技术资料

The influence of membrane potential on the effects of the enantiomers and the racemate of Bay K 8644 [1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluor-methylphenyl)-p yri dine-5-carboxylate] on force of contraction and on action potentials were studied in guinea-pig papillary muscles in order to detect possible changes in the direction of drug action or in potency. Membrane potential was varied by changing the potassium concentration ([K+]o) in the bathing solution. At normal resting potential,(-)-Bay K 8644 enhanced force of contraction and prolonged the action potential duration measured at 50% of repolarization (APD) to the same extent as the racemate and with similar pD2 values. After membrane depolarization by raising [K+]o from 5.4 to 17.4 mmol/l,the (-)-enantiomer and the racemate prolonged the APD to a similar degree but enhanced force to a lesser extent. The maximum rate of depolarization of slow action potentials,Vmax,was increased at the highest concentrations (10(-5) mol/l). The effects of (+)-Bay K 8644 were more complicated. At high concentrations (10(-5) mol/l) it decreased force of contraction and APD,the pD2 values were one order of magnitude lower than for the (-)-enantiomer and the racemate. A high concentration (+)-Bay K 8644 (10(-5) mol/l) virtually abolished contractile activity at all membrane potentials,the extent of shortening in APD increased with membrane depolarization in elevated [K+]o. Vmax of slow action potentials was decreased.(ABSTRACT TRUNCATED AT 250 WORDS) View Publication

We have generated Tie2Cre+alpha4(f/f) mice with documented alpha4-integrin ablation in hematopoietic and endothelial cells. A prominent feature in this model is a sustained,significant increase in circulating progenitors at levels higher than the levels seen with Tie2Cre+VCAM-1(f/f) mice. To test whether phenotypic differences are due to contributions by ligands other than VCAM-1 in bone marrow,or to alpha4-deficient endothelial cells or pericytes,we carried out transplantation experiments using these mice as donors or as recipients. Changes in progenitor biodistribution after transplantation were seen only with alpha4-deficient donor cells,suggesting that these cells were necessary and sufficient to reproduce the phenotype with no discernible contribution by alpha4-deficient nonhematopoietic cells. Because several similarities are seen after transplantation between our results and those with CXCR4-/- donor cells,the data suggest that VLA4/VCAM-1 and CXCR4/CXCL12 pathways contribute to a nonredundant,ongoing signaling required for bone marrow retention of progenitor cells during homeostasis. View Publication

Neurotrophins are a family of closely related proteins that were identified initially as survival factors for sensory and sympathetic neurons,and have since been shown to control many aspects of survival,development and function of neurons in both the peripheral and the central nervous systems. Each of the four mammalian neurotrophins has been shown to activate one or more of the three members of the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases (TrkA,TrkB and TrkC). In addition,each neurotrophin activates p75 neurotrophin receptor (p75NTR),a member of the tumour necrosis factor receptor superfamily. Through Trk receptors,neurotrophins activate Ras,phosphatidyl inositol-3 (PI3)-kinase,phospholipase C-gamma1 and signalling pathways controlled through these proteins,such as the MAP kinases. Activation of p75NTR results in activation of the nuclear factor-kappaB (NF-kappaB) and Jun kinase as well as other signalling pathways. Limiting quantities of neurotrophins during development control the number of surviving neurons to ensure a match between neurons and the requirement for a suitable density of target innervation. The neurotrophins also regulate cell fate decisions,axon growth,dendrite growth and pruning and the expression of proteins,such as ion channels,transmitter biosynthetic enzymes and neuropeptide transmitters that are essential for normal neuronal function. Continued presence of the neurotrophins is required in the adult nervous system,where they control synaptic function and plasticity,and sustain neuronal survival,morphology and differentiation. They also have additional,subtler roles outside the nervous system. In recent years,three rare human genetic disorders,which result in deleterious effects on sensory perception,cognition and a variety of behaviours,have been shown to be attributable to mutations in brain-derived neurotrophic factor and two of the Trk receptors. View Publication

The nuclear proto-oncogene c-myb plays crucial roles in the growth,survival,and differentiation of hematopoietic cells. We established three lines of erythropoietin receptor-transgenic mice and found that one of them exhibited anemia,thrombocythemia,and splenomegaly. These abnormalities were independent of the function of the transgenic erythropoietin receptor and were observed exclusively in mice harboring the transgene homozygously,suggesting transgenic disruption of a certain gene. The transgene was inserted 77 kb upstream of the c-myb gene,and c-Myb expression was markedly decreased in megakaryocyte/erythrocyte lineage-restricted progenitors (MEPs) of the homozygous mutant mice. In the bone marrows and spleens of the mutant mice,numbers of megakaryocytes were increased and numbers of erythroid progenitors were decreased. These abnormalities were reproducible in vitro in a coculture assay of MEPs with OP9 cells but eliminated by the retroviral expression of c-Myb in MEPs. The erythroid/megakaryocytic abnormalities were reconstituted in mice in vivo by transplantation of mutant mouse bone marrow cells. These results demonstrate that the transgene insertion into the c-myb gene far upstream regulatory region affects the gene expression at the stage of MEPs,leading to an imbalance between erythroid and megakaryocytic cells,and suggest that c-Myb is an essential regulator of the erythroid-megakaryocytic lineage bifurcation. View Publication

Apoptosis of B cell chronic lymphocytic leukemia (B-CLL) cells is regulated by the PI-3K-Akt pathway. In the present work,we have analyzed the mechanisms of Akt phosphorylation in B-CLL cells. Freshly isolated cells present basal Akt phosphorylation,which is PI-3K-dependent,as incubation with the PI-3K inhibitor LY294002 decreased Ser-473 and Thr-308 phosphorylation in most samples analyzed (seven out of 10). In three out of 10 cases,inhibition of protein kinase C (PKC) inhibited basal Akt phosphorylation. Stromal cell-derived factor-1alpha,IL-4,and B cell receptor activation induced PI-3K-dependent Akt phosphorylation. PMA induced the phosphorylation of Akt at Ser-473 and Thr-308 and the phosphorylation of Akt substrates,independently of PI-3K in B-CLL cells. In contrast,PKC-mediated phosphorylation of Akt was PI-3K-dependent in normal B cells. Finally,a specific inhibitor of PKCbeta blocked the phosphorylation and activation of Akt by PMA in B-CLL cells. Taken together,these results suggest a model in which Akt could be activated by two different pathways (PI-3K and PKCbeta) in B-CLL cells. View Publication

Hmgb3 is an X-linked member of a family of sequence-independent chromatin-binding proteins that is preferentially expressed in hematopoietic stem cells (HSC). Hmgb3-deficient mice (Hmgb3(-/Y)) contain normal numbers of HSCs,capable of self-renewal and hematopoietic repopulation,but fewer common lymphoid (CLP) and common myeloid progenitors (CMP). In this study,we tested the hypothesis that Hmgb3(-/Y) HSCs are biased toward self-renewal at the expense of progenitor production. Wild-type and Hmgb3(-/Y) CLPs and CMPs proliferate and differentiate equally in vitro,indicating that CLP and CMP function normally in Hmgb3(-/Y) mice. Hmgb3(-/Y) HSCs exhibit constitutive activation of the canonical Wnt signaling pathway,which regulates stem cell self-renewal. Increased Wnt signaling in Hmgb3(-/Y) HSCs corresponds to increased expression of Dvl1,a positive regulator of the canonical Wnt pathway. To induce hematopoietic stress and a subsequent response from HSCs,we treated Hmgb3(-/Y) mice with 5-fluorouracil. Hmgb3(-/Y) mice exhibit a faster recovery of functional HSCs after administration of 5-fluorouracil compared with wild-type mice,which may be due to the increased Wnt signaling. Furthermore,the recovery of HSC number in Hmgb3(-/Y) mice occurs more rapidly than CLP and CMP recovery. From these data,we propose a model in which Hmgb3 is required for the proper balance between HSC self-renewal and differentiation. View Publication

Thymus-derived CD4+ CD25+ T regulatory cells (Tregs) are essential for the maintenance of self-tolerance. What critical factors and conditions are required for the extra-thymic development of Tregs remains an important question. In this study,we show that the anti-inflammatory extracellular matrix protein,thrombospondin-1,promoted the generation of human peripheral regulatory T cells through the ligation of one of its receptor,CD47. CD47 stimulation by mAb or a thrombospondin-1 peptide induced naive or memory CD4+ CD25- T cells to become suppressive. The latter expressed increased amounts of CTLA-4,OX40,GITR,and Foxp3 and inhibited autologous Th0,Th1,and Th2 cells. Their regulatory activity was contact dependent,TGF-beta independent,and partially circumvented by IL-2. This previously unknown mechanism to induce human peripheral Tregs in response to inflammation may participate to the limitation of collateral damage induced by exacerbated responses to self or foreign Ags and thus be relevant for therapeutic intervention in autoimmune diseases and transplantation. View Publication

NK cells play an important role in the innate immune response. We have isolated NK cells from human lymphoid tissues and found that these cells express the CD4 molecule on their surface at levels higher than those found on peripheral blood NK cells. To study the functional role of the CD4 molecule on NK cells,we developed an in vitro system by which we are able to obtain robust CD4 expression on NK cells derived from blood. CD4+ NK cells efficiently mediate NK cell cytotoxicity,and CD4 expression does not appear to alter lytic function. CD4+ NK cells are more likely to produce the cytokines gamma-IFN and TNF-alpha than are CD4- NK cells. Ligation of CD4 further increases the number of NK cells producing these cytokines. NK cells expressing CD4 are also capable of migrating toward the CD4-specific chemotactic factor IL-16,providing another function for the CD4 molecule on NK cells. Thus,the CD4 molecule is present and functional on NK cells and plays a role in innate immune responses as a chemotactic receptor and by increasing cytokine production,in addition to its well-described function on T cells as a coreceptor for Ag responsive cell activation. View Publication

Nonmammalian glycan structures from helminths act as Th2 adjuvants. Some of these structures are also common on plant glycoproteins. We hypothesized that glycan structures present on peanut glycoallergens act as Th2 adjuvants. Peanut Ag (PNAg),but not deglycosylated PNAg,activated monocyte-derived dendritic cells (MDDCs) as measured by MHC/costimulatory molecule up-regulation,and by their ability to drive T cell proliferation. Furthermore,PNAg-activated MDDCs induced 2- to 3-fold more IL-4- and IL-13-secreting Th2 cells than immature or TNF/IL-1-activated MDDCs when cultured with naive CD4+ T cells. Human MDDCs rapidly internalized Ag in a calcium- and glycan-dependent manner consistent with recognition by C-type lectin. Dendritic cell (DC)-specific ICAM-grabbing nonintegrin (DC-SIGN) (CD209) was shown to recognize PNAg by enhanced uptake in transfected cell lines. To identify the DC-SIGN ligand from unfractionated PNAg,we expressed the extracellular portion of DC-SIGN as an Fc-fusion protein and used it to immunoprecipitate PNAg. A single glycoprotein was pulled down in a calcium-dependent manner,and its identity as Ara h 1 was proven by immunolabeling and mass spectrometry. Purified Ara h 1 was found to be sufficient for the induction of MDDCs that prime Th2-skewed T cell responses. Both PNAg and purified Ara h 1 induced Erk 1/2 phosphorylation of MDDCs,consistent with previous reports on the effect of Th2 adjuvants on DCs. View Publication

Therapeutic mAbs that target tumor-associated Ags on the surface of malignant cells have proven to be an effective and specific option for the treatment of certain cancers. However,many of these protein markers of carcinogenesis are not expressed on the cells' surface. Instead these tumor-associated Ags are processed into peptides that are presented at the cell surface,in the context of MHC class I molecules,where they become targets for T cells. To tap this vast source of tumor Ags,we generated a murine IgG2a mAb,3.2G1,endowed with TCR-like binding specificity for peptide-HLA-A*0201 (HLA-A2) complex and designated this class of Ab as TCR mimics (TCRm). The 3.2G1 TCRm recognizes the GVL peptide (GVLPALPQV) from human chorionic gonadotropin beta presented by the peptide-HLA-A*0201 complex. When used in immunofluorescent staining reactions using GVL peptide-loaded T2 cells,the 3.2G1 TCRm specifically stained the cells in a peptide and Ab concentration-dependent manner. Staining intensity correlated with the extent of cell lysis by complement-dependent cytotoxicity (CDC),and a peptide concentration-dependent threshold level existed for the CDC reaction. Staining of human tumor lines demonstrated that 3.2G1 TCRm was able to recognize endogenously processed peptide and that the breast cancer cell line MDA-MB-231 highly expressed the target epitope. The 3.2G1 TCRm-mediated CDC and Ab-dependent cellular cytotoxicity of a human breast carcinoma line in vitro and inhibited in vivo tumor implantation and growth in nude mice. These results provide validation for the development of novel TCRm therapeutic reagents that specifically target and kill tumors via recognition and binding to MHC-peptide epitopes. View Publication