Lack of Activation Marker Induction and Chemokine Receptor Switch in Human Neonatal Myeloid Dendritic Cells in Response to Human Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) infects and causes disease in infants and reinfects with reduced disease throughout life without significant antigenic change. In contrast, reinfection by influenza A virus (IAV) largely requires antigenic change. The adaptive immune response depends on antigen presentation by dendritic cells (DC), which may be too immature in young infants to induce a fully protective immune response against RSV reinfections.

Differential Responses by Human Respiratory Epithelial Cell Lines to Respiratory Syncytial Virus Reflect Distinct Patterns of Infection Control.

Respiratory syncytial virus (RSV) infects small foci of respiratory epithelial cells via infected droplets. Infection induces expression of type I and III interferons (IFNs) and proinflammatory cytokines, the balance of which may restrict viral replication and affect disease severity. We explored this balance by infecting two respiratory epithelial cell lines with low doses of recombinant RSV expressing green fluorescent protein (rgRSV).

Respiratory syncytial virus infection induces a subset of types I and III interferons in human dendritic cells.

Whether respiratory syncytial virus (RSV) induces severe infantile pulmonary disease may depend on viral strain and expression of types I and III interferons (IFNs). These IFNs impact disease severity by inducing expression of many anti-viral IFN-stimulated genes (ISGs). To investigate the impact of RSV strain on IFN and ISG expression, we stimulated human monocyte-derived DCs (MDDCs) with either RSV A2 or Line 19 and measured expression of types I and III IFNs and ISGs.

High-throughput quantitative real-time RT-PCR assay for determining expression profiles of types I and III interferon subtypes.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-α subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-λ2 and -λ3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

Human metapneumovirus SH and G glycoproteins inhibit macropinocytosis-mediated entry into human dendritic cells and reduce CD4+ T cell activation.

Unlabelled: Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We used gene-deletion viruses to evaluate the role of the attachment G and small hydrophobic SH glycoproteins on virus uptake by primary human monocyte-derived dendritic cells (MDDC) in vitro and on subsequent MDDC maturation and activation of autologous T cells.

Subtypes of type I IFN differentially enhance cytokine expression by suboptimally stimulated CD4(+) T cells.

Human type I interferons (IFNs) include IFN-β and 12 subtypes of IFN-α. During viral infection, infiltrating memory CD4(+) T cells are exposed to IFNs, but their impact on memory T-cell function is poorly understood. To address this, we pretreated PBMCs with different IFNs for 16 h before stimulation with Staphylococcus aureus enterotoxin B and measured cytokine expression by flow cytometry.

Expression profiles of human interferon-alpha and interferon-lambda subtypes are ligand- and cell-dependent.

Recent genome-wide association studies suggest distinct roles for 12 human interferon-alpha (IFN-α) and 3 IFN-λ subtypes that may be elucidated by defining the expression patterns of these sets of genes. To overcome the impediment of high homology among each of the sets, we designed a quantitative real-time PCR assay that incorporates the use of molecular beacon and locked nucleic acid (LNA) probes, and in some instances, LNA oligonucleotide inhibitors. We then measured IFN subtype expression by human peripheral blood mononuclear cells and by purified monocytes, myeloid dendritic cells (mDC), plasmacytoid dendritic cells (pDC), and monocyte-derived macrophages (MDM), and -dendritic cells (MDDC) in response to poly I:C, lipopolysaccharide (LPS), imiquimod and CpG oligonucleotides.

Low CCR7-mediated migration of human monocyte derived dendritic cells in response to human respiratory syncytial virus and human metapneumovirus.

Human respiratory syncytial virus (HRSV) and, to a lesser extent, human metapneumovirus (HMPV) and human parainfluenza virus type 3 (HPIV3), can re-infect symptomatically throughout life without significant antigenic change, suggestive of incomplete or short-lived immunity. In contrast, re-infection by influenza A virus (IAV) largely depends on antigenic change, suggestive of more complete immunity. Antigen presentation by dendritic cells (DC) is critical in initiating the adaptive immune response.

Respiratory syncytial virus interferon antagonist NS1 protein suppresses and skews the human T lymphocyte response.

We recently demonstrated that the respiratory syncytial virus (RSV) NS1 protein, an antagonist of host type I interferon (IFN-I) production and signaling, has a suppressive effect on the maturation of human dendritic cells (DC) that was only partly dependent on released IFN-I. Here we investigated whether NS1 affects the ability of DC to activate CD8+ and CD4+ T cells. Human DC were infected with RSV deletion mutants lacking the NS1 and/or NS2 genes and assayed for the ability to activate autologous T cells in vitro, which were analyzed by multi-color flow cytometry.

Effects of human respiratory syncytial virus, metapneumovirus, parainfluenza virus 3 and influenza virus on CD4+ T cell activation by dendritic cells.

Background: Human respiratory syncytial virus (HRSV), and to a lesser extent human metapneumovirus (HMPV) and human parainfluenza virus type 3 (HPIV3), re-infect symptomatically throughout life without antigenic change, suggestive of incomplete immunity. One causative factor is thought to be viral interference with dendritic cell (DC)-mediated stimulation of CD4+ T cells.

Methodology, Principal Findings: We infected human monocyte-derived DC with purified HRSV, HMPV, HPIV3, or influenza A virus (IAV) and compared their ability to induce activation and proliferation of autologous CD4+ T cells in vitro.

Investigating the role of the interleukin-23/-17A axis in rheumatoid arthritis.

Objective: IL-23 is a pro-inflammatory cytokine proposed to be central to the development of autoimmune disease. We investigated whether IL-23, together with the downstream mediator IL-17A, was present and functional in RA in humans.

Methods: RA synovial cells were cultured in the presence or absence of antibodies directed against IL-23p19 or -23R and -17.

Systematic method for determining an ideal housekeeping gene for real-time PCR analysis.

To standardize the amount of biological material between samples (e.g., number of cells or amount of tissue) for quantitative real-time reverse transcriptase PCR (qRT-PCR), the cycle of the target gene at which expression is detected (the cycle threshold, or Ct) is divided by the Ct of a gene either thought to be unaffected by experimental conditions or similarly expressed among donors.

Resting CD4+ effector memory T cells are precursors of bystander-activated effectors: a surrogate model of rheumatoid arthritis synovial T-cell function.

Background: Previously we described a system whereby human peripheral blood T cells stimulated for 8 days in a cytokine cocktail acquired effector function for contact-dependent induction of proinflammatory cytokines from monocytes. We termed these cells cytokine-activated (Tck) cells and found that the signalling pathways elicited in the responding monocytes were identical whether they were placed in contact with Tck cells or with T cells isolated from rheumatoid arthritis (RA) synovial tissue.

Methods: Here, using magnetic beads and fluorescence-activated cell sorting, we extensively phenotype the Tck effector cells and conclude that effector function resides within the CD4+CD45RO+, CCR7-, CD49dhigh population, and that these cells are derived from the effector memory CD4+ T cells in resting blood.

Chemokine transport across human vascular endothelial cells.

Leukocyte migration across vascular endothelium is mediated by chemokines that are either synthesized by the endothelium or transferred across the endothelium from the tissue. The mechanism of transfer of two chemokines, CXCL10 (interferon gamma-inducible protein [IP]-10) and CCL2 (macrophage chemotactic protein [MCP]-1), was compared across dermal and lung microvessel endothelium and saphenous vein endothelium. The rate of transfer depended on both the type of endothelium and the chemokine.

Expression of chemokines on the surface of different human endothelia.

Expression of chemokines at the endothelial surface depends on their rate of synthesis, the capacity of the endothelium to bind chemokines and the rate of clearance from the surface. The aim of this study was to establish how these factors depend on the chemokine and the tissue of origin of the endothelium. Human lung and dermal microvascular endothelium, saphenous and umbilical vein endothelium, and a bone marrow endothelial line were assayed in vitro.