技术资料

OBJECTIVE: To investigate the kinetics of myocardial engraftment of bone marrow-derived mononuclear cells (BMNCs) after intracoronary injection using 99mTc-d,l-hexamethylpropylene amine oxime (99mTc-HMPAO) nuclear imaging in patients with acute and chronic anterior myocardial infarction. DESIGN: Nuclear imaging-derived tracking of BMNCs at 2 and 20 h after injection in the left anterior descending (LAD) coronary artery. SETTING: Academical cardiocentre. PATIENTS: Five patients with acute (mean (SD) age 58 (11) years; ejection fraction range 33-45%) and five patients with chronic (mean (SD) age 50 (6) years; ejection fraction range 28-34%) anterior myocardial infarction. INTERVENTIONS: A total of 24.2 x 10(8)-57.0 x 10(8) BMNCs (20% labelled with 700-1000 MBq 99mTc-HMPAO) were injected in the LAD coronary artery. RESULTS: At 2 h after BMNC injection,myocardial activity was observed in all patients with acute (range 1.31-5.10%) and in all but one patient with chronic infarction (range 1.10-3.0%). At 20 h,myocardial engraftment was noted only in three patients with acute myocardial infarction,whereas no myocardial activity was noted in any patient with chronic infarction. CONCLUSIONS: Engraftment of BMNCs shows dynamic changes within the first 20 h after intracoronary injection. Persistent myocardial engraftment was noted only in a subset of patients with acute myocardial infarction. View Publication

Differences in clinical outcome of simian immunodeficiency virus (SIV) infection in disease-resistant African sooty mangabeys (SM) and disease-susceptible Asian rhesus macaques (RM) prompted us to examine the role of regulatory T cells (Tregs) in these two animal models. Results from a cross-sectional study revealed maintenance of the frequency and absolute number of peripheral Tregs in chronically SIV-infected SM while a significant loss occurred in chronically SIV-infected RM compared to uninfected animals. A longitudinal study of experimentally SIV-infected animals revealed a transient increase in the frequency of Tregs from baseline values following acute infection in RM,but no change in the frequency of Tregs occurred in SM during this period. Further examination revealed a strong correlation between plasma viral load (VL) and the level of Tregs in SIV-infected RM but not SM. A correlation was also noted in SIV-infected RM that control VL spontaneously or in response to antiretroviral chemotherapy. In addition,immunofluorescent cell count assays showed that while Treg-depleted peripheral blood mononuclear cells from RM led to a significant enhancement of CD4+ and CD8+ T-cell responses to select pools of SIV peptides,there was no detectable T-cell response to the same pool of SIV peptides in Treg-depleted cells from SIV-infected SM. Our data collectively suggest that while Tregs do appear to play a role in the control of viremia and the magnitude of the SIV-specific immune response in RM,their role in disease resistance in SM remains unclear. View Publication

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid with growth-factor-like activities [van Corven,Groenink,Jalink,Eichholtz & Moolenaar (1989) Cell 45,45-54]. We have examined various structural analogues of LPA for their ability to stimulate DNA synthesis in quiescent fibroblasts. When the acyl-chain length is varied,the rank order of mitogenic potency is: 1-oleoyl LPA congruent to 1-palmitoyl LPA greater than 1-myristoyl LPA greater than 1-lauroyl LPA greater than 1-decanoyl LPA; the last compound shows almost no activity over the concentration range tested (1-100 microM). An ether-linked LPA (1-O-hexadecylglycerol 3-phosphate) has much decreased mitogenic activity as compared with the ester-linked analogue at concentrations less than 25 microM,and becomes cytotoxic at higher concentrations. Hexadecylphosphate,which lacks a glycerol backbone,has negligible activity. On a molar basis,diacyl phosphatidic acid (PA) is about equally potent as the corresponding LPA analogue,showing similar acyl-chain-length dependence; the data argue against the possibility that the mitogenic action of PA is due to contaminating traces of LPA. Although the short-chain analogues of LPA and PA fail to antagonize the action of long-chain (L)PAs,the polyanionic drug suramin inhibits LPA- and PA-induced,DNA synthesis in a reversible and dose-dependent manner,at concentrations [IC50 (concn. giving 50% inhibition) approximately 70 microM] that do not affect epidermal-growth-factor-induced DNA synthesis. Suramin appears to act in the early G0/G1 phase of the cell cycle,blocking immediate responses to LPA such as phosphoinositide hydrolysis. We conclude that both LPA and PA can function as growth-promoting phospholipids,with the fatty acid chain length being a major determinant of mitogenic potency. View Publication

Mouse embryonic stem (mES) cells have short duration of their cell cycle and are capable of proliferating in the absence of growth factors. To find out which signaling pathways contribute to the regulation of the mES cell cycle,we used pharmacological inhibitors of MAP and PI3 kinase cascades. The MAP kinase inhibitors as well as serum withdrawal did not affect mES cell cycle distribution,whereas the inhibitor of PI3K activity,LY294002,induced accumulation of cells in G(1) phase followed by apoptotic cell death. Serum withdrawal also causes apoptosis,but it does not change the content and activity of cell cycle regulators. In contrast,in mES cells treated with LY294002,the activities of Cdk2 and E2F were significantly decreased. Interestingly,LY294002had a much stronger effect on cell cycle distribution in low serum conditions,implying that serum can promote G(1)--textgreaterS transition of mES cells by a LY294002-resistant mechanism. Thus,proliferation of mES cells is maintained by at least two separate mechanisms: a LY294002-sensitive pathway,which is active even in the absence of serum,and LY294002-resistant,but serum-dependent,pathway. View Publication

Parthenogenetic embryonic stem (ES) cells with two oocyte-derived genomes (uniparental) have been proposed as a source of autologous tissue for transplantation. The therapeutic applicability of any uniparental cell type is uncertain due to the consequences of genomic imprinting that in mammalian uniparental tissues causes unbalanced expression of imprinted genes. We transplanted uniparental fetal liver cells into lethally irradiated adult mice to test their capacity to replace adult hematopoietic tissue. Both maternal (gynogenetic) and paternal (androgenetic) derived cells conveyed long-term,multilineage reconstitution of hematopoiesis in recipients,with no associated pathologies. We also establish that uniparental ES cells can differentiate into transplantable hematopoietic progenitors in vitro that contribute to long-term hematopoiesis in recipients. Hematopoietic tissue in recipients maintained fidelity of parent-of-origin methylation marks at the Igf2/H19 locus; however,variability occurred in the maintenance of parental-specific methylation marks at other loci. In summary,despite genomic imprinting and its consequences on development that are particularly evident in the androgenetic phenotype,uniparental cells of both parental origins can form adult-transplantable stem cells and can repopulate an adult organ. View Publication

Histone deacetylase inhibitors such as sodium butyrate are known to regulate the differentiation of a variety of cells. Mesenchymal stem cells (MSCs) differentiate into osteoblasts and adipocytes under transcriptional control of Runx2 and PPARgamma2,respectively. How these two transcription factors are regulated by sodium butyrate in order to specify the alternate cell fates remains a pivotal question. Sodium butyrate stimulated osteogenic differentiation and increased expression of Runx2 and genes regulated by Runx2 when cells were induced to undergo osteogenic differentiation. Sodium butyrate suppressed the adipogenic differentiation and decreased the expression of PPARgamma2 and LPL when MSCs were treated under conditions that promote adipogenic differentiation. Sodium butyrate also decreased the ratio of RANKL/OPG gene expression by MSCs. Analysis of MSCs induced in the presence of sodium butyrate revealed an immediate increase in ERK phosphorylation by sodium butyrate. The MEK-specific inhibitor,PD98059 but not p38- or JNK-specific inhibitor and the transfection with dominant negative ERK expressing plasmids blocked the sodium butyrate-induced regulation of MSC differentiation and increase in the RANKL/OPG ratio. Our results suggest that sodium butyrate modulates MSC differentiation and the RANKL/OPG ratio via activating ERK,and could be applied for in vivo bone growth using MSCs. View Publication

PURPOSE: The Ras-Raf-mitogen-activated protein kinase kinase (MEK) pathway is overactive in many human cancers and is thus a target for novel therapeutics. We have developed a highly potent and selective inhibitor of MEK1/2. The purpose of these studies has been to show the biological efficacy of ARRY-142886 (AZD6244) in enzymatic,cellular,and animal models. EXPERIMENTAL DESIGN: The ability of ARRY-142886 to inhibit purified MEK1 as well as other kinases was evaluated. Its effects on extracellular signal-regulated kinase (ERK) phosphorylation and proliferation in several cell lines were also determined. Finally,the inhibitor was tested in HT-29 (colorectal) and BxPC3 (pancreatic) xenograft tumor models. RESULTS: The IC(50) of ARRY-142886 was determined to be 14 nmol/L against purified MEK1. This activity is not competitive with ATP,which is consistent with the high specificity of compound for MEK1/2. Basal and epidermal growth factor-induced ERK1/2 phosphorylation was inhibited in several cell lines as well as 12-O-tetradecanoylphorbol-13-acetate-induced ERK1/2 phosphorylation in isolated peripheral blood mononuclear cells. Treatment with ARRY-142886 resulted in the growth inhibition of several cell lines containing B-Raf and Ras mutations but had no effect on a normal fibroblast cell line. When dosed orally,ARRY-142886 was capable of inhibiting both ERK1/2 phosphorylation and growth of HT-29 xenograft tumors in nude mice. Tumor regressions were also seen in a BxPC3 xenograft model. In addition,tumors remained responsive to growth inhibition after a 7-day dosing holiday. CONCLUSIONS: ARRY-142886 is a potent and selective MEK1/2 inhibitor that is highly active in both in vitro and in vivo tumor models. This compound is currently being investigated in clinical studies. View Publication

In the context of pancreatic cancer,metastasis remains the most critical determinant of resectability,and hence survival. The objective of this study was to determine whether Hedgehog (Hh) signaling plays a role in pancreatic cancer invasion and metastasis because this is likely to have profound clinical implications. In pancreatic cancer cell lines,Hh inhibition with cyclopamine resulted in down-regulation of snail and up-regulation of E-cadherin,consistent with inhibition of epithelial-to-mesenchymal transition,and was mirrored by a striking reduction of in vitro invasive capacity (P textless 0.0001). Conversely,Gli1 overexpression in immortalized human pancreatic ductal epithelial cells led to a markedly invasive phenotype (P textless 0.0001) and near total down-regulation of E-cadherin. In an orthotopic xenograft model,cyclopamine profoundly inhibited metastatic spread; only one of seven cyclopamine-treated mice developed pulmonary micrometastases versus seven of seven mice with multiple macrometastases in control animals. Combination of gemcitabine and cyclopamine completely abrogated metastases while also significantly reducing the size of primary" tumors. Gli1 levels were up-regulated in tissue samples of metastatic human pancreatic cancer samples compared with matched primary tumors. Aldehyde dehydrogenase (ALDH) overexpression is characteristic for both hematopoietic progenitors and leukemic stem cells; cyclopamine preferentially reduced "ALDH-high" cells by approximately 3-fold (P = 0.048). We confirm pharmacologic Hh pathway inhibition as a valid therapeutic strategy for pancreatic cancer and show for the first time its particular efficacy against metastatic spread. By targeting specific cellular subpopulations likely involved in tumor initiation at metastatic sites� View Publication

View Publication

NK and T cells are important for combating CMV infection. Some NK and T cells express leukocyte Ig-like receptor-1 (LIR-1),an inhibitory receptor recognizing MHC class I and the CMV-encoded homolog UL18. We previously demonstrated an early increase in LIR-1-expressing blood lymphocytes in lung-transplanted patients later developing CMV disease. We now show that NK and T cells account for the observed LIR-1 augmentation. Coincubation of PBMC from CMV-seropositive donors with virus-infected lung fibroblasts led to a T cell-dependent secretion of IFN-gamma,produced mainly by LIR-1(+) T cells and by NK cells. Cytokine production during coculture with fibroblasts infected with virus containing the UL18 gene was augmented compared with the UL18 deletion virus,suggesting a stimulatory role for UL18. However,purified UL18Fc proteins inhibited IFN-gamma production of LIR-1(+) T cells. We propose that cytokine production in the transplant induces NK and T cells to express LIR-1,which may predispose to CMV disease by MHC/LIR-1-mediated suppression. Although the UL18/LIR-1 interaction could inhibit T cell responses,this unlikely plays a role in response to infected cells. Instead,our data point to an activating role for viral UL18 during infection,where indirect intracellular effects cannot be excluded. View Publication

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

T cell activation is predicated on the interaction between the T cell receptor and peptide-major histocompatibility (pMHC) ligands. The factors that determine the stimulatory potency of a pMHC molecule remain unclear. We describe results showing that a peptide exhibiting many hallmarks of a weak agonist stimulates T cells to proliferate more than the wild-type agonist ligand. An in silico approach suggested that the inability to form the central supramolecular activation cluster (cSMAC) could underlie the increased proliferation. This conclusion was supported by experiments that showed that enhancing cSMAC formation reduced stimulatory capacity of the weak peptide. Our studies highlight the fact that a complex interplay of factors determines the quality of a T cell antigen. View Publication