参考文献

Drug resistance resulting from emergence of imatinib-resistant BCR-ABL point mutations is a significant problem in advanced-stage chronic myelogenous leukemia (CML). The BCR-ABL inhibitor,nilotinib (AMN107),is significantly more potent against BCR-ABL than imatinib,and is active against many imatinib-resistant BCR-ABL mutants. Phase 1/2 clinical trials show that nilotinib can induce remissions in patients who have previously failed imatinib,indicating that sequential therapy with these 2 agents has clinical value. However,simultaneous,rather than sequential,administration of 2 BCR-ABL kinase inhibitors is attractive for many reasons,including the theoretical possibility that this could reduce emergence of drug-resistant clones. Here,we show that exposure of a variety of BCR-ABL+ cell lines to imatinib and nilotinib results in additive or synergistic cytotoxicity,including testing of a large panel of cells expressing BCR-ABL point mutations causing resistance to imatinib in patients. Further,using a highly quantifiable bioluminescent in vivo model,drug combinations were at least additive in antileukemic activity,compared with each drug alone. These results suggest that despite binding to the same site in the same target kinase,the combination of imatinib and nilotinib is highly efficacious in these models,indicating that clinical testing of combinations of BCR-ABL kinase inhibitors is warranted. View Publication

In the adult,platelets are derived from unipotential megakaryocyte colony-forming cells (Meg-CFCs) that arise from bipotential megakaryocyte/erythroid progenitors (MEPs). To better define the developmental origin of the megakaryocyte lineage,several aspects of megakaryopoiesis,including progenitors,maturing megakaryocytes,and circulating platelets,were examined in the murine embryo. We found that a majority of hemangioblast precursors during early gastrulation contains megakaryocyte potential. Combining progenitor assays with immunohistochemical analysis,we identified 2 waves of MEPs in the yolk sac associated with the primitive and definitive erythroid lineages. Primitive MEPs emerge at E7.25 along with megakaryocyte and primitive erythroid progenitors,indicating that primitive hematopoiesis is bilineage in nature. Subsequently,definitive MEPs expand in the yolk sac with Meg-CFCs and definitive erythroid progenitors. The first GP1bbeta-positive cells in the conceptus were identified in the yolk sac at E9.5,while large,highly reticulated platelets were detected in the embryonic bloodstream beginning at E10.5. At this time,the number of megakaryocyte progenitors begins to decline in the yolk sac and expand in the fetal liver. We conclude that the megakaryocyte lineage initially originates from hemangioblast precursors during early gastrulation and is closely associated both with primitive and with definitive erythroid lineages in the yolk sac prior to the transition of hematopoiesis to intraembryonic sites. View Publication

Recent population studies provide clues that the use of metformin may be associated with reduced incidence and improved prognosis of certain cancers. This drug is widely used in the treatment of type 2 diabetes,where it is often referred to as an insulin sensitizer" because it not only lowers blood glucose but also reduces the hyperinsulinemia associated with insulin resistance. As insulin and insulin-like growth factors stimulate proliferation of many normal and transformed cell types� View Publication

Lysophosphatidic acid (LPA) is an extracellular lipid mediator that regulates cortical development. Here we examined how LPA influences the cell fate of cortical neuroblasts using a neurosphere culture system. We generated neurospheres in the presence of basic fibroblast growth factor (bFGF). Treatment with LPA throughout the culture period significantly reduced the number of cells in the neurospheres. When dissociated single cells derived from neurospheres were induced to differentiate by adherence on coverslips,the proportion of MAP2-positive neurons was higher in LPA-treated neurospheres than in those treated with bFGF alone,and the proportion of myelin basic protein-positive oligodendrocytes was lower. Consistent with this finding,LPA raised the ratio of beta-tubulin type III-positive young neurons and reduced the ratio of CD140a-positive oligodendrocyte precursors in neurospheres. These effects of LPA were inhibited by pretreatment of neurospheres with pertussis toxin or an LPA(1)-preferring antagonist,Ki16425. Moreover,LPA-induced enhancement of neuronal differentiation was not observed in neurospheres derived from lpa(1)-null mice. These results suggest that LPA promotes the commitment of neuroblasts to the neural lineage through the LPA(1)-G(i/o) pathway. View Publication

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin,glucagon,somatostatin,pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm,gut-tube endoderm,pancreatic endoderm and endocrine precursor--en route to cells that express endocrine hormones. The hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal beta-cells,they release C-peptide in response to multiple secretory stimuli,but only minimally to glucose. Production of these hES cell-derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy. View Publication

The interleukin-12 (IL-12) cytokine induces the differentiation of naive T cells to the T helper cell type 1 (Th1) phenotype and is integral to the pathogenesis of Th1-mediated immunologic disorders. A more recently discovered IL-12 family member,IL-23,shares the p40 protein subunit with IL-12 and plays a critical role in the generation of effector memory T cells and IL-17-producing T cells. We introduce a novel compound,STA-5326,that down-regulates both IL-12 p35 and IL-12/IL-23 p40 at the transcriptional level,and inhibits the production of both IL-12 and IL-23 cytokines. Oral administration of STA-5326 led to a suppression of the Th1 but not Th2 immune response in mice. In vivo studies using a CD4+CD45Rbhigh T-cell transfer severe combined immunodeficiency (SCID) mouse inflammatory bowel disease model demonstrated that oral administration of STA-5326 markedly reduced inflammatory histopathologic changes in the colon. A striking decrease in interferon-gamma (IFN-gamma) production was observed in ex vivo culture of lamina propria cells harvested from animals treated with STA-5326,indicating a down-regulation of the Th1 response by STA-5326. These results suggest that STA-5326 has potential for use in the treatment of Th1-related autoimmune or immunologic disorders. STA-5326 currently is being evaluated in phase 2 clinical trials in patients with Crohn disease and rheumatoid arthritis. View Publication

The human pre-T-cell receptor alpha (TCRalpha; pTalpha) gene encodes a polypeptide which associates with the TCRbeta chain and CD3 molecules to form the pre-TCR complex. The surface expression of the pre-TCR is pTalpha dependent,and signaling through this complex triggers an early alphabeta T-cell developmental checkpoint inside the thymus,known as beta-selection. E2A transcription factors,which are involved at multiple stages of T-cell development,regulate the transcription of the pTalpha gene. Here we show that the regulatory protein Tax of the human T-cell leukemia virus type 1 (HTLV-1) efficiently suppresses the E47-mediated activation of the pTalpha promoter. Furthermore,we report that in Tax lentivirally transduced human MOLT-4 T cells,which constitutively express the pTalpha gene,the amount of pTalpha transcripts decreases. Such a decrease is not observed in MOLT-4 cells transduced by a vector encoding the Tax mutant K88A,which is unable to interact with p300. These data underline that Tax inhibits pTalpha transcription by recruiting this coactivator. Finally,we show that the expression of Tax in human immature thymocytes results in a decrease of pTalpha gene transcription but does not modify the level of E47 transcripts. These observations indicate that Tax,by silencing E proteins,down-regulates pTalpha gene transcription during early thymocyte development. They further provide evidence that Tax can interfere with an important checkpoint during T-cell differentiation in the thymus. View Publication

Toll-like receptors (TLRs) play a key role in pathogen recognition and regulation of the innate and adaptive immune responses. Although TLR expression and signaling have been investigated in blood cells,it is currently unknown whether their bone marrow ancestors express TLRs and respond to their ligands. Here we found that TLRs (e.g. TLR4,TLR7 and TLR8) were expressed by freshly isolated human bone marrow (BM) hematopoietic CD34+ progenitor cells. Incubation of these primitive cells with TLR ligands such as immunostimulatory small interfering RNAs and R848,a specific ligand for TLR7/8,induced cytokine production (e.g. IL1-beta,IL6,IL8,TNF-alpha,GM-CSF). Moreover,TLR7/8 signaling induced the differentiation of BM CD34+ progenitors into cells with the morphology of macrophages and monocytic dendritic precursors characterized by the expression of CD13,CD14 and/or CD11c markers. By contrast,R848 ligand did not induce the expression of glycophorin A,an early marker for erythropoiesis. Collectively,the data indicate for the first time that human BM CD34+ progenitor cells constitutively express functional TLR7/TLR8,whose ligation can induce leukopoiesis without the addition of any exogenous cytokines. Thus,TLR signaling may regulate BM cell development in humans. View Publication

Combined deletion of chromosomes 1p and 19q is associated with improved prognosis and responsiveness to therapy in patients with anaplastic oligodendroglioma. The deletions usually involve whole chromosome arms,suggesting a t(1;19)(q10;p10). Using stem cell medium,we cultured a few tumors. Paraffin-embedded tissue was obtained from 21 Mayo Clinic patients and 98 patients enrolled in 2 North Central Cancer Treatment Group (NCCTG) low-grade glioma trials. Interphase fusion of CEP1 and 19p12 probes detected the t(1;19). 1p/19q deletions were evaluated by fluorescence in situ hybridization. Upon culture,one oligodendroglioma contained an unbalanced 45,XX,t(1;19)(q10;p10). CEP1/19p12 fusion was observed in all metaphases and 74% of interphase nuclei. Among Mayo Clinic oligodendrogliomas,the prevalence of fusion was 81%. Among NCCTG patients,CEP1/19p12 fusion prevalence was 55%,47%,and 0% among the oligodendrogliomas,mixed oligoastrocytomas,and astrocytomas,respectively. Ninety-one percent of NCCTG gliomas with 1p/19q deletion and 12% without 1p/19q deletion had CEP1/19p12 fusion (P textless 0.001,chi(2) test). The median overall survival (OS) for all patients was 8.1 years without fusion and 11.9 years with fusion (P = 0.003). The median OS for patients with low-grade oligodendroglioma was 9.1 years without fusion and 13.0 years with fusion (P = 0.01). Similar significant median OS differences were observed for patients with combined 1p/19q deletions. The absence of alterations was associated with a significantly shorter OS for patients who received higher doses of radiotherapy. Our results strongly suggest that a t(1;19)(q10;p10) mediates the combined 1p/19q deletion in human gliomas. Like combined 1p/19q deletion,the 1;19 translocation is associated with superior OS and progression-free survival in low-grade glioma patients. View Publication

We report the systematic use of large-scale cDNA microarrays to study the gene expression profiles of primary human peripheral blood monocytes (MONO) in comparison with in vitro-differentiated,M-CSF-induced MONO-derived macrophages (MAC) and primary human alveolar MAC (AM),obtained by bronchoalveolar lavage from the lungs of normal volunteers. These studies revealed large-scale differences in the gene expression profile between both MAC types (MAC and AM) and MONO. In addition,large differences were observed in the gene expression profiles of the two MAC types. Specifically,21% of genes on the array (2904 out of 13,582) were differentially expressed between AM and MONO,and 2229 out of 13,583 probes were differentially expressed between MAC and AM. Our expression data show remarkable differences in gene expression between different MAC subpopulations and emphasize the heterogeneity of different MAC populations. This study underscores the need to scrutinize models of MAC biology for relevance to specific disease processes. View Publication

Monitoring genome-wide,cell-specific responses to human disease,although challenging,holds great promise for the future of medicine. Patients with injuries severe enough to develop multiple organ dysfunction syndrome have multiple immune derangements,including T cell apoptosis and anergy combined with depressed monocyte antigen presentation. Genome-wide expression analysis of highly enriched circulating leukocyte subpopulations,combined with cell-specific pathway analyses,offers an opportunity to discover leukocyte regulatory networks in critically injured patients. Severe injury induced significant changes in T cell (5,693 genes),monocyte (2,801 genes),and total leukocyte (3,437 genes) transcriptomes,with only 911 of these genes common to all three cell populations (12%). T cell-specific pathway analyses identified increased gene expression of several inhibitory receptors (PD-1,CD152,NRP-1,and Lag3) and concomitant decreases in stimulatory receptors (CD28,CD4,and IL-2Ralpha). Functional analysis of T cells and monocytes confirmed reduced T cell proliferation and increased cell surface expression of negative signaling receptors paired with decreased monocyte costimulation ligands. Thus,genome-wide expression from highly enriched cell populations combined with knowledge-based pathway analyses leads to the identification of regulatory networks differentially expressed in injured patients. Importantly,application of cell separation,genome-wide expression,and cell-specific pathway analyses can be used to discover pathway alterations in human disease. View Publication

Thyroid neoplasia is frequently associated with rearranged during transfection (RET) proto-oncogene mutations that cause hyperactivation of RET kinase activity. Selective inhibition of RET-mediated signaling should lead to an efficacious therapy. SU5416 is a potent inhibitor of vascular endothelial cell growth factor receptor,c-Kit,and FLT-3 receptor tyrosine kinases presently used in clinical trials. We found that SU5416 inhibits RET with similar potency,both in cell-free assays and in cells,thus causing proliferation arrest in oncogenic RET-transfected cells and in papillary thyroid carcinoma (PTC) cells expressing the RET/PTC1 oncogene,but not in RET-negative control cells. SU5416 inhibited RET-mediated signaling through the extracellular signal regulated kinase (ERK) and JNK pathways. In addition,we show that a naturally occurring MEN2 mutation at codon 804 confers resistance to SU5416,but not to the related compound SU4984. We provide a possible explanation to these results by using molecular docking. Finally,SU5416 was also assessed against an array of 52 tyrosine and serine/threonine kinases. View Publication