技术资料

The dynamic interplay between dendritic cells (DCs) and human immunodeficiency virus type-1 (HIV-1) is thought to result in viral dissemination and evasion of antiviral immunity. Although initial observations suggested that the C-type lectin receptor (CLR) DC-SIGN was responsible for the trans-infection function of the virus,subsequent studies demonstrated that trans-infection of CD4(+) T cells with HIV-1 can also occur through DC-SIGN-independent mechanisms. We demonstrate that a cell surface molecule designated DCIR (for DC immunoreceptor),a member of a recently described family of DC-expressing CLRs,can participate in the capture of HIV-1 and promote infection in trans and in cis of autologous CD4(+) T cells from human immature monocyte-derived DCs. The contribution of DCIR to these processes was revealed using DCIR-specific siRNAs and a polyclonal antibody specific for the carbohydrate recognition domain of DCIR. Data from transfection experiments indicated that DCIR acts as a ligand for HIV-1 and is involved in events leading to productive virus infection. Finally,we show that the neck domain of DCIR is important for the DCIR-mediated effect on virus binding and infection. These results point to a possible role for DCIR in HIV-1 pathogenesis by supporting the productive infection of DCs and promoting virus propagation. 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

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

The lectin galectin-9 may help establish and maintain chronic hepatitis C virus infection. Galectin-9 is elevated in the liver and sera of hepatitis C virus patients,induces apoptosis of hepatitis C virus-specific T cells,and increases inhibitory regulatory T cells. Kupffer cells stain strongly for galectin-9 protein in hepatitis C virus patients. In the current study,we determined stimuli that induce galectin-9 production by monocytes and macrophages in hepatitis C virus infection. With the use of real-time PCR and flow cytometry,we analyzed galectin-9 mRNA and protein from human monocytes cocultured with hepatitis C virus-infected cells or noninfectious hepatitis C virus subgenomic replicon cells. We focused on finding the stimuli for galectin-9 production. Additionally,we measured galectin-9 during monocyte-to-macrophage maturation. Finally,we examined galectin-9 in peripheral monocytes from hepatitis C virus patients using flow cytometry. Galectin-9 mRNA increased 8-fold when primary monocytes were exposed to hepatitis C virus--infected cells. Maximum induction required proximity or contact and did not require IFN-γ or hepatitis C virus virions. Coculture of monocytes with subgenomic replicon cells increased galectin-9 5-fold,and purified exosomes from infected cells stimulated galectin-9 production. Stimulation of monocyte TLR3,-7,and -8 increased galectin-9 production. Differentiation of monocytes to macrophages increased galectin-9,and nonclassic monocytes from hepatitis C virus patients had the highest levels of galectin-9. Hepatitis C virus-infected cells stimulated monocytes to produce galectin-9 in close proximity,possibly,in part,as a result of exosomes and endosomal TLRs. Differentiation of monocytes to macrophages increased galectin-9. Nonclassic monocytes from hepatitis C virus patients express the highest galectin-9 levels,suggesting they may contribute to elevated galectin-9 and adaptive immune inhibition in hepatitis C virus infection. View Publication

Immunotherapy treatments for cancer are becoming increasingly successful,however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy,precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1),MelanA,Wilms' Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8-1.4 x 10(6)). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes,and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3),tyrosinase (n = 3) and WT1(126-134) (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1(950-958) epitope. In the future the pMHC array may be used provide point of care T-cell analyses,predict patient response to conventional therapy and direct personalised immunotherapy for patients. View Publication

Human monocytes are a heterogeneous cell population classified into three different subsets: Classical CD14++CD16-,intermediate CD14++CD16+,and non-classical CD14+CD16++ monocytes. These subsets are distinguished by their differential expression of CD14 and CD16,and unique gene expression profile. So far,the variation in inter-cellular gene expression within the monocyte subsets is largely unknown. In this study,the cellular variation within each human monocyte subset from a single healthy donor was described by using a novel single-cell PCR gene-expression analysis tool. We investigated 86 different genes mainly encoding cell surface markers,and proteins involved in immune regulation. Within the three human monocyte subsets,our descriptive findings show multimodal expression of key immune response genes,such as CD40,NFⱪB1,RELA,TLR4,TLR8 and TLR9. Furthermore,we discovered one subgroup of cells within the classical monocytes,which showed alterations of 22 genes e.g. IRF8,CD40,CSF1R,NFⱪB1,RELA and TNF. Additionally one subgroup within the intermediate and non-classical monocytes also displayed distinct gene signatures by altered expression of 8 and 6 genes,respectively. Hence the three monocyte subsets can be further subdivided according to activation status and differentiation,independently of the traditional classification based on cell surface markers. Demonstrating the use and the ability to discover cell heterogeneity within defined populations of human monocytes is of great importance,and can be useful in unravelling inter-cellular variation in leukocyte populations,identifying subpopulations involved in disease pathogenesis and help tailor new therapies. View Publication

BACKGROUND: The cellular sulfonation pathway modulates key steps of virus replication. This pathway comprises two main families of sulfonate-conjugating enzymes: Golgi sulfotransferases,which sulfonate proteins,glycoproteins,glycolipids and proteoglycans; and cytosolic sulfotransferases (SULTs),which sulfonate various small molecules including hormones,neurotransmitters,and xenobiotics. Sulfonation controls the functions of numerous cellular factors such as those involved in cell-cell interactions,cell signaling,and small molecule detoxification. We previously showed that the cellular sulfonation pathway regulates HIV-1 gene expression and reactivation from latency. Here we show that a specific cellular sulfotransferase can regulate HIV-1 replication in primary human monocyte-derived macrophages (MDMs) by yet another mechanism,namely reverse transcription. METHODS: MDMs were derived from monocytes isolated from donor peripheral blood mononuclear cells (PBMCs) obtained from the San Diego Blood Bank. After one week in vitro cell culture under macrophage-polarizing conditions,MDMs were transfected with sulfotranserase-specific or control siRNAs and infected with HIV-1 or SIV constructs expressing a luciferase reporter. Infection levels were subsequently monitored by luminescence. Western blotting was used to assay siRNA knockdown and viral protein levels,and qPCR was used to measure viral RNA and DNA products. RESULTS: We demonstrate that the cytosolic sulfotransferase SULT1A1 is highly expressed in primary human MDMs,and through siRNA knockdown experiments,we show that this enzyme promotes infection of MDMs by single cycle VSV-G pseudotyped human HIV-1 and simian immunodeficiency virus vectors and by replication-competent HIV-1. Quantitative PCR analysis revealed that SULT1A1 affects HIV-1 replication in MDMs by modulating the kinetics of minus-strand DNA elongation during reverse transcription. CONCLUSIONS: These studies have identified SULT1A1 as a cellular regulator of HIV-1 reverse transcription in primary human MDMs. The normal substrates of this enzyme are small phenolic-like molecules,raising the possibility that one or more of these substrates may be involved. Targeting SULT1A1 and/or its substrate(s) may offer a novel host-directed strategy to improve HIV-1 therapeutics. View Publication

Langerhans cell histiocytosis (LCH) is a rare disease with an unknown etiology characterized by heterogeneous lesions containing CD207+CD1a+ cells that can arise in almost any tissue and cause significant morbidity and mortality. Precursors of pathological Langerhans cells have yet to be defined. Our aim was to identify circulating CD207+CD1a+ cells and their inducers in LCH. Expression of CD207 and CD1a in the blood myeloid compartment as well as thymic stromal lymphopoietin (TSLP) and transforming growth factor β (TGF-β) plasma levels were measured in 22 pediatric patients with active disease (AD) or nonactive disease (NAD). In patients with AD vs those with NAD,the myeloid compartment showed an increased CD11b (CD11bhigh plus CD11b+) fraction (39.7 ± 3.6 vs 18.6 ± 1.9),a higher percentage of circulating CD11bhighCD11c+CD207+ cells (44.5 ± 11.3 vs 3.2 ± 0.5),and the presence of CD11chighCD207+CD1a+ cells (25.0 ± 9.1 vs 2.3 ± 0.5). Blood CD207+CD1a+ cells were not observed in adult controls or umbilical cord. Increased TSLP and TGF-β levels were detected in patients with AD. Interestingly,plasma from patients with AD induces CD207 expression on CD14+ monocytes. We conclude that CD207+CD1a+ cells are circulating in patients with active LCH,and TSLP and TGF-β are potential drivers of Langerhans-like cells in vivo. View Publication

The present study shows that alumina nanotopography affects monocyte/macrophage behavior. Human mononuclear cells cultured on alumina membranes with pore diameters of 20 and 200 nm were evaluated in terms of cell adhesion,viability,morphology,and release of proinflammatory cytokines. After 24 hours,cell adhesion was assessed by means of light microscopy and cell viability by measuring LDH release. The inflammatory response was evaluated by quantifying interleukin-1β and tumour necrosis factor-α. Finally,scanning electron microscopy was used to study cell morphology. Results showed pronounced differences in cell number,morphology,and cytokine release depending on the nanoporosity. Few but highly activated cells were found on the 200 nm porous alumina,while relatively larger number of cells were found on the 20 nm porous surface. However,despite their larger number,the cells adhering on the 20 nm surface exhibited reduced pro-inflammatory activity. The data of this paper implies that nanotopography could be exploited for controlling the inflammatory response to implants. View Publication

P-selectin glycoprotein ligand-1 (PSGL-1) is a homodimeric transmembrane mucin on leukocytes. During inflammation,reversible interactions of PSGL-1 with selectins mediate leukocyte rolling on vascular surfaces. The transmembrane domain of PSGL-1 is required for dimerization,and the cytoplasmic domain propagates signals that activate β(2) integrins to slow rolling on integrin ligands. Leukocytes from knock-in ΔCD" mice express a truncated PSGL-1 that lacks the cytoplasmic domain. Unexpectedly� View Publication

OBJECTIVE Atherosclerosis is a major cause of cardiovascular disease. Monocyte-endothelial cell interactions are partly mediated by expression of monocyte CX3CR1 and endothelial cell fractalkine (CX3CL1). Interrupting the interaction between this ligand-receptor pair should reduce monocyte binding to the endothelial wall and reduce atherosclerosis. We sought to reduce atherosclerosis by preventing monocyte-endothelial cell interactions through use of a long-acting CX3CR1 agonist. METHODS In this study,the chemokine domain of CX3CL1 was fused to the mouse Fc region to generate a long-acting soluble form of CX3CL1 suitable for chronic studies. CX3CL1-Fc or saline was injected twice a week (30 mg/kg) for 4 months into Ldlr knockout (KO) mice on an atherogenic western diet. RESULTS CX3CL1-Fc-treated Ldlr KO mice showed decreased en face aortic lesion surface area and reduced aortic root lesion size with decreased necrotic core area. Flow cytometry analyses of CX3CL1-Fc-treated aortic wall cell digests revealed a decrease in M1-like polarized macrophages and T cells. Moreover,CX3CL1-Fc administration reduced diet-induced atherosclerosis after switching from an atherogenic to a normal chow diet. In vitro monocyte adhesion studies revealed that CX3CL1-Fc treatment caused fewer monocytes to adhere to a human umbilical vein endothelial cell monolayer. Furthermore,a dorsal window chamber model demonstrated that CX3CL1-Fc treatment decreased in vivo leukocyte adhesion and rolling in live capillaries after short-term ischemia-reperfusion. CONCLUSION These results indicate that CX3CL1-Fc can inhibit monocyte/endothelial cell adhesion as well as reduce atherosclerosis. View Publication

Increasing evidence suggests that the deposition of amyloid plaques,composed primarily of the amyloid-beta protein (Abeta),within the cerebrovasculature is a frequent occurrence in Alzheimer's disease and may play a significant role in disease progression. Accordingly,the pathogenic mechanisms by which Abeta can alter vascular function may have therapeutic implications. Despite observations that Abeta elicits a number of physiological responses in endothelial cells,ranging from alteration of protein expression to cell death,the Abeta species accountable for these responses remains unexplored. In the current study,we show that isolated soluble Abeta aggregation intermediates activate human brain microvascular endothelial cells for both adhesion and subsequent transmigration of monocyte cells in the absence of endothelial cell death and monolayer disruption. In contrast,unaggregated Abeta monomer and mature Abeta fibril fail to induce any change in endothelial adhesion or transmigration. Correlations between average Abeta aggregate size and observed increases in adhesion illustrate that smaller soluble aggregates are more potent activators of endothelium. These results support previous studies demonstrating heightened neuronal activity of soluble Abeta aggregates,including Abeta-derived diffusible ligands,oligomers,and protofibrils,and further show that soluble aggregates also selectively exhibit activity in a vascular cell model. View Publication