参考文献

Killer immunoglobulin-like receptors (KIR) bind self-major histocompatibility complex class I molecules,allowing natural killer (NK) cells to recognize aberrant cells that have down-regulated class I. NK cells express variable numbers and combinations of highly homologous clonally restricted KIR genes,but uniformly express KIR2DL4. We show that NK clones express both 2DL4 alleles and either one or both alleles of the clonally restricted KIR 3DL1 and 3DL2 genes. Despite allele-independent expression,3DL1 alleles differed in the core promoter by only one or two nucleotides. Allele-specific 3DL1 gene expression correlated with promoter and 5' gene DNA hypomethylation in NK cells in vitro and in vivo. The DNA methylase inhibitor,5-aza-2'-deoxycytidine,induced KIR DNA hypomethylation and heterogeneous expression of multiple KIR genes. Thus,NK cells use DNA methylation to maintain clonally restricted expression of highly homologous KIR genes and alleles. View Publication

We have previously examined the specificities of 28 commercially available compounds,reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases [Davies,Reddy,Caivano and Cohen (2000) Biochem. J. 351,95-105]. In the present study,we have extended this analysis to a further 14 compounds. Of these,indirubin-3'-monoxime,SP 600125,KT 5823 and ML-9 were found to inhibit a number of protein kinases and conclusions drawn from their use in cell-based assays are likely to be erroneous. Kenpaullone,Alsterpaullone,Purvalanol,Roscovitine,pyrazolopyrimidine 1 (PP1),PP2 and ML-7 were more specific,but still inhibited two or more protein kinases with similar potency. Our results suggest that the combined use of Roscovitine and Kenpaullone may be useful for identifying substrates and physiological roles of cyclin-dependent protein kinases,whereas the combined use of Kenpaullone and LiCl may be useful for identifying substrates and physiological roles of glycogen synthase kinase 3. The combined use of SU 6656 and either PP1 or PP2 may be useful for identifying substrates of Src family members. Epigallocatechin 3-gallate,one of the main polyphenolic constituents of tea,inhibited two of the 28 protein kinases in the panel,dual-specificity,tyrosine-phosphorylated and regulated kinase 1A (DYRK1A; IC(50)=0.33 microM) and p38-regulated/activated kinase (PRAK; IC(50)=1.0 microM). View Publication

Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however,the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects,with a median of 14 individual epitopic regions targeted per person (range,2 to 42),and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median,4,245) among all study participants. However,the number of epitopic regions targeted,the protein subunits recognized,and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals,with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid,sensitive,specific,and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response,even if a comprehensive pan-genome screening approach is applied. View Publication

Detailed equilibrium binding studies were conducted on a monoclonal antibody directed against Pb(II) complexed with a protein conjugate of diethylenetriaminepentaacetic acid (DTPA). Binding curves obtained with DTPA and a cyclohexyl derivative of DTPA in the presence and absence of metal ions were consistent with the anticipated one-site homogeneous binding model. Binding curves obtained with aminobenzyl-DTPA or its complexes with Ca(II),Sr(II),and Ba(II) were highly sigmoidal,characterized by Hill coefficients of 2.3-6.5. Binding curves obtained with the Pb(II) and In(III) complexes of aminobenzyl-DTPA were hyperbolic,but in each case the apparent affinity of the antibody for the chelator-metal complex was higher in the presence of excess chelator than it was in the presence of excess metal ion. In the presence of excess chelator,the equilibrium dissociation constant for the binding of aminobenzyl-DTPA-Pb(II) to the antibody was 9.5 x 10(-)(10) M. Binding curves obtained with the Hg(II) and Cd(II) complexes of aminobenzyl-DTPA were biphasic,indicative of negative cooperativity. Further binding studies demonstrated that aminobenzyl-DTPA-Hg(II) opposed the binding of additional chelator-metal complexes to the antibody more strongly than did aminobenzyl-DTPA-Cd(II). The Fab fragment differed from the intact antibody only in that the apparent affinity of the Fab was generally lower for a given chelator-metal complex. These data are interpreted in terms of a model in which (i) aminobenzyl-DTPA and its complexes bind both to the antigen binding site and to multiple charged sites on the surface of the compact immunoglobulin; and (ii) the bound,highly charged ligands interact in a complicated fashion through the apolar core of the folded antibody. View Publication

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Granzyme B (grB) is a serine proteinase released by cytotoxic lymphocytes (CLs) to kill abnormal cells. GrB-mediated apoptotic pathways are conserved in nucleated cells; hence,CLs require mechanisms to protect against ectopic or misdirected grB. The nucleocytoplasmic serpin,proteinase inhibitor 9 (PI-9),is a potent inhibitor of grB that protects cells from grB-mediated apoptosis in model systems. Here we show that PI-9 is present in CD4(+) cells,CD8(+) T cells,NK cells,and at lower levels in B cells and myeloid cells. PI-9 is up-regulated in response to grB production and degranulation,and associates with grB-containing granules in activated CTLs and NK cells. Intracellular complexes of PI-9 and grB are evident in NK cells,and overexpression of PI-9 enhances CTL potency,suggesting that cytoplasmic grB,which may threaten CL viability,is rapidly inactivated by PI-9. Because dendritic cells (DCs) acquire characteristics similar to those of target cells to activate naive CD8(+) T cells and therefore may also require protection against grB,we investigated the expression of PI-9 in DCs. PI-9 is evident in thymic DCs (CD3(-),CD4(+),CD8(-),CD45(+)),tonsillar DCs,and DC subsets purified from peripheral blood (CD16(+) monocytes and CD123(+) plasmacytoid DCs). Furthermore,PI-9 is expressed in monocyte-derived DCs and is up-regulated upon TNF-alpha-induced maturation of monocyte-derived DCs. In conclusion,the presence and subcellular localization of PI-9 in leukocytes and DCs are consistent with a protective role against ectopic or misdirected grB during an immune response. View Publication

T lymphocytes are exposed to hypoxia during their development and also when they migrate to hypoxic pathological sites such as tumors and wounds. Although hypoxia can affect T cell development and function,the mechanisms by which immune cells sense and respond to changes in O(2)-availability are poorly understood. K(+) channels encoded by the Kv1.3 subtype of the voltage-dependent Kv1 gene family are highly expressed in lymphocytes and are involved in the control of membrane potential and cell function. In this study,we investigate the sensitivity of Kv1.3 channels to hypoxia in freshly isolated human T lymphocytes and leukemic Jurkat T cells. Acute exposure to hypoxia (20 mmHg,2 min) inhibits Kv1.3 currents in both cell types by 20%. Prolonged exposure to hypoxia (1% O(2) for 24 h) selectively decreases Kv1.3 protein levels in Jurkat T cells by 47%,but not Kvbeta2 and SK2 Ca-activated K(+) channel subunit levels. The decrease in Kv1.3 protein levels occurs with no change in Kv1.3 mRNA expression and is associated with a significant decrease in K(+) current density. A decrease in Kv1.3 polypeptide levels similar to that obtained during hypoxia is produced by Kv1.3 channel blockage. Our results indicate that hypoxia produces acute and long-term inhibition of Kv1.3 channels in T lymphocytes. This effect could account for the inhibition of lymphocyte proliferation during hypoxia. Indeed,we herein present evidence showing that hypoxia selectively inhibits TCR-mediated proliferation and that this inhibition is associated with a decrease in Kv1.3 proteins. View Publication

We disclose here a new structural class of low-molecular-weight inhibitors of NF-kappa B activation that were designed and synthesized by starting from quinazoline derivative 6a. Structure-activity relationship (SAR) studies based on 6a elucidated the structural requirements essential for the inhibitory activity toward NF-kappa B transcriptional activation,and led to the identification of the 6-amino-4-phenethylaminoquinazoline skeleton as the basic framework. In this series of compounds,11q,containing the 4-phenoxyphenethyl moiety at the C(4)-position,showed strong inhibitory effects on both NF-kappa B transcriptional activation and TNF-alpha production. Furthermore,11q exhibited an anti-inflammatory effect on carrageenin-induced paw edema in rats. View Publication

The steps to leukemia following an in utero fusion of MLL (HRX,ALL-1) to a partner gene in humans are not known. Introduction of the Mll-AF9 fusion gene into embryonic stem cells results in leukemia in mice with cell-type specificity similar to humans. In this study we used myeloid colony assays,immunophenotyping,and transplantation to evaluate myelopoiesis in Mll-AF9 mice. Colony assays demonstrated that both prenatal and postnatal Mll-AF9 tissues have significantly increased numbers of CD11b(+)/CD117(+)/Gr-1(+/-) myeloid cells,often in compact clusters. The self-renewal capacity of prenatal myeloid progenitors was found to decrease following serial replating of colony-forming cells. In contrast,early postnatal myeloid progenitors increased following replating; however,the enhanced self-renewal of early postnatal myeloid progenitor cells was limited and did not result in long-term cell lines or leukemia in vivo. Unlimited replating,long-term CD11b/Gr-1(+) myeloid cell lines,and the ability to produce early leukemia in vivo in transplantation experiments,were found only in mice with overt leukemia. Prenatal Mll-AF9 tissues had reduced total (mature and progenitor) CD11b/Gr-1(+) cells compared with wild-type tissues. Colony replating,immunophenotyping,and cytochemistry suggest that any perturbation of cellular differentiation from the prenatal stage onward is partial and largely reversible. We describe a novel informative in vitro and in vivo model system that permits study of the stages in the pathogenesis of Mll fusion gene leukemia,beginning in prenatal myeloid cells,progressing to a second stage in the postnatal period and,finally,resulting in overt leukemia in adult animals. View Publication

Essential thrombocythemia (ET) and polycythemia vera (PV) are clonal myeloproliferative disorders that are often difficult to distinguish from other causes of elevated blood cell counts. Assays that could reliably detect clonal hematopoiesis would therefore be extremely valuable for diagnosis. We previously reported 3 X-chromosome transcription-based clonality assays (TCAs) involving the G6PD,IDS,and MPP1 genes,which together were informative in about 65% of female subjects. To increase our ability to detect clonality,we developed simple TCA for detecting the transcripts of 2 additional X-chromosome genes: Bruton tyrosine kinase (BTK) and 4-and-a-half LIM domain 1 (FHL1). The combination of TCA established the presence or absence of clonal hematopoiesis in about 90% of female subjects. We show that both genes are subject to X-chromosome inactivation and are polymorphic in all major US ethnic groups. The 5 TCAs were used to examine clonality in 46 female patients along with assays for erythropoietin-independent erythroid colonies (EECs) and granulocyte PRV-1 mRNA levels to discriminate polycythemias and thrombocytoses. Of these,all 19 patients with familial polycythemia or thrombocytosis had polyclonal hematopoiesis,whereas 22 of 26 patients with clinical evidence of myeloproliferative disorder and 1 patient with clinically obscure polycythemia were clonal. Interestingly,interferon alpha therapy in 2 patients with PV was associated with reversion of clonal to polyclonal hematopoiesis. EECs were observed in 14 of 14 patients with PV and 4 of 12 with ET,and increased granulocyte PRV-1 mRNA levels were found in 9 of 13 patients with PV and 2 of 12 with ET. Thus,these novel clonality assays are useful in the diagnosis and follow-up of polycythemic conditions and disorders with increased platelet levels. View Publication

T lymphocytes with unusually high expression of the voltage-gated Kv1.3 channel (Kv1.3(high) cells) have been implicated in the pathogenesis of experimental autoimmune encephalomyelitis,an animal model for multiple sclerosis. We have developed a fluoresceinated analog of ShK (ShK-F6CA),the most potent known inhibitor of Kv1.3,for detection of Kv1.3(high) cells by flow cytometry. ShK-F6CA blocked Kv1.3 at picomolar concentrations with a Hill coefficient of 1 and exhibited textgreater80-fold specificity for Kv1.3 over Kv1.1 and other K(V) channels. In flow cytometry experiments,ShK-F6CA specifically stained Kv1.3-expressing cells with a detection limit of approximately 600 channels per cell. Rat and human T cells that had been repeatedly stimulated 7-10 times with antigen were readily distinguished on the basis of their high levels of Kv1.3 channels (textgreater600 channels/cell) and ShK-F6CA staining from resting T cells or cells that had undergone 1-3 rounds of activation. Functional Kv1.3 expression levels increased substantially in a myelin-specific rat T cell line following myelin antigen stimulation,peaking at 15-20 h and then declining to baseline over the next 7 days,in parallel with the acquisition and loss of encephalitogenicity. Both calcium- and protein kinase C-dependent pathways were required for the antigen-induced Kv1.3 up-regulation. ShK-F6CA might be useful for rapid and quantitative detection of Kv1.3(high) expressing cells in normal and diseased tissues,and to visualize the distribution of functional channels in intact cells. View Publication

Natural killer (NK) cell differentiation from pluripotent CD34(+) human hematopoietic stem cells or oligopotent lymphoid progenitors has already been reported. In the present study,long-term cultures of the CD56(-)/CD34(-) myeloid-like adherent cell fraction (ACF) from umbilical cord blood (UCB),characterized by the expression of CD14(+) as well as other myeloid markers,were set up with flt3 ligand (FL) and interleukin-15 (IL-15). The UCB/ACF gradually expressed the CD56 marker,which reached fairly high levels (approximately 90% of the cells were CD56(+)) by day 15. FL plus IL-15-driven ACF/CD56(+) cells progressively expressed a mature NK functional program lysing both NK- and lymphokine-activate killer (LAK)-sensitive tumor targets and producing high levels of interferon-gamma (IFN-gamma),granulocyte-macrophage colony-stimulating factor,tumor necrosis factor alpha,and IL-10 upon stimulation with IL-12 and IL-18. Similar results were obtained when highly purified CD14(+) cells from UCB were cultured with FL and IL-15. In contrast,UCB/CD34(+) cells cultured under the same conditions showed a delayed expression of CD56 and behaved functionally differently in that they exhibited NK but not LAK cytotoxicity and produced significantly fewer cytokines. Kinetic studies on the phenotype of UCB/ACF or UCB/CD14(+) cells cultured in the presence of FL and IL-15 showed a rapid decrease in CD14 expression after day 5,which reached levels of zero by day 20. Approximately 60% of the CD56(+) derived from the UCB/ACF or the UCB/CD14(+) cells coexpressed CD14 by day 5. Taken together,our data support the role of CD14(+) myeloid-like cells within UCB as a novel progenitor for lymphoid NK cells. View Publication