技术资料

The Wiskott-Aldrich syndrome protein (WASP) is a product of the gene defective in an Xid disorder,Wiskott-Aldrich syndrome. WASP expression is limited to hemopoietic cells,and WASP regulates the actin cytoskeleton. It has been reported that monocytes/macrophages from WASP-deficient Wiskott-Aldrich syndrome patients are severely defective in chemotaxis,resulting in recurrent infection. However,the molecular basis of such chemotactic defects is not understood. Recently,the WASP N-terminal region was found to bind to the three mammalian verprolin homologs: WASP interacting protein (WIP); WIP and CR16 homologous protein (WICH)/WIP-related protein (WIRE); and CR16. Verprolin was originally found to play an important role in the regulation of actin cytoskeleton in yeast. We have shown that WASP,WIP,and WICH/WIRE are expressed predominantly in the human monocyte cell line THP-1 and that WIP and WICH/WIRE are involved in monocyte chemotaxis. When WASP binding to verprolins was blocked,chemotactic migration of monocytes was impaired in both THP-1 cells and primary human monocytes. Increased expression of WASP and WIP enhanced monocyte chemotaxis. Blocking WASP binding to verprolins impaired cell polarization but not actin polymerization. These results indicate that a complex of WASP with mammalian verprolins plays an important role in chemotaxis of monocytes. Our results suggest that WASP and mammalian verprolins function as a unit in monocyte chemotaxis and that the activity of this unit is critical to establish cell polarization. In addition,our results also indicate that the WASP-verprolin complex is involved in other functions such as podosome formation and phagocytosis. View Publication

Apoptosis is a key pathogenic mechanism in sepsis that induces extensive death of lymphocytes and dendritic cells,thereby contributing to the immunosuppression that characterizes the septic disorder. Numerous animal studies indicate that prevention of apoptosis in sepsis improves survival and may represent a potential therapy for this highly lethal disorder. Recently,novel cell-penetrating peptide constructs such as HIV-1 TAT basic domain and related peptides have been developed to deliver bioactive cargoes and peptides into cells. In the present study,we investigated the effects of sepsis-induced apoptosis in Bcl-x(L) transgenic mice and in wild-type mice treated with an antiapoptotic TAT-Bcl-x(L) fusion protein and TAT-BH4 peptide. Lymphocytes from Bcl-x(L) transgenic mice were resistant to sepsis-induced apoptosis,and these mice had a approximately 3-fold improvement in survival. TAT-Bcl-x(L) and TAT-BH4 prevented Escherichia coli-induced human lymphocyte apoptosis ex vivo and markedly decreased lymphocyte apoptosis in an in vivo mouse model of sepsis. In conclusion,TAT-conjugated antiapoptotic Bcl-2-like peptides may offer a novel therapy to prevent apoptosis in sepsis and improve survival. View Publication

IL-12 is an immunoregulatory cytokine,which promotes Th1 cell differentiation and is a major inducer of IFN-gamma. IFN-beta,a Type I IFN used in the treatment of multiple sclerosis,has been shown to significantly increase the expression of the anti-inflammatory cytokine IL-10,a major suppressor of Th1 cytokines. The beneficial immunomodulatory effects of IFN-beta may in part be a result of its ability to suppress IL-12. However,IL-12 and IFN-beta signal via the STAT4 pathway. Our aim was to investigate the relationship between IL-12 and IFN-beta by observing the effect of prior exposure to IL-12 or IFN-beta on the ability of T cells to subsequently respond to the other cytokine. We report that IFN-beta increases IL-12-induced STAT4 phosphorylation and up-regulates IL-12 receptor beta1 and beta2 expression. However,despite this up-regulation,IFN-beta suppressed IL-12-induced IFN-gamma expression. Our results suggest that this may be a result of the parallel induction of IL-10 by IFN-beta. View Publication

NK and T cells are important for combating CMV infection. Some NK and T cells express leukocyte Ig-like receptor-1 (LIR-1),an inhibitory receptor recognizing MHC class I and the CMV-encoded homolog UL18. We previously demonstrated an early increase in LIR-1-expressing blood lymphocytes in lung-transplanted patients later developing CMV disease. We now show that NK and T cells account for the observed LIR-1 augmentation. Coincubation of PBMC from CMV-seropositive donors with virus-infected lung fibroblasts led to a T cell-dependent secretion of IFN-gamma,produced mainly by LIR-1(+) T cells and by NK cells. Cytokine production during coculture with fibroblasts infected with virus containing the UL18 gene was augmented compared with the UL18 deletion virus,suggesting a stimulatory role for UL18. However,purified UL18Fc proteins inhibited IFN-gamma production of LIR-1(+) T cells. We propose that cytokine production in the transplant induces NK and T cells to express LIR-1,which may predispose to CMV disease by MHC/LIR-1-mediated suppression. Although the UL18/LIR-1 interaction could inhibit T cell responses,this unlikely plays a role in response to infected cells. Instead,our data point to an activating role for viral UL18 during infection,where indirect intracellular effects cannot be excluded. View Publication

Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes mellitus or rheumatoid arthritis (RA) are mainly CD4+ CCR7- CD45RA- effector memory T cells (T(EM) cells) with elevated Kv1.3 potassium channel expression. In contrast,T cells with other antigen specificities from these patients,or autoreactive T cells from healthy individuals and disease controls,express low levels of Kv1.3 and are predominantly naïve or central-memory (T(CM)) cells. In T(EM) cells,Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvbeta2,SAP97,ZIP,p56(lck),and CD4. Although Kv1.3 inhibitors [ShK(L5)-amide (SL5) and PAP1] do not prevent immunological synapse formation,they suppress Ca2+-signaling,cytokine production,and proliferation of autoantigen-specific T(EM) cells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted. View Publication

In this study,we examined the effects of isoform-specific functional inhibitors of lysophosphatidic acid acyltransferase (LPAAT),which converts lysophosphatidic acid to phosphatidic acid,on multiple myeloma (MM) cell growth and survival. The LPAAT-beta inhibitors CT-32176,CT-32458,and CT-32615 induced textgreater95% growth inhibition (P textless 0.01) in MM.1S,U266,and RPMI8226 MM cell lines,as well as MM cells from patients (IC(50),50-200 nM). We further characterized this LPAAT-beta inhibitory effect using CT-32615,the most potent inhibitor of MM cell growth. CT-32615 triggered apoptosis in MM cells via caspase-8,caspase-3,caspase-7,and poly (ADP-ribose) polymerase cleavage. Neither interleukin 6 nor insulin-like growth factor I inhibited CT-32615-induced apoptosis. Dexamethasone and immunomodulatory derivatives of thalidomide (IMiDs),but not proteasome inhibitor PS-341,augmented MM cell apoptosis triggered by LPAAT-beta inhibitors. CT-32615-induced apoptosis was associated with phosphorylation of p53 and c-Jun NH(2)-terminal kinase (JNK); conversely,JNK inhibitor SP600125 and dominant-negative JNK inhibited CT-32615-induced apoptosis. Importantly,CT-32615 inhibited tumor necrosis factor-alpha-triggered nuclear factor-kappaB activation but did not affect either tumor necrosis factor-alpha-induced p38 mitogen-activated protein kinase phosphorylation or interleukin 6-triggered signal transducers and activators of transcription 3 phosphorylation. Finally,although binding of MM cells to bone marrow stromal cells augments MM cell growth and protects against dexamethasone-induced apoptosis,CT-32615 induced apoptosis even of adherent MM cells. Our data therefore demonstrate for the first time that inhibiting LPAAT-beta induces cytotoxicity in MM cells in the bone marrow milieu,providing the framework for clinical trials of these novel agents in MM. View Publication

MSCs have received attention for their therapeutic potential in a number of disease states,including bone formation,diabetes,stem cell engraftment after marrow transplantation,graft-versus-host disease,and heart failure. Despite this diverse interest,the molecular signals regulating MSC trafficking to sites of injury are unclear. MSCs are known to transiently home to the freshly infarcted myocardium. To identify MSC homing factors,we determined chemokine expression pattern as a function of time after myocardial infarction (MI). We merged these profiles with chemokine receptors expressed on MSCs but not cardiac fibroblasts,which do not home after MI. This analysis identified monocyte chemotactic protein-3 (MCP-3) as a potential MSC homing factor. Overexpression of MCP-3 1 month after MI restored MSC homing to the heart. After serial infusions of MSCs,cardiac function improved in MCP-3-expressing hearts (88.7%,p textless .001) but not in control hearts (8.6%,p = .47). MSC engraftment was not associated with differentiation into cardiac myocytes. Rather,MSC engraftment appeared to result in recruitment of myofibroblasts and remodeling of the collagen matrix. These data indicate that MCP-3 is an MSC homing factor; local overexpression of MCP-3 recruits MSCs to sites of injured tissue and improves cardiac remodeling independent of cardiac myocyte regeneration. View Publication

Despite considerable success in treating newly diagnosed childhood acute lymphoblastic leukemia (ALL),relapsed disease remains a significant clinical challenge. Using a NOD/SCID mouse xenograft model,we report that immunostimulatory DNA oligonucleotides containing CpG motifs (CpG ODNs) stimulate significant immune activity against primary human ALL cells in vivo. The administration of CpG ODNs induced a significant reduction in systemic leukemia burden,mediated continued disease control,and significantly improved survival of mice with established human ALL. The death of leukemia cells in vivo was independent of the ability of ALL cells to respond directly to CpG ODNs and correlated with the production of IL-12p70,IFN-alpha,and IFN-gamma by the host. In addition,depletion of natural killer cells by anti-asialo-GM1 treatment significantly reduced the in vivo antileukemic activity of CpG ODN. This antileukemia effect was not limited to the xenograft model because natural killer cell-dependent killing of ALL by human peripheral blood mononuclear cells (PBMCs) was also increased by CpG ODN stimulation. These results suggest that CpG ODNs have potential as therapeutic agents for the treatment of ALL. View Publication

Macroautophagy (hereafter referred to as autophagy) is a well-conserved intracellular degradation process. Recent studies examining cells lacking the autophagy genes Atg5 and Atg7 have demonstrated that autophagy plays essential roles in cell survival during starvation,in innate cell clearance of microbial pathogens,and in neural cell maintenance. However,the role of autophagy in T lymphocyte development and survival is not known. Here,we demonstrate that autophagosomes form in primary mouse T lymphocytes. By generating Atg5-/- chimeric mice,we found that Atg5-deficient T lymphocytes underwent full maturation. However,the numbers of total thymocytes and peripheral T and B lymphocytes were reduced in Atg5 chimeras. In the periphery,Atg5-/- CD8+ T lymphocytes displayed dramatically increased cell death. Furthermore,Atg5-/- CD4+ and CD8+ T cells failed to undergo efficient proliferation after TCR stimulation. These results demonstrate a critical role for Atg5 in multiple aspects of lymphocyte development and function and suggest that autophagy may be essential for both T lymphocyte survival and proliferation. View Publication

NK cells are important for innate resistance to tumors and viruses. Engagement of activating Ly-49 receptors expressed by NK cells leads to rapid NK cell activation resulting in target cell lysis and cytokine production. The ITAM-containing DAP12 adapter protein stably associates with activating Ly-49 receptors,and couples receptor recognition with generation of NK responses. Activating Ly-49s are potent stimulators of murine NK cell functions,yet how they mediate such activities is not well understood. We demonstrate that these receptors trigger LFA-1-dependent tight conjugation between NK cells and target cells. Furthermore,we show that activating Ly-49 receptor engagement leads to rapid DAP12-dependent up-regulation of NK cell LFA-1 adhesiveness to ICAM-1 that is also dependent on tyrosine kinases of the Syk and Src families. These results indicate for the first time that activating Ly-49s control adhesive properties of LFA-1,and by DAP12-dependent inside-out signaling. Ly-49-driven mobilization of LFA-1 adhesive function may represent a fundamental proximal event during NK cell interactions with target cells involving activating Ly-49 receptors,leading to target cell death. View Publication

Chronic hepatitis C virus (HCV) infection is characterized by diminished numbers and function of HCV-reactive T cells and impaired responses to immunization. Because host response to viral infection likely involves TLR signaling,we examined whether chronic HCV infection impairs APC response to TLR ligand and contributes to the origin of dysfunctional T cells. Freshly purified myeloid dendritic cells (MDC) and plasmacytoid DC (PDC) obtained from subjects with chronic HCV infection and healthy controls were exposed to TLR ligands (poly(I:C),R-848,or CpG),in the presence or absence of cytokine (TNF-alpha or IL-3),and examined for indices of maturation and for their ability to activate allogeneic naive CD4 T cells to proliferate and secrete IFN-gamma. TLR ligand was observed to enhance both MDC and PDC activation of naive CD4 T cells. Although there was increased CD83 and CD86 expression on MDC from HCV-infected persons,the ability of MDC to activate naive CD4 T cells in the presence or absence of poly(I:C) or TNF-alpha did not differ between HCV-infected and healthy control subjects. In contrast,PDC from HCV-infected persons had reduced activation marker (HLA-DR) and cytokine (IFN-alpha) expression upon R-848 stimulation,and these were associated with impaired activation of naive CD4 T cells. These data indicate that an impaired PDC responsiveness to TLR ligation may play an important role in the fundamental and unexplained failure to induce new T cell responses to HCV Ags and to other new Ags as a consequence of HCV infection. View Publication

Chaetocin,a thiodioxopiperazine natural product previously unreported to have anticancer effects,was found to have potent antimyeloma activity in IL-6-dependent and -independent myeloma cell lines in freshly collected sorted and unsorted patient CD138(+) myeloma cells and in vivo. Chaetocin largely spares matched normal CD138(-) patient bone marrow leukocytes,normal B cells,and neoplastic B-CLL (chronic lymphocytic leukemia) cells,indicating a high degree of selectivity even in closely lineage-related B cells. Furthermore,chaetocin displays superior ex vivo antimyeloma activity and selectivity than doxorubicin and dexamethasone,and dexamethasone- or doxorubicin-resistant myeloma cell lines are largely non-cross-resistant to chaetocin. Mechanistically,chaetocin is dramatically accumulated in cancer cells via a process inhibited by glutathione and requiring intact/unreduced disulfides for uptake. Once inside the cell,its anticancer activity appears mediated primarily through the imposition of oxidative stress and consequent apoptosis induction. Moreover,the selective antimyeloma effects of chaetocin appear not to reflect differential intracellular accumulation of chaetocin but,instead,heightened sensitivity of myeloma cells to the cytotoxic effects of imposed oxidative stress. Considered collectively,chaetocin appears to represent a promising agent for further study as a potential antimyeloma therapeutic. View Publication