参考文献

The termination of activation signals is a critical step in the control of the immune response; perturbation of inhibitory feedback pathways results in profound immune defects culminating in autoimmunity and overwhelming inflammation. FcgammaRIIB receptor is a well described inhibitory receptor. The ligation of B-cell receptor (BCR) and FcgammaRIIB leads to the inhibition of B-cell activation. Numerous studies have demonstrated that the SH2-domain-containing inositol 5-phosphatase SHIP (referred hereto as SHIP-1) is essential in this process. The cDNA encoding a second SH2-domain-containing inositol 5-phosphatase,SHIP-2,has been cloned [Pesesse,Deleu,De Smedt,Drayer and Erneux (1997) Biochem. Biophys. Res. Commun. 239,697-700]. Here we report the distribution of SHIP-2 in mouse tissues: a Western blot analysis of mouse tissues reveals that SHIP-2 is expressed in both haemopoietic and non-haemopoietic cells. In addition to T-cell and B-cell lines,spleen,thymus and lung are shown to coexpress SHIP-1 and SHIP-2. Moreover,SHIP-2 is detected in fibroblasts,heart and different brain areas. SHIP-2 shows a maximal tyrosine phosphorylation and association to Shc after ligation of BCR to FcgammaRIIB but not after stimulation of BCR alone. Our results therefore suggest a possible role for SHIP-2 in the negative regulation of immunocompetent cells. View Publication

To study molecular events involved in B lymphocyte development and V(D)J rearrangement,we have established an efficient system for the differentiation of embryonic stem (ES) cells into mature Ig-secreting B lymphocytes. Here,we show that B lineage cells generated in vitro from ES cells are functionally analogous to normal fetal liver-derived or bone marrow-derived B lineage cells at three important developmental stages: first,they respond to Flt-3 ligand during an early lymphopoietic progenitor stage; second,they become targets for Abelson murine leukemia virus (A-MuLV) infection at a pre-B cell stage; third,they secrete Ig upon stimulation with lipopolysaccharide at a mature mitogen-responsive stage. Moreover,the ES cell-derived A-MuLV-transformed pre-B (EAB) cells are phenotypically and functionally indistinguishable from standard A-MuLV-transformed pre-B cells derived from infection of mouse fetal liver or bone marrow. Notably,EAB cells possess functional V(D)J recombinase activity. In particular,the generation of A-MuLV transformants from ES cells will provide an advantageous system to investigate genetic modifications that will help to elucidate molecular mechanisms in V(D)J recombination and in A-MuLV-mediated transformation. View Publication

Because hematopoietic stem cells are rich in aldehyde dehydrogenase (ALDH) activity,we developed a fluorescent substrate for ALDH,termed BODIPY aminoacetaldehyde (BAAA),and tested its potential for isolating primitive human hematopoietic cells. A population of cells with low orthogonal light scattering and bright fluorescence intensity (SSC(lo)ALDH(br) cells) could be readily fractionated from human umbilical cord blood cells costained with BAAA and the multidrug-resistance inhibitor verapamil. The SSC(lo)ALDH(br) population was depleted of lineage-committed cells,40-90% pure for CD34(+)CD38(lo/-) cells,and enriched 50- to 100-fold for primitive hematopoietic progenitors detected in short- and long-term culture analyses. Together,these observations indicate that fractionating human hematopoietic stem cells on the basis of ALDH activity using BAAA is an effective method for isolating primitive human hematopoietic progenitors. This technique may be useful for isolating stem cells from other tissues as well. View Publication

Bispecific antibodies directed against tumor-associated target antigens and to surface receptors mediating T-cell activation,such as the TCR/CD3 complex and the costimulatory receptor CD28,are capable of mediating T-cell activation resulting in tumor cell killing. In this study,we used the B-cell-associated antigens CD19 and CD20 as target structures on human leukemic cells. We found that a combination of bispecific antibody fragments (bsFab2) with target x CD3 and target x CD28 specificity induces vigorous autologous T-cell activation and killing of malignant cells in peripheral blood and bone marrow cultures from patients with chronic lymphocytic leukemia and follicular lymphoma. The bsFab2 targeting CD20 were considerably more effective than those binding to CD19. The colony-forming capacity of treated bone marrow was impaired due to large amounts of tumor necrosis factor alpha produced during bsFab2-induced T-cell activation. Neutralizing tumor necrosis factor alpha antibodies were found to reverse this negative effect without affecting T-cell activation and tumor cell killing. CD20 x CD28 bsFab2,when used alone rather than in combination,markedly improved the recognition of leukemic cells by allogeneic T cells. Therefore,these reagents may be capable of enhancing the immunogenicity of leukemic cells in general and,in particular,of increasing the antileukemic activity of allogeneic donor buffy coat cells in relapsed bone marrow transplanted patients. View Publication

In this report,we sought to optimize gene transfer into primitive human umbilical cord blood (UCB) cells. Initially,we found that fresh UCB isolated with the CD34brCD38 CD33 phenotype were highly enriched for hematopoietic progenitors detected in extended long-term cultures (8-week LTCs). In addition,following ex vivo gene transfer,this population possessed virtually all the 8-week LTC activity of the cultured cells. A multiparameter FACS assay was developed to efficiently screen the effects of alternative retroviral vector gene transfer procedures on the transduction efficiency and maintenance of CD34brCD38 CD33 cells. Proliferation of the CD34brCD38 CD33 cells was found to be a prerequisite for efficient transduction. However,in all conditions tested,proliferation of the CD34brCD38 CD33 cells was associated with a progressive loss of primitive cell properties including a reduction in CD34 expression,an increase in CD38/CD33 expression,and a decline in the ability to sustain 8-week LTCs. These observations indicate that it will be necessary to define conditions that more effectively support the self-renewal capacity of CD34brCD38 CD33 cells to optimize retroviral vector gene transfer in these cells. Evaluating these conditions and reagents will be facilitated by the multiparameter FACS assay described in this report. 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

PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed,and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs,as determined in the modified Boyden chamber,as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly,in addition to inhibition of Src kinase,PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells,including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR,PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases,the activities of the epidermal growth factor receptor,fibroblast growth factor receptor-1,and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872,whereas PDGF alpha-receptor was susceptible,albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events,creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity,such as vascular remodeling and restenosis. View Publication

A novel homology model of the kinase domain of Janus kinase (JAK) 3 was used for the structure-based design of dimethoxyquinazoline compounds with potent and specific inhibitory activity against JAK3. The active site of JAK3 in this homology model measures roughly 8 A x 11 A x 20 A,with a volume of approximately 530 A3 available for inhibitor binding. Modeling studies indicated that 4-(phenyl)-amino-6,7-dimethoxyquinazoline (parent compound WHI-258) would likely fit into the catalytic site of JAK3 and that derivatives of this compound that contain an OH group at the 4' position of the phenyl ring would more strongly bind to JAK3 because of added interactions with Asp-967,a key residue in the catalytic site of JAK3. These predictions were consistent with docking studies indicating that compounds containing a 4'-OH group,WHI-P131 [4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline],WHI-P154 [4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline],and WHI-P97 [4-(3',5'-dibromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazolin e],were likely to bind favorably to JAK3,with estimated K(i)s ranging from 0.6 to 2.3 microM. These compounds inhibited JAK3 in immune complex kinase assays in a dose-dependent fashion. In contrast,compounds lacking the 4'-OH group,WHI-P79 [4-(3'-bromophenyl)-amino-6,7-dimethoxyquinazoline],WHI-P111 [4-(3'-bromo-4'-methylphenyl)-amino-6,7-dimethoxyquinazoline],WHI-P112 [4-(2',5'-dibromophenyl)-amino-6,7-dimethoxyquinazoline],WHI-P132 [4-(2'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline],and WHI-P258 [4-(phenyl)-amino-6,7-dimethoxyquinazoline],were predicted to bind less strongly,with estimated K(i)s ranging from 28 to 72 microM. These compounds did not show any significant JAK3 inhibition in kinase assays. Furthermore,the lead dimethoxyquinazoline compound,WHI-P131,which showed potent JAK3-inhibitory activity (IC50 of 78 microM),did not inhibit JAK1 and JAK2,the ZAP/SYK family tyrosine kinase SYK,the TEC family tyrosine kinase BTK,the SRC family tyrosine kinase LYN,or the receptor family tyrosine kinase insulin receptor kinase,even at concentrations as high as 350 microM. WHI-P131 induced apoptosis in JAK3-expressing human leukemia cell lines NALM-6 and LC1;19 but not in melanoma (M24-MET) or squamous carcinoma (SQ20B) cells. Leukemia cells were not killed by dimethoxyquinazoline compounds that were inactive against JAK3. WHI-P131 inhibited the clonogenic growth of JAK3-positive leukemia cell lines DAUDI,RAMOS,LC1;19,NALM-6,MOLT-3,and HL-60 (but not JAK3-negative BT-20 breast cancer,M24-MET melanoma,or SQ20B squamous carcinoma cell lines) in a concentration-dependent fashion. Potent and specific inhibitors of JAK3 such as WHI-P131 may provide the basis for the design of new treatment strategies against acute lymphoblastic leukemia,the most common form of childhood cancer. View Publication

We have generated rat monoclonal antibodies specific for the mouse eotaxin receptor,C-C chemokine receptor 3 (CCR3). Several anti-CCR3 mAbs proved to be useful for in vivo depletion of CCR3-expressing cells and immunofluorescent staining. In vivo CCR3 mAbs of the IgG2b isotype substantially depleted blood eosinophil levels in Nippostrongyus brasiliensis-infected mice. Repeated anti-CCR3 mAb treatment in these mice significantly reduced tissue eosinophilia in the lung tissue and bronchoalveolar lavage fluid. Flow cytometry revealed that mCCR3 was expressed on eosinophils but not on stem cells,dendritic cells,or cells from the thymus,lymph node,or spleen of normal mice. Unlike human Th2 cells,mouse Th2 cells did not express detectable levels of CCR3 nor did they give a measurable response to eotaxin. None of the mAbs were antagonists or agonists of CCR3 calcium mobilization. To our knowledge,the antibodies described here are the first mAbs reported to be specific for mouse eosinophils and to be readily applicable for the detection,isolation,and in vivo depletion of eosinophils. View Publication

OBJECTIVE To investigate the inhibition of matrix metalloproteinase 1 (MMP-1),MMP-8,and MMP-13 by doxycycline,and to determine whether the variable hemopexin-like domain of each MMP was responsible for the differences in susceptibility to doxycycline inhibition among these collagenases. METHODS Recombinant human MMP-1 (collagenase 1),MMP-8 (collagenase 2),and MMP-13 (collagenase 3),truncated forms of MMP-8 and MMP-13 lacking the hemopexin-like domain,and a mutant form of truncated MMP-13 were used in these studies. The activity of the full-length MMP in the presence of doxycycline was tested against type II collagen,a natural substrate for the enzymes. A small peptolide substrate was used to determine which structural features of the MMPs were related to sensitivity to doxycycline inhibition. RESULTS The activity of MMP-13 and MMP-8 against type II collagen was inhibited by 50-60% by 30 microM doxycycline,while that of MMP-1 was inhibited only 18% by 50 microM doxycycline. In contrast,in experiments with the peptolide substrate,neither full-length nor truncated MMP-13 was inhibited until the concentration of the drug exceeded 90 microM. MMP-8 and truncated MMP-8 were sensitive to inhibition by 30 microM doxycycline,while MMP-1 was slightly inhibited (14%) by 90 microM doxycycline. For MMP-8,inhibition was reversible upon dilution and was independent of the order in which the reagents were added. Kinetic analysis of the inhibition constant (K(i)) of MMP-8 (K(i) = 36 microM) and truncated MMP-8 (K(i) = 77 microM) indicated that inhibition was noncompetitive. CONCLUSION Significant inhibition of MMP-13 and MMP-8 activity against collagen occurred in vitro at concentrations that were near the concentrations achieved in serum after oral dosing. Studies with truncated enzymes and 2 substrates suggest that doxycycline disrupts the conformation of the hemopexin-like domain of MMP-13 and the catalytic domain of MMP-8. View Publication