搜索结果: 'EasySep Direct'

Interferon regulatory factor 3 (IRF-3) is essential for innate intracellular immune defenses that limit virus replication,but these defenses fail to suppress human immunodeficiency virus (HIV) infection,which can ultimately associate with opportunistic coinfections and the progression to AIDS. Here,we examined antiviral defenses in CD4+ cells during virus infection and coinfection,revealing that HIV type 1 (HIV-1) directs a global disruption of innate immune signaling and supports a coinfection model through suppression of IRF-3. T cells responded to paramyxovirus infection to activate IRF-3 and interferon-stimulated gene expression,but they failed to mount a response against HIV-1. The lack of response associated with a marked depletion of IRF-3 but not IRF-7 in HIV-1-infected cells,which supported robust viral replication,whereas ectopic expression of active IRF-3 suppressed HIV-1 infection. IRF-3 depletion was dependent on a productive HIV-1 replication cycle and caused the specific disruption of Toll-like receptor and RIG-I-like receptor innate immune signaling that rendered cells permissive to secondary virus infection. IRF-3 levels were reduced in vivo within CD4+ T cells from patients with acute HIV-1 infection but not from long-term nonprogressors. Our results indicate that viral suppression of IRF-3 promotes HIV-1 infection by disrupting IRF-3-dependent signaling pathways and innate antiviral defenses of the host cell. IRF-3 may direct an innate antiviral response that regulates HIV-1 replication and viral set point while governing susceptibility to opportunistic virus coinfections. View Publication

How HIV controllers (HICs) maintain undetectable viremia without therapy is unknown. The strong CD8(+) T-cell HIV suppressive capacity found in many,but not all,HICs may contribute to long-lasting viral control. However,other earlier defense mechanisms may be involved. Here,we examined intrinsic HIC cell resistance to HIV-1 infection. After in vitro challenge,monocyte-derived macrophages and anti-CD3-activated CD4(+) T cells from HICs showed low HIV-1 susceptibility. CD4 T-cell resistance was independent of HIV-1 coreceptors and affected also SIVmac infection. CD4(+) T cells from HICs expressed ex vivo higher levels of p21(Waf1/Cip1),which has been involved in the control of HIV-1 replication,than cells from control subjects. However,HIV restriction in anti-CD3-activated CD4(+) T cells and macrophages was not associated with p21 expression. Restriction inhibited accumulation of reverse transcripts,leading to reduction of HIV-1 integrated proviruses. The block could be overcome by high viral inocula,suggesting the action of a saturable mechanism. Importantly,cell-associated HIV-1 DNA load was extremely low in HICs and correlated with CD4(+) T-cell permissiveness to infection. These results point to a contribution of intrinsic cell resistance to the control of infection and the containment of viral reservoir in HICs. View Publication

BACKGROUND Patients with gene expression profiling-defined high-risk myeloma in relapse have poor outcomes with current therapies. We tested whether natural killer cells expanded by co-culture with K562 cells transfected with 41BBL and membrane-bound interleukin-15 could kill myeloma cells with a high-risk gene expression profile in vitro and in a unique model which recapitulates human myeloma. DESIGN AND METHODS OPM2 and high-risk primary myeloma tumors were grown in human fetal bone implanted into non-obese diabetic severe combined immunodeficiency mice with a deficient interleukin-2 receptor gamma chain. These mice are devoid of endogenous natural killer and T-cell activity and were used to determine whether adoptively transferred expanded natural killer cells could inhibit myeloma growth and myeloma-associated bone destruction. RESULTS Natural killer cells from healthy donors and myeloma patients expanded a median of 804- and 351-fold,respectively,without significant T-cell expansion. Expanded natural killer cells killed both allogeneic and autologous primary myeloma cells avidly via a perforin-mediated mechanism in which the activating receptor NKG2D,natural cytotoxicity receptors,and DNAX-accessory molecule-1 played a central role. Adoptive transfer of expanded natural killer cells inhibited the growth of established OPM2 and high-risk primary myeloma tumors grown in the murine model. The transferred,expanded natural killer cells proliferated in vivo in an interleukin-2 dose-dependent fashion,persisted up to 4 weeks,were readily detectable in the human bone,inhibited myeloma growth and protected bone from myeloma-induced osteolysis. CONCLUSIONS These studies provide the rationale for testing expanded natural killer cells in humans. 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

The immune system can act as an extrinsic suppressor of tumors. Therefore,tumor progression depends in part on mechanisms that downmodulate intrinsic immune surveillance. Identifying these inhibitory pathways may provide promising targets to enhance antitumor immunity. Here,we show that Stat3 is constitutively activated in diverse tumor-infiltrating immune cells,and ablating Stat3 in hematopoietic cells triggers an intrinsic immune-surveillance system that inhibits tumor growth and metastasis. We observed a markedly enhanced function of dendritic cells,T cells,natural killer (NK) cells and neutrophils in tumor-bearing mice with Stat3(-/-) hematopoietic cells,and showed that tumor regression requires immune cells. Targeting Stat3 with a small-molecule drug induces T cell- and NK cell-dependent growth inhibition of established tumors otherwise resistant to direct killing by the inhibitor. Our findings show that Stat3 signaling restrains natural tumor immune surveillance and that inhibiting hematopoietic Stat3 in tumor-bearing hosts elicits multicomponent therapeutic antitumor immunity. View Publication

NK cells play an important role in the innate immune response. We have isolated NK cells from human lymphoid tissues and found that these cells express the CD4 molecule on their surface at levels higher than those found on peripheral blood NK cells. To study the functional role of the CD4 molecule on NK cells,we developed an in vitro system by which we are able to obtain robust CD4 expression on NK cells derived from blood. CD4+ NK cells efficiently mediate NK cell cytotoxicity,and CD4 expression does not appear to alter lytic function. CD4+ NK cells are more likely to produce the cytokines gamma-IFN and TNF-alpha than are CD4- NK cells. Ligation of CD4 further increases the number of NK cells producing these cytokines. NK cells expressing CD4 are also capable of migrating toward the CD4-specific chemotactic factor IL-16,providing another function for the CD4 molecule on NK cells. Thus,the CD4 molecule is present and functional on NK cells and plays a role in innate immune responses as a chemotactic receptor and by increasing cytokine production,in addition to its well-described function on T cells as a coreceptor for Ag responsive cell activation. 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

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

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

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

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

Expansion of hematopoietic progenitor cells (HPC) ex vivo remains an important focus in fundamental and clinical research. The aim of this study was to determine whether the implementation of such expansion phase in a two-phase culture strategy prior to the induction of megakaryocyte (Mk) differentiation would increase the yield of Mks produced in cultures. Toward this end,we first characterized the functional properties of five cytokine cocktails to be tested in the expansion phase on the growth and differentiation kinetics of CD34+-enriched cells,and on their capacity to expand clonogenic progenitors in cultures. Three of these cocktails were chosen based on their reported ability to induce HPC expansion ex vivo,while the other two represented new cytokine combinations. These analyses revealed that none of the cocktails tested could prevent the differentiation of CD34+ cells and the rapid expansion of lineage-positive cells. Hence,we sought to determine the optimum length of time for the expansion phase that would lead to the best final Mk yields. Despite greater expansion of CD34+ cells and overall cell growth with a longer expansion phase,the optimal length for the expansion phase that provided greater Mk yield at near maximal purity was found to be 5 days. Under such settings,two functionally divergent cocktails were found to significantly increase the final yield of Mks. Surprisingly,these cocktails were either deprived of thrombopoietin or of stem cell factor,two cytokines known to favor megakaryopoiesis and HPC expansion,respectively. Based on these results,a short resource-efficient two-phase culture protocol for the production of Mks near purity (textgreater95%) from human CD34+ CB cells has been established. View Publication