技术资料

BACKGROUND: The unfolded protein response (UPR) is one of the pathways triggered to ensure quality control of the proteins assembled in the endoplasmic reticulum (ER) when cell homeostasis is compromised. This mechanism is primarily composed of three transmembrane proteins serving as stress sensors: PKR-like ER kinase (PERK),activating transcription factor 6 (ATF6),and inositol-requiring enzyme 1 (IRE1). These three proteins' synergic action elicits translation and transcriptional downstream pathways,leading to less protein production and activating genes that encode important proteins in folding processes,including chaperones. Previous reports showed that viruses have evolved mechanisms to curtail or customize this UPR signaling for their own benefit. However,HIV infection's effect on the UPR has scarcely been investigated. METHODS: This work investigated UPR modulation by HIV infection by assessing UPR-related protein expression under in vitro and in vivo conditions via Western blotting. Antiretroviral (ARV) drugs' influence on this stress response was also considered. RESULTS: In in vitro and in vivo analyses,our results confirm that HIV infection activates stress-response components and that ARV therapy contributes to changes in the UPR's activation profile. CONCLUSIONS: This is the first report showing UPR-related protein expression in HIV target cells derived directly from HIV-infected patients receiving different ARV therapies. Thus,two mechanisms may occur simultaneously: interference by HIV itself and the ARV drugs' pharmacological effects as UPR activators. New evidence of how HIV modulates the UPR to enhance its own replication and secure infection success is also presented. View Publication

Spleen tyrosine kinase (Syk) is an attractive drug target in autoimmune,inflammatory,and oncology disease indications. The most advanced Syk inhibitor,R406,1 (or its prodrug form fostamatinib,2),has shown efficacy in multiple therapeutic indications,but its clinical progress has been hampered by dose-limiting adverse effects that have been attributed,at least in part,to the off-target activities of 1. It is expected that a more selective Syk inhibitor would provide a greater therapeutic window. Herein we report the discovery and optimization of a novel series of imidazo[1,2-a]pyrazine Syk inhibitors. This work culminated in the identification of GS-9973,68,a highly selective and orally efficacious Syk inhibitor which is currently undergoing clinical evaluation for autoimmune and oncology indications. View Publication

Hematopoietic progenitor cells arising from bone marrow (BM) are known to contribute to the formation and expansion of tumor vasculature. However,whether different subsets of these cells have different roles in this process is unclear. To investigate the roles of BM-derived progenitor cell subpopulations in the formation of tumor vasculature in a Ewing's sarcoma model,we used a functional assay based on endothelial cell and pericyte differentiation in vivo. Fluorescence-activated cell sorting of human cord blood/BM or mouse BM from green fluorescent protein transgenic mice was used to isolate human CD34+/CD38(-),CD34+/CD45+,and CD34(-)/CD45+ cells and mouse Sca1+/Gr1+,Sca1(-)/Gr1+,VEGFR1+,and VEGFR2+ cells. Each of these progenitor subpopulations was separately injected intravenously into nude mice bearing Ewing's sarcoma tumors. Tumors were resected 1 week later and analyzed using immunohistochemistry and confocal microscopy for the presence of migrated progenitor cells expressing endothelial,pericyte,or inflammatory cell surface markers. We showed two distinct patterns of stem cell infiltration. Human CD34+/CD45+ and CD34+/CD38(-) and murine VEGFR2+ and Sca1+/Gr1+ cells migrated to Ewing's tumors,colocalized with the tumor vascular network,and differentiated into cells expressing either endothelial markers (mouse CD31 or human vascular endothelial cadherin) or the pericyte markers desmin and alpha-smooth muscle actin. By contrast,human CD34(-)/CD45+ and mouse Sca1(-)/Gr1+ cells migrated predominantly to sites outside of the tumor vasculature and differentiated into monocytes/macrophages expressing F4/80 or CD14. Our data indicate that only specific BM stem/progenitor subpopulations participate in Ewing's sarcoma tumor vasculogenesis. View Publication

The purpose of these studies is to determine why an immunogenic tumor grows unchecked in the anterior chamber (a.c.) of the eye. The OVA-expressing EL4 tumor,E.G7-OVA,was injected into the a.c. or skin of immunocompetent and immunodeficient mice. Tumor growth and tumor-specific immune responses were monitored. Ocular tumor-infiltrating leukocytes were characterized phenotypically and functionally. Growth of E.G7-OVA was inhibited when limiting numbers of cells were injected in the skin but not in the a.c. of C57BL/6 mice,although both routes primed OVA-specific immune responses,which prevented the growth of a subsequent injection with E.G7-OVA in the skin or opposite eye. Tumor regression was OVA-specific because growth of the parental EL-4 tumor was not inhibited in primed mice. E.G7-OVA growth in the skin was not inhibited in immunodeficient Rag(-/-) or CD8 T cell-deficient mice,suggesting that CD8(+) CTLs mediate tumor elimination. CD8(+) T cell numbers were significantly increased in eyes of mice primed with E.G7-OVA,but few were detected in primary ocular tumors. Nevertheless,growth of E.G7-OVA was retarded in the a.c. of TCR-transgenic OT-I mice,and CD8(+) T cell numbers were increased within eyes,suggesting that tumor-specific CD8(+) CTLs migrated into and controlled primary ocular tumor growth. E.G7-OVA did not lose antigenicity or become immunosuppressive after 13 days of growth in the eye. However,CD11b(+) cells accumulated in primary ocular tumors and contained potent immunosuppressive activity when assayed in vitro. Thus,CD11b(+) cells that accumulate within the eye as tumors develop in the a.c. may contribute to immune evasion by primary ocular tumors by inhibiting CTLs within the eye. View Publication

Nonmammalian glycan structures from helminths act as Th2 adjuvants. Some of these structures are also common on plant glycoproteins. We hypothesized that glycan structures present on peanut glycoallergens act as Th2 adjuvants. Peanut Ag (PNAg),but not deglycosylated PNAg,activated monocyte-derived dendritic cells (MDDCs) as measured by MHC/costimulatory molecule up-regulation,and by their ability to drive T cell proliferation. Furthermore,PNAg-activated MDDCs induced 2- to 3-fold more IL-4- and IL-13-secreting Th2 cells than immature or TNF/IL-1-activated MDDCs when cultured with naive CD4+ T cells. Human MDDCs rapidly internalized Ag in a calcium- and glycan-dependent manner consistent with recognition by C-type lectin. Dendritic cell (DC)-specific ICAM-grabbing nonintegrin (DC-SIGN) (CD209) was shown to recognize PNAg by enhanced uptake in transfected cell lines. To identify the DC-SIGN ligand from unfractionated PNAg,we expressed the extracellular portion of DC-SIGN as an Fc-fusion protein and used it to immunoprecipitate PNAg. A single glycoprotein was pulled down in a calcium-dependent manner,and its identity as Ara h 1 was proven by immunolabeling and mass spectrometry. Purified Ara h 1 was found to be sufficient for the induction of MDDCs that prime Th2-skewed T cell responses. Both PNAg and purified Ara h 1 induced Erk 1/2 phosphorylation of MDDCs,consistent with previous reports on the effect of Th2 adjuvants on DCs. View Publication

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis that in many cases is caused by mutations of the ELANE gene,which encodes neutrophil elastase (NE). Recent data suggest a model in which ELANE mutations result in NE protein misfolding,induction of endoplasmic reticulum (ER) stress,activation of the unfolded protein response (UPR),and ultimately a block in granulocytic differentiation. To test this model,we generated transgenic mice carrying a targeted mutation of Elane (G193X) reproducing a mutation found in SCN. The G193X Elane allele produces a truncated NE protein that is rapidly degraded. Granulocytic precursors from G193X Elane mice,though without significant basal UPR activation,are sensitive to chemical induction of ER stress. Basal and stress granulopoiesis after myeloablative therapy are normal in these mice. Moreover,inaction of protein kinase RNA-like ER kinase (Perk),one of the major sensors of ER stress,either alone or in combination with G193X Elane,had no effect on basal granulopoiesis. However,inhibition of the ER-associated degradation (ERAD) pathway using a proteosome inhibitor resulted in marked neutropenia in G193X Elane. The selective sensitivity of G913X Elane granulocytic cells to ER stress provides new and strong support for the UPR model of disease patho-genesis in SCN. View Publication

Monocytes have long been considered a heterogeneous group of cells both in terms of morphology and function. In humans,three distinct subsets have been described based on their differential expression of the cell surface markers CD14 and CD16. However,the relationship between these subsets and the production of cytokines has for the most part been based on ELISA measurements,making it difficult to draw conclusions as to their functional profile on the cellular level. In the present study,we have investigated lipoteichoic acid (LTA) and lipopolysaccharide (LPS) induced cytokine secretion by monocytes using the FluoroSpot technique. This method measures the number of cytokine secreting cells on the single cell level and uses fluorescent detection,allowing for the simultaneous analysis of two cytokines from the same population of isolated cells. By this approach,human monocytes from healthy volunteers could be divided into several subgroups as IL-1β,IL-6,TNF-α and MIP-1β were secreted by larger populations of responding cells (25.9-39.2%) compared to the smaller populations of GM-CSF (9.1%),IL-10 (1.3%) and IL-12p40 (1.2%). Furthermore,when studying co-secretion in FluoroSpot,an intricate relationship between the monocytes secreting IL-1β and/or IL-6 and those secreting TNF-α,MIP-1β,GM-CSF,IL-10 and IL-12p40 was revealed. In this way,dissecting the secretion pattern of the monocytes in response to TLR-2 or TLR-4 stimulation,several subpopulations with distinct cytokine secreting profiles could be identified. View Publication

IL-27,which is produced by activated APCs,bridges innate and adaptive immunity by regulating the development of Th cells. Recent evidence supports a role for IL-27 in the activation of monocytic cells in terms of inflammatory responses. Indeed,proinflammatory and anti-inflammatory activities are attributed to IL-27,and IL-27 production itself is modulated by inflammatory agents such as LPS. IL-27 primes LPS responses in monocytes; however,the molecular mechanism behind this phenomenon is not understood. In this study,we demonstrate that IL-27 priming results in enhanced LPS-induced IL-6,TNF-α,MIP-1α,and MIP-1β expression in human primary monocytes. To elucidate the molecular mechanisms responsible for IL-27 priming,we measured levels of CD14 and TLR4 required for LPS binding. We determined that IL-27 upregulates TLR4 in a STAT3- and NF-κB-dependent manner. Immunofluorescence microscopy revealed enhanced membrane expression of TLR4 and more distinct colocalization of CD14 and TLR4 upon IL-27 priming. Furthermore,IL-27 priming enhanced LPS-induced activation of NF-κB family members. To our knowledge,this study is the first to show a role for IL-27 in regulating TLR4 expression and function. This work is significant as it reveals new mechanisms by which IL-27 can enhance proinflammatory responses that can occur during bacterial infections. View Publication

OBJECTIVE: We used genetically engineered mouse hematopoietic progenitor cells (HPCs) to investigate the therapeutic effects of human apoAI on atherosclerosis in apoE(-/-) mice. METHODS AND RESULTS: Lentiviral constructs expressing either human apoAI (LV-apoAI) or green fluorescent protein (LV-GFP) cDNA under a macrophage specific promoter (CD68) were generated and used for ex vivo transduction of mouse HPCs and macrophages. The transduction efficiency was textgreater25% for HPCs and textgreater70% for macrophages. ApoAI was found in the macrophage culture media,mostly associated with the HDL fraction. Interestingly,a significant increase in mRNA and protein levels for ATP binding cassette A1 (ABCA1) and ABCG1 were found in apoAI-expressing macrophages after acLDL loading. Expression of apoAI significantly increased cholesterol efflux in wild-type and apoE(-/-) macrophages. HPCs transduced with LV-apoAI ex vivo and then transplanted into apoE(-/-) mice caused a 50% reduction in atherosclerotic lesion area compared to GFP controls,without influencing plasma HDL-C levels. CONCLUSIONS: Lentiviral transduction of apoAI into HPCs reduces atherosclerosis in apoE(-/-) mice. Expression of apoAI in macrophages improves cholesterol trafficking in wild-type apoE-producing macrophages and causes upregulation of ABCA1 and ABCG1. These novel observations set the stage for a cell therapy approach to atherosclerosis regression,exploiting the cooperation between apoE and apoAI to maximize cholesterol exit from the plaque. View Publication

We have generated mice null for IFN-beta and report the diverse consequences of IFN-beta for both the innate and adaptive arms of immunity. Despite no abnormalities in the proportional balance of CD4 and CD8 T cell populations in the peripheral blood,thymus,and spleen of IFN-beta-/- mice,activated lymph node and splenic T lymphocytes exhibit enhanced T cell proliferation and decreased tumor necrosis factor alpha production,relative to IFN-beta+/+ mice. Notably,constitutive and induced expression of tumor necrosis factor alpha is reduced in the spleen and bone marrow (BM) macrophages,respectively,of IFN-beta-/- mice. We also observe an altered splenic architecture in IFN-beta-/- mice and a reduction in resident macrophages. We identify a potential defect in B cell maturation in IFN-beta-/- mice,associated with a decrease in B220+ve/high/CD43-ve BM-derived cells and a reduction in BP-1,IgM,and CD23 expression. Circulating IgM-,Mac-1-,and Gr-1-positive cells are also substantially decreased in IFN-beta-/- mice. The decrease in the numbers of circulating macrophages and granulocytes likely reflects defective maturation of primitive BM hematopoiesis in mice,shown by the reduction of colony-forming units,granulocyte-macrophage. We proceeded to evaluate the in vivo growth of malignant cells in the IFN-beta-/- background and give evidence that Lewis lung carcinoma-specific tumor growth is more aggressive in IFN-beta-/- mice. Taken altogether,our data suggest that,in addition to the direct growth-inhibitory effects on tumor cells,IFN-beta is required during different stages of maturation in the development of the immune system. View Publication

Histoplasma capsulatum (Hc) is a facultative intracellular fungus that modulates the intraphagosomal environment to survive within macrophages (Mphi). In the present study,we sought to quantify the intraphagosomal pH under conditions in which Hc yeasts replicated or were killed. Human Mphi that had ingested both viable and heat-killed or fixed yeasts maintained an intraphagosomal pH of approximately 6.4-6.5 over a period of several hours. These results were obtained using a fluorescent ratio technique and by electron microscopy using the 3-(2,4-dinitroanilo)-3'-amino-N-methyldipropylamine reagent. Mphi that had ingested Saccharomyces cerevisae,a nonpathogenic yeast that is rapidly killed and degraded by Mphi,also maintained an intraphagosomal pH of approximately 6.5 over a period of several hours. Stimulation of human Mphi fungicidal activity by coculture with chloroquine or by adherence to type 1 collagen matrices was not reversed by bafilomycin,an inhibitor of the vacuolar ATPase. Human Mphi cultured in the presence of bafilomycin also completely degraded heat-killed Hc yeasts,whereas mouse peritoneal Mphi digestion of yeasts was completely reversed in the presence of bafilomycin. However,bafilomycin did not inhibit mouse Mphi fungistatic activity induced by IFN-gamma. Thus,human Mphi do not require phagosomal acidification to kill and degrade Hc yeasts,whereas mouse Mphi do require acidification for fungicidal but not fungistatic activity. View Publication

IkappaB kinase (IKK) complex plays a key regulatory role in macrophages for NF-kappaB activation during both innate and adaptive immune responses. Because IKK1-/- mice died at birth,we differentiated functional macrophages from embryonic day 15.5 IKK1 mutant embryonic liver. The embryonic liver-derived macrophage (ELDM) showed enhanced phagocytotic clearance of bacteria,more efficient antigen-presenting capacity,elevated secretion of several key proinflammatory cytokines and chemokines,and known NFkappaB target genes. Increased NFkappaB activity in IKK1 mutant ELDM was the result of prolonged degradation of IkappaBalpha in response to infectious pathogens. The delayed restoration of IkappaBalpha in pathogen-activated IKK1-/- ELDM was a direct consequence of uncontrolled IKK2 kinase activity. We hypothesize that IKK1 plays a checkpoint role in the proper control of IkappaBalpha kinase activity in innate and adaptive immunity. View Publication