技术资料

The anti-My-10 mouse monoclonal antibody was raised against the immature human myeloid cell line KG-1a and was selected for nonreactivity with mature human granulocytes. Anti-My-10 immunoprecipitated a KG-1a cell surface protein with an apparent Mr of approximately 115 kD. We describe the binding of this antibody to human hematopoietic cell types and show that My-10 is expressed specifically on immature normal human marrow cells,including hematopoietic progenitor cells. My-10 is also expressed by leukemic marrow cells from a subpopulation of patients. Thus,this antibody allows the identification and purification of hematopoietic progenitor cells from normal human marrow and the subclassification of leukemia. View Publication

The My-10 glycoprotein is an hematopoietic cell surface antigen expressed specifically by undifferentiated (blast) cells,constituting 1%-4% of normal adult bone marrow leukocytes. We used several immunological and in vitro culture methods to analyze the expression of this unique antigen on a variety of lymphohematopoietic progenitor cells. Colony-forming cells (CFC) for granulocyte-monocyte colonies (CFC-GM) and erythroid colonies (BFU-E) were predominantly My-10 positive. CFC with higher proliferative potential were more strongly My-10 positive than CFC with lower proliferative potential,and those for mixed-lineage and blast cell colonies were even more uniformly My-10 positive. Cells maintaining CFC-GM number in short-term marrow culture (pre-CFC) were found to be My-10 positive,as were lymphoid precursors defined by their content of intranuclear terminal deoxynucleotidyl transferase. More mature erythroid precursors (CFU-E) were heterogeneous for antigen expression and lost My-10 antigen progressively,in parallel with advancing maturational stage. The My-10 antigen permits rapid identification and purification of hematopoietic progenitor cells for further study or potential clinical application. The disappearance of the My-10 antigen,moreover,may be a probe for differentiation-linked cellular events. View Publication

Donor-specific antibodies (DSAs) targeting mismatched human leukocyte antigen (HLA) molecules are one of the principal threats to long-term graft survival in solid organ transplantation. However,many patients with long-term circulating DSAs do not manifest rejection responses,suggesting a degree of heterogeneity in their pathogenicity and related functional activity. Immunologic risk stratification of transplant recipients is complicated by challenges intrinsic to defining alloantibody responses that are potentially pathogenic versus those that are not. Thus,a comprehensive understanding of how human alloantibodies target and interact with donor HLA molecules is vital for the development and evaluation of new strategies aimed at reducing antibody-mediated rejection responses. In this study,we employ hydrogen–deuterium exchange–mass spectrometry (HDX–MS),molecular dynamics (MD) simulations,and advanced biochemical and biophysical methodologies to thoroughly characterize a panel of human monoclonal alloantibodies and define the influence of Fc-region biology,antibody binding kinetics,target antigen density,and structural characteristics on their ability to potentiate the forms of immune effector mechanisms that are strongly implicated in transplant rejection. Our findings have significant implications for our understanding of the key biological determinants that underlie the pathogenicity or lack thereof of human alloantibodies. View Publication

Glycogen synthase kinase 3 (GSK-3) functions in the regulation of glycogen metabolism,in the cell cycle,and in immune responses and is targeted by some viruses to favor the viral life cycle. Inhibition of GSK-3 by 6-bromoindirubin-3'-acetoxime (BIO-acetoxime),a synthetic derivative of a compound from the Mediterranean mollusk Hexaplex trunculus,protects cells from varicella infection. In this study,we examined the effects of BIO-acetoxime against herpes simplex virus-1 (HSV-1) infection in human oral epithelial cells,which represent a natural target cell type. The results revealed that BIO-acetoxime relieves HSV-1-induced cytopathic effects and apoptosis. We also found that BIO-acetoxime reduced viral yields and the expression of different classes of viral proteins. Furthermore,addition of BIO-acetoxime before,simultaneously with or after HSV-1 infection significantly reduced viral yields. Collectively,BIO-acetoxime may suppress viral gene expression and protect oral epithelial cells from HSV-1 infection. These results suggest the possible involvement of GSK-3 in HSV-1 infection. View Publication

An attractive target for therapeutic intervention is constitutively activated,mutant FLT3,which is expressed in a subpopulation of patients with acute myelocyic leukemia (AML) and is generally a poor prognostic indicator in patients under the age of 65 years. PKC412 is one of several mutant FLT3 inhibitors that is undergoing clinical testing,and which is currently in late-stage clinical trials. However,the discovery of drug-resistant leukemic blast cells in PKC412-treated patients with AML has prompted the search for novel,structurally diverse FLT3 inhibitors that could be alternatively used to override drug resistance. Here,we report the potent and selective antiproliferative effects of the novel mutant FLT3 inhibitor NVP-AST487 on primary patient cells and cell lines expressing FLT3-ITD or FLT3 kinase domain point mutants. NVP-AST487,which selectively targets mutant FLT3 protein kinase activity,is also shown to override PKC412 resistance in vitro,and has significant antileukemic activity in an in vivo model of FLT3-ITD(+) leukemia. Finally,the combination of NVP-AST487 with standard chemotherapeutic agents leads to enhanced inhibition of proliferation of mutant FLT3-expressing cells. Thus,we present a novel class of FLT3 inhibitors that displays high selectivity and potency toward FLT3 as a molecular target,and which could potentially be used to override drug resistance in AML. View Publication

OBJECTIVE Long-term treatment with tyrosine kinase inhibitors (TKI) represents an effective cure for chronic myeloid leukemia (CML) patients and discontinuation of TKI therapy is now proposed to patient with deep molecular responses. However,evidence demonstrating that TKI are unable to fully eradicate dormant leukemic stem cells (LSC) indicate that new therapeutic strategies are needed to control LSC and to prevent relapse. In this study we investigated the metabolic pathways responsible for CML surviving to imatinib exposure and its potential therapeutic utility to improve the efficacy of TKI against stem-like CML cells. METHODS Using complementary cell-based techniques,metabolism was characterized in a large panel of BCR-ABL+ cell lines as well as primary CD34+ stem-like cells from CML patients exposed to TKI and L-Asparaginases. Colony forming cell (CFC) assay and flow cytometry were used to identify CML progenitor and stem like-cells. Preclinical models of leukemia dormancy were used to test the effect of treatments. RESULTS Although TKI suppressed glycolysis,compensatory glutamine-dependent mitochondrial oxidation supported ATP synthesis and CML cell survival. Glutamine metabolism was inhibited by L-asparaginases such as Kidrolase or Erwinase without inducing predominant CML cell death. However,clinically relevant concentrations of TKI render CML cells susceptible to Kidrolase. The combination of TKI with Lasparaginase reactivates the intinsic apoptotic pathway leading to efficient CML cell death. CONCLUSION Targeting glutamine metabolism with the FDA-approved drug,Kidrolase in combination with TKI that suppress glycolysis represents an effective and widely applicable therapeutic strategy for eradicating stem-like CML cells. View Publication

Wine polyphenols were examined for their capacity to protect the lipid and protein moieties of porcine low density lipoproteins (LDL) during oxidation. The efficiency of resveratrol (3,4',5,trihydroxystilbene) and defined flavonoids was compared to that of a wine extract (WE) containing 0.5 g/g proanthocyanidols. The efficiency of resveratrol for protecting polyunsaturated fatty acids (PUFA) was higher than that of flavonoids in copper-induced oxidation and lower in AAPH (radical initiator)-induced oxidation. The LDL receptor activity was evaluated by flow cytometry using LDL labeled with fluorescein isothiocyanate (FITC) and Chinese hamster ovary cells (CHO-K1). The incubation of CHO-K1 with FITC-LDL oxidized for 16 h reduced the proportion of fluorescent cells from 97% to 4%. At a concentration of 40 microM,resveratrol and flavonoids completely restored the uptake of copper-oxidized LDL and AAPH-oxidized LDL respectively. Total fluorescence could also be obtained with 20 mg/L of WE with both oxidation systems. These data are consistent with previous findings relative to the formation of degradative products from PUFA. They confirm that resveratrol was more effective than flavonoids as a chelator of copper and less effective as a free-radical scavenger. Moreover,they show that WE,which contained monomeric and oligomeric forms of flavonoids and phenolic acids,protected LDL by both mechanisms. View Publication

View Publication

Reactive oxygen (ROS) and nitrogen oxide (RNOS) species are produced as by-products of oxidative metabolism. A major function for ROS and RNOS is immunological host defense. Recent evidence indicate that ROS and RNOS may also function as signaling molecules. However,high levels of ROS and RNOS have been considered to potentially damage cellular macromolecules and have been implicated in the pathogenesis and progression of various chronic diseases. alpha-Lipoic acid and dihydrolipoic acid exhibit direct free radical scavenging properties and as a redox couple,with a low redox potential of -0.32 V,is a strong reductant. Several studies provided evidence that alpha-lipoic acid supplementation decreases oxidative stress and restores reduced levels of other antioxidants in vivo. However,there is also evidence indicating that alpha-lipoic acid and dihydrolipoic acid may exert prooxidant properties in vitro. alpha-Lipoic acid and dihydrolipoic acid were shown to promote the mitochondrial permeability transition in permeabilized hepatocytes and isolated rat liver mitochondria. Dihydrolipoic acid also stimulated superoxide anion production in rat liver mitochondria and submitochondrial particles. alpha-Lipoic acid was recently shown to stimulate glucose uptake into 3T3-L1 adipocytes by increasing intracellular oxidant levels and/or facilitating insulin receptor autophosphorylation presumably by oxidation of critical thiol groups present in the insulin receptor beta-subunit. Whether alpha-lipoic acid and/or dihydrolipoic acid-induced oxidative protein modifications contribute to their versatile effects observed in vivo warrants further investigation. View Publication

The antioxidant properties of butein,isolated from Dalbergia odorifera T. Chen,were investigated in this study. Butein inhibited iron-induced lipid peroxidation in rat brain homogenate in a concentration-dependent manner with an IC50,3.3+/-0.4 microM. It was as potent as alpha-tocopherol in reducing the stable free radical diphenyl-2-picrylhydrazyl (DPPH) with an IC0.200,9.2+/-1.8 microM. It also inhibited the activity of xanthine oxidase with an IC50,5.9+/-0.3 microM. Besides,butein scavenged the peroxyl radical derived from 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) in aqueous phase,but not that from 2,2-azobis(2,4-dimethylvaleronitrile) (AMVN) in hexane. Furthermore,butein inhibited copper-catalyzed oxidation of human low-density lipoprotein (LDL),as measured by conjugated dienes and thiobarbituric acid-reactive substance (TBARS) formations,and electrophoretic mobility in a concentration-dependent manner. Spectral analysis revealed that butein was a chelator of ferrous and copper ions. It is proposed that butein serves as a powerful antioxidant against lipid and LDL peroxidation by its versatile free radical scavenging actions and metal ion chelation. View Publication

Glioblastoma multiforme (GBM) is the most common and aggressive brain cancer,and despite treatment advances,patient prognosis remains poor. During routine animal studies,we serendipitously observed that fenbendazole,a benzimidazole antihelminthic used to treat pinworm infection,inhibited brain tumor engraftment. Subsequent in vitro and in vivo experiments with benzimidazoles identified mebendazole as the more promising drug for GBM therapy. In GBM cell lines,mebendazole displayed cytotoxicity,with half-maximal inhibitory concentrations ranging from 0.1 to 0.3 µM. Mebendazole disrupted microtubule formation in GBM cells,and in vitro activity was correlated with reduced tubulin polymerization. Subsequently,we showed that mebendazole significantly extended mean survival up to 63% in syngeneic and xenograft orthotopic mouse glioma models. Mebendazole has been approved by the US Food and Drug Administration for parasitic infections,has a long track-record of safe human use,and was effective in our animal models with doses documented as safe in humans. Our findings indicate that mebendazole is a possible novel anti-brain tumor therapeutic that could be further tested in clinical trials. View Publication

The immunosuppressive effects of microcolin A,a lipopeptide extracted from the marine blue green alga Lyngbya majuscula were investigated. Microcolin A suppressed concanavalin A (IC50 = 5.8 nM),phytohemagglutinin (IC50 = 12.5 nM) and lipopolysaccharide (IC50 = 8.0 nM) induced proliferation of murine splenocytes. Mixed lymphocyte reaction (IC50 = 5.0 nM),anti-IgM (mu-chain specific) (IC50 = 10.0 nM),and phorbol 12-myristate 13-acetate plus ionomycin (IC50 = 5.8 nM) stimulation of murine splenocytes were all similarly suppressed by microcolin A. The inhibitory activity of microcolin A was time-dependent and reversible and was not associated with a reduction in cell viability. Moreover,microcolin A not only inhibited IL-2 production and IL-2 receptor expression by concanavalin A activated splenocytes,but also suppressed in vitro antibody responsiveness to keyhole limpet hemocyanin. These results indicate that microcolin A is a potent immunosuppressive and antiproliferative agent. View Publication