技术资料

We investigated the involvement of the urokinase-type plasminogen-activator receptor (uPAR) in granulocyte-colony-stimulating factor (G-CSF)-induced mobilization of CD34+ hematopoietic stem cells (HSCs) from 16 healthy donors. Analysis of peripheral blood mononuclear cells (PBMNCs) showed an increased uPAR expression after G-CSF treatment in CD33+ myeloid and CD14+ monocytic cells,whereas mobilized CD34+ HSCs remained uPAR negative. G-CSF treatment also induced an increase in serum levels of soluble uPAR (suPAR). Cleaved forms of suPAR (c-suPAR) were released in vitro by PBMNCs and were also detected in the serum of G-CSF-treated donors. c-suPAR was able to chemoattract CD34+ KG1 leukemia cells and CD34+ HSCs,as documented by their in vitro migratory response to a chemotactic suPAR-derived peptide (uPAR84-95). uPAR84-95 induced CD34+ KG1 and CD34+ HSC migration by activating the high-affinity fMet-Leu-Phe (fMLP) receptor (FPR). In addition,uPAR84-95 inhibited CD34+ KG1 and CD34+ HSC in vitro migration toward the stromal-derived factor 1 (SDF1),thus suggesting the heterologous desensitization of its receptor,CXCR4. Finally,uPAR84-95 treatment significantly increased the output of clonogenic progenitors from long-term cultures of CD34+ HSCs. Our findings demonstrate that G-CSF-induced upregulation of uPAR on circulating CD33+ and CD14+ cells is associated with increased uPAR shedding,which leads to the appearance of serum c-suPAR. c-suPAR could contribute to the mobilization of HSCs by promoting their FPR-mediated migration and by inducing CXCR4 desensitization. View Publication

Wnts are important regulators of dopamine (DA) neuron differentiation in the developing ventral mesencephalon and could thus serve as potential tools in the treatment of Parkinson's disease. In this study,we investigate whether established intracellular Wnt signalling components could modulate the development of DA neurons. Two chemical inhibitors of glycogen synthase kinase (GSK)-3beta,indirubin-3-monoxime and kenpaullone,were found to increase neuronal differentiation in ventral mesencephalon precursor cultures. In addition,the GSK-3beta-specific inhibitor kenpaullone increased the size of the DA neuron population through conversion of precursors expressing the orphan nuclear receptor-related factor 1 into tyrosine hydroxylase positive neurons,thereby mimicking an effect of Wnts. We show that GSK-3beta inhibitors stabilized beta-catenin and that overexpression of beta-catenin in ventral mesencephalic precursors resulted in increased DA differentiation. The three- to fivefold increase in DA differentiation of precursor cells by GSK-3beta inhibitors suggests that such compounds could be used to improve stem/precursor cell therapy approaches in Parkinson's disease. View Publication

Thiazolidinediones (TZDs) increase peripheral tissue insulin sensitivity in patients with type 2 diabetes mellitus by activating the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). In bone marrow stromal cell cultures and in vivo,activation of PPARgamma by high doses (20 mg/kg/day) of TZDs has been reported to alter stem cell differentiation by promoting commitment of progenitor cells to the adipocytic lineage while inhibiting osteoblastogenesis. Here,we have examined the in vivo effects of low-dose rosiglitazone (3 mg/kg/day) on bone,administered to mice by gavage for 90 days. Rosiglitazone-treated mice had increased weight when compared with controls,with no significant alterations in serum levels of glucose,calcium or parathyroid hormone (PTH). Bone mineral density (BMD) at the lumbar vertebrae (L1-L4),ilium/sacrum,and total body was diminished by rosiglitazone treatment. Histologically,bone was characterized by decreased trabecular bone volume and increased marrow space with no significant change in bone marrow adipocity. Decreased osteoblast number and activity due to increased apoptotic death of osteoblasts and osteocytes was apparent while osteoclast parameters and serum levels of osteocalcin,alkaline phosphatase activity,and leptin were unaltered by rosiglitazone treatment. Therefore,the imbalance in bone remodeling that follows rosiglitazone administration arises from increased apoptotic death of osteogenic cells and diminished bone formation leading to the observed decrease in trabecular bone volume and BMD. These novel in vivo effects of TZDs on bone are of clinical relevance as patients with type 2 diabetes mellitus and other insulin resistant states treated with these agents may potentially be at increased risk of osteoporosis. View Publication

SU5416 is a multi-targeted kinase inhibitor that potentially has the ability to directly block tumor growth by inhibiting Kit signaling,as well as blocking angiogenesis by inhibiting vascular endothelial growth factor receptor (VEGFR) signaling. Previous work has demonstrated that SU5416 efficiently blocks Kit-mediated growth of small cell lung cancer (SCLC) in vitro. To determine the drug's effect on in vivo growth of SCLC,we studied its activity,alone and in combination with carboplatin,in chemotherapy-resistant H526,and chemotherapy-sensitive H209 murine xenograft models. SU5416 efficiently inhibited Kit activity in vivo when administered on a twice-weekly schedule. When administered over a 3-week period to animals bearing established tumors,it inhibited growth by at least 70%. It was at least as effective as carboplatin in suppressing growth of H526 xenografts. However,the combination with carboplatin was not superior to the most active single agent in either xenograft model at the doses and schedule utilized. SU5416 clearly inhibited growth in part by inhibiting angiogenesis,with microvessel density dropping by approximately 50% in treated xenografts. In addition to the recognized mechanism of inhibition of VEGFR,we uncovered a novel mechanism of angiogenesis suppression by demonstrating reduced VEGF expression in SU5416-treated xenografts. In vitro,stem cell factor treatment of the H526 cell line enhanced expression of VEGF,which was efficiently blocked with SU5416. Thus,we have demonstrated that SU5416 can inhibit SCLC growth by directly inhibiting tumor cell proliferation and by inhibiting angiogenesis,in part by inhibiting Kit-mediated VEGF expression. These data suggest that kinase inhibitors that target both Kit and VEGFR could play an important role in the treatment of SCLC,as well as other malignancies that express Kit. View Publication

There are several different technical approaches to the isolation of hematopoietic stem cells (HSCs) with long-term repopulating ability,but these have problems in terms of yield,complexity,or cell viability. Simpler strategies for HSC isolation are needed. We have enriched primitive hematopoietic progenitors from murine bone marrow of mice from different genetic backgrounds by lineage depletion followed by selection of cells with high aldehyde dehydrogenase activity using the Aldefluor reagent (BD Biosciences,Oxford,U.K.). Lin- ALDH(bright) cells comprised 26.8 +/- 1.0% of the total Lin- population of C57BL6 mice,and 23.5 +/- 1.0% of the Lin- population of BALB/c mice expressed certain cell-surface markers typical of primitive hematopoietic progenitors. In vitro hematopoietic progenitor function was substantially higher in the Lin- ALDH(bright) population compared with the Lin- ALDH(low) cells. These cells have higher telomerase activity and the lowest percentage of cells in S phase. These data strongly suggest that progenitor enrichment from Lin- cells on the basis of ALDH is a valid method whose simplicity of application makes it advantageous over conventional separations. View Publication

Overwhelming evidence from leukemia research has shown that the clonal population of neoplastic cells exhibits marked heterogeneity with respect to proliferation and differentiation. There are rare stem cells within the leukemic population that possess extensive proliferation and self-renewal capacity not found in the majority of the leukemic cells. These leukemic stem cells are necessary and sufficient to maintain the leukemia. Interestingly,the BCR/ABL fusion gene,which is present in chronic myelogenous leukemia (CML),was also detected in the endothelial cells of patients with CML,suggesting that CML might originate from hemangioblastic progenitor cells that can give rise to both blood cells and endothelial cells. Here we isolated fetal liver kinase-1-positive (Flk1+) cells carrying the BCR/ABL fusion gene from the bone marrow of 17 Philadelphia chromosome-positive (Ph+) patients with CML and found that these cells could differentiate into malignant blood cells and phenotypically defined endothelial cells at the single-cell level. These findings provide direct evidence for the first time that rearrangement of the BCR/ABL gene might happen at or even before the level of hemangioblastic progenitor cells,thus resulting in detection of the BCR/ABL fusion gene in both blood and endothelial cells. View Publication

The role of human NK cells in viral infections is poorly understood. We used a cytokine flow-cytometry assay to simultaneously investigate the IFN-gamma response of NK and T lymphocytes to influenza A virus (fluA). When PBMCs from fluA-immune adult donors were incubated with fluA,IFN-gamma was produced by both CD56(dim) and CD56(bright) subsets of NK cells,as well as by fluA-specific T cells. Purified NK cells did not produce IFN-gamma in response to fluA,while depletion of T lymphocytes reduced to background levels the fluA-induced IFN-gamma production by NK cells,which indicates that T cells are required for the IFN-gamma response of NK cells. The fluA-induced IFN-gamma production of NK cells was suppressed by anti-IL-2 Ab,while recombinant IL-2 replaced the helper function of T cells for IFN-gamma production by NK cells. This indicates that IL-2 produced by fluA-specific T cells is involved in the T cell-dependent IFN-gamma response of NK cells to fluA. Taken together,these results suggest that at an early stage of recurrent viral infection,NK-mediated innate immunity to the virus is enhanced by preexisting virus-specific T cells. View Publication

Monocyte cytokines (ie,monokines) induce natural killer (NK) cells to produce interferon-gamma (IFN-gamma),which is critical for monocyte clearance of infectious pathogens and tumor surveillance. Human CD56bright NK cells produce far more IFN-gamma in response to monokines than do CD56dim NK cells. The kinases and phosphatases involved in regulating IFN-gamma production by monokine-activated NK cells are not clearly identified. SHIP1 is a 5' inositol phosphatase that dephosphorylates the phosphatidylinositol-3 kinase (PI-3K) product PI3,4,5P3. Here,we show that constitutive expression of SHIP1 is distinctly lower in CD56bright NK cells compared with CD56dim NK cells,suggesting it could be an important negative regulator of IFN-gamma production in monokine-activated NK cells. Indeed,overexpression of SHIP1 in CD56bright NK cells followed by monokine activation substantially lowered IFN-gamma production. This effect was not seen when NK cells were infected with a SHIP1 mutant containing an inactive catalytic domain. Finally,NK cells in SHIP1-/- mice produced more IFN-gamma in response to monokines in vivo than did NK cells from wild-type mice. Collectively,these results demonstrate that SHIP1 negatively regulates monokine-induced NK cell IFN-gamma production in vitro and in vivo and provide the first molecular explanation for an important functional distinction observed between CD56bright and CD56dim human NK subsets. View Publication

OBJECTIVE To investigate the long-term effect of cryopreservation on hepatocyte function,as well as attempt to improve cell viability and function through the utilization of the hypothermic preservation solution,HypoThermosol (HTS),as the carrier solution. SUMMARY BACKGROUND DATA Advances in the field of bioartificial liver support have led to an increasing demand for successful,efficient means of cryopreservation of hepatocytes. METHODS Fresh rat hepatocytes were cryopreserved in suspension in culture media (Media-cryo group) or HTS (HTS-cryo group),both supplemented with 10% DMSO. Following storage up to 2 months in liquid nitrogen,cells were thawed and maintained in a double collagen gel culture for 14 days. Hepatocyte yield and viability were assessed up to 14 days postthaw. Serial measurements of albumin secretion,urea synthesis,deethylation of ethoxyresorufin (CYT P450 activity),and responsiveness to stimulation with interleukin-6 (IL-6) were performed. RESULTS Immediate postthaw viability was 60% in Media-cryo and 79% in HTS-cryo,in comparison with control (90%). Albumin secretion,urea synthesis and CYT P450 activity yielded 33%,55%,and 59% in Media-cryo and 71%,80%,and 88% in HTS-cryo,respectively,compared with control (100%). Assessment of cellular response to IL-6 following cryopreservation revealed a similar pattern of up-regulation in fibrinogen production and suppression of albumin secretion compared with nonfrozen controls. CONCLUSIONS This study demonstrates that isolated rat hepatocytes cryopreserved using HTS showed high viability,long-term hepatospecific function,and response to cytokine challenge. These results may represent an important step forward to the utilization of cryopreserved isolated hepatocytes in bioartificial liver devices. View Publication

The Notch signaling pathway controls several cell fate decisions during lymphocyte development,from T-cell lineage commitment to the peripheral differentiation of B and T lymphocytes. Deltex-1 is a RING finger ubiquitin ligase which is conserved from Drosophila to humans and has been proposed to be a regulator of Notch signaling. Its pattern of lymphoid expression as well as gain-of-function experiments suggest that Deltex-1 regulates both B-cell lineage and splenic marginal-zone B-cell commitment. Deltex-1 was also found to be highly expressed in germinal-center B cells. To investigate the physiological function of Deltex-1,we generated a mouse strain lacking the Deltex-1 RING finger domain,which is essential for its ubiquitin ligase activity. Deltex-1(Delta/Delta) mice were viable and fertile. A detailed histological analysis did not reveal any defects in major organs. T- and B-cell development was normal,as were humoral responses against T-dependent and T-independent antigens. These data indicate that the Deltex-1 ubiquitin ligase activity is dispensable for mouse development and immune function. Possible compensatory mechanisms,in particular those from a fourth Deltex gene identified during the course of this study,are also discussed. View Publication

Recent studies indicate that dysregulation of the Akt/PKB family of serine/threonine kinases is a prominent feature of many human cancers. The Akt/PKB family is composed of three members termed Akt1/PKBalpha,Akt2/PKBbeta,and Akt3/PKBgamma. It is currently not known to what extent there is functional overlap between these family members. We have recently identified small molecule inhibitors of Akt. These compounds have pleckstrin homology domain-dependent,isozyme-specific activity. In this report,we present data showing the relative contribution that inhibition of the different isozymes has on the apoptotic response of tumor cells to a variety of chemotherapies. In multiple cell backgrounds,maximal induction of caspase-3 activity is achieved when both Akt1 and Akt2 are inhibited. This induction is not reversed by overexpression of functionally active Akt3. The level of caspase-3 activation achieved under these conditions is equivalent to that observed with the phosphatidylinositol-3-kinase inhibitor LY294002. We also show that in different tumor cell backgrounds inhibition of mammalian target of rapamycin,a downstream substrate of Akt,is less effective in inducing caspase-3 activity than inhibition of Akt1 and Akt2. This shows that the survival phenotype conferred by Akt can be mediated by signaling pathways independent of mammalian target of rapamycin in some tumor cell backgrounds. Finally,we show that inhibition of both Akt1 and Akt2 selectively sensitizes tumor cells,but not normal cells,to apoptotic stimuli. View Publication

We provide evidence that tumor cells can induce apoptosis of NK cells by engaging the natural cytotoxicity receptors (NCR) NKp30,NKp44,and NKp46. Indeed,the binding between NCR on NK cells and their putative ligands on tumor target cells led to NK cell apoptosis,and this event was abolished by blocking NCR/NCR-ligand interaction by anti-NCR-specific mAbs. The engagement of NCR induced up-regulation of Fas ligand (FasL) mRNA,FasL protein synthesis,and release. In turn,FasL interacting with Fas at NK cell surface causes NK cell suicide,as apoptosis of NK cells was inhibited by blocking FasL/Fas interaction with specific mAbs. Interestingly,NK cell apoptosis,but not killing of tumor target cells,is inhibited by cyclosporin A,suggesting that apoptosis and cytolysis are regulated by different biochemical pathways. These findings indicate that NCR are not only triggering molecules essential for antitumor activity,but also surface receptors involved in NK cell suicide. View Publication