A robust mouse model of HPIV-3 infection and efficacy of GS-441524 against virus-induced lung pathology

Human parainfluenza virus type 3 (HPIV-3) can cause severe respiratory tract infections. There are no convenient small-animal infection models. Here, we show viral replication in the upper and lower airways of AG129 mice (double IFNα/β and IFNγ receptor knockout mice) upon intranasal inoculation. By multiplex fluorescence RNAscope and immunohistochemistry followed by confocal microscopy, we demonstrate viral tropism to ciliated cells and club cells of the bronchiolar epithelium. HPIV-3 causes a marked lung pathology. No virus transmission of the virus was observed by cohousing HPIV-3-infected AG129 mice with other mice. Oral treatment with GS-441524, the parent nucleoside of remdesivir, reduced infectious virus titers in the lung, with a relatively normal histology. Intranasal treatment also affords an antiviral effect. Thus, AG129 mice serve as a robust preclinical model for developing therapeutic and prophylactic strategies against HPIV-3. We suggest further investigation of GS-441524 and its prodrug forms to treat HPIV-3 infection in humans

Examining the constructs about the supervisor\u27s difficulty scale in supporting the return to work of people with mental health disorders

Within the framework of the multidisciplinary RECS project and with the aim of describing the particle flux transfer from the continental shelf to the deep basin, an array of five mooring lines equipped with a total of five pairs of PPS3/3 sequential-sampling sediment traps and RCM-7/8 current meters were deployed 30 m above the bottom from March 2003 to March 2004 inside and outside the Blanes Canyon. One mooring line was located in the upper canyon at 600 m depth, one in the canyon axis at 1700 m depth and other two close to the canyon walls at 900 m depth. A fifth mooring line was deployed in the continental open slope at 1500 m water depth. The highest near-bottomdownwardparticle flux (14.50 g m-2 d-1)wasrecorded at the trap located in the upper canyon (M1), where continental inputs associated with the presence of the Tordera River are most relevant. On the other hand, the downward fluxes (4.35 g m-2 d-1) in the canyon axis (M2) were of the same order as those found in the western flank (M3) of the canyon. Both values were clearly higher than the value (1.95 g m-2 d-1) recorded at the eastern canyon wall (M4). The open slope (M5) mass flux (5.42 mg m-2 d-1) recorded by the sediment trap located outside the canyon system was three orders of magnitude lower than the other values registered by the inner canyon stations. The relevance of our data is that it explains how the transport pathway in the canyon occurs through its western flank, where a more active and persistent current toward the open ocean was recorded over the entire year of the experiment. Off-shelf sediment transport along the canyon axis showed clear differences during the period of the study, with some important events leading to strong intensifications of the current coupled with large transport of particle fluxes to the deepest parts of the canyon. Such events are primarily related to increases in river discharge and the occurrence of strong storms and cascading events during the winter. In summary, in this study it is shown that the dynamics of thewater masses and the currents in the study area convert the sharp western flank of the Blanes Canyon in a more active region that favors erosion processes than the eastern flank, which has a smoother topography and where the absence of erosional conditions yields to steadier sedimentary processes.Peer ReviewedPostprint (published version

Particle fluxes dynamics in Blanes submarine canyon (Northwestern Mediterranean)

Within the framework of the multidisciplinary RECS project and with the aim of describing the particle flux transfer from the continental shelf to the deep basin, an array of five mooring lines equipped with a total of five pairs of PPS3/3 sequential-sampling sediment traps and RCM-7/8 current meters were deployed 30 m above the bottom from March 2003 to March 2004 inside and outside the Blanes Canyon. One mooring line was located in the upper canyon at 600 m depth, one in the canyon axis at 1700 m depth and other two close to the canyon walls at 900 m depth. A fifth mooring line was deployed in the continental open slope at 1500 m water depth. The highest near-bottomdownwardparticle flux (14.50 g m-2 d-1)wasrecorded at the trap located in the upper canyon (M1), where continental inputs associated with the presence of the Tordera River are most relevant. On the other hand, the downward fluxes (4.35 g m-2 d-1) in the canyon axis (M2) were of the same order as those found in the western flank (M3) of the canyon. Both values were clearly higher than the value (1.95 g m-2 d-1) recorded at the eastern canyon wall (M4). The open slope (M5) mass flux (5.42 mg m-2 d-1) recorded by the sediment trap located outside the canyon system was three orders of magnitude lower than the other values registered by the inner canyon stations. The relevance of our data is that it explains how the transport pathway in the canyon occurs through its western flank, where a more active and persistent current toward the open ocean was recorded over the entire year of the experiment. Off-shelf sediment transport along the canyon axis showed clear differences during the period of the study, with some important events leading to strong intensifications of the current coupled with large transport of particle fluxes to the deepest parts of the canyon. Such events are primarily related to increases in river discharge and the occurrence of strong storms and cascading events during the winter. In summary, in this study it is shown that the dynamics of thewater masses and the currents in the study area convert the sharp western flank of the Blanes Canyon in a more active region that favors erosion processes than the eastern flank, which has a smoother topography and where the absence of erosional conditions yields to steadier sedimentary processes.Peer ReviewedPostprint (published version

Respiratory Syncytial Virus Induced Type I IFN Production by pDC Is Regulated by RSV-Infected Airway Epithelial Cells, RSV-Exposed Monocytes and Virus Specific Antibodies

<div><p>Innate immune responses elicited upon virus exposure are crucial for the effective eradication of viruses, the onset of adaptive immune responses and for establishing proper immune memory. Respiratory syncytial virus (RSV) is responsible for a high disease burden in neonates and immune compromised individuals, causing severe lower respiratory tract infections. During primary infections exuberant innate immune responses may contribute to disease severity. Furthermore, immune memory is often insufficient to protect during RSV re-exposure, which results in frequent symptomatic reinfections. Therefore, identifying the cell types and pattern recognition receptors (PRRs) involved in RSV-specific innate immune responses is necessary to understand incomplete immunity against RSV. We investigated the innate cellular response triggered upon infection of epithelial cells and peripheral blood mononuclear cells. We show that CD14⁺ myeloid cells and epithelial cells are the major source of IL-8 and inflammatory cytokines, IL-6 and TNF-α, when exposed to live RSV Three routes of RSV-induced IFN-α production can be distinguished that depend on the cross-talk of different cell types and the presence or absence of virus specific antibodies, whereby pDC are the ultimate source of IFN-α. RSV-specific antibodies facilitate direct TLR7 access into endosomal compartments, while in the absence of antibodies, infection of monocytes or epithelial cells is necessary to provide an early source of type I interferons, required to engage the IFN-α,β receptor (IFNAR)-mediated pathway of IFN-α production by pDC. However, at high pDC density infection with RSV causes IFN-α production without the need for a second party cell. Our study shows that cellular context and immune status are factors affecting innate immune responses to RSV. These issues should therefore be addressed during the process of vaccine development and other interventions for RSV disease.</p> </div

Particle fluxes dynamics in Blanes submarine canyon (Northwestern Mediterranean)

Within the framework of the multidisciplinary RECS project and with the aim of describing the particle flux transfer from the continental shelf to the deep basin, an array of five mooring lines equipped with a total of five pairs of PPS3/3 sequential-sampling sediment traps and RCM-7/8 current meters were deployed 30 m above the bottom from March 2003 to March 2004 inside and outside the Blanes Canyon. One mooring line was located in the upper canyon at 600 m depth, one in the canyon axis at 1700 m depth and other two close to the canyon walls at 900 m depth. A fifth mooring line was deployed in the continental open slope at 1500 m water depth. The highest near-bottomdownwardparticle flux (14.50 g m-2 d-1)wasrecorded at the trap located in the upper canyon (M1), where continental inputs associated with the presence of the Tordera River are most relevant. On the other hand, the downward fluxes (4.35 g m-2 d-1) in the canyon axis (M2) were of the same order as those found in the western flank (M3) of the canyon. Both values were clearly higher than the value (1.95 g m-2 d-1) recorded at the eastern canyon wall (M4). The open slope (M5) mass flux (5.42 mg m-2 d-1) recorded by the sediment trap located outside the canyon system was three orders of magnitude lower than the other values registered by the inner canyon stations. The relevance of our data is that it explains how the transport pathway in the canyon occurs through its western flank, where a more active and persistent current toward the open ocean was recorded over the entire year of the experiment. Off-shelf sediment transport along the canyon axis showed clear differences during the period of the study, with some important events leading to strong intensifications of the current coupled with large transport of particle fluxes to the deepest parts of the canyon. Such events are primarily related to increases in river discharge and the occurrence of strong storms and cascading events during the winter. In summary, in this study it is shown that the dynamics of thewater masses and the currents in the study area convert the sharp western flank of the Blanes Canyon in a more active region that favors erosion processes than the eastern flank, which has a smoother topography and where the absence of erosional conditions yields to steadier sedimentary processes.Peer Reviewe

CD14⁺ monocytes inhibit IFN-α production triggered by Ab-RSV via TLR7 in pDC.

<p>(<b>A</b>) IFN-α production in CD14⁺ cell depleted PBMCs induced by Ab-RSV complexes, TLR9 ligand ODN 2216 and TLR7 ligand imiquimod is decreased by blocking endosomal acidification with 50nM Bafilomycin A₁. (<b>B</b>) Ab-RSV-induced IFN-α production in CD14⁺ cell depleted PBMCs was abrogated in the presence of immune regulatory sequence (IRS) 661 (1.4 µM) a specific blocking agent for endosomal TLR7 and not by a scrambled control nucleotide. (<b>C</b>) pDC are the source of Ab-RSV induced, TLR7 mediated production of IFN-α, as shown by intracellular staining for IFN-α in Lineage (CD3^neg., CD14^neg., CD19^neg., CD16^neg., CD56^neg., Lin-1), MHC-II^high, BDCA-4⁺ cells. The inhibitor brefeldin A was added at different time points post infection (the time points when BFA was added are given on the X-axis). Cytokines were allowed to accumulate for 10 hrs. after addition of BFA. (<b>D</b>) Purified pDC (obtained by negative selection removing CD3⁺, CD19⁺ and CD16⁺ cells from fresh PBMC, followed by FACS purification of the BDCA-4⁺ cell population, which resulted in > 95% pure pDC) produce IFN-α upon infection with RSV. This response is abrogated after UV inactivation of RSV. In AS, both live RSV and UV-inactivated RSV induced IFN-α production to a similar extent. One representative experiment out of two performed with pDC isolated from two different donors is shown. (<b>E</b>) IFN-α production by Ab-RSV in purified pDC is blocked by IRS661 (1.4µM). (<b>F</b>) TLR1,-2 (PAM3CSK4, Peptidoglycan) and TLR4 (LPS) ligands suppress TLR9-triggered (ODN 2216) IFN-α production, but do not affect TLR7 (Gardiquimod) induced IFN-α production. All data represent mean ± SEM of triplicate measurements within 1 donor and analyzed using one way ANOVA followed by a Bonferroni post-test. ns not significant, *P< 0.05, **P <0.01. Experiments were performed in 3 different donors with similar results.</p

IFN-α production induced by live RSV in PBMC depends on IFNAR signalling.

<p>PBMC were exposed to live RSV in the presence of interferon-α/β receptor (IFNAR) blocking antibody (5µg/ml), isotype control Ab or no Ab. Levels of IFN-α were determined via intracellular staining (<b>A</b>) or in 20hrs. supernatant by ELISA (<b>B</b>). IFN-α was trapped intracellular by BFA treatment initiated 6 hrs. after RSV infection, or at t=0 for the ODN 2216 control stimulus, because of different kinetics of anti-viral and ODN elicited IFN-α response. For both stimuli, intracellular staining for IFN-α was performed in CD3^neg., CD16^neg., CD19^neg., CD56^neg., MHCII⁺, BDCA-4⁺ cells after 10 hrs. BFA treatment. Experiments were performed in 3 different donors with similar results. Data represent the mean ± SEM of triplicate measurements within 1 donor and were analyzed using one way ANOVA followed by a Bonferroni post-test. ns not significant, **P <0.01.</p

IFN-α production induced by live RSV in pDC depends on CD14.

<p>The role of the TLR4/CD14/MD2 complex in the IFN-α and IL-6 response by PBMC after RSV infection (strain A2, MOI 5) was investigated (<b>A</b>) with a blocking monoclonal antibody specific for human TLR4 (20µg/ml), (<b>B</b>) the MD2 antagonist lipopolysaccharide from the bacterium <i>Rhodobacter </i><i>sphaeroides</i> (1µg/ml) or (<b>C</b>) via neutralization of CD14 with monoclonal antibody MY4 (10µg/ml). (<b>A</b>-<b>C</b>) Cytokine responses were measured by ELISA. Control stimuli; LPS: 10ng/ml, ODN: 5µg/ml. (<b>D</b>) A549 epithelial cells were co-cultured with PBMC, CD14⁺ cell depleted PBMC, pDC depleted PBMC, or CD14⁺ and BDCA-4⁺ double depleted PBMC, in the presence of RSV at MOI 5, ODN 2216 or no stimulus, for a period of 20 hrs. IL-6 and IFN-α production were measured by ELISA. Experiments were performed in 3 different donors with similar results. Data represent the mean ± SEM of triplicate measurements within 1 donor and were analyzed using one way ANOVA followed by a Bonferroni post-test. ns not significant, *P<0.05, **P<0.01. </p

Virus specific polyclonal antibodies in human serum increase RSV binding to monocytes and B cells.

<p>(<b>A</b>) The effect of autologous serum, IgG-depleted serum and palivizumab in FCS on binding of RSV to monocytes, B cells and NK cells was measured after 1 hour incubation at 4°C. (<b>B</b>) Polyclonal antibodies (IVIG) increase binding of RSV to CD14^+/CD16^neg. cells. Data shown represent the mean ± SEM of triplicate measurements in two different donors (for A and B) and were analyzed using the Kruskal-Wallis test followed by Dunn's Multiple Comparison analysis, *P< 0.05, **P <0.01. Experiments were performed in 3 additional donors with similar results.</p

RSV-specific antibodies inhibit RSV-induced IFN-α production in PBMC, but enhance IFN-α production in CD14⁺ cell depleted PBMC.

<p>(<b>A</b>) Inhibition of RSV infection in pDC. Lineage^neg., MHC-II^high, BDCA-4⁺ pDC are partially infected with rgRSV224 after a period of 20 hours. Infection is blocked after UV inactivation, after neutralization in 10% fresh human serum or in 5µg/ml Palivizumab. Similar results were obtained with sera from all donors used during our studies. IFN-α in supernatant of RSV-A2 exposed PBMC-CD14⁺ cell cultures (<b>B</b>) and rgRSV224 exposed PBMC-CD14⁺ cells or PBMC, (<b>C</b>) was measured after 20 hrs. The role of virus specific antibodies on the cytokine response was tested by removing IgGs from AS with protein G Sepharose^® beads and reconstitution with 2 mg/ml IVIG (<b>B</b>) or 5 µg/ml palivizumab (<b>B</b>, <b>C</b>). Experiments represent the mean ± SEM of experiments performed in 4 different donors and were analyzed using one way ANOVA followed by a Bonferroni post-test, **P <0.01.</p