技术资料

The application of genome-wide expression analysis to a large-scale,multicentered program in critically ill patients poses a number of theoretical and technical challenges. We describe here an analytical and organizational approach to a systematic evaluation of the variance associated with genome-wide expression analysis specifically tailored to study human disease. We analyzed sources of variance in genome-wide expression analyses performed with commercial oligonucleotide arrays. In addition,variance in gene expression in human blood leukocytes caused by repeated sampling in the same subject,among different healthy subjects,among different leukocyte subpopulations,and the effect of traumatic injury,were also explored. We report that analytical variance caused by sample processing was acceptably small. Blood leukocyte gene expression in the same individual over a 24-h period was remarkably constant. In contrast,genome-wide expression varied significantly among different subjects and leukocyte subpopulations. Expectedly,traumatic injury induced dramatic changes in apparent gene expression that were greater in magnitude than the analytical noise and interindividual variance. We demonstrate that the development of a nation-wide program for gene expression analysis with careful attention to analytical details can reduce the variance in the clinical setting to a level where patterns of gene expression are informative among different healthy human subjects,and can be studied with confidence in human disease. View Publication

Through the regulation of human leukocyte antigen (HLA)-DM (DM) in B cells,HLA-DO (DO) modulates positively or negatively the presentation of specific peptides. Transduction of DO into human blood monocyte-derived dendritic cells (MoDC) has been proposed as a mean of modifying the peptide repertoire of major histocompatibility complex class II molecules. However,maturation of DC induced by inflammatory stimuli or possibly the adenoviral vector itself triggers acidification of vesicles and shuts down transcription of the class II transactivator gene as well as de novo biosynthesis of class II-related molecules and DM activity. In these conditions,it is unclear that transduced DO could alter the peptide repertoire. Our Western blot and reverse transcriptase-polymerase chain reaction analyses revealed that human DC derived from blood monocytes express small amounts of DOalpha. Transduction of DObeta alone resulted in the accumulation of a small pool of DO in DM(+) CD63(+) vesicles and at the plasma membrane of mature DC. The cell-surface increase in class II-associated invariant chain peptide (CLIP)/class II complexes is in line with an inhibitory role of DO on DM. Cotransduction of DOalpha and DObeta only slightly increased CLIP and DO levels at the cell surface. Together with the fact that a large fraction of transduced DO remains in the endoplasmic reticulum,this suggests that DM is limiting in these conditions. DO expression did not affect a mixed lymphocyte reaction but reduced presentation of the exogenous gp100 antigen to a specific T cell clone. These results show that transduced DO modulates antigen presentation in human mature MoDC,evoking the possible use of this chaperone for immunotherapy. View Publication

CD154 (CD40 ligand) expression on CD4 T cells is normally tightly controlled,but abnormal or dysregulated expression of CD154 has been well documented in autoimmune diseases,such as systemic lupus erythematosus. Beyond regulation by NFAT proteins,little is known about the transcriptional activation of the CD154 promoter. We identified a species-conserved purine-rich sequence located adjacent to the CD154 transcriptional promoter proximal NFAT site,which binds early growth response (Egr) transcription factors. Gel shift assays and chromatin immunoprecipitation assays reveal that Egr-1,Egr-3,and NFAT1 present in primary human CD4 T cells are capable of binding this combinatorial site in vitro and in vivo,respectively. Multimerization of this NFAT/Egr sequence in the context of a reporter gene demonstrates this sequence is transcriptionally active upon T cell activation in primary human CD4 T cells. Overexpression of Egr-1,but not Egr-3,is capable of augmenting transcription of this reporter gene as well as that of an intact CD154 promoter. Conversely,overexpression of small interfering RNA specific for Egr-1 in primary human CD4 T cells inhibits CD154 expression. Similarly,upon activation,CD154 message is notably decreased in splenic CD4 T cells from Egr-1-deficient mice compared with wild-type controls. Our data demonstrate that Egr-1 is required for CD154 transcription in primary CD4 T cells. This has implications for selective targeting of Egr family members to control abnormal expression of CD154 in autoimmune diseases such as systemic lupus erythematosus. View Publication

Chronic myeloid leukemia is a clonal multilineage myeloproliferative disease of stem cell origin characterized by the presence of the Bcr/Abl oncoprotein,a constitutively active tyrosine kinase. In previous studies,we have provided evidence that Bcr/Abl overexpression in leukemic cells increased their susceptibility to NK-mediated lysis by different mechanisms. In the present study,using UT-7/9 cells,a high level Bcr/Abl transfectant of UT-7 cells,we show that the treatment of Bcr/Abl target by imatinib mesylate (IM),a specific Abl tyrosine kinase inhibitor,hampers the formation of the NK/target immunological synapse. The main effect of IM involves an induction of surface GM1 ganglioside on Bcr/Abl transfectants that prevents the redistribution of MHC-related Ag molecules in lipid rafts upon interaction with NK cells. IM also affects cell surface glycosylation of targets,as assessed by binding of specific lectins resulting in the subsequent modulation of their binding to lectin type NK receptor,particularly NKG2D. In addition,we demonstrate that the tyrosine kinase activity repression results in a decrease of MHC-related Ags-A/B and UL-16-binding protein expression on Bcr/Abl transfectants UT-7/9. We show that NKG2D controls the NK-mediated lysis of UT-7/9 cells,and IM treatment inhibits this activating pathway. Taken together,our results show that the high expression of Bcr/Abl in leukemic cells controls the expression of NKG2D receptor ligands and membrane GM1 via a tyrosine kinase-dependent mechanism and that the modulation of these molecules by IM interferes with NK cell recognition and cytolysis of the transfectants. View Publication

NK cell transcript 4 (NK4),now denoted as IL-32,was originally identified as a transcript whose expression was increased in activated NK cells. It has been very recently demonstrated that NK4 is secreted from several cells upon the stimulation of some inflammatory cytokines such as IL-18,IL-1beta,IFN-gamma and IL-12. Furthermore,NK4 induces production of tumor necrosis factor,macrophage inflammatory protein (MIP)-2 and IL-8 in monocytic cell lines,indicating that this factor would be involved in the inflammatory responses. Based on these findings,NK4 was renamed IL-32. However,the biological activities of IL-32 on other cell types remained undetermined. Furthermore,it was still argued whether IL-32 acts on cells from outside or inside the cells. In this article,we first report that expression of IL-32 was up-regulated in activated T cells and NK cells,and that IL-32beta was the predominantly expressed isoform in activated T cells. IL-32 was specifically expressed in T cells undergoing apoptosis and enforced expression of IL-32-induced apoptosis,whereas its down-regulation rescued the cells from apoptosis in HeLa cells. IL-32 existing in the supernatant would be derived from the cytoplasm of apoptotic cells. These results strongly indicated that IL-32 would be involved in activation-induced cell death in T cells,probably via its intracellular actions. Our present findings expand our understanding of the biological function of IL-32 and argue that IL-32 may act on cells,not only from the outside but also from the inside. View Publication

Perifosine is a synthetic novel alkylphospholipid,a new class of antitumor agents which targets cell membranes and inhibits Akt activation. Here we show that baseline phosphorylation of Akt in multiple myeloma (MM) cells is completely inhibited by perifosine [octadecyl-(1,1-dimethyl-piperidinio-4-yl)-phosphate] in a time- and dose-dependent fashion,without inhibiting phosphoinositide-dependent protein kinase 1 phosphorylation. Perifosine induces significant cytotoxicity in both MM cell lines and patient MM cells resistant to conventional therapeutic agents. Perifosine does not induce cytotoxicity in peripheral blood mononuclear cells. Neither exogenous interleukin-6 (IL-6) nor insulinlike growth factor 1 (IGF-1) overcomes Perifosine-induced cytotoxicity. Importantly,Perifosine induces apoptosis even of MM cells adherent to bone marrow stromal cells. Perifosine triggers c-Jun N-terminal kinase (JNK) activation,followed by caspase-8/9 and poly (ADP)-ribose polymerase cleavage. Inhibition of JNK abrogates perifosine-induced cytotoxicity,suggesting that JNK plays an essential role in perifosine-induced apoptosis. Interestingly,phosphorylation of extracellular signal-related kinase (ERK) is increased by perifosine; conversely,MEK inhibitor synergistically enhances Perifosine-induced cytotoxicity in MM cells. Furthermore,perifosine augments dexamethasone,doxorubicin,melphalan,and bortezomib-induced MM cell cytotoxicity. Finally,perifosine demonstrates significant antitumor activity in a human plasmacytoma mouse model,associated with down-regulation of Akt phosphorylation in tumor cells. Taken together,our data provide the rationale for clinical trials of perifosine to improve patient outcome in MM. View Publication

Protein tyrosine phosphatase 1B (PTP-1B) is a ubiquitously expressed cytosolic phosphatase with the ability to dephosphorylate JAK2 and TYK2,and thereby down-regulate cytokine receptor signaling. Furthermore,PTP-1B levels are up-regulated in certain chronic myelogenous leukemia patients,which points to a potential role for PTP-1B in myeloid development. The results presented here show that the absence of PTP-1B affects murine myelopoiesis by modifying the ratio of monocytes to granulocytes in vivo. This bias toward monocytic development is at least in part due to a decreased threshold of response to CSF-1,because the PTP-1B -/- bone marrow presents no abnormalities at the granulocyte-monocyte progenitor level but produces significantly more monocytic colonies in the presence of CSF-1. This phenomenon is not due to an increase in receptor levels but rather to enhanced phosphorylation of the activation loop tyrosine. PTP-1B -/- cells display increased inflammatory activity in vitro and in vivo through the constitutive up-regulation of activation markers as well as increased sensitivity to endotoxin. Collectively,our data indicate that PTP-1B is an important modulator of myeloid differentiation and macrophage activation in vivo and provide a demonstration of a physiological role for PTP-1B in immune regulation. View Publication

Protein kinase C theta (PKC theta) is unique among PKC isozymes in its translocation to the center of the immune synapse in T cells and its unique downstream signaling. Here we show that the hematopoietic protein tyrosine phosphatase (HePTP) also accumulates in the immune synapse in a PKC theta-dependent manner upon antigen recognition by T cells and is phosphorylated by PKC theta at Ser-225,which is required for lipid raft translocation. Immune synapse translocation was completely absent in antigen-specific T cells from PKC theta-/- mice. In intact T cells,HePTP-S225A enhanced T-cell receptor (TCR)-induced NFAT/AP-1 transactivation,while the acidic substitution mutant was as efficient as wild-type HePTP. We conclude that HePTP is phosphorylated in the immune synapse by PKC theta and thereby targeted to lipid rafts to temper TCR signaling. This represents a novel mechanism for the active immune synapse recruitment and activation of a phosphatase in TCR signaling. View Publication

CD74 is an integral membrane protein that was thought to function mainly as an MHC class II chaperone. However,CD74 was recently shown to have a role as an accessory-signaling molecule. Our studies demonstrated that CD74 regulates B-cell differentiation by inducing a pathway leading to the activation of transcription mediated by the NF-kappaB p65/RelA homodimer and its coactivator,TAF(II)105. Here,we show that CD74 stimulation with anti-CD74 antibody leads to an induction of a signaling cascade resulting in NF-kappaB activation,entry of the stimulated cells into the S phase,elevation of DNA synthesis,cell division,and augmented expression of BCL-X(L). These studies therefore demonstrate that surface CD74 functions as a survival receptor. View Publication

Human and mouse studies indicate that TLRs are important in mycobacterial infections. We investigated TLR gene expression in fresh unstimulated blood and bronchoalveolar lavage from patients with pulmonary tuberculosis using a well-validated,real-time PCR. A human splice variant of TLR1,designated hsTLR1,was found in all donors tested. hsTLR1 mRNA lacks exon 2,which is a 77-bp region of the 5'-untranslated region,but contains the same coding sequence as TLR1. Compared with the matched controls,whole blood from patients had increased levels of mRNA encoding TLR2 (p = 0.0006),TLR1 (p = 0.004),hsTLR1 (p = 0.0003),TLR6 (p textless 0.0001),and TLR4 (p = 0.0002). By contrast,expression of these TLRs was not increased in bronchoalveolar lavage. An increased level of hsTLR1 mRNA was found in both CD3- (p = 0.0078) and CD4+ cells (p = 0.028),resulting in an increased ratio of hsTLR1 mRNA to TLR1 and to TLR6 mRNA. An in vitro study in THP1 cells suggested that this relative increase in hsTLR1 might be attributable to a direct effect of mycobacterial components because it could be mimicked by mycobacterial preparations in the absence of IFN-gamma or T cells and by the TLR1/2 agonist Pam3CysK4. Half-life studies using blood from patients with pulmonary tuberculosis and THP1 cells exposed to Myobacterium tuberculosis in vitro showed p38 MAPK-independent stabilization of mRNAs encoding hsTLR1 and TLR1. We conclude that M. tuberculosis exerts direct effects on patterns of TLR expression,partly via changes in mRNA half-life. The significance of these changes in the pathogenesis of disease deserves further investigation. View Publication

The ability of acute myeloid leukemic (AML) blasts to differentiate into leukemic dendritic cells (DC) thus acquiring the potential to present known and unknown leukemic antigens efficiently,holds promise as a possible new treatment for AML patients with minimal residual disease. Recent advances in culture methods have made the clinical use of leukemic DC feasible. However,additional measures appear to be essential in order to potentiate vaccines and to overcome the intrinsic tolerant state of the patients immune system. This review describes ways to improve AML-DC vaccines and discusses critical aspects concerning the development of clinical vaccination protocols. View Publication

Using whole-cell patch-clamp,fluorescence microscopy and flow cytometry,we demonstrate a switch in potassium channel expression during differentiation of human B cells from naive to memory cells. Naive and IgD(+)CD27(+) memory B cells express small numbers of the voltage-gated Kv1.3 and the Ca(2+)-activated intermediate-conductance IKCa1 channel when quiescent,and increase IKCa1 expression 45-fold upon activation with no change in Kv1.3 levels. In contrast,quiescent class-switched memory B cells express high levels of Kv1.3 ( approximately 2000 channels/cell) and maintain their Kv1.3(high) expression after activation. Consistent with their channel phenotypes,proliferation of naive and IgD(+)CD27(+) memory B cells is suppressed by the specific IKCa1 inhibitor TRAM-34 but not by the potent Kv1.3 blocker Stichodactyla helianthus toxin,whereas the proliferation of class-switched memory B cells is suppressed by Stichodactyla helianthus toxin but not TRAM-34. These changes parallel those reported for T cells. Therefore,specific Kv1.3 and IKCa1 inhibitors may have use in therapeutic manipulation of selective lymphocyte subsets in immunological disorders. View Publication