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To advance T cell-based HIV vaccine development,it is necessary to evaluate the immune correlates of a protective CD8(+) T-cell response. We have developed an assay that assesses the capacity ex vivo of HIV-specific CD8(+) T cells to suppress HIV-1 infection of autologous CD4(+) T cells. This assay directly reflects the ultimate effector function of CD8(+) T cells,the elimination of infected cells,and accurately differentiates the effective CD8(+) T-cell response in spontaneous HIV controllers from ineffective responses in other patients. In this article,we describe all the steps from cell purification to assessment of viral replication by HIV-p24 ELISA and analysis,along with conditions for cell culturing,and how to choose the viral infectious dose that gives the most reliable results. We also depict the conditions of a rapid assay on the basis of flow cytometry analysis of intracellular HIV-Gag products. These procedures take 14-17 d when the p24 ELISA assay is used,or 6 d with the intracellular Gag assay. View Publication

Caveolin 1 (Cav-1) is a major protein of a specific membrane lipid raft known as caveolae. Cav-1 interacts with the gp41 of the human immunodeficiency virus (HIV) envelope,but the role of Cav-1 in HIV replication and pathogenesis is not known. In this report,we demonstrate that HIV infection in primary human monocyte-derived macrophages (MDMs),THP-1 macrophages,and U87-CD4 cells results in a dramatic upregulation of Cav-1 expression mediated by HIV Tat. The activity of p53 is essential for Tat-induced Cav-1 expression,as our findings show enhanced phosphorylation of serine residues at amino acid positions 15 and 46 in the presence of Tat with a resulting Cav-1 upregulation. Furthermore,inhibition of p38 mitogen-activated protein kinase (MAPK) blocked phosphorylation of p53 in the presence of Tat. Infection studies of Cav-1-overexpressing cells reveal a significant reduction of HIV production. Taken together,these results suggest that HIV infection enhances the expression of Cav-1,which subsequently causes virus reduction,suggesting that Cav-1 may contribute to persistent infection in macrophages. View Publication

Extracorporeal photochemotherapy (ECP) is widely used to treat cutaneous T-cell lymphoma,graft-versus-host disease,and allografted organ rejection. Its clinical and experimental efficacy in cancer immunotherapy and autoreactive disorders suggests a novel mechanism. This study reveals that ECP induces a high percentage of processed monocytes to enter the antigen-presenting dendritic cell (DC) differentiation pathway,within a single day,without added cytokines,as determined by enhanced expression of relevant genes. The resulting DCs are capable of processing and presentation of exogenous and endogenous antigen and are largely maturationally synchronized,as assessed by the level of expression of costimulatory surface molecules. Principal component analysis of the ECP-induced monocyte transcriptome reveals that activation or suppression of more than 1100 genes produces a reproducible distinctive molecular signature,common to ECP-processed monocytes from normal subjects,and those from patients. Because ECP induces normal monocytes to enter the DC differentiation pathway,this phenomenon is independent of disease state. The efficiency with which ECP stimulates new functional DCs supports the possibility that these cells participate prominently in the clinical successes of the treatment. Appropriately modified by future advances,ECP may potentially offer a general source of therapeutic DCs. View Publication

It has recently been shown that nucleosome distribution,histone modifications and RNA polymerase II (Pol II) occupancy show preferential association with exons (exon-intron marking")� View Publication

In elderly subjects and in patients with chronic inflammatory diseases,there is an increased subset of monocytes with a CD14(+)CD16(+) phenotype,whose origin and functional relevance has not been well characterized. In this study,we determined whether prolonged survival of human CD14(++)CD16(-) monocytes promotes the emergence of senescent cells,and we analyzed their molecular phenotypic and functional characteristics. We used an in vitro model to prolong the life span of healthy monocytes. We determined cell senescence,intracellular cytokine expression,ability to interact with endothelial cells,and APC activity. CD14(+)CD16(+) monocytes were senescent cells with shortened telomeres (215 ± 37 relative telomere length) versus CD14(++)CD16(-) cells (339 ± 44 relative telomere length; p textless 0.05) and increased expression of β-galactosidase (86.4 ± 16.4% versus 10.3 ± 7.5%,respectively; p = 0.002). CD14(+)CD16(+) monocytes exhibited features of activated cells that included expression of CD209,release of cytokines in response to low-intensity stimulus,and increased capacity to sustain lymphocyte proliferation. Finally,compared with CD14(++)CD16(-) cells,CD14(+)CD16(+) monocytes showed elevated expression of chemokine receptors and increased adhesion to endothelial cells (19.6 ± 8.1% versus 5.3 ± 4.1%; p = 0.033). In summary,our data indicated that the senescent CD14(+)CD16(+) monocytes are activated cells,with increased inflammatory activity and ability to interact with endothelial cells. Therefore,accumulation of senescent monocytes may explain,in part,the development of chronic inflammation and atherosclerosis in elderly subjects and in patients with chronic inflammatory diseases. View Publication

Transcriptionally activated monocytes are recruited to the heart after acute myocardial infarction (AMI). After AMI in mice and humans,the number of extracellular vesicles (EVs) increased acutely. In humans,EV number correlated closely with the extent of myocardial injury. We hypothesized that EVs mediate splenic monocyte mobilization and program transcription following AMI. Some plasma EVs bear endothelial cell (EC) integrins,and both proinflammatory stimulation of ECs and AMI significantly increased VCAM-1-positive EV release. Injected EC-EVs localized to the spleen and interacted with,and mobilized,splenic monocytes in otherwise naive,healthy animals. Analysis of human plasma EV-associated miRNA showed 12 markedly enriched miRNAs after AMI; functional enrichment analyses identified 1,869 putative mRNA targets,which regulate relevant cellular functions (e.g.,proliferation and cell movement). Furthermore,gene ontology termed positive chemotaxis as the most enriched pathway for the miRNA-mRNA targets. Among the identified EV miRNAs,EC-associated miRNA-126-3p and -5p were highly regulated after AMI. miRNA-126-3p and -5p regulate cell adhesion- and chemotaxis-associated genes,including the negative regulator of cell motility,plexin-B2. EC-EV exposure significantly downregulated plexin-B2 mRNA in monocytes and upregulated motility integrin ITGB2. These findings identify EVs as a possible novel signaling pathway by linking ischemic myocardium with monocyte mobilization and transcriptional activation following AMI. View Publication

Droplet single-cell RNA-sequencing (dscRNA-seq) has enabled rapid,massively parallel profiling of transcriptomes. However,assessing differential expression across multiple individuals has been hampered by inefficient sample processing and technical batch effects. Here we describe a computational tool,demuxlet,that harnesses natural genetic variation to determine the sample identity of each droplet containing a single cell (singlet) and detect droplets containing two cells (doublets). These capabilities enable multiplexed dscRNA-seq experiments in which cells from unrelated individuals are pooled and captured at higher throughput than in standard workflows. Using simulated data,we show that 50 single-nucleotide polymorphisms (SNPs) per cell are sufficient to assign 97% of singlets and identify 92% of doublets in pools of up to 64 individuals. Given genotyping data for each of eight pooled samples,demuxlet correctly recovers the sample identity of<99% of singlets and identifies doublets at rates consistent with previous estimates. We apply demuxlet to assess cell-type-specific changes in gene expression in 8 pooled lupus patient samples treated with interferon (IFN)-β and perform eQTL analysis on 23 pooled samples. View Publication

Proteasome inhibitors benefit patients with multiple myeloma and B cell-dependent autoimmune disorders but exert toxicity from inhibition of proteasomes in other cells. Toxicity should be minimized by reversible inhibition of the immunoproteasome β5i subunit while sparing the constitutive β5c subunit. Here we report β5i-selective inhibition by asparagine-ethylenediamine (AsnEDA)-based compounds and present the high-resolution cryo-EM structural analysis of the human immunoproteasome. Despite inhibiting noncompetitively,an AsnEDA inhibitor binds the active site. Hydrophobic interactions are accompanied by hydrogen bonding with β5i and β6 subunits. The inhibitors are far more cytotoxic for myeloma and lymphoma cell lines than for hepatocarcinoma or non-activated lymphocytes. They block human B-cell proliferation and promote apoptotic cell death selectively in antibody-secreting B cells,and to a lesser extent in activated human T cells. Reversible,β5i-selective inhibitors may be useful for treatment of diseases involving activated or neoplastic B cells or activated T cells. View Publication

Mobile sensing based on the integration of microfluidic device and smartphone,so-called MS2 technology,has enabled many applications over recent years,and continues to stimulate growing interest in both research communities and industries. In particular,it has been envisioned that MS2 technology can be developed for various cell functional assays to enable basic research and clinical applications. Toward this direction,in this paper,we describe the development of a MS2-based cell functional assay for testing cell migration (the Mkit). The system is constructed as an integrated test kit,which includes microfluidic chips,a smartphone-based imaging platform,the phone apps for image capturing and data analysis,and a set of reagent and accessories for performing the cell migration assay. We demonstrated that the Mkit can effectively measure purified neutrophil and cancer cell chemotaxis. Furthermore,neutrophil chemotaxis can be tested from a drop of whole blood using the Mkit with red blood cell (RBC) lysis. The effects of chemoattractant dose and gradient profile on neutrophil chemotaxis were also tested using the Mkit. In addition to research applications,we demonstrated the effective use of the Mkit for on-site test at the hospital and for testing clinical samples from chronic obstructive pulmonary disease patient. Thus,this developed Mkit provides an easy and integrated experimental platform for cell migration related research and potential medical diagnostic applications. View Publication

Mucosal Langerhans cells (LCs) originate from pre-dendritic cells and monocytes. However,the mechanisms involved in their in situ development remain unclear. Here,we demonstrate that the differentiation of murine mucosal LCs is a two-step process. In the lamina propria,signaling via BMP7-ALK3 promotes translocation of LC precursors to the epithelium. Within the epithelium,TGF-β1 finalizes LC differentiation,and ALK5 is crucial to this process. Moreover,the local microbiota has a major impact on the development of mucosal LCs,whereas LCs in turn maintain mucosal homeostasis and prevent tissue destruction. These results reveal the differential and sequential role of TGF-β1 and BMP7 in LC differentiation and highlight the intimate interplay of LCs with the microbiota. View Publication

MicroRNAs play an important role in T cell responses. However,how microRNAs regulate CD8 T cell memory remains poorly defined. Here,we found that miR-150 negatively regulates CD8 T cell memory in vivo. Genetic deletion of miR-150 disrupted the balance between memory precursor and terminal effector CD8 T cells following acute viral infection. Moreover,miR-150-deficient memory CD8 T cells were more protective upon rechallenge. A key circuit whereby miR-150 repressed memory CD8 T cell development through the transcription factor c-Myb was identified. Without miR-150,c-Myb was upregulated and anti-apoptotic targets of c-Myb,such as Bcl-2 and Bcl-xL,were also increased,suggesting a miR-150-c-Myb survival circuit during memory CD8 T cell development. Indeed,overexpression of non-repressible c-Myb rescued the memory CD8 T cell defects caused by overexpression of miR-150. Overall,these results identify a key role for miR-150 in memory CD8 T cells through a c-Myb-controlled enhanced survival circuit. View Publication

We report that SHIP(-/-) mice,compared to SHIP(+/+) mice,are Th2 skewed with elevated serum IgE and twice as many splenic CD4(+) Th2 cells that,when stimulated with anti-CD3,produce more IL-4 and less IFN-$\gamma$. Exploring the reason for this Th2 skewing,we found that freshly isolated SHIP(-/-) splenic and bone marrow basophils are present in elevated numbers and secrete far more IL-4 in response to IL-3 or to Fc$\epsilon$RI stimulation than do WT basophils. These SHIP(-/-) basophils markedly skew wild-type macrophage colony stimulating factor-derived macrophages toward an M2 phenotype,stimulate OT-II CD4(+) Th cells to differentiate into Th2 cells,and trigger SHIP(+/+) B cells to become IgE-producing cells. All these effects are completely abrogated with neutralizing anti-IL-4 Ab. Exploring the cell signaling pathways responsible for hyperproduction of IL-4 by SHIP(-/-) basophils,we found that IL-3-induced activation of the PI3K pathway is significantly enhanced and that PI3K inhibitors,especially a p110$\alpha$ inhibitor,dramatically suppresses IL-4 production from these cells. In vivo studies,in which basophils were depleted from mast cell-deficient SHIP(+/+) and SHIP(-/-) mice,confirmed the central role that basophils play in the Th2 skewing of naive SHIP-deficient mice. Taken together,these studies demonstrate that SHIP is a potent negative regulator of IL-4 production from basophils and thus may be a novel therapeutic target for Th1- and Th2-related diseases. View Publication