技术资料

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

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

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

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

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

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

Although natural Abs (NAbs) are present from birth,little is known about what drives their selection and whether they have housekeeping functions. The prototypic T15-NAb,first identified because of its protective role in infection,is representative of a special type of NAb response that specifically recognizes and forms complexes with apoptotic cells and which promotes cell-corpse engulfment by phagocytes. We now show that this T15-NAb IgM-mediated clearance process is dependent on the recruitment of C1q and mannose-binding lectin,which have known immune modulatory activities that also provide eat me" signals for enhancing phagocytosis. Further investigation revealed that the addition of T15-NAb significantly suppressed in vitro LPS-induced TNF-alpha and IL-6 secretion by the macrophage-like cell line� View Publication

A major mechanism of hypercoagulability in the antiphospholipid syndrome (APS) is antiphospholipid Ab-mediated upregulation of tissue factor (TF) on monocytes via activation of TLRs,p38 MAPK,and NF-kappaB pathways. We examined whether monocyte signaling pathways are differentially activated by IgG from patients with vascular thrombosis (VT) alone compared with IgG from patients with pregnancy morbidity (PM) alone. We purified IgG from 49 subjects. A human monocyte cell line and ex vivo healthy monocytes were treated with 100 microg/ml IgG for 6 h,and cell extracts were examined by immunoblot using Abs to p38 MAPK and NF-kappaB. To further investigate intracellular signaling pathways induced by these IgGs,specific inhibitors of p38 MAPK,NF-kappaB,TLR4,and TLR2 were used to determine their effect on TF activity. Only IgG from patients with VT but no PM (VT+/PM-) caused phosphorylation of NF-kappaBand p38 MAPK and upregulation of TF activity in monocytes. These effects were not seen with IgG from patients with PM alone (VT-/PM+),anti-phospholipid Ab-positive patients without APS,or healthy controls. TF upregulation caused by the VT+/PM- samples was reduced by inhibitors of p38 MAPK,NF-kappaB,and TLR4. The effects of VT+/PM- IgG on signaling and TF upregulation were concentrated in the fraction that bound beta-2-glycoprotein I. Our findings demonstrate that IgGs from patients with diverse clinical manifestations of APS have differential effects upon phosphorylation of NF-kappaB and p38 MAPK and TF activity that may be mediated by differential activation of TLR4. View Publication

In this study,we examined whether bacterial pathogen-associated molecular patterns recognized by toll-like receptors (TLRs) can modify the CCR7-dependent migration of human monocytes. MonoMac-1 (MM-1) cells and freshly isolated human monocytes were cultivated in the presence of agonists for TLR4 (which senses lipopolysaccharides from gram-negative bacteria),TLR1/2 (which senses peptidoglycan from gram-positive bacteria),and TLR9 (which recognizes bacterial DNA rich in unmethylated CpG DNA). CCR7 mRNA transcription was measured using quantitative reverse transcription polymerase chain reaction and protein expression was examined using flow cytometry. CCR7 function was monitored using migration and transmigration assays in response to CCL19/CCL21,which are natural ligands for CCR7. Our results show that TLR4 strongly increases monocyte migratory capacity in response to CCL19 in chemotaxis and transmigration assays in a model that mimics the human blood-brain barrier,whereas TLR1/2 and 9 have no effect. Examination of monocyte migration in response to TLRs that are activated by bacterial components would contribute to understanding the excessive monocyte migration that characterizes the pathogenesis of bacterial infections and/or neuroinflammatory diseases. View Publication

Proof of drug-target engagement in physiologically-relevant contexts is a key pillar of successful therapeutic target validation. We developed two orthogonal technologies,the cellular thermal shift assay (CETSA) and a covalent chemical probe reporter approach (harnessing sulfonyl fluoride tyrosine labeling and subsequent click chemistry) to measure the occupancy of the mRNA-decapping scavenger enzyme DcpS by a small molecule inhibitor in live cells. Enzyme affinity determined using isothermal dose response fingerprinting (ITDRFCETSA) and the concentration required to occupy 50% of the enzyme (OC50) using the chemical probe reporter assay were very similar. In this case,the chemical probe method worked well due to the long offset kinetics of the reversible inhibitor (determined using a fluorescent dye-tagged probe). This work suggests that CETSA could become the first choice assay to determine in-cell target engagement due to its simplicity. 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

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