参考文献

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

We have established a system for directed differentiation of human embryonic stem (hES) cells into myeloid dendritic cells (DCs). As a first step,we induced hemopoietic differentiation by coculture of hES cells with OP9 stromal cells,and then,expanded myeloid cells with GM-CSF using a feeder-free culture system. Myeloid cells had a CD4+CD11b+CD11c+CD16+CD123(low)HLA-DR- phenotype,expressed myeloperoxidase,and included a population of M-CSFR+ monocyte-lineage committed cells. Further culture of myeloid cells in serum-free medium with GM-CSF and IL-4 generated cells that had typical dendritic morphology; expressed high levels of MHC class I and II molecules,CD1a,CD11c,CD80,CD86,DC-SIGN,and CD40; and were capable of Ag processing,triggering naive T cells in MLR,and presenting Ags to specific T cell clones through the MHC class I pathway. Incubation of DCs with A23187 calcium ionophore for 48 h induced an expression of mature DC markers CD83 and fascin. The combination of GM-CSF with IL-4 provided the best conditions for DC differentiation. DCs obtained with GM-CSF and TNF-alpha coexpressed a high level of CD14,and had low stimulatory capacity in MLR. These data clearly demonstrate that hES cells can be used as a novel and unique source of hemopoietic and DC precursors as well as DCs at different stages of maturation to address essential questions of DC development and biology. In addition,because ES cells can be expanded without limit,they can be seen as a potential scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance. View Publication

G(q/11) protein-coupled muscarinic receptors are known to regulate the release of soluble amyloid precursor protein (sAPPalpha) produced by alpha-secretase processing; however,their signaling mechanisms remain to be elucidated. It has been reported that a muscarinic agonist activates nuclear factor (NF)-kappaB,a transcription factor that has been shown to play an important role in the Alzheimer disease brain,and that NF-kappaB activation is regulated by intracellular Ca2+ level. In the present study,we investigated whether NF-kappaB activation plays a role in muscarinic receptor-mediated sAPPalpha release enhancement and contributes to a changed capacitative Ca2+ entry (CCE),which was suggested to be involved in the muscarinic receptor-mediated stimulation of sAPPalpha release. Muscarinic receptor-mediated NF-kappaB activation was confirmed by observing the translocation of the active subunit (p65) of NF-kappaB to the nucleus by the muscarinic agonist,oxotremorine M (oxoM),in SH-SY5Y neuroblastoma cells expressing muscarinic receptors that are predominantly of the M3 subtype. NF-kappaB activation and sAPPalpha release enhancement induced by oxoM were inhibited by NF-kappaB inhibitors,such as an NF-kappaB peptide inhibitor (SN50),an IkappaB alpha kinase inhibitor (BAY11-7085),a proteasome inhibitor (MG132),the inhibitor of proteasome activity and IkappaB phosphorylation,pyrrolidine dithiocarbamate,the novel NF-kappaB activation inhibitor (6-amino-4-(4-phenoxyphenylethylamino) quinazoline),and by an intracellular Ca2+ chelator (TMB-8). Furthermore,both oxoM-induced NF-kappaB activation and sAPPalpha release were antagonized by CCE inhibitors (gadolinium or SKF96365) but not by voltage-gated Ca2+-channel blockers. On the other hand,treatment of cells with NF-kappaB inhibitors (SN50,BAY11-7085,MG132,or pyrrolidine dithiocarbamate) did not inhibit muscarinic receptor-mediated CCE. These findings provide evidence for the involvement of NF-kappaB regulated by CCE in muscarinic receptor-mediated sAPPalpha release enhancement. 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

Niemann-Pick type C2 (NPC2) protein has been characterized as a cholesterol-binding protein. Its loss leads to NPC2 disease,an inherited neurodegenerative disorder. When analyzing gene expression profile,we noticed high expression of both NPC2 and its receptor,mannose 6-phosphate receptor (MPR),in murine hematopoietic stem cells. NPC2 protein,in the presence of thrombopoietin (TPO),causes an increase in CFU-GEMM (colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte) and a decrease in CFU-GM (colony-forming unit-granulocyte-macrophage) colony number in colony-forming cell (CFC) assays. This effect is independent of cholesterol binding but does require the presence of MPR. With M07e cells,a TPO-dependent hematopoietic leukemia cell line,NPC2 can inhibit TPO-induced differentiation and enhance TPO-mediated anti-apoptosis effects. Strikingly,these results are not observed under the standard 20% O(2) level of the standard incubator,but rather at 7% O(2),the physiological oxygen level of bone marrow. Furthermore,NPC2 protein upregulates hypoxia inducible factor 1-alpha protein level at 7% O(2),but not at 20% O(2). Our results demonstrate that NPC2 protein plays a role in hematopoiesis at the physiologic bone marrow level of O(2). 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

An acquired somatic mutation,Jak2V617F,was recently discovered in most patients with polycythemia vera (PV),chronic idiopathic myelofibrosis (CIMF),and essential thrombocythemia (ET). To investigate the role of this mutation in vivo,we transplanted bone marrow (BM) transduced with a retrovirus expressing either Jak2 wild-type (wt) or Jak2V617F into lethally irradiated syngeneic recipient mice. Expression of Jak2V617F,but not Jak2wt,resulted in clinicopathologic features that closely resembled PV in humans. These included striking elevation in hemoglobin level/hematocrit,leukocytosis,megakaryocyte hyperplasia,extramedullary hematopoiesis resulting in splenomegaly,and reticulin fibrosis in the bone marrow. Histopathologic and flow cytometric analyses showed an increase in maturing myeloid lineage progenitors,although megakaryocytes showed decreased polyploidization and staining for acetylcholinesterase. In vitro analysis of primary cells showed constitutive activation of Stat5 and cytokine-independent growth of erythroid colony-forming unit (CFU-E) and erythropoietin hypersensitivity,and Southern blot analysis for retroviral integration indicated that the disease was oligoclonal. Furthermore,we observed strain-specific differences in phenotype,with Balb/c mice demonstrating markedly elevated leukocyte counts,splenomegaly,and reticulin fibrosis compared with C57Bl/6 mice. We conclude that Jak2V617F expression in bone marrow progenitors results in a PV-like syndrome with myelofibrosis and that there are strain-specific modifiers that may in part explain phenotypic pleiotropy of Jak2V617F-associated myeloproliferative disease in humans. 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

Antagonists to alpha4 integrin show promise for several autoimmune and inflammatory diseases but may exhibit mechanism-based toxicities. We tested the capacity of blockade of alpha4 integrin signaling to perturb functions involved in inflammation,while limiting potential adverse effects. We generated and characterized mice bearing a Y991A mutation in alpha4 integrin [alpha4(Y991A) mice],which blocks paxillin binding and inhibits alpha4 integrin signals that support leukocyte migration. In contrast to the embryonic-lethal phenotype of alpha4 integrin-null mice,mice bearing the alpha4(Y991A) mutation were viable and fertile; however,they exhibited defective recruitment of mononuclear leukocytes into thioglycollate-induced peritonitis. Alpha4 integrins are essential for definitive hematopoiesis; however,the alpha4(Y991A) mice had intact lymphohematopoiesis and,with the exception of reduced Peyer's patches,normal architecture and cellularity of secondary lymphoid tissues. We conclude that interference with alpha4 integrin signaling can selectively impair mononuclear leukocyte recruitment to sites of inflammation while sparing vital functions of alpha4 integrins in development and hematopoiesis. View Publication

The therapeutic potential of hematopoietic stem cell (HSC) gene therapy can be fully exploited only by reaching efficient gene transfer into HSCs without compromising their biologic properties. Although HSCs can be transduced by HIV-derived lentiviral vectors (LVs) in short ex vivo culture,they display low permissivity to the vector,requiring cytokine stimulation to reach high-frequency transduction. Using stringent assays of competitive xenograft repopulation,we show that early-acting cytokines synergistically enhanced human HSC gene transfer by LVs without impairing engraftment and repopulation capacity. Using S-phase suicide assays,we show that transduction enhancement by cytokines was not dependent on cell cycle progression and that LVs can transduce quiescent HSCs. Pharmacologic inhibition of the proteasome during transduction dramatically enhanced HSC gene transfer,allowing the reach of very high levels of vector integration in their progeny in vivo. Thus,LVs are effectively restricted at a postentry step by the activity of this proteolytic complex. Unexpectedly,cytokine stimulation rapidly and substantially down-regulated proteasome activity in hematopoietic progenitors,highlighting one mechanism by which cytokines may enhance permissiveness to LV gene transfer. These findings demonstrate that antiviral responses ultimately mediated by proteasomes strongly limit the efficiency of HSC transduction by LVs and establish improved conditions for HSC-based gene therapy. View Publication