技术资料

Interleukin-10 (IL-10) has potent immunoregulatory effects on the maturation and the antigen-presenting cell (APC) function of dendritic cells (DCs). The molecular basis underlying these effects in DCs,however,is ill defined. It is well established that the transcription factor NF-kappaB is a key regulator of DC development,maturation,and APC function. This study was initiated to determine the effects of IL-10 on the NF-kappaB signaling pathway in immature DCs. IL-10 pretreatment of myeloid DCs cultured from bone marrow resulted in reduced DNA binding and nuclear translocation of NF-kappaB after anti-CD40 antibody or lipopolysaccharide (LPS) stimulation. Furthermore,inhibited NF-kappaB activation was characterized by reduced degradation,phosphorylation,or both of IkappaBalpha and IkappaBepsilon but not IkappaBbeta and by reduced phosphorylation of Ser536,located in the trans-activation domain of p65. Notably,IL-10-mediated inhibition of NF-kappaB coincided with suppressed IkappaB kinase (IKK) activity in vitro. Furthermore,IL-10 blocked inducible Akt phosphorylation,and inhibitors of phosphatidylinositol 3-kinase (PI3K) effectively suppressed the activation of Akt,IKK,and NF-kappaB. These findings demonstrate that IL-10 targets IKK activation in immature DCs and that suppressing the PI3K pathway in part mediates blockade of the pathway. View Publication

Splitomicin (1) and 41 analogues were prepared and evaluated in cell-based Sir2 inhibition and toxicity assays and an in vitro Sir2 inhibition assay. Lactone ring or naphthalene (positions 7-9) substituents decrease activity,but other naphthalene substitutions (positions 5 and 6) are well-tolerated. The hydrolytically unstable aromatic lactone is important for activity. Lactone hydrolysis rates were used as a measure of reactivity; hydrolysis rates correlate with inhibitory activity. The most potent Sir2 inhibitors were structurally similar to and had hydrolysis rates similar to 1. View Publication

Overexpression of fibroblast growth factor receptor (FGFR) tyrosine kinases has been found in many human breast cancers and has been associated with poor patient prognosis. In order to understand the mechanism by which FGFR mediates breast cancer cell proliferation,we used a low molecular weight compound,PD173074,that selectively inhibits FGFR tyrosine kinase activity and autophosphorylation. This potential anticancer agent caused a G1 growth arrest of MDA-MB-415,MDA-MB-453 and SUM 52 breast cancer cells. Our analyses revealed that FGFR signaling links to the cell cycle machinery via D-type cyclins. PD173074-mediated inhibition of FGFR activity caused downregulation of cyclin D1 and cyclin D2 expression,inhibition of cyclin D/cdk4 activity and,as a consequence,reduction of pRB phosphorylation. Retroviral-mediated ectopic expression of cyclin D1 prevented pRB hypophosphorylation and the cell cycle G1 block in PD173074-treated cells,suggesting a central role for D cyclins in proliferation of FGFR-driven breast cancer cells. The repression of FGFR activity caused downregulation of MAPK in MDA-MB-415 and MDA-MB-453 cells. In SUM 52 cells,both MAPK and PI3K signaling pathways were suppressed. In conclusion,results shown here describe a mechanism by which FGFR promotes proliferation of breast cancer cells. View Publication

Mammalian presenilins (PS) consist of two highly homologous proteins,PS1 and PS2. Because of their indispensable activity in the gamma-secretase cleavage of amyloid precursor protein to generate Abeta peptides,inhibition of PS gamma-secretase activity is considered a potential therapy for Abeta blockage and Alzheimer's disease intervention. However,a variety of other substrates are also subject to PS-dependent processing,and it is thus imperative to understand the consequences of PS inactivation in vivo. Here we report a pivotal role of PS in hematopoiesis. Mice heterozygous for PS1 and homozygous for PS2 (PS1(+/)(-)PS2(-)(/)(-)) developed splenomegaly with severe granulocyte infiltration. This was preceded by an overrepresentation of granulocytic cells in the bone marrow and a greatly increased multipotent granulocyte-monocyte progenitor in the spleen. In contrast,hematopoietic stem cells and T- and B-lymphocytes were not affected. Importantly,treatment of wild-type splenocytes with a gamma-secretase inhibitor directly promoted the granulocyte-macrophage colony-forming unit (GM-CFU). These results establish a critical role of PS in myelopoiesis. Our finding that this activity can be directly modulated by its gamma-secretase activity has important safety implications concerning these inhibitors. View Publication

The galactose-alpha-1,3-galactose (alphaGal) carbohydrate epitope is expressed on porcine,but not human cells,and therefore represents a major target for preformed human anti-pig natural Abs (NAb). Based on results from pig-to-primate animal models,NAb binding to porcine endothelial cells will likely induce complement activation,lysis,and hyperacute rejection in pig-to-human xenotransplantation. Human NK cells may also contribute to innate immune responses against xenografts,either by direct recognition of activating molecules on target cells or by FcgammaRIII-mediated xenogeneic Ab-dependent cellular cytotoxicity (ADCC). The present study addressed the question as to whether the lack of alphaGal protects porcine endothelial cells from NAb/complement-induced lysis,direct xenogeneic NK lysis,NAb-dependent ADCC,and adhesion of human NK cells under shear stress. Homologous recombination,panning,and limiting dilution cloning were used to generate an alphaGal-negative porcine endothelial cell line,PED2*3.51. NAb/complement-induced xenogeneic lysis of PED2*3.51 was reduced by an average of 86% compared with the alphaGal-positive phenotype. PED2*3.51 resisted NK cell-mediated ADCC with a reduction of lysis ranging from 30 to 70%. However,direct xenogeneic lysis of PED2*3.51,mediated either by freshly isolated or IL-2-activated human NK cells or the NK cell line NK92,was not reduced. Furthermore,adhesion of IL-2-activated human NK cells did not rely on alphaGal expression. In conclusion,removal of alphaGal leads to a clear reduction in complement-induced lysis and ADCC,but does not resolve adhesion of NK cells and direct anti-porcine NK cytotoxicity,indicating that alphaGal is not a dominant target for direct human NK cytotoxicity against porcine cells. View Publication

Amphoterin (HMGB1) is a 30-kD heparin-binding protein involved in process extension and migration of cells by a mechanism involving the receptor for advanced glycation end products (RAGE). High levels of amphoterin are released to serum during septic shock. We have studied the expression of amphoterin in monocytes and the role of amphoterin and RAGE in monocyte transendothelial migration. Un-activated monocytes in suspension did not reveal amphoterin on their surface,but adherent monocytes exported amphoterin to the cell surface. Immunohistochemical staining of arterial thrombi in vivo revealed amphoterin in mononuclear cells and in surrounding extracellular matrix. Amphoterin was secreted from phorbol ester and interferon-gamma (IFN-gamma)-activated macrophages,and the secretion was inhibited by blocking the adenosine 5'-triphosphate (ATP)-binding cassette transporter-1,a member of the multidrug resistance protein family. Amphoterin was specifically adhesive for monocytes in peripheral blood leukocyte adhesion assay. Adhesion caused an extensive spreading of cells,which was inhibited by the dominant-negative RAGE receptor (soluble ectodomain of RAGE),and adhesion up-regulated chromogranin expression in monocytes,also suggesting a RAGE-dependent interaction. Monocyte transendothelial migration was efficiently inhibited by anti-amphoterin and anti-RAGE antibodies and by the soluble RAGE. We suggest that amphoterin is an autocrine/paracrine regulator of monocyte invasion through the endothelium. View Publication

Histone acetylation is a diagnostic feature of transcriptionally active genes. The proper recruitment and function of histone acetyltransferases (HATs) and deacetylases (HDACs) are key regulatory steps for gene expression and cell cycle. Functional defects of either of these enzymes may lead to several diseases,including cancer. HATs and HDACs thus are potential therapeutic targets. Here we report that garcinol,a polyisoprenylated benzophenone derivative from Garcinia indica fruit rind,is a potent inhibitor of histone acetyltransferases p300 (IC50 approximately 7 microm) and PCAF (IC50 approximately 5 microm) both in vitro and in vivo. The kinetic analysis shows that it is a mixed type of inhibitor with an increased affinity for PCAF compared with p300. HAT activity-dependent chromatin transcription was strongly inhibited by garcinol,whereas transcription from DNA template was not affected. Furthermore,it was found to be a potent inducer of apoptosis,and it alters (predominantly down-regulates) the global gene expression in HeLa cells. View Publication

Although extracellular nucleotides support a wide range of biologic responses of mature blood cells,little is known about their effect on blood cell progenitor cells. In this study,we assessed whether receptors for extracellular nucleotides (P2 receptors [P2Rs]) are expressed on human hematopoietic stem cells (HSCs),and whether activation by their natural ligands,adenosine triphosphate (ATP) and uridine triphosphate (UTP),induces HSC proliferation in vitro and in vivo. Our results demonstrated that CD34(+) HSCs express functional P2XRs and P2YRs of several subtypes. Furthermore,stimulation of CD34(+) cells with extracellular nucleotides caused a fast release of Ca(2+) from intracellular stores and an increase in ion fluxes across the plasma membrane. Functionally,ATP and,to a higher extent,UTP acted as potent early acting growth factors for HSCs,in vitro,because they strongly enhanced the stimulatory activity of several cytokines on clonogenic CD34(+) and lineage-negative CD34(-) progenitors and expanded more primitive CD34(+)-derived long-term culture-initiating cells. Furthermore,xenogenic transplantation studies showed that short-term preincubation with UTP significantly expanded the number of marrow-repopulating HSCs in nonobese diabetic/severe combined immunodeficiency mice. Our data suggest that extracellular nucleotides may provide a novel and powerful tool to modulate HSC functions. View Publication

Like their human counterparts,mouse plasmacytoid dendritic cells (pDCs) play a central role in innate immunity against viral infections,but their capacity to prime T cells in vivo remains unknown. We show here that virus-activated pDCs differentiate into antigen-presenting cells able to induce effector/memory CD8(+) T-cell responses in vivo against both epitopic peptides and endogenous antigen,whereas pDCs activated by synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG) acquire only the ability to recall antigen-experienced T-cell responses. We also show that immature pDCs are unable to induce effector or regulatory CD8(+) T-cell responses. Thus,murine pDCs take part in both innate and adaptive immune responses by directly priming naive CD8(+) T cells during viral infection. View Publication

Cell enrichment methods that deal with larger volumes of peripheral blood and BM are needed for increased sensitivity of detection,characterization and quantification of isolated tumor cells (ITC). This study was designed to evaluate a new procedure,the RosetteSep-Applied Imaging Rare Event (RARE) detection method,which depletes the majority of the erythrocytes and leucocytes in a peripheral blood (PB) sample,thereby negatively enriching tumor cells if present. This enrichment procedure allows for increased sensitivity,by analyzing a 5-10 fold larger volume of blood,compared with a direct immunocytochemical (ICC) technique,with minimal impact on laboratory workload. Model experiments showed comparable tumor cell recoveries between the two tested methods,both in PB and BM. Clinical samples were evaluated using paired PB and BM samples from 95 carcinoma patients. Analysis of PB results showed that 25.3% had textgreater or = 1 tumor cell detected by the RARE procedure,compared with 5.2% after direct ICC analysis,analyzing a 10-fold larger volume by the RARE procedure. The direct ICC analysis of BM from the same patients revealed 16.8% positive. The ITC detection differed both quantitatively and qualitatively between BM and PB,as samples with high numbers of ITC in BM were still negative in PB. The clinical significance of ITC in blood still needs to be established. However,the easy access of peripheral blood,and the increased sensitivity obtained by increasing the sample volume with the RARE procedure,suggests that the value of peripheral blood analysis should be tested in parallel in studies where ITC detection in BM is performed. View Publication

We used primary peripheral blood T cells,a population that exists in G(0) and can be stimulated to enter the cell cycle synchronously,to define more precisely the effects of nicotine on pathways that control cell cycle entry and progression. Our data show that nicotine decreased the ability of T cells to transit through the G(0)/G(1) boundary (acquire competence) and respond to progression signals. These effects were due to nuclear factor of activated T cells c2 (NFATc2)-dependent repression of cyclin-dependent kinase 4 (CDK4) expression. Growth arrest at the G(0)/G(1) boundary was further enforced by inhibition of cyclin D2 expression and by increased expression and stabilization of p27Kip1. Intriguingly,T cells from habitual users of tobacco products and from NFATc2-deficient mice constitutively expressed CDK4 and were resistant to the antiproliferative effects of nicotine. These results indicate that nicotine impairs T cell cycle entry through NFATc2-dependent mechanisms and suggest that,in the face of chronic nicotine exposure,selection may favor cells that can evade these effects. We postulate that cross talk between nicotinic acetylcholine receptors and growth factor receptor-activated pathways offers a novel mechanism by which nicotine may directly impinge on cell cycle progression. This offers insight into possible reasons that underlie the unique effects of nicotine on distinct cell types and identifies new targets that may be useful control tobacco-related diseases. View Publication

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report,the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented,based upon their specificity,binding requirements,and biological activity. Initial screening by ELISA,using highly purified virus as the coating antigen,resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb,F26G15,is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients,but not normal human serum,is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests,studies of SARS-CoV pathogenesis and vaccine development. ?? 2004 Elsevier B.V. All rights reserved. View Publication