技术资料

Anthrax lethal toxin (LT) is a critical virulence factor that cleaves and inactivates MAPK kinases (MAPKKs) in host cells and has been proposed as a therapeutic target in the treatment of human anthrax infections. Despite the potential use of anti-toxin agents in humans,the standard activity assays for anthrax LT are currently based on cytotoxic actions of anthrax LT that are cell-,strain-,and species-specific,which have not been demonstrated to occur in human cells. We now report that T cell proliferation and IL-2 production inversely correlate with anthrax LT levels in human cell assays. The model CD4+ T cell tumor line,Jurkat,is a susceptible target for the specific protease action of anthrax LT. Anthrax LT cleaves and inactivates MAPKKs in Jurkat cells,whereas not affecting proximal or parallel TCR signal transduction pathways. Moreover,anthrax LT specifically inhibits PMA/ionomycin- and anti-CD3-induced IL-2 production in Jurkat cells. An inhibitor of the protease activity of anthrax LT completely restores IL-2 production by anthrax LT-treated Jurkat cells. Anthrax LT acts on primary CD4+ T cells as well,cleaving MAPKKs and leading to a 95% reduction in anti-CD3-induced proliferation and IL-2 production. These findings not only will be useful in the development of new human cell-based bioassays for the activity of anthrax LT,but they also suggest new mechanisms that facilitate immune evasion by Bacillus anthracis. Specifically,anthrax LT inhibits IL-2 production and proliferative responses in CD4+ T cells,thereby blocking functions that are pivotal in the regulation of immune responses. View Publication

Patients with sepsis are immune compromised,as evidenced by their failure to clear their primary infection and their propensity to develop secondary infections with pathogens that are often not particularly virulent in normal healthy individuals. A potential mechanism for immunosuppression in sepsis is lymphocyte apoptosis,which may occur by either a death receptor or a mitochondrial-mediated pathway. A prospective study of blood samples from 71 patients with sepsis,55 nonseptic patients,and 6 healthy volunteers was undertaken to quantitate lymphocyte apoptosis and determine cell death pathways and mechanisms of apoptosis. Apoptosis was evaluated by flow cytometry and Western blotting. Lymphocyte apoptosis was increased in CD4 and CD8 T cells,B cells (CD20),and NK cells (CD56) in septic vs nonseptic patients. Samples taken sequentially from 10 patients with sepsis showed that the degree of CD3 T cell apoptosis correlated with the activity of his/her sepsis. In septic patients,apoptotic lymphocytes were positive for active caspases 8 and 9,consistent with death occurring by both mitochondrial-mediated and receptor-mediated pathways. In support of the concept that both death pathways were operative,lymphocyte apoptosis occurred in cells with markedly decreased Bcl-2 (an inhibitor of mitochondrial-mediated apoptosis) as well as cells with normal concentrations of Bcl-2. In conclusion,apoptosis occurs in a broad range of lymphocyte subsets in patients with sepsis and correlates with the activity of the disease. Lymphocyte loss occurs by both death receptor and mitochondrial-mediated apoptosis,suggesting that there may be multiple triggers for lymphocyte apoptosis. View Publication

Human organ-specific microvascular endothelial cells (ECs) were established and used in the present study to investigate their susceptibility to natural killer cell line (NKL)-induced lysis. Our data indicate that although IL-2-stimulated NKL (NKL2) cells adhered to the human peripheral (HPLNEC.B3),mesenteric lymph node (HMLNEC),brain (HBrMEC),and lung (HLMEC) and skin (HSkMEC.2) ECs,they significantly killed these cells quite differently. A more pronounced lysis of OSECs was also observed when IL-2-stimulated,purified peripheral blood NK cells were used as effector cells. In line with the correlation observed between adhesion pattern and the susceptibility to NKL2-mediated killing,we demonstrated using different chelators that the necessary adhesion step was governed by an Mg(2+)-dependent,but Ca(2+)-independent,mechanism as opposed to the subsequent Ca(2+)-dependent killing. To identify the cytotoxic pathway used by NKL2 cells,the involvement of the classical and alternate pathways was examined. Blocking of the Ca(2+)-dependent cytotoxicity pathway by EGTA/MgCl(2) significantly inhibited endothelial target cell killing,suggesting a predominant role for the perforin/granzyme pathway. Furthermore,using confocal microscopy,we demonstrated that the interaction between NKL2 effectors and ECs induced cytochrome c release and Bid translocation in target cells,indicating an involvement of the mitochondrial pathway in NKL2-induced EC death. In addition,although all tested cells were sensitive to the cytotoxic action of TNF,no susceptibility to TRAIL or anti-Fas mAb was observed. The present studies emphasize that human NK cell cytotoxicity toward ECs may be a potential target to block vascular injury. View Publication

Kawasaki disease (KD) is an acute vasculitis of infants and young children,preferentially affecting the coronary arteries. Intravenous infusion of high dose Ig (IVIG) effectively reduces systemic inflammation and prevents coronary artery lesions in KD. To investigate the mechanisms underlying the therapeutic effects of IVIG,we examined gene expression profiles of PBMC and purified monocytes obtained from acute patients before and after IVIG therapy. The results suggest that IVIG suppresses activated monocytes and macrophages by altering various functional aspects of the genes of KD patients. Among the 18 commonly decreased transcripts in both PBMC and purified monocytes,we selected six genes,FCGR1A,FCGR3A,CCR2,ADM,S100A9,and S100A12,and confirmed the microarray results by real-time RT-PCR. Moreover,the expressions of FcgammaRI and FcgammaRIII on monocytes were reduced after IVIG. Plasma S100A8/A9 heterocomplex,but not S100A9,levels were elevated in patients with acute KD compared with those in febrile controls. Furthermore,S100A8/A9 was rapidly down-regulated in response to IVIG therapy. Persistent elevation of S100A8/A9 after IVIG was found in patients who later developed coronary aneurysms. These results indicate that the effects of IVIG in KD may be mediated by suppression of an array of immune activation genes in monocytes,including those activating FcgammaRs and the S100A8/A9 heterocomplex. View Publication

Chalcones are precursor compounds for flavonoid synthesis in plants,and they can also be synthesized in laboratory. Previous study has documented some of the pharmacological applications of these compounds. Estrogen has long been associated with the initiation and promotion of breast cancer. Inhibiting estrogen synthesis can be effective in the prevention and treatment of the disease. Since most breast cancers received estrogen supplied from local tissues,we employed a breast cancer cell line expressing aromatase to screen for the inhibitory potentials of five hydroxychalcones,i.e. 2-hydroxychalcone,2'-hydroxychalcone,4-hydroxychalcone,4,2',4'-trihydroxy-chalcone (isoquiritigenin),3,4,2',4'-tetrahydroxychalcone (butein). In the preliminary results,butein was found to be the strongest inhibitor among the tested compounds,and its IC(50) value was 3.75 microM. Subsequent enzyme kinetic study revealed that butein acted on aromatase with a mixed type of inhibition and the K(i) value was determined to be 0.32 microM. Cell proliferation assay indicated that the cell number increased by 10 nM-testosterone treatment was significantly reduced by 5 microM butein,and the administration of flutamide could not reverse the effect. The present study illustrated that butein was an aromatase inhibitor and a potential natural alternative for the chemoprevention or therapy of breast cancer. View Publication

Imatinib,a Bcr-Abl tyrosine kinase inhibitor,is a highly effective therapy for patients with chronic myelogenous leukemia (CML). Despite durable responses in most chronic phase patients,relapses have been observed and are much more prevalent in patients with advanced disease. The most common mechanism of acquired imatinib resistance has been traced to Bcr-Abl kinase domain mutations with decreased imatinib sensitivity. Thus,alternate Bcr-Abl kinase inhibitors that have activity against imatinib-resistant mutants would be useful for patients who relapse on imatinib therapy. Two such Bcr-Abl inhibitors are currently being evaluated in clinical trials: the improved potency,selective Abl inhibitor AMN107 and the highly potent dual Src/Abl inhibitor BMS-354825. In the current article,we compared imatinib,AMN107,and BMS-354825 in cellular and biochemical assays against a panel of 16 kinase domain mutants representing textgreater90% of clinical isolates. We report that AMN107 and BMS-354825 are 20-fold and 325-fold more potent than imatinib against cells expressing wild-type Bcr-Abl and that similar improvements are maintained for all imatinib-resistant mutants tested,with the exception of T315I. Thus,both inhibitors hold promise for treating imatinib-refractory CML. View Publication

OBJECTIVE: To compare the suppressive effect of mesenchymal stem cells (MSC),derived from normal individuals or severe aplastic anemia patients (SAA),on T-cell activation. PATIENTS AND METHODS: We studied bone marrow MSC from 19 healthy donors and 23 SAA patients in different phases of the disease: at diagnosis (n = 3),following immunosuppressive therapy (IS) (n = 16),or after a bone marrow transplant (BMT) (n = 4). MSC were tested for T-cell suppression in the following assays: mixed lymphocyte reaction (MLR),phytohemaglutinin (PHA)-primed cultures,activation surface markers,gamma-IFN production,hematopoietic colony formation (CFC),production of cyclic ADP-ribose (cADPR). RESULTS: The abnormalities of SAA MSC included: 1) significantly lower suppression of T-cell proliferation induced by alloantigens (p = 0.009) or PHA (p = 0.006); 2) impaired capacity to suppress CD38 expression on PHA-primed T cells (p = 0.001); 3) impaired ability to suppress gamma-IFN production in PHA cultures,resulting in an 11-fold higher gamma-IFN concentration; 4) no preventive effect on T cell-mediated inhibition of CFC; and 5) significantly reduced (p = 0.009) production of cADPR,a universal calcium mobilizer. MSC-mediated suppression of PHA-induced T-cell proliferation was restored to control levels in 3 of 4 patients post-BMT. CONCLUSION: The ability of MSC to downregulate T-cell priming,proliferation,and cytokine release is deficient in patients with SAA,persists indefinitely after immunosuppressive therapy,but seems to be restored after BMT. Whether these abnormalities are relevant to the pathogenesis of aplastic anemia remains to be determined. View Publication

A series of 30 N10-substituted phenoxazines were synthesized and screened as potential inhibitors of Akt. In cellular assays at 5 mum,17 compounds inhibited insulin-like growth factor 1 (IGF-I)-stimulated phosphorylation of Akt (Ser-473) by at least 50% but did not inhibit IGF-I-stimulated phosphorylation of Erk-1/2 (Thr-202/Tyr-204). Substitutions at the 2-position (Cl or CF3) did not alter inhibitory activity,whereas N10-substitutions with derivatives having acetyl (20B) or morpholino (12B) side chain lost activity compared with propyl or butyl substituents (7B and 14B). Inhibition of Akt phosphorylation was associated with the inhibition of IGF-I stimulation of the mammalian target of rapamycin phosphorylation (Ser-2448 and Ser-2481),phosphorylation of p70 S6 kinase (Thr-389),and ribosomal protein S6 (Ser-235/236) in Rh1,Rh18,and Rh30 cell lines. The two most potent compounds 10-[4'-(N-diethylamino)butyl]-2-chlorophenoxazine (10B) and 10-[4'-[(beta-hydroxyethyl)piperazino]butyl]-2-chlorophenoxazine (15B) (in vitro,IC50 approximately 1-2 microM) were studied further. Inhibition of Akt phosphorylation correlated with inhibition of its kinase activity as determined in vitro after immunoprecipitation. Akt inhibitory phenoxazines did not inhibit the activity of recombinant phosphatidylinositol 3'-kinase,PDK1,or SGK1 but potently inhibited the kinase activity of recombinant Akt and Akt deltaPH,a mutant lacking the pleckstrin homology domain. Akt inhibitory phenoxazines blocked IGF-I-stimulated nuclear translocation of Akt in Rh1 cells and suppressed growth of Rh1,Rh18,and Rh30 cells (IC50 2-5 microM),whereas inactive" derivatives were textgreater or = 10-fold less potent inhibitors of cell growth. In contrast to rapamycin analogs� View Publication

The hematopoietic stem cell (HSC) gives rise to all mature,terminally differentiated cells of the blood. Here we show that calmodulin-dependent protein kinase IV (CaMKIV) is present in c-Kit+ ScaI+ Lin(-/low) hematopoietic progenitor cells (KLS cells) and that its absence results in hematopoietic failure,characterized by a diminished KLS cell population and by an inability of these cells to reconstitute blood cells upon serial transplantation. KLS cell failure in the absence of CaMKIV is correlated with increased apoptosis and proliferation of these cells in vivo and in vitro. In turn,these cell biological defects are correlated with decreases in CREB-serine 133 phosphorylation as well as in CREB-binding protein (CBP) and Bcl-2 levels. Re-expression of CaMKIV in Camk4-/- KLS cells results in the rescue of the proliferation defects in vitro as well as in the restoration of CBP and Bcl-2 to wild type levels. These studies show that CaMKIV is a regulator of HSC homeostasis and suggest that its effects may be in part mediated via regulation of CBP and Bcl-2. View Publication

DNA methylation regulates gene expression in normal and malignant cells. The possibility to reactivate epigenetically silenced genes has generated considerable interest in the development of DNA methyltransferase inhibitors. Here,we provide a detailed characterization of RG108,a novel small molecule that effectively blocked DNA methyltransferases in vitro and did not cause covalent enzyme trapping in human cell lines. Incubation of cells with low micromolar concentrations of the compound resulted in significant demethylation of genomic DNA without any detectable toxicity. Intriguingly,RG108 caused demethylation and reactivation of tumor suppressor genes,but it did not affect the methylation of centromeric satellite sequences. These results establish RG108 as a DNA methyltransferase inhibitor with fundamentally novel characteristics that will be particularly useful for the experimental modulation of epigenetic gene regulation. View Publication

To realize the full potential of targeted protein kinase inhibitors for the treatment of cancer,it is important to address the emergence of drug resistance in treated patients. Mutant forms of BCR-ABL,KIT,and the EGF receptor (EGFR) have been found that confer resistance to the drugs imatinib,gefitinib,and erlotinib. The mutations weaken or prevent drug binding,and interestingly,one of the most common sites of mutation in all three kinases is a highly conserved gatekeeper" threonine residue near the kinase active site. We have identified existing clinical compounds that bind and inhibit drug-resistant mutant variants of ABL� View Publication

Several recent reports suggest that there is far more plasticity that previously believed in the developmental potential of bone-marrow-derived cells (BMCs) that can be induced by extracellular developmental signals of other lineages whose nature is still largely unknown. In this study,we demonstrate that bone-marrow-derived mesenchymal stem cells (MSCs) co-cultured with mouse proliferating or fixed (by paraformaldehyde or methanol) neural stem cells (NSCs) generate neural stem cell-like cells with a higher expression of Sox-2 and nestin when grown in NS-A medium supplemented with N2,NSC conditioned medium (NSCcm) and bFGF. These neurally induced MSCs eventually differentiate into beta-III-tubulin and GFAP expressing cells with neuronal and glial morphology when grown an additional week in Neurobasal/B27 without bFGF. We conclude that juxtacrine interaction between NSCs and MSCs combined with soluble factors released from NSCs are important for generation of neural-like cells from bone-marrow-derived adherent MSCs. View Publication