ab. Collectively,these observations are the first to demonstrate that ASM can present SAg to CD4(+) T cells,and that this MHC class II-mediated cooperative ASM/T cell interaction elicits release of IL-13 that,in turn,evokes proasthmatic changes in ASM constrictor and relaxant responsiveness. Thus,a new immuno-regulatory role for ASM is identified that potentially contributes to the pathogenesis of nonallergic (intrinsic) asthma and,accordingly,may underlie the reported association between microbial SAg exposure,T cell activation,and severe asthma. View Publication

During pregnancy the uterine decidua is populated by large numbers of natural killer (NK) cells with a phenotype CD56(superbright)CD16(-)CD9(+)KIR(+) distinct from both subsets of peripheral blood NK cells. Culture of highly purified CD16(+)CD9(-) peripheral blood NK cells in medium containing TGFbeta1 resulted in a transition to CD16(-)CD9(+) NK cells resembling decidual NK cells. Decidual stromal cells,when isolated and cultured in vitro,were found to produce TGFbeta1. Incubation of peripheral blood NK cells with conditioned medium from decidual stromal cells mirrored the effects of TGFbeta1. Similar changes may occur upon NK cell entry into the decidua or other tissues expressing substantial TGFbeta. In addition,Lin(-)CD34(+)CD45(+) hematopoietic stem/progenitor cells could be isolated from decidual tissue. These progenitors also produced NK cells when cultured in conditioned medium from decidual stromal cells supplemented with IL-15 and stem cell factor. View Publication

Activated tyrosine kinases have been frequently implicated in the pathogenesis of cancer,including acute myeloid leukemia (AML),and are validated targets for therapeutic intervention with small-molecule kinase inhibitors. To identify novel activated tyrosine kinases in AML,we used a discovery platform consisting of immunoaffinity profiling coupled to mass spectrometry that identifies large numbers of tyrosine-phosphorylated proteins,including active kinases. This method revealed the presence of an activated colony-stimulating factor 1 receptor (CSF1R) kinase in the acute megakaryoblastic leukemia (AMKL) cell line MKPL-1. Further studies using siRNA and a small-molecule inhibitor showed that CSF1R is essential for the growth and survival of MKPL-1 cells. DNA sequence analysis of cDNA generated by 5'RACE from CSF1R coding sequences identified a novel fusion of the RNA binding motif 6 (RBM6) gene to CSF1R gene generated presumably by a t(3;5)(p21;q33) translocation. Expression of the RBM6-CSF1R fusion protein conferred interleukin-3 (IL-3)-independent growth in BaF3 cells,and induces a myeloid proliferative disease (MPD) with features of megakaryoblastic leukemia in a murine transplant model. These findings identify a novel potential therapeutic target in leukemogenesis,and demonstrate the utility of phosphoproteomic strategies for discovery of tyrosine kinase alleles. View Publication

The catalytic domain of the flavin-dependent human histone demethylase lysine-specific demethylase 1 (LSD1) belongs to the family of amine oxidases including polyamine oxidase and monoamine oxidase (MAO). We previously assessed monoamine oxidase inhibitors (MAOIs) for their ability to inhibit the reaction catalyzed by LSD1 [Lee,M. G.,et al. (2006) Chem. Biol. 13,563-567],demonstrating that trans-2-phenylcyclopropylamine (2-PCPA,tranylcypromine,Parnate) was the most potent with respect to LSD1. Here we show that 2-PCPA is a time-dependent,mechanism-based irreversible inhibitor of LSD1 with a KI of 242 microM and a kinact of 0.0106 s-1. 2-PCPA shows limited selectivity for human MAOs versus LSD1,with kinact/KI values only 16-fold and 2.4-fold higher for MAO B and MAO A,respectively. Profiles of LSD1 activity and inactivation by 2-PCPA as a function of pH are consistent with a mechanism of inactivation dependent upon enzyme catalysis. Mass spectrometry supports a role for FAD as the site of covalent modification by 2-PCPA. These results will provide a foundation for the design of cyclopropylamine-based inhibitors that are selective for LSD1 to probe its role in vivo. View Publication

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Disruption of pathways leading to programmed cell death plays a major role in most malignancies,including multiple myeloma (MM). ABT-737 is a BH3 mimetic small-molecule inhibitor that binds with high affinity to Bcl-2 and Bcl-xL,preventing the sequestration of proapoptotic molecules and shifting the cell survival/apoptosis balance toward apoptosis induction. In this study,we show that ABT-737 is cytotoxic to MM cell lines,including those resistant to conventional therapies,and primary tumor cells. Flow cytometric analysis of intracellular levels of Bcl-2 family proteins demonstrates a clear inversion of the Bax/Bcl-2 ratio leading to induction of apoptosis. Activation of the mitochondrial apoptosis pathway was indicated by mitochondrial membrane depolarization and caspase cleavage. Additionally,several signaling pathways known to be important for MM cell survival are disrupted following treatment with ABT-737. The impact of ABT-737 on survival could not be overcome by the addition of interleukin-6,vascular endothelial growth factor or insulin-like growth factor,suggesting that ABT-737 may be effective in preventing the growth and survival signals provided by the microenvironment. These data indicate that therapies targeting apoptotic pathways may be effective in MM treatment and warrant clinical evaluation of ABT-737 and similar drugs alone or in combination with other agents in the setting of MM. View Publication

In order to investigate the biologic processes underlying and resulting from the megakaryocytic hyperplasia that characterizes idiopathic myelofibrosis (IMF),peripheral blood CD34+ cells isolated from patients with IMF,polycythemia vera (PV),and G-CSF-mobilized healthy volunteers were cultured in the presence of stem cell factor and thrombopoietin. IMF CD34+ cells generated 24-fold greater numbers of megakaryocytes (MKs) than normal CD34+ cells. IMF MKs were also shown to have a delayed pattern of apoptosis and to overexpress the antiapoptotic protein bcl-xL. MK hyperplasia in IMF is,therefore,likely a consequence of both the increased ability of IMF progenitor cells to generate MKs and a decreased rate of MK apoptosis. Media conditioned (CM) by CD61+ cells generated in vitro from CD34+ cells were then assayed for the levels of growth factors and proteases. Higher levels of transforming growth factor-beta (TGF-beta) and active matrix metalloproteinase-9 (MMP9) were observed in media conditioned with IMF CD61+ cells than normal or PV CD61+ cells. Both normal and IMF CD61+ cells produced similar levels of VEGF. MK-derived TGF-B and MMP-9,therefore,likely contribute to the development of many pathological epiphenomena associated with IMF. View Publication

15-Methyl PGE2 and 16,16-dimethyl PGE2 were found (1) to be 40 and 100 times,respectively,more potent than PGE2 after intravenous administration in inhibiting histamine-stimulated gastric secretion in dogs with a denervated (Heidenhain) gastric pouch,(2) to be active orally and intrajejunally,whereas PGE2 was inactive,and (3) to exert antisecretory activity for longer duration than PGE2. 16,16-Dimethyl PGE2 was about 2.5 times more potent than 15-methyl PGE2. Volume,acid concentration,and output,and pepsin output (but not concentration) were reduced in a dose-dependent manner. In the rat,16,16-dimethyl PGE2 also inhibited gastric secretion and prevented the formation of ulcers produced by various methods: gastric ulcers (Shay,and steroid induced) and duodenal ulcers (secretogogue induced). In this species,1l816-dimethyl PGE2 was 2 to 50 times more potent than PGE2,depending on the endpoint,and was active orally. These prostaglandins appear to inhibit gastric acid secretion by acting directly on the parietal cells,and making these unresponsive to most stimulants. Vomiting was a side effect of the prostaglandin analogues in the dog,but almost exclusively when these were given orally. After intravenous or intrajejunal administration at doses inhibiting gastric secretion by 80%,vomiting was seen only once. These results suggest that 15-methyl PGE2 and 16,16-dimethyl PGE2 may be of value in the treatment of peptic ulcer. View Publication

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is an important component of tumor angiogenesis. Rho and Rho-associated kinase (ROCK) are key regulators of focal adhesion,stress fiber formation,and thus cell motility. Inhibitors of this pathway have been shown to inhibit endothelial cell motility and angiogenesis. In this study,we investigated the antiangiogenic effect of fasudil,one of the ROCK inhibitors. Fasudil inhibited VEGF-induced endothelial cell migration,viability,and tube formation in vitro in human umbilical vein endothelial cells. VEGF-induced endothelial cell migration was reduced by fasudil associated with loss of stress fiber formation,focal adhesion assembly,and with the suppression of tyrosine phosphorylation of focal adhesion proteins. Furthermore,fasudil inhibited VEGF-induced phosphorylation of myosin light chain,which is one of the main substrates of ROCK. Therefore,the effect of fasudil was suggested to be ROCK dependent. Fasudil not only inhibited VEGF-induced cell proliferation but also reversed the protective effect of VEGF on apoptosis,which resulted in the decrease of cell viability. Moreover,fasudil inhibited VEGF-induced angiogenesis in a directed in vivo angiogenesis assay. These data are the first demonstration that fasudil has antiangiogenic properties. Therefore,fasudil might be useful for the treatment of angiogenesis-related diseases,especially cancer. View Publication

After more than 40 years of clinical use,the mechanisms of action of valproate in epilepsy,bipolar disorder and migraine are still not fully understood. However,recent findings reviewed here shed new light on the cellular effects of valproate. Beyond the enhancement of gamma-aminobutyric acid-mediated neurotransmission,valproate has been found to affect signalling systems like the Wnt/beta-catenin and ERK pathways and to interfere with inositol and arachidonate metabolism. Nevertheless,the clinical relevance of these effects is not always clear. Valproate treatment also produces marked alterations in the expression of multiple genes,many of which are involved in transcription regulation,cell survival,ion homeostasis,cytoskeletal modifications and signal transduction. These alterations may well be relevant to the therapeutic effects of valproate,and result from its enhancement of activator protein-1 DNA binding and direct inhibition of histone deacetylases,and possibly additional,yet unknown,mechanism(s). Most likely,both immediate biochemical and longer-term genomic influences underlie the effects of valproate in all three indications. View Publication

A first step in hematopoiesis is the specification of the lymphoid and myeloid lineages from multipotent progenitor cells in the bone marrow. Using a conditional ablation strategy in adult mice,we show that this differentiation step requires Patched (Ptch),the cell surface-bound receptor for Hedgehog (Hh). In the absence of Ptch,the development of T- and B-lymphoid lineages is blocked at the level of the common lymphoid progenitor in the bone marrow. Consequently,the generation of peripheral T and B cells is abrogated. Cells of the myeloid lineage develop normally in Ptch mutant mice. Finally,adoptive transfer experiments identified the stromal cell compartment as a critical Ptch-dependent inducer of lymphoid versus myeloid lineage commitment. Our data show that Ptch acts as a master switch for proper diversification of hematopoietic stem cells in the adult organism. View Publication

JAK2V617F and MPLW515L/K represent recently identified mutations in myeloproliferative disorders (MPD) that cause dysregulated JAK-STAT signaling,which is implicated in MPD pathogenesis. We developed TG101209,an orally bioavailable small molecule that potently inhibits JAK2 (IC(50)=6 nM),FLT3 (IC(50)=25 nM) and RET (IC(50)=17 nM) kinases,with significantly less activity against other tyrosine kinases including JAK3 (IC(50)=169 nM). TG101209 inhibited growth of Ba/F3 cells expressing JAK2V617F or MPLW515L mutations with an IC(50) of approximately 200 nM. In a human JAK2V617F-expressing acute myeloid leukemia cell line,TG101209-induced cell cycle arrest and apoptosis,and inhibited phosphorylation of JAK2V617F,STAT5 and STAT3. Therapeutic efficacy of TG101209 was demonstrated in a nude mouse model. Furthermore,TG101209 suppressed growth of hematopoietic colonies from primary progenitor cells harboring JAK2V617F or MPL515 mutations. View Publication