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ATCC type a2
Type A2, supplied by ATCC, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Modification:

Article Title: The beta2 integrin CD11c distinguishes a subset of cytotoxic pulmonary T cells with potent antiviral effects in vitro and in vivo
Article Snippet: Virus and animals Human RSV, type A2 from ATCC (Rockville, MD) free of mycoplasma contamination was used. .. The virus was cultured on HEp-2 cells from ATCC in Dulbecco's modified Eagle Medium (Invitrogen, Paisley, UK) containing 5% heat inactivated fetal calf serum and 1 % Penicillin / Streptomycin both from Sigma (Gillingham, UK).

Cell Culture:

Article Title: The beta2 integrin CD11c distinguishes a subset of cytotoxic pulmonary T cells with potent antiviral effects in vitro and in vivo
Article Snippet: Virus and animals Human RSV, type A2 from ATCC (Rockville, MD) free of mycoplasma contamination was used. .. The virus was cultured on HEp-2 cells from ATCC in Dulbecco's modified Eagle Medium (Invitrogen, Paisley, UK) containing 5% heat inactivated fetal calf serum and 1 % Penicillin / Streptomycin both from Sigma (Gillingham, UK).

Mouse Assay:

Article Title: The beta2 integrin CD11c distinguishes a subset of cytotoxic pulmonary T cells with potent antiviral effects in vitro and in vivo
Article Snippet: Virus and animals Human RSV, type A2 from ATCC (Rockville, MD) free of mycoplasma contamination was used. .. Female BALB/c AnNCrl mice, 8 to 12 weeks of age, free of specific pathogens, were obtained from Charles River Laboratories (Margate, UK) and kept under specific pathogen free conditions.

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  • 83
    ATCC plaque purified rsv a2
    IL-6 promotes resolution of <t>RSV-mediated</t> disease. 8 week old BALB/c mice were infected with 8 x 10 5 ffu of <t>RSV</t> A2 i.n. (A) IL-6 was measured by ELISA in the BAL, lung tissue and serum. (B-H) RSV infected mice were given 0.5 mg of either HRPN (IgG 1 ) or MP5-20F3 (αIL-6) i.p. on day -1 p.i. and 0.25 mg i.p. every other day after that. (B) The percentage of weight on day 0 was measured daily, area under the curve (AUC) was used to test statistical significance, (C) Albumin was measured in the BAL by ELISA. (D) The number of BAL cells was counted and (E) lymphocytes and (F) Neutrophil (PMN) frequencies were determined by H E staining. (G) Lung neutrophils (defined as Ly6G + CD11b + CD90 - CD19 - autofluorescence - ) were enumerated by flow cytometry. (H) Viral load in the lungs was determined by focus forming assay. For (A) * represents BAL, # represents lung and † represents serum. Data is representative of n = 2 independent repeats of n = 5 mice per time point except B which represents 20 mice per group.
    Plaque Purified Rsv A2, supplied by ATCC, used in various techniques. Bioz Stars score: 83/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    ATCC hrsv a2
    SHe-specific IgG binds to <t>HRSV-infected</t> cells A–C A549 cells were infected overnight with 0.5 MOI <t>HRSV</t> A2 (A), mock-infected (B). In parallel, A549 cells were incubated with 5 MOI HRSV A2 at 4°C for 2 h to allow virus attachment without cell entry (C). After infection or virus attachment, the cells were fixed and stained with SHe-KLH mouse immune serum, KLH mouse immune serum, or a HRSV F-specific mouse monoclonal antibody. Reactivity of mouse IgG was revealed with a green fluorescently labeled secondary antibody. In addition, cells were stained with a polyclonal goat anti-HRSV immune serum, followed by a red fluorescently labeled secondary anti-goat antibody. Nuclei were stained with DAPI (blue color). Confocal images were recorded with a Zeiss SP5 confocal microscope. The upper row of each figure shows the overlay of the red (anti-HRSV), green (indicated serum or antibodies) and the blue (DAPI) signal. The scale bars in the right lower corner of the overlay images indicate 20 μm. D Quantification of virion-associated immunoreactivity of F, SHe-KLH, and KLH mouse IgG on cell-attached HRSV A2 virus particles. This quantification is based on image analysis of multiple micrographs obtained with the attachment and immunostaining experiment shown in (B). The graph shows for each cell-attached virion the ratio of a/b, with ‘a’ being the total pixel intensities of either bound HRSV F-specific monoclonal IgG antibodies (F mAb), SHe-KLH or KLH immune serum IgG and with ‘b’ being the total pixel intensities of bound goat anti-HRSV IgG antibodies. The virions were identified as anti-RSV IgG-positive regions. Multiples images were used for this analysis. Horizontal bars represent the median.
    Hrsv A2, supplied by ATCC, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ATCC human rsv a2
    Co-infection elicited reduction in the number of CD11b or CD11c myeloid cells. Six groups of BALB/c mice were intranasally treated with 32 μl of normal saline (uninfected), <t>RSV</t> A2 (RSV(R)), Pr/H3N2 (IAV (I)) or co-treated simultaneously (I + R) or one after the other at 24 hours interval (IR and RI). On days 2, 4 and 7 splenic cells (n = 5) were processed for flow cytometric analysis. to determine the frequency of NK1.1 gated on CD3 negative cells. (A) Proportion of CD11b myeloid cells, which was significantly low in the I + R group on day 2 was increased by day 7. (B) Proportions of CD11c myeloid cells were lower in I + R group compared to other groups on days 2 and 7. Unless indicated by lines, a vs uninfected, b vs IAV (I), c vs RSV (R), d vs IR, e vs RI. No data was available for IR and RI as well as for IAV (I), RSV(R) and I + R groups on day 2 and day 4 respectively.
    Human Rsv A2, supplied by ATCC, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC f prausnitzii strains
    a , Hierarchical clustering of F. <t>prausnitzii</t> strains and type strains of other species of the family Ruminococcaceae based on the gene orthologues content. Orthologous protein products were grouped using OrthoMCL. Clustering performed with Euclidean distances using Ward.D2 algorithm. F. prausnitzii clade highlighted in red. Clustering identified 245 non-paralogous single copy genes constituting the core genome of the family. b , Maximum-likelihood phylogenetic tree of the family Ruminococcaceae based on concatenated alignments of 245 highly conserved proteins. Phylogeny inference done with PROTGAMMABLOSUM62 model, 100 bootstrap replicates. Phylogroups I, IIa, and IIb highlighted in red, purple and light blue respectively
    F Prausnitzii Strains, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    IL-6 promotes resolution of RSV-mediated disease. 8 week old BALB/c mice were infected with 8 x 10 5 ffu of RSV A2 i.n. (A) IL-6 was measured by ELISA in the BAL, lung tissue and serum. (B-H) RSV infected mice were given 0.5 mg of either HRPN (IgG 1 ) or MP5-20F3 (αIL-6) i.p. on day -1 p.i. and 0.25 mg i.p. every other day after that. (B) The percentage of weight on day 0 was measured daily, area under the curve (AUC) was used to test statistical significance, (C) Albumin was measured in the BAL by ELISA. (D) The number of BAL cells was counted and (E) lymphocytes and (F) Neutrophil (PMN) frequencies were determined by H E staining. (G) Lung neutrophils (defined as Ly6G + CD11b + CD90 - CD19 - autofluorescence - ) were enumerated by flow cytometry. (H) Viral load in the lungs was determined by focus forming assay. For (A) * represents BAL, # represents lung and † represents serum. Data is representative of n = 2 independent repeats of n = 5 mice per time point except B which represents 20 mice per group.

    Journal: PLoS Pathogens

    Article Title: Early IL-6 signalling promotes IL-27 dependent maturation of regulatory T cells in the lungs and resolution of viral immunopathology

    doi: 10.1371/journal.ppat.1006640

    Figure Lengend Snippet: IL-6 promotes resolution of RSV-mediated disease. 8 week old BALB/c mice were infected with 8 x 10 5 ffu of RSV A2 i.n. (A) IL-6 was measured by ELISA in the BAL, lung tissue and serum. (B-H) RSV infected mice were given 0.5 mg of either HRPN (IgG 1 ) or MP5-20F3 (αIL-6) i.p. on day -1 p.i. and 0.25 mg i.p. every other day after that. (B) The percentage of weight on day 0 was measured daily, area under the curve (AUC) was used to test statistical significance, (C) Albumin was measured in the BAL by ELISA. (D) The number of BAL cells was counted and (E) lymphocytes and (F) Neutrophil (PMN) frequencies were determined by H E staining. (G) Lung neutrophils (defined as Ly6G + CD11b + CD90 - CD19 - autofluorescence - ) were enumerated by flow cytometry. (H) Viral load in the lungs was determined by focus forming assay. For (A) * represents BAL, # represents lung and † represents serum. Data is representative of n = 2 independent repeats of n = 5 mice per time point except B which represents 20 mice per group.

    Article Snippet: Plaque purified RSV A2 (obtained from ATCC) was grown in HEp-2 cells, and viral titre determined by focus forming assay as described [ ].

    Techniques: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Cytometry, Focus Forming Assay

    An IL-6/IL-27 dependent pathway matures regulatory T cells after RSV infection. 8 week old BALB/c female mice were infected with 8 x 10 5 ffu of RSV A2 and dosed with either αIL-6 or isotype control antibody i.p. between days -1 and 3 p.i. (A) Treg expression of KLRG1 and Neuropilin alongside (B) CTLA-4 and GITR in the airways and (C) Ki67 expression in the lungs. (D) CD4 + GITR + CD25 + Tregs were FACS isolated from BAL and lungs at day 4 p.i. and co-cultured in increasing concentrations with proliferation dye stained, activated naïve splenic CD4 + T cells for 5 days. Proliferation of these “effector” T cells, relative to effector CD4 T cells cultured without Tregs, was then calculated. Representative plots from the 1:4 Treg:Effector cell ratio are depicted. The dilution at which Tregs from each condition significantly suppressed effector cells compared to effector cells on their own is shown in brackets in the legend. (E) CD25 + splenic Tregs were activated in vitro with αCD3/28 in the presence or absence of 50 ng/ml of rIL-27 and KLRG1 and GITR expression were measured 48 hours later. (A-D) Data represents n = 6 mice pooled from 2 independent repeats. (E) represents 5 mice from 3 independent experiments.

    Journal: PLoS Pathogens

    Article Title: Early IL-6 signalling promotes IL-27 dependent maturation of regulatory T cells in the lungs and resolution of viral immunopathology

    doi: 10.1371/journal.ppat.1006640

    Figure Lengend Snippet: An IL-6/IL-27 dependent pathway matures regulatory T cells after RSV infection. 8 week old BALB/c female mice were infected with 8 x 10 5 ffu of RSV A2 and dosed with either αIL-6 or isotype control antibody i.p. between days -1 and 3 p.i. (A) Treg expression of KLRG1 and Neuropilin alongside (B) CTLA-4 and GITR in the airways and (C) Ki67 expression in the lungs. (D) CD4 + GITR + CD25 + Tregs were FACS isolated from BAL and lungs at day 4 p.i. and co-cultured in increasing concentrations with proliferation dye stained, activated naïve splenic CD4 + T cells for 5 days. Proliferation of these “effector” T cells, relative to effector CD4 T cells cultured without Tregs, was then calculated. Representative plots from the 1:4 Treg:Effector cell ratio are depicted. The dilution at which Tregs from each condition significantly suppressed effector cells compared to effector cells on their own is shown in brackets in the legend. (E) CD25 + splenic Tregs were activated in vitro with αCD3/28 in the presence or absence of 50 ng/ml of rIL-27 and KLRG1 and GITR expression were measured 48 hours later. (A-D) Data represents n = 6 mice pooled from 2 independent repeats. (E) represents 5 mice from 3 independent experiments.

    Article Snippet: Plaque purified RSV A2 (obtained from ATCC) was grown in HEp-2 cells, and viral titre determined by focus forming assay as described [ ].

    Techniques: Infection, Mouse Assay, Expressing, FACS, Isolation, Cell Culture, Staining, In Vitro

    IL-27 acts independently of IL-10R signalling to regulate weight loss and T cell responses to RSV. 8 week old BALB/c mice were dosed as in Fig 7 , and in addition were dosed with αIL-10R i.p. on days -1, 2, 5 and 8 p.i. and i.n. on day 3 p.i. or isotype control. Mice were then infected with 2 x 10 5 ffu of RSV A2 on day 0. (A) Schematic of dosing and weight change over time following infection. At day 10 p.i. the (B) total lung cell counts, (C) representative flow plots and enumeration of RSV tetramer specific CD8 + T cells and antigen experienced CD4 + T cells in the lungs and (D) IFN-γ + and TNF + CD8 and CD4 T cells in the lungs following RSV peptide stimulation were determined. Data represents n = 5 mice in each group and representative of 2 independent experiments.

    Journal: PLoS Pathogens

    Article Title: Early IL-6 signalling promotes IL-27 dependent maturation of regulatory T cells in the lungs and resolution of viral immunopathology

    doi: 10.1371/journal.ppat.1006640

    Figure Lengend Snippet: IL-27 acts independently of IL-10R signalling to regulate weight loss and T cell responses to RSV. 8 week old BALB/c mice were dosed as in Fig 7 , and in addition were dosed with αIL-10R i.p. on days -1, 2, 5 and 8 p.i. and i.n. on day 3 p.i. or isotype control. Mice were then infected with 2 x 10 5 ffu of RSV A2 on day 0. (A) Schematic of dosing and weight change over time following infection. At day 10 p.i. the (B) total lung cell counts, (C) representative flow plots and enumeration of RSV tetramer specific CD8 + T cells and antigen experienced CD4 + T cells in the lungs and (D) IFN-γ + and TNF + CD8 and CD4 T cells in the lungs following RSV peptide stimulation were determined. Data represents n = 5 mice in each group and representative of 2 independent experiments.

    Article Snippet: Plaque purified RSV A2 (obtained from ATCC) was grown in HEp-2 cells, and viral titre determined by focus forming assay as described [ ].

    Techniques: Mouse Assay, Infection, Flow Cytometry

    IL-6 depletion results in enhanced virus specific T cell responses. 8 week old BALB/c mice were infected with 8 x 10 5 ffu of RSV A2 i.n. and given 0.5 mg of either HRPN (IgG 1 ) or MP5-20F3 (αIL-6) i.p. on day -1 p.i. and 0.25 mg i.p. every other day after that. Mice were euthanized at days 4, 7 and 14 p.i. Flow cytometry was used to determine (A) the number of K b M2 82-90 + CD8 + T cells, (B) the proportion of IFN-γ + and TNF + lung CD8 + T cells following M2 82-90 stimulation, (C) the number of CD11a + CD49d + CD4 + T cells and (D-F) the proportion of IFN-γ + at different days p.i., and TNF + , IL-2 + , IL-17 + , IL-13 + and IL-4 + CD4 + T cells at day 7 p.i. following stimulation with RSV F 51-66 , P 39-55 and G 181-197 peptides ex vivo in the lungs and lymph nodes. (G) IFN-γ and IL-17 were measured in lung tissue at day 7 p.i. by ELISA. Representative FACS plots are lungs at day 7 p.i.. Data is representative of n = 2 independent repeats of n = 5 mice per time point.

    Journal: PLoS Pathogens

    Article Title: Early IL-6 signalling promotes IL-27 dependent maturation of regulatory T cells in the lungs and resolution of viral immunopathology

    doi: 10.1371/journal.ppat.1006640

    Figure Lengend Snippet: IL-6 depletion results in enhanced virus specific T cell responses. 8 week old BALB/c mice were infected with 8 x 10 5 ffu of RSV A2 i.n. and given 0.5 mg of either HRPN (IgG 1 ) or MP5-20F3 (αIL-6) i.p. on day -1 p.i. and 0.25 mg i.p. every other day after that. Mice were euthanized at days 4, 7 and 14 p.i. Flow cytometry was used to determine (A) the number of K b M2 82-90 + CD8 + T cells, (B) the proportion of IFN-γ + and TNF + lung CD8 + T cells following M2 82-90 stimulation, (C) the number of CD11a + CD49d + CD4 + T cells and (D-F) the proportion of IFN-γ + at different days p.i., and TNF + , IL-2 + , IL-17 + , IL-13 + and IL-4 + CD4 + T cells at day 7 p.i. following stimulation with RSV F 51-66 , P 39-55 and G 181-197 peptides ex vivo in the lungs and lymph nodes. (G) IFN-γ and IL-17 were measured in lung tissue at day 7 p.i. by ELISA. Representative FACS plots are lungs at day 7 p.i.. Data is representative of n = 2 independent repeats of n = 5 mice per time point.

    Article Snippet: Plaque purified RSV A2 (obtained from ATCC) was grown in HEp-2 cells, and viral titre determined by focus forming assay as described [ ].

    Techniques: Mouse Assay, Infection, Flow Cytometry, Cytometry, Ex Vivo, Enzyme-linked Immunosorbent Assay, FACS

    Airway IL-27 promotes virus specific T cell suppression in respiratory viral infection. 8 week old BALB/c mice were infected with 8 x 10 5 ffu of RSV A2 and dosed with either αIL-27 or isotype control antibody i.n. between days -1 and 3 p.i. (A) Weight was measured daily. (B-H) At day 10 p.i. mice were euthanized and (B) Lung virus specific CD8 and CD4 T cell responses, (C-D) airway virus specific production of IFN-γ, TNF and IL-10 by CD4 and/or CD8 T cells, (E) IL-17A + CD4 + T cells following polyclonal stimulation, (F) airway and (G) lung Treg numbers and expression of KLRG1 + and IL-10 and (H) T-bet expression by lung Tregs, KLRG1 + Tregs and natural killer (NK) cells were all measured. For Treg IL-10 measurements cells were stimulated with PMA/I in the presence of BFA prior to intracellular staining. Data represents n = 5 mice per group and is representative of n = 2 independent experiments.

    Journal: PLoS Pathogens

    Article Title: Early IL-6 signalling promotes IL-27 dependent maturation of regulatory T cells in the lungs and resolution of viral immunopathology

    doi: 10.1371/journal.ppat.1006640

    Figure Lengend Snippet: Airway IL-27 promotes virus specific T cell suppression in respiratory viral infection. 8 week old BALB/c mice were infected with 8 x 10 5 ffu of RSV A2 and dosed with either αIL-27 or isotype control antibody i.n. between days -1 and 3 p.i. (A) Weight was measured daily. (B-H) At day 10 p.i. mice were euthanized and (B) Lung virus specific CD8 and CD4 T cell responses, (C-D) airway virus specific production of IFN-γ, TNF and IL-10 by CD4 and/or CD8 T cells, (E) IL-17A + CD4 + T cells following polyclonal stimulation, (F) airway and (G) lung Treg numbers and expression of KLRG1 + and IL-10 and (H) T-bet expression by lung Tregs, KLRG1 + Tregs and natural killer (NK) cells were all measured. For Treg IL-10 measurements cells were stimulated with PMA/I in the presence of BFA prior to intracellular staining. Data represents n = 5 mice per group and is representative of n = 2 independent experiments.

    Article Snippet: Plaque purified RSV A2 (obtained from ATCC) was grown in HEp-2 cells, and viral titre determined by focus forming assay as described [ ].

    Techniques: Infection, Mouse Assay, Expressing, Staining

    IL-27 promotes IL-6 dependent resolution of RSV disease. 8 week old BALB/c female mice were infected with 8 x 10 5 ffu of RSV A2 and dosed with either αIL-6 or isotype control antibody i.p. between days -1 and 3 p.i. (A) Mice received either rIL-27 or PBS i.n. on days 1–4 p.i. and were weighed until day 10 p.i. (B) Representative H E staining of lung tissue using a 10X objective at day 10 p.i., black bar represents 200 μm. (C) virus specific CD4 + and CD8 + T cells in the lung were enumerated at days 4 and 10 p.i. (D) At day 10 p.i. IFN-γ + CD4 and CD8 T cells in the lungs following RSV peptide simulation and (E) IFN-γ and IL-17A in lung homogenate were determined. At day 4 p.i. (F) Lung viral load, (G) IL-10 in the airways, (H) IL-10 + Tr1 cells and (I) Foxp3 + Tregs in the BAL, lungs and lung draining lymph nodes were determined. (A-E) Data representative of n = 5 mice per group from 2 independent experiments. (F) Represents n = 10 mice per group combined from 2 independent repeats. (G-I) Data represents n = 6 mice pooled from 2 independent repeats.

    Journal: PLoS Pathogens

    Article Title: Early IL-6 signalling promotes IL-27 dependent maturation of regulatory T cells in the lungs and resolution of viral immunopathology

    doi: 10.1371/journal.ppat.1006640

    Figure Lengend Snippet: IL-27 promotes IL-6 dependent resolution of RSV disease. 8 week old BALB/c female mice were infected with 8 x 10 5 ffu of RSV A2 and dosed with either αIL-6 or isotype control antibody i.p. between days -1 and 3 p.i. (A) Mice received either rIL-27 or PBS i.n. on days 1–4 p.i. and were weighed until day 10 p.i. (B) Representative H E staining of lung tissue using a 10X objective at day 10 p.i., black bar represents 200 μm. (C) virus specific CD4 + and CD8 + T cells in the lung were enumerated at days 4 and 10 p.i. (D) At day 10 p.i. IFN-γ + CD4 and CD8 T cells in the lungs following RSV peptide simulation and (E) IFN-γ and IL-17A in lung homogenate were determined. At day 4 p.i. (F) Lung viral load, (G) IL-10 in the airways, (H) IL-10 + Tr1 cells and (I) Foxp3 + Tregs in the BAL, lungs and lung draining lymph nodes were determined. (A-E) Data representative of n = 5 mice per group from 2 independent experiments. (F) Represents n = 10 mice per group combined from 2 independent repeats. (G-I) Data represents n = 6 mice pooled from 2 independent repeats.

    Article Snippet: Plaque purified RSV A2 (obtained from ATCC) was grown in HEp-2 cells, and viral titre determined by focus forming assay as described [ ].

    Techniques: Mouse Assay, Infection, Staining

    Early, but not late, IL-6 signalling is required for the resolution of RSV induced immunopathology. 8 week old BALB/c female mice were infected with 8 x 10 5 ffu of RSV A2 i.n. and dosed with either αIL-6 or isotype control antibody as shown in A. (B) Weight loss was monitored daily, area under the curve (AUC) was used to test statistical significance. (C) At day 14 p.i. airway albumin was measured by ELISA. (D-G) At the same timepoint virus specific CD8 + T cells (D), antigen experienced CD4 + T cells (E), virus specific IFN-γ + CD4 + T cells (F) and the proportion of those cells that were IL-10 + (G) were determined in the lungs by flow cytometry. A-E represent n = 10 mice per group from 2 independent experiments. F and G are n = 5 mice per group and are representative of 2 independent experiments.

    Journal: PLoS Pathogens

    Article Title: Early IL-6 signalling promotes IL-27 dependent maturation of regulatory T cells in the lungs and resolution of viral immunopathology

    doi: 10.1371/journal.ppat.1006640

    Figure Lengend Snippet: Early, but not late, IL-6 signalling is required for the resolution of RSV induced immunopathology. 8 week old BALB/c female mice were infected with 8 x 10 5 ffu of RSV A2 i.n. and dosed with either αIL-6 or isotype control antibody as shown in A. (B) Weight loss was monitored daily, area under the curve (AUC) was used to test statistical significance. (C) At day 14 p.i. airway albumin was measured by ELISA. (D-G) At the same timepoint virus specific CD8 + T cells (D), antigen experienced CD4 + T cells (E), virus specific IFN-γ + CD4 + T cells (F) and the proportion of those cells that were IL-10 + (G) were determined in the lungs by flow cytometry. A-E represent n = 10 mice per group from 2 independent experiments. F and G are n = 5 mice per group and are representative of 2 independent experiments.

    Article Snippet: Plaque purified RSV A2 (obtained from ATCC) was grown in HEp-2 cells, and viral titre determined by focus forming assay as described [ ].

    Techniques: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    IL-6 promotes the maturation of regulatory CD4 + T cells in the lungs. 8 week old BALB/c mice were infected with 8 x 10 5 ffu of RSV A2 i.n. and given 0.5 mg of either HRPN (IgG 1 ) or MP5-20F3 (αIL-6) i.p. on day -1 p.i. and 0.25 mg i.p. every other day after that. (A) IL-10 was measured in the airways and lungs at multiple timepoints post infection by ELISA. (B-E) At day 7 p.i. (B) The frequency of IL-10 + in lung IFN-γ + CD4 + T cells at day 7 p.i. was determined after RSV F 51-66 , P 39-55 and G 181-197 peptide stimulation. (C) The frequency of lung Foxp3 + CD4 + T cells. (D) The proportion and number of IL-10 + after PMA and ionomycin stimulation and (E) KLRG1 + Tregs in the lung. Data is representative of n = 2 independent repeats of n = 5 mice per time point.

    Journal: PLoS Pathogens

    Article Title: Early IL-6 signalling promotes IL-27 dependent maturation of regulatory T cells in the lungs and resolution of viral immunopathology

    doi: 10.1371/journal.ppat.1006640

    Figure Lengend Snippet: IL-6 promotes the maturation of regulatory CD4 + T cells in the lungs. 8 week old BALB/c mice were infected with 8 x 10 5 ffu of RSV A2 i.n. and given 0.5 mg of either HRPN (IgG 1 ) or MP5-20F3 (αIL-6) i.p. on day -1 p.i. and 0.25 mg i.p. every other day after that. (A) IL-10 was measured in the airways and lungs at multiple timepoints post infection by ELISA. (B-E) At day 7 p.i. (B) The frequency of IL-10 + in lung IFN-γ + CD4 + T cells at day 7 p.i. was determined after RSV F 51-66 , P 39-55 and G 181-197 peptide stimulation. (C) The frequency of lung Foxp3 + CD4 + T cells. (D) The proportion and number of IL-10 + after PMA and ionomycin stimulation and (E) KLRG1 + Tregs in the lung. Data is representative of n = 2 independent repeats of n = 5 mice per time point.

    Article Snippet: Plaque purified RSV A2 (obtained from ATCC) was grown in HEp-2 cells, and viral titre determined by focus forming assay as described [ ].

    Techniques: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay

    IL-6 upregulates IL-27 production in myeloid cells after viral exposure. (A-C) 8 week old BALB/c female mice were infected with 8 x 10 5 ffu of RSV A2 and dosed with either αIL-6 or isotype control antibody i.p. between days -1 and 3 p.i. (A) IL-27 in the BAL and lungs after infection. (B) IL-27 + , IL-6 + and TNF + alveolar macrophages in the BAL. (C) IL-27 + neutrophils, Ly6C + monocytes, CD11b + and CD11b - DCs in the lungs. Data is n = 5 mice per group and representative of 2 independent experiments. (D) IL-6 and IL-27 concentrations in the supernatant from primary murine alveolar macrophages at 24 hours p.i. with RSV A2 in the presence of 50 ng/ml rIL-6 or blocking αIL-6R. Data represent n = 3 repeats per condition and are representative of 3 independent experiments. (E) At day 4 p.i. BAL alveolar macrophages (Siglec F + CD11c + AF + ) were FACS isolated from RSV infected mice and expression of Il6 , Il27p28 and Il10 relative to Gapdh determined by RT-qPCR. (F) 8 week old BALB/c female mice were infected with 8 x 10 5 ffu of RSV A2 i.n. and given either αIL-6R or isotype control antibody on day -1 p.i. At day 4 p.i. IL-6, IL-27, IL-10 and IFN-γ were measured in the airways by ELISA. Dotted lines on the graphs represent the concentrations observed in uninfected mice. Data except E is n = 5 mice per group, and representative of 2 independent repeats. F is from n = 6 mice combined from 2 independent repeats.

    Journal: PLoS Pathogens

    Article Title: Early IL-6 signalling promotes IL-27 dependent maturation of regulatory T cells in the lungs and resolution of viral immunopathology

    doi: 10.1371/journal.ppat.1006640

    Figure Lengend Snippet: IL-6 upregulates IL-27 production in myeloid cells after viral exposure. (A-C) 8 week old BALB/c female mice were infected with 8 x 10 5 ffu of RSV A2 and dosed with either αIL-6 or isotype control antibody i.p. between days -1 and 3 p.i. (A) IL-27 in the BAL and lungs after infection. (B) IL-27 + , IL-6 + and TNF + alveolar macrophages in the BAL. (C) IL-27 + neutrophils, Ly6C + monocytes, CD11b + and CD11b - DCs in the lungs. Data is n = 5 mice per group and representative of 2 independent experiments. (D) IL-6 and IL-27 concentrations in the supernatant from primary murine alveolar macrophages at 24 hours p.i. with RSV A2 in the presence of 50 ng/ml rIL-6 or blocking αIL-6R. Data represent n = 3 repeats per condition and are representative of 3 independent experiments. (E) At day 4 p.i. BAL alveolar macrophages (Siglec F + CD11c + AF + ) were FACS isolated from RSV infected mice and expression of Il6 , Il27p28 and Il10 relative to Gapdh determined by RT-qPCR. (F) 8 week old BALB/c female mice were infected with 8 x 10 5 ffu of RSV A2 i.n. and given either αIL-6R or isotype control antibody on day -1 p.i. At day 4 p.i. IL-6, IL-27, IL-10 and IFN-γ were measured in the airways by ELISA. Dotted lines on the graphs represent the concentrations observed in uninfected mice. Data except E is n = 5 mice per group, and representative of 2 independent repeats. F is from n = 6 mice combined from 2 independent repeats.

    Article Snippet: Plaque purified RSV A2 (obtained from ATCC) was grown in HEp-2 cells, and viral titre determined by focus forming assay as described [ ].

    Techniques: Mouse Assay, Infection, Blocking Assay, FACS, Isolation, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    SHe-specific IgG binds to HRSV-infected cells A–C A549 cells were infected overnight with 0.5 MOI HRSV A2 (A), mock-infected (B). In parallel, A549 cells were incubated with 5 MOI HRSV A2 at 4°C for 2 h to allow virus attachment without cell entry (C). After infection or virus attachment, the cells were fixed and stained with SHe-KLH mouse immune serum, KLH mouse immune serum, or a HRSV F-specific mouse monoclonal antibody. Reactivity of mouse IgG was revealed with a green fluorescently labeled secondary antibody. In addition, cells were stained with a polyclonal goat anti-HRSV immune serum, followed by a red fluorescently labeled secondary anti-goat antibody. Nuclei were stained with DAPI (blue color). Confocal images were recorded with a Zeiss SP5 confocal microscope. The upper row of each figure shows the overlay of the red (anti-HRSV), green (indicated serum or antibodies) and the blue (DAPI) signal. The scale bars in the right lower corner of the overlay images indicate 20 μm. D Quantification of virion-associated immunoreactivity of F, SHe-KLH, and KLH mouse IgG on cell-attached HRSV A2 virus particles. This quantification is based on image analysis of multiple micrographs obtained with the attachment and immunostaining experiment shown in (B). The graph shows for each cell-attached virion the ratio of a/b, with ‘a’ being the total pixel intensities of either bound HRSV F-specific monoclonal IgG antibodies (F mAb), SHe-KLH or KLH immune serum IgG and with ‘b’ being the total pixel intensities of bound goat anti-HRSV IgG antibodies. The virions were identified as anti-RSV IgG-positive regions. Multiples images were used for this analysis. Horizontal bars represent the median.

    Journal: EMBO Molecular Medicine

    Article Title: Protection and mechanism of action of a novel human respiratory syncytial virus vaccine candidate based on the extracellular domain of small hydrophobic protein

    doi: 10.15252/emmm.201404005

    Figure Lengend Snippet: SHe-specific IgG binds to HRSV-infected cells A–C A549 cells were infected overnight with 0.5 MOI HRSV A2 (A), mock-infected (B). In parallel, A549 cells were incubated with 5 MOI HRSV A2 at 4°C for 2 h to allow virus attachment without cell entry (C). After infection or virus attachment, the cells were fixed and stained with SHe-KLH mouse immune serum, KLH mouse immune serum, or a HRSV F-specific mouse monoclonal antibody. Reactivity of mouse IgG was revealed with a green fluorescently labeled secondary antibody. In addition, cells were stained with a polyclonal goat anti-HRSV immune serum, followed by a red fluorescently labeled secondary anti-goat antibody. Nuclei were stained with DAPI (blue color). Confocal images were recorded with a Zeiss SP5 confocal microscope. The upper row of each figure shows the overlay of the red (anti-HRSV), green (indicated serum or antibodies) and the blue (DAPI) signal. The scale bars in the right lower corner of the overlay images indicate 20 μm. D Quantification of virion-associated immunoreactivity of F, SHe-KLH, and KLH mouse IgG on cell-attached HRSV A2 virus particles. This quantification is based on image analysis of multiple micrographs obtained with the attachment and immunostaining experiment shown in (B). The graph shows for each cell-attached virion the ratio of a/b, with ‘a’ being the total pixel intensities of either bound HRSV F-specific monoclonal IgG antibodies (F mAb), SHe-KLH or KLH immune serum IgG and with ‘b’ being the total pixel intensities of bound goat anti-HRSV IgG antibodies. The virions were identified as anti-RSV IgG-positive regions. Multiples images were used for this analysis. Horizontal bars represent the median.

    Article Snippet: HRSV A2 (VR-1540) and HRSV B1 (VR-1580) were obtained from ATCC (ATCC, Rockville).

    Techniques: Infection, Incubation, Staining, Labeling, Microscopy, Immunostaining

    Suppression of HRSV replication by SHe immune serum depends on alveolar macrophages A Schematic overview of the protocol used to investigate the role of alveolar macrophages in SHe-antibody-mediated protection against HRSV. Three days before infection, anesthetized BALB/c mice were treated intranasally with PBS (PBS) or clodronate-loaded liposomes (Cl. Lip.). One day before and one day after HRSV A2 infection, the mice received KLH (KLH) or SHe-KLH immune serum (SHe-KLH) via the intranasal route. On day 5 after challenge with 5 × 10 5 PFU of HRSV A2, mice were sacrificed and the pulmonary viral load was determined by plaque assay. B Treatment with clodronate-loaded liposomes selectively reduces the number of alveolar macrophages in the lungs. Mice were treated with PBS or clodronate-loaded liposomes 3 days before BAL fluid collection. Two days later, these mice were additionally treated with PBS or KLH immune serum. The number and type of cells in the BAL fluid was determined by flow cytometry. The graph represents the number of CD8 + T cells (CD8+), CD4 + T cells (CD4+), neutrophils (neut.), eosinophils (eos.), resident alveolar macrophages (rAM), infiltrating monocytes (Mon.), and dendritic cells (DC) in BAL fluid. PBS/PBS: passive transfer of PBS and treatment with PBS; PBS/Cl. lip.: passive transfer of PBS and treatment with clodronate-loaded liposomes; serum/PBS: passive transfer of KLH immune serum and treatment with PBS; Serum/Cl. lip.: passive transfer of KLH immune serum and treatment with clodronate-loaded liposomes. C Treatment of mice with clodronate-loaded liposomes impairs SHe-immune serum-mediated suppression of HRSV replication. The graph shows the number of PFU per lung of each mouse on day 5 after challenge. Horizontal bars represent the means (one-way ANOVA Dunn's multiple comparisons test). The graph is representative for two independent experiments. D Depletion of alveolar macrophages does not affect body weight upon HRSV infection. The graph shows the relative body weight of each animal on day 5 after infection and horizontal bars depict the mean. E Depletion of alveolar macrophages does not decrease the amount of SHe-specific IgG in the lung. The graph shows the SHe-specific IgG endpoint titers in cleared lung homogenates prepared on day 5 after challenge with HRSV A2, as determined by a SHe peptide ELISA. Horizontal bars represent the mean. F, G BALB/c mice ( n = 16 per group) were immunized three times with KLH or SHe-KLH in combination with incomplete Freund's adjuvant. Immunizations were performed intraperitoneally with 3-week interval. Nineteen days after the last immunization, half of the mice were treated with, respectively, PBS (KLH PBS and SHe-KLH PBS) or clodronate-loaded liposomes (KLH Cl. Lip. and SHe-KLH Cl. Lip.). Three days later, all mice were challenged with 1 × 10 6 PFU RSV A2. Five days after challenge, the lungs were collected for HRSV titration. (F) SHe-specific IgG endpoint titers of sera collected 2 weeks after the last immunization, as tested by SHe peptide ELISA. Horizontal bars represent the mean. (G) The reduction of HRSV replication in SHe-KLH-vaccinated mice depends on alveolar macrophages. The graph shows the number of PFU per lung for each mouse. Horizontal bars represent the means (one-way ANOVA Tukey's multiple comparisons test).

    Journal: EMBO Molecular Medicine

    Article Title: Protection and mechanism of action of a novel human respiratory syncytial virus vaccine candidate based on the extracellular domain of small hydrophobic protein

    doi: 10.15252/emmm.201404005

    Figure Lengend Snippet: Suppression of HRSV replication by SHe immune serum depends on alveolar macrophages A Schematic overview of the protocol used to investigate the role of alveolar macrophages in SHe-antibody-mediated protection against HRSV. Three days before infection, anesthetized BALB/c mice were treated intranasally with PBS (PBS) or clodronate-loaded liposomes (Cl. Lip.). One day before and one day after HRSV A2 infection, the mice received KLH (KLH) or SHe-KLH immune serum (SHe-KLH) via the intranasal route. On day 5 after challenge with 5 × 10 5 PFU of HRSV A2, mice were sacrificed and the pulmonary viral load was determined by plaque assay. B Treatment with clodronate-loaded liposomes selectively reduces the number of alveolar macrophages in the lungs. Mice were treated with PBS or clodronate-loaded liposomes 3 days before BAL fluid collection. Two days later, these mice were additionally treated with PBS or KLH immune serum. The number and type of cells in the BAL fluid was determined by flow cytometry. The graph represents the number of CD8 + T cells (CD8+), CD4 + T cells (CD4+), neutrophils (neut.), eosinophils (eos.), resident alveolar macrophages (rAM), infiltrating monocytes (Mon.), and dendritic cells (DC) in BAL fluid. PBS/PBS: passive transfer of PBS and treatment with PBS; PBS/Cl. lip.: passive transfer of PBS and treatment with clodronate-loaded liposomes; serum/PBS: passive transfer of KLH immune serum and treatment with PBS; Serum/Cl. lip.: passive transfer of KLH immune serum and treatment with clodronate-loaded liposomes. C Treatment of mice with clodronate-loaded liposomes impairs SHe-immune serum-mediated suppression of HRSV replication. The graph shows the number of PFU per lung of each mouse on day 5 after challenge. Horizontal bars represent the means (one-way ANOVA Dunn's multiple comparisons test). The graph is representative for two independent experiments. D Depletion of alveolar macrophages does not affect body weight upon HRSV infection. The graph shows the relative body weight of each animal on day 5 after infection and horizontal bars depict the mean. E Depletion of alveolar macrophages does not decrease the amount of SHe-specific IgG in the lung. The graph shows the SHe-specific IgG endpoint titers in cleared lung homogenates prepared on day 5 after challenge with HRSV A2, as determined by a SHe peptide ELISA. Horizontal bars represent the mean. F, G BALB/c mice ( n = 16 per group) were immunized three times with KLH or SHe-KLH in combination with incomplete Freund's adjuvant. Immunizations were performed intraperitoneally with 3-week interval. Nineteen days after the last immunization, half of the mice were treated with, respectively, PBS (KLH PBS and SHe-KLH PBS) or clodronate-loaded liposomes (KLH Cl. Lip. and SHe-KLH Cl. Lip.). Three days later, all mice were challenged with 1 × 10 6 PFU RSV A2. Five days after challenge, the lungs were collected for HRSV titration. (F) SHe-specific IgG endpoint titers of sera collected 2 weeks after the last immunization, as tested by SHe peptide ELISA. Horizontal bars represent the mean. (G) The reduction of HRSV replication in SHe-KLH-vaccinated mice depends on alveolar macrophages. The graph shows the number of PFU per lung for each mouse. Horizontal bars represent the means (one-way ANOVA Tukey's multiple comparisons test).

    Article Snippet: HRSV A2 (VR-1540) and HRSV B1 (VR-1580) were obtained from ATCC (ATCC, Rockville).

    Techniques: Infection, Mouse Assay, Plaque Assay, Flow Cytometry, Cytometry, Peptide ELISA, Titration

    Human sera with high HRSV-neutralizing activity lack high levels of SHe-specific IgG A–D SHe peptide ELISA of human (A) and mouse (C) sera. Human reference sera to HRSV were obtained from the NIH Biodefense and Emerging Infections Research Resources Repository (NIAID, NIH). Antiserum NR-4020: pooled human reference serum; NR-4021: pooled human sera with high HRSV neutralization titer; NR-4022: pooled human sera with intermediate HRSV neutralization titer: NR-4023: pooled human sera with low HRSV neutralization titer; and NR-21973: human reference Ig to HRSV (1% Ig in PBS). The mouse sera represent pooled pre-immune sera (pre.), pooled immune sera from mice immunized three times intraperitoneally with SHe-KLH or KLH in combination with incomplete Freund's adjuvant, and pooled sera of mice infected with HRSV A2 (HRSV). HRSV-specific ELISA of human (B) and mouse (D) sera. The ELISA plates in (B) and (D) were coated with the supernatant of HRSV A2 infected cells and tested with the sera used in (A) and (C).

    Journal: EMBO Molecular Medicine

    Article Title: Protection and mechanism of action of a novel human respiratory syncytial virus vaccine candidate based on the extracellular domain of small hydrophobic protein

    doi: 10.15252/emmm.201404005

    Figure Lengend Snippet: Human sera with high HRSV-neutralizing activity lack high levels of SHe-specific IgG A–D SHe peptide ELISA of human (A) and mouse (C) sera. Human reference sera to HRSV were obtained from the NIH Biodefense and Emerging Infections Research Resources Repository (NIAID, NIH). Antiserum NR-4020: pooled human reference serum; NR-4021: pooled human sera with high HRSV neutralization titer; NR-4022: pooled human sera with intermediate HRSV neutralization titer: NR-4023: pooled human sera with low HRSV neutralization titer; and NR-21973: human reference Ig to HRSV (1% Ig in PBS). The mouse sera represent pooled pre-immune sera (pre.), pooled immune sera from mice immunized three times intraperitoneally with SHe-KLH or KLH in combination with incomplete Freund's adjuvant, and pooled sera of mice infected with HRSV A2 (HRSV). HRSV-specific ELISA of human (B) and mouse (D) sera. The ELISA plates in (B) and (D) were coated with the supernatant of HRSV A2 infected cells and tested with the sera used in (A) and (C).

    Article Snippet: HRSV A2 (VR-1540) and HRSV B1 (VR-1580) were obtained from ATCC (ATCC, Rockville).

    Techniques: Activity Assay, Peptide ELISA, Neutralization, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay

    Immunization with SHe conjugate vaccine reduces HRSV replication in BALB/c mice SHe-KLH immunization induces SHe-specific serum IgG antibodies. BALB/c mice were immunized 3 times intraperitoneally with 20 μg KLH or SHe-KLH combined with incomplete Freund's adjuvant or with PBS. Serum was collected one day before the first immunization and 20 days after each immunization. Endpoint IgG titers of pooled sera from each group ( n = 6, except PBS group: n = 3) tested in a SHe peptide ELISA. Individual SHe-specific serum IgG titers obtained 20 days after the second boost immunization. Horizontal bars represent the median. SHe-specific serum IgG, IgG1, and IgG2 endpoint titers in pooled sera after the second boost immunization. SHe-KLH immune serum does not neutralize HRSV in vitro . An HRSV A2 in vitro neutralization assay using the indicated dilutions of pooled sera, obtained after the second boost vaccination of BALB/c mice immunized as in (A), was performed. RSV: serum from BALB/c mice that were previously infected with HRSV A2. The amount of viral antigen was quantified by ELISA using goat anti-HRSV immune serum. The graph shows the O.D. for each sample. Vaccination with SHe-KLH reduces HRSV A2 replication in the lungs of challenged BALB/c mice. The graph shows the number of PFU per lung for each mouse, sampled 5 days after challenge with 10 6 PFU of HRSV A2. Horizontal bars depict the median value (Dunn's multiple comparisons test). Challenged SHe-KLH-immunized BALB/c mice do not display weight loss. The graphs are representative for two independent experiments, and horizontal bars depict the mean value.

    Journal: EMBO Molecular Medicine

    Article Title: Protection and mechanism of action of a novel human respiratory syncytial virus vaccine candidate based on the extracellular domain of small hydrophobic protein

    doi: 10.15252/emmm.201404005

    Figure Lengend Snippet: Immunization with SHe conjugate vaccine reduces HRSV replication in BALB/c mice SHe-KLH immunization induces SHe-specific serum IgG antibodies. BALB/c mice were immunized 3 times intraperitoneally with 20 μg KLH or SHe-KLH combined with incomplete Freund's adjuvant or with PBS. Serum was collected one day before the first immunization and 20 days after each immunization. Endpoint IgG titers of pooled sera from each group ( n = 6, except PBS group: n = 3) tested in a SHe peptide ELISA. Individual SHe-specific serum IgG titers obtained 20 days after the second boost immunization. Horizontal bars represent the median. SHe-specific serum IgG, IgG1, and IgG2 endpoint titers in pooled sera after the second boost immunization. SHe-KLH immune serum does not neutralize HRSV in vitro . An HRSV A2 in vitro neutralization assay using the indicated dilutions of pooled sera, obtained after the second boost vaccination of BALB/c mice immunized as in (A), was performed. RSV: serum from BALB/c mice that were previously infected with HRSV A2. The amount of viral antigen was quantified by ELISA using goat anti-HRSV immune serum. The graph shows the O.D. for each sample. Vaccination with SHe-KLH reduces HRSV A2 replication in the lungs of challenged BALB/c mice. The graph shows the number of PFU per lung for each mouse, sampled 5 days after challenge with 10 6 PFU of HRSV A2. Horizontal bars depict the median value (Dunn's multiple comparisons test). Challenged SHe-KLH-immunized BALB/c mice do not display weight loss. The graphs are representative for two independent experiments, and horizontal bars depict the mean value.

    Article Snippet: HRSV A2 (VR-1540) and HRSV B1 (VR-1580) were obtained from ATCC (ATCC, Rockville).

    Techniques: Mouse Assay, Peptide ELISA, In Vitro, Neutralization, Infection, Enzyme-linked Immunosorbent Assay

    Reduction of HRSV replication by SHe immune serum depends on Fcγ receptors I and/or III Schematic overview of the protocol used to investigate the role of Fcγ receptors in SHe-antibody-mediated reduction of HRSV replication. One day before and one day after HRSV A2 infection, wild-type (WT, 6 mice per group) or Fcγ receptor I and III double knockout (FcγR I/III −/− , 5 mice per group) BALB/c mice were treated intranasally with 35 μl of PBS (PBS), KLH (KLH) or SHe-KLH immune serum (SHe-KLH). Five days after challenge with 5 × 10 5 PFU of HRSV A2, the lungs were collected and HRSV A2 titers were determined by plaque assay. Transfer of SHe-KLH immune serum reduces HRSV replication in wild-type, but not in FcγR I/III −/− mice. The graph shows the number of PFU per lung of each mouse, and horizontal bars represent the means (one-way ANOVA Dunn's multiple comparisons test). SHe-specific IgG levels in lung homogenates of wild-type and FcγR I/III −/− mice are comparable. The graph shows the SHe-specific endpoint IgG titer in lung homogenates prepared 5 days after infection of each mouse treated with SHe-KLH immune serum, with horizontal bars representing the mean. The graphs are representative for two independent experiments.

    Journal: EMBO Molecular Medicine

    Article Title: Protection and mechanism of action of a novel human respiratory syncytial virus vaccine candidate based on the extracellular domain of small hydrophobic protein

    doi: 10.15252/emmm.201404005

    Figure Lengend Snippet: Reduction of HRSV replication by SHe immune serum depends on Fcγ receptors I and/or III Schematic overview of the protocol used to investigate the role of Fcγ receptors in SHe-antibody-mediated reduction of HRSV replication. One day before and one day after HRSV A2 infection, wild-type (WT, 6 mice per group) or Fcγ receptor I and III double knockout (FcγR I/III −/− , 5 mice per group) BALB/c mice were treated intranasally with 35 μl of PBS (PBS), KLH (KLH) or SHe-KLH immune serum (SHe-KLH). Five days after challenge with 5 × 10 5 PFU of HRSV A2, the lungs were collected and HRSV A2 titers were determined by plaque assay. Transfer of SHe-KLH immune serum reduces HRSV replication in wild-type, but not in FcγR I/III −/− mice. The graph shows the number of PFU per lung of each mouse, and horizontal bars represent the means (one-way ANOVA Dunn's multiple comparisons test). SHe-specific IgG levels in lung homogenates of wild-type and FcγR I/III −/− mice are comparable. The graph shows the SHe-specific endpoint IgG titer in lung homogenates prepared 5 days after infection of each mouse treated with SHe-KLH immune serum, with horizontal bars representing the mean. The graphs are representative for two independent experiments.

    Article Snippet: HRSV A2 (VR-1540) and HRSV B1 (VR-1580) were obtained from ATCC (ATCC, Rockville).

    Techniques: Infection, Mouse Assay, Double Knockout, Plaque Assay

    SHe immune serum reduces pulmonary HRSV replication and morbidity One day before and one day after HRSV A2 infection, BALB/c mice were treated intranasally with 35 μl of PBS (PBS), KLH (KLH) or SHe-KLH immune serum (SHe-KLH). Five days after challenge with 5 × 10 5 PFU of HRSV A2, the lungs were collected to determine HRSV A2 titers. The graph shows the number of HRSV plaques per lung for each mouse 5 days after infection. Horizontal bars represent the mean (one-way ANOVA Dunn's multiple comparisons test). Passively transferred SHe-KLH immune serum reduces body weight loss in HRSV-challenged mice. One day before and one day after infection with 1 × 10 7 PFU of HRSV A2, BALB/c mice were treated intranasally with 35 μl, KLH, or SHe-KLH immune serum. In addition, one group of mice received serum from mice that had been infected with HRSV. Mice that were not treated and not infected (NI) were used as additional control. The graph represents the average relative (compared to day 0) body weight ± SEM of all mice in each group ( n = 6, two-way ANOVA Tukey's multiple comparisons test, the indicated P -value's concern differences between the KLH and SHe-KLH groups).

    Journal: EMBO Molecular Medicine

    Article Title: Protection and mechanism of action of a novel human respiratory syncytial virus vaccine candidate based on the extracellular domain of small hydrophobic protein

    doi: 10.15252/emmm.201404005

    Figure Lengend Snippet: SHe immune serum reduces pulmonary HRSV replication and morbidity One day before and one day after HRSV A2 infection, BALB/c mice were treated intranasally with 35 μl of PBS (PBS), KLH (KLH) or SHe-KLH immune serum (SHe-KLH). Five days after challenge with 5 × 10 5 PFU of HRSV A2, the lungs were collected to determine HRSV A2 titers. The graph shows the number of HRSV plaques per lung for each mouse 5 days after infection. Horizontal bars represent the mean (one-way ANOVA Dunn's multiple comparisons test). Passively transferred SHe-KLH immune serum reduces body weight loss in HRSV-challenged mice. One day before and one day after infection with 1 × 10 7 PFU of HRSV A2, BALB/c mice were treated intranasally with 35 μl, KLH, or SHe-KLH immune serum. In addition, one group of mice received serum from mice that had been infected with HRSV. Mice that were not treated and not infected (NI) were used as additional control. The graph represents the average relative (compared to day 0) body weight ± SEM of all mice in each group ( n = 6, two-way ANOVA Tukey's multiple comparisons test, the indicated P -value's concern differences between the KLH and SHe-KLH groups).

    Article Snippet: HRSV A2 (VR-1540) and HRSV B1 (VR-1580) were obtained from ATCC (ATCC, Rockville).

    Techniques: Infection, Mouse Assay

    The reduction of HRSV in SHe-KLH-immunized mice is not short living Two groups of 8 BALB/c mice were immunized three times intraperitoneally with KLH or SHe-KLH combined with incomplete Freund's adjuvant (KLH IFA and SHe-KLH IFA). In parallel, two groups of 7 mice were immunized two times intraperitoneally with KLH or SHe-KLH in combination with Sigma Adjuvant System (KLH SAS and SHe-KLH SAS). As a third immunization, the latter two groups received PBS without adjuvant. As negative control, one group of 8 mice was vaccinated with PBS without adjuvant (PBS). All immunizations were performed every 2 weeks. Serum was collected 20 days after each immunization. Six weeks after the last immunization with incomplete Freund's adjuvant and 8 weeks after the last immunization with Sigma Adjuvant System, the mice were challenged with 1 × 10 6 PFU HRSV A2. Six days after challenge, the lungs were collected to determine the pulmonary HRSV titer by plaque assay. SHe-KLH immunization induces SHe-specific serum IgG1 antibodies. The graph shows the SHe-specific IgG1 serum endpoint titers of each mouse at 3 weeks before viral challenge as determined by SHe peptide ELISA. Horizontal bars indicate the mean IgG1 titers. SHe-KLH immunization induces SHe-specific serum IgG2a antibodies. The graph shows the SHe-specific IgG2a serum endpoint titers of each mouse at 3 weeks before viral challenge as determined by SHe peptide ELISA. Horizontal bars indicate the mean IgG2a titers. Vaccination with SHe-KLH reduces HRSV A2 replication in the lungs of challenged BALB/c mice. The graph shows the number of PFU per lung for each mouse, sampled 6 days after challenge with 1 × 10 6 PFU of HRSV A2. Horizontal bars represent the median (one-way ANOVA Dunn's multiple comparisons test). Vaccination with SHe-KLH is not associated with enhanced body weight loss upon HRSV infection. The graph shows the relative body weight at day 6 post-infection calculated as the ratio between body weight at day 6 and body weight at day 0. Horizontal bars represent the median.

    Journal: EMBO Molecular Medicine

    Article Title: Protection and mechanism of action of a novel human respiratory syncytial virus vaccine candidate based on the extracellular domain of small hydrophobic protein

    doi: 10.15252/emmm.201404005

    Figure Lengend Snippet: The reduction of HRSV in SHe-KLH-immunized mice is not short living Two groups of 8 BALB/c mice were immunized three times intraperitoneally with KLH or SHe-KLH combined with incomplete Freund's adjuvant (KLH IFA and SHe-KLH IFA). In parallel, two groups of 7 mice were immunized two times intraperitoneally with KLH or SHe-KLH in combination with Sigma Adjuvant System (KLH SAS and SHe-KLH SAS). As a third immunization, the latter two groups received PBS without adjuvant. As negative control, one group of 8 mice was vaccinated with PBS without adjuvant (PBS). All immunizations were performed every 2 weeks. Serum was collected 20 days after each immunization. Six weeks after the last immunization with incomplete Freund's adjuvant and 8 weeks after the last immunization with Sigma Adjuvant System, the mice were challenged with 1 × 10 6 PFU HRSV A2. Six days after challenge, the lungs were collected to determine the pulmonary HRSV titer by plaque assay. SHe-KLH immunization induces SHe-specific serum IgG1 antibodies. The graph shows the SHe-specific IgG1 serum endpoint titers of each mouse at 3 weeks before viral challenge as determined by SHe peptide ELISA. Horizontal bars indicate the mean IgG1 titers. SHe-KLH immunization induces SHe-specific serum IgG2a antibodies. The graph shows the SHe-specific IgG2a serum endpoint titers of each mouse at 3 weeks before viral challenge as determined by SHe peptide ELISA. Horizontal bars indicate the mean IgG2a titers. Vaccination with SHe-KLH reduces HRSV A2 replication in the lungs of challenged BALB/c mice. The graph shows the number of PFU per lung for each mouse, sampled 6 days after challenge with 1 × 10 6 PFU of HRSV A2. Horizontal bars represent the median (one-way ANOVA Dunn's multiple comparisons test). Vaccination with SHe-KLH is not associated with enhanced body weight loss upon HRSV infection. The graph shows the relative body weight at day 6 post-infection calculated as the ratio between body weight at day 6 and body weight at day 0. Horizontal bars represent the median.

    Article Snippet: HRSV A2 (VR-1540) and HRSV B1 (VR-1580) were obtained from ATCC (ATCC, Rockville).

    Techniques: Mouse Assay, Immunofluorescence, Negative Control, Plaque Assay, Peptide ELISA, Infection

    Co-infection elicited reduction in the number of CD11b or CD11c myeloid cells. Six groups of BALB/c mice were intranasally treated with 32 μl of normal saline (uninfected), RSV A2 (RSV(R)), Pr/H3N2 (IAV (I)) or co-treated simultaneously (I + R) or one after the other at 24 hours interval (IR and RI). On days 2, 4 and 7 splenic cells (n = 5) were processed for flow cytometric analysis. to determine the frequency of NK1.1 gated on CD3 negative cells. (A) Proportion of CD11b myeloid cells, which was significantly low in the I + R group on day 2 was increased by day 7. (B) Proportions of CD11c myeloid cells were lower in I + R group compared to other groups on days 2 and 7. Unless indicated by lines, a vs uninfected, b vs IAV (I), c vs RSV (R), d vs IR, e vs RI. No data was available for IR and RI as well as for IAV (I), RSV(R) and I + R groups on day 2 and day 4 respectively.

    Journal: Heliyon

    Article Title: Virus genes and host correlates of pathology are markedly reduced during respiratory syncytial and influenza virus co-infection in BALB/c mice

    doi: 10.1016/j.heliyon.2018.e01094

    Figure Lengend Snippet: Co-infection elicited reduction in the number of CD11b or CD11c myeloid cells. Six groups of BALB/c mice were intranasally treated with 32 μl of normal saline (uninfected), RSV A2 (RSV(R)), Pr/H3N2 (IAV (I)) or co-treated simultaneously (I + R) or one after the other at 24 hours interval (IR and RI). On days 2, 4 and 7 splenic cells (n = 5) were processed for flow cytometric analysis. to determine the frequency of NK1.1 gated on CD3 negative cells. (A) Proportion of CD11b myeloid cells, which was significantly low in the I + R group on day 2 was increased by day 7. (B) Proportions of CD11c myeloid cells were lower in I + R group compared to other groups on days 2 and 7. Unless indicated by lines, a vs uninfected, b vs IAV (I), c vs RSV (R), d vs IR, e vs RI. No data was available for IR and RI as well as for IAV (I), RSV(R) and I + R groups on day 2 and day 4 respectively.

    Article Snippet: Human RSV A2 (ATCC-1540) was obtained from the National Institute for Biological Standards and Control (NIBSC), Potters Bar, Hertfordshire, EN6 3QG, WHO International Laboratory for Biological Standard, UK Official Medicine Control Laboratory, and was propagated on Hep-2 cell line.

    Techniques: Infection, Mouse Assay, Flow Cytometry

    Co-infection elicited very low numbers of CD8 and CD8+CD25 frequencies during the course of infection. Six groups of BALB/c mice were intranasally treated with 32 μl of normal saline (uninfected), RSV A2 (RSV(R)), Pr/H3N2 (IAV (I)) or co-treated simultaneously (I + R) or one after the other at 24 hours interval (IR and RI). On days 2, 4 and 7 splenic cells (n = 5) were processed for flow cytometric analysis. (A) CD8 T cells proportions were very low in I + R group compared to other groups. (B) The frequency of CD8+CD25 T cells was very low in I + R group compared to other groups. Unless indicated by lines, a vs uninfected, b vs IAV (I), c vs RSV (R), d vs IR, e vs RI. No data was available for IR and RI as well as for IAV (I), RSV(R) and I + R groups on day 2 and day 4 respectively.

    Journal: Heliyon

    Article Title: Virus genes and host correlates of pathology are markedly reduced during respiratory syncytial and influenza virus co-infection in BALB/c mice

    doi: 10.1016/j.heliyon.2018.e01094

    Figure Lengend Snippet: Co-infection elicited very low numbers of CD8 and CD8+CD25 frequencies during the course of infection. Six groups of BALB/c mice were intranasally treated with 32 μl of normal saline (uninfected), RSV A2 (RSV(R)), Pr/H3N2 (IAV (I)) or co-treated simultaneously (I + R) or one after the other at 24 hours interval (IR and RI). On days 2, 4 and 7 splenic cells (n = 5) were processed for flow cytometric analysis. (A) CD8 T cells proportions were very low in I + R group compared to other groups. (B) The frequency of CD8+CD25 T cells was very low in I + R group compared to other groups. Unless indicated by lines, a vs uninfected, b vs IAV (I), c vs RSV (R), d vs IR, e vs RI. No data was available for IR and RI as well as for IAV (I), RSV(R) and I + R groups on day 2 and day 4 respectively.

    Article Snippet: Human RSV A2 (ATCC-1540) was obtained from the National Institute for Biological Standards and Control (NIBSC), Potters Bar, Hertfordshire, EN6 3QG, WHO International Laboratory for Biological Standard, UK Official Medicine Control Laboratory, and was propagated on Hep-2 cell line.

    Techniques: Infection, Mouse Assay, Flow Cytometry

    Co-infection elicited high proportions of NKT cells. Six groups of BALB/c mice were intranasally treated with 32 μl of normal saline (uninfected), RSV A2 (RSV(R)), Pr/H3N2 (IAV (I)) or co-treated simultaneously (I + R) or one after the other at 24 hours interval (IR and RI). On days 2, 4 and 7 splenic cells (n = 5) were processed for flow cytometric analysis. (A) Proportions of NK1.1 cells gated on CD3 on day 2. (B) Frequency of NKT cells on day 4. (C) Frequency of NKT cells on day 7. Unless indicated by lines, a vs uninfected, b vs IAV (I), c vs RSV (R), d vs IR, e vs RI.

    Journal: Heliyon

    Article Title: Virus genes and host correlates of pathology are markedly reduced during respiratory syncytial and influenza virus co-infection in BALB/c mice

    doi: 10.1016/j.heliyon.2018.e01094

    Figure Lengend Snippet: Co-infection elicited high proportions of NKT cells. Six groups of BALB/c mice were intranasally treated with 32 μl of normal saline (uninfected), RSV A2 (RSV(R)), Pr/H3N2 (IAV (I)) or co-treated simultaneously (I + R) or one after the other at 24 hours interval (IR and RI). On days 2, 4 and 7 splenic cells (n = 5) were processed for flow cytometric analysis. (A) Proportions of NK1.1 cells gated on CD3 on day 2. (B) Frequency of NKT cells on day 4. (C) Frequency of NKT cells on day 7. Unless indicated by lines, a vs uninfected, b vs IAV (I), c vs RSV (R), d vs IR, e vs RI.

    Article Snippet: Human RSV A2 (ATCC-1540) was obtained from the National Institute for Biological Standards and Control (NIBSC), Potters Bar, Hertfordshire, EN6 3QG, WHO International Laboratory for Biological Standard, UK Official Medicine Control Laboratory, and was propagated on Hep-2 cell line.

    Techniques: Infection, Mouse Assay, Flow Cytometry

    Co-infection elicited very high numbers of CD4 and CD4+CD25 frequencies towards the later stages of infection. Six groups of BALB/c mice were intranasally treated with 32 μl of normal saline (uninfected), RSV A2 (RSV(R)), Pr/H3N2 (IAV (I)) or co-treated simultaneously (I + R) or one after the other at 24 hours interval (IR and RI). On days 2, 4 and 7 splenic cells (n = 5) were processed for flow cytometric analysis. (A) CD4 T cells proportions were high on days 2 and 7 in I + R group. (B) The frequency of CD4+CD25 T cells was highest in I + R on day 7. Unless indicated by lines, a vs uninfected, b vs IAV (I), c vs RSV (R), d vs IR, e vs RI. No data was available for IR and RI as well as for IAV (I), RSV(R) and I + R groups on day 2 and day 4 respectively.

    Journal: Heliyon

    Article Title: Virus genes and host correlates of pathology are markedly reduced during respiratory syncytial and influenza virus co-infection in BALB/c mice

    doi: 10.1016/j.heliyon.2018.e01094

    Figure Lengend Snippet: Co-infection elicited very high numbers of CD4 and CD4+CD25 frequencies towards the later stages of infection. Six groups of BALB/c mice were intranasally treated with 32 μl of normal saline (uninfected), RSV A2 (RSV(R)), Pr/H3N2 (IAV (I)) or co-treated simultaneously (I + R) or one after the other at 24 hours interval (IR and RI). On days 2, 4 and 7 splenic cells (n = 5) were processed for flow cytometric analysis. (A) CD4 T cells proportions were high on days 2 and 7 in I + R group. (B) The frequency of CD4+CD25 T cells was highest in I + R on day 7. Unless indicated by lines, a vs uninfected, b vs IAV (I), c vs RSV (R), d vs IR, e vs RI. No data was available for IR and RI as well as for IAV (I), RSV(R) and I + R groups on day 2 and day 4 respectively.

    Article Snippet: Human RSV A2 (ATCC-1540) was obtained from the National Institute for Biological Standards and Control (NIBSC), Potters Bar, Hertfordshire, EN6 3QG, WHO International Laboratory for Biological Standard, UK Official Medicine Control Laboratory, and was propagated on Hep-2 cell line.

    Techniques: Infection, Mouse Assay, Flow Cytometry

    Influenza A virus (IAV) M, PB1 and PB2 genes expressions across the experimental groups. Six groups of BALB/c mice were intranasally treated with 32 μl of normal saline (uninfected), RSV A2 (RSV(R)), Pr/H3N2 (IAV (I)) or co-treated simultaneously (I + R) or one after the other at 24 hours interval (IR and RI). (A) On days 2, 4 and 7, mRNA was extracted from lungs (n = 5) for real time PCR analysis of IAV genes. (B) mRNA expression level of M gene (C) mRNA expression level of PB1 (D) mRNA expression level of PB2. IAV M, PB1 and PB2 genes were expressed early on infection in I + R group and this was sustained up until the 4 th day. CT values were normalized with GADPH, mRNA expression level was calculated using 2 −ΔΔCT method and presented as Log10 fold increase relative to uninfected controls. mRNA expressions for IAV M, PB1 and PB2 were not detected in RSV (single virus) infected mice. * vs Day 2 and # vs Day 4 for within group comparison while a vs RI, b vs IR and c vs I + R for comparing between groups of the same day (p

    Journal: Heliyon

    Article Title: Virus genes and host correlates of pathology are markedly reduced during respiratory syncytial and influenza virus co-infection in BALB/c mice

    doi: 10.1016/j.heliyon.2018.e01094

    Figure Lengend Snippet: Influenza A virus (IAV) M, PB1 and PB2 genes expressions across the experimental groups. Six groups of BALB/c mice were intranasally treated with 32 μl of normal saline (uninfected), RSV A2 (RSV(R)), Pr/H3N2 (IAV (I)) or co-treated simultaneously (I + R) or one after the other at 24 hours interval (IR and RI). (A) On days 2, 4 and 7, mRNA was extracted from lungs (n = 5) for real time PCR analysis of IAV genes. (B) mRNA expression level of M gene (C) mRNA expression level of PB1 (D) mRNA expression level of PB2. IAV M, PB1 and PB2 genes were expressed early on infection in I + R group and this was sustained up until the 4 th day. CT values were normalized with GADPH, mRNA expression level was calculated using 2 −ΔΔCT method and presented as Log10 fold increase relative to uninfected controls. mRNA expressions for IAV M, PB1 and PB2 were not detected in RSV (single virus) infected mice. * vs Day 2 and # vs Day 4 for within group comparison while a vs RI, b vs IR and c vs I + R for comparing between groups of the same day (p

    Article Snippet: Human RSV A2 (ATCC-1540) was obtained from the National Institute for Biological Standards and Control (NIBSC), Potters Bar, Hertfordshire, EN6 3QG, WHO International Laboratory for Biological Standard, UK Official Medicine Control Laboratory, and was propagated on Hep-2 cell line.

    Techniques: Mouse Assay, Real-time Polymerase Chain Reaction, Expressing, Infection

    Respiratory syncytial virus (RSV) NS1 and NS2 genes expressions across the experimental groups. Six groups of BALB/c mice were intranasally treated with 32 μl of normal saline (uninfected), RSV A2 (RSV(R)), Pr/H3N2 (IAV (I)) or co-treated simultaneously (I + R) or one after the other at 24 hours interval (IR and RI). On days 2, 4 and 7, mRNA was extracted from lungs (n = 5) for real time PCR analysis of RSV genes. (A) mRNA expression level of NS1 gene (B) mRNA expression level of NS2. RSV NS1 and NS2 expressions were downregulated by day 7 in I + R group. CT values were normalized with GADPH, mRNA expression level was calculated using 2 −ΔΔCT method and presented as Log10 fold increase relative to uninfected controls. mRNA expressions for RSV NS1 and NS2 were not detected in IAV (single virus) infected mice. * vs Day 2 and # vs Day 4 for within group comparison while a vs RI, b vs IR and c vs I + R for comparing between groups of the same day (p

    Journal: Heliyon

    Article Title: Virus genes and host correlates of pathology are markedly reduced during respiratory syncytial and influenza virus co-infection in BALB/c mice

    doi: 10.1016/j.heliyon.2018.e01094

    Figure Lengend Snippet: Respiratory syncytial virus (RSV) NS1 and NS2 genes expressions across the experimental groups. Six groups of BALB/c mice were intranasally treated with 32 μl of normal saline (uninfected), RSV A2 (RSV(R)), Pr/H3N2 (IAV (I)) or co-treated simultaneously (I + R) or one after the other at 24 hours interval (IR and RI). On days 2, 4 and 7, mRNA was extracted from lungs (n = 5) for real time PCR analysis of RSV genes. (A) mRNA expression level of NS1 gene (B) mRNA expression level of NS2. RSV NS1 and NS2 expressions were downregulated by day 7 in I + R group. CT values were normalized with GADPH, mRNA expression level was calculated using 2 −ΔΔCT method and presented as Log10 fold increase relative to uninfected controls. mRNA expressions for RSV NS1 and NS2 were not detected in IAV (single virus) infected mice. * vs Day 2 and # vs Day 4 for within group comparison while a vs RI, b vs IR and c vs I + R for comparing between groups of the same day (p

    Article Snippet: Human RSV A2 (ATCC-1540) was obtained from the National Institute for Biological Standards and Control (NIBSC), Potters Bar, Hertfordshire, EN6 3QG, WHO International Laboratory for Biological Standard, UK Official Medicine Control Laboratory, and was propagated on Hep-2 cell line.

    Techniques: Mouse Assay, Real-time Polymerase Chain Reaction, Expressing, Infection

    Respiratory syncytial virus (RSV) F, G and M genes expressions across the experimental groups. Six groups of BALB/c mice were intranasally treated with 32 μl of normal saline (uninfected), RSV A2 (RSV(R)), Pr/H3N2 (IAV (I)) or co-treated simultaneously (I + R) or one after the other at 24 hours interval (IR and RI). On days 2, 4 and 7, mRNA was extracted from lungs (n = 5) for real time PCR analysis of RSV genes. (A) mRNA expression level of F gene (B) mRNA expression level of M (C) mRNA expression level of G. RSV F and G expression were markedly downregulated in I + R group by day 7 while RSV M expression was very low throughout the infection period in I + R group. CT values were normalized with GADPH, mRNA expression level was calculated using 2 −ΔΔCT method and presented as Log10 fold increase relative to uninfected controls. mRNA expressions for RSV F, M and G were not detected in IAV (single virus) infected mice. * vs Day 2 and # vs Day 4 for within group comparison while a vs RI, b vs IR and c vs I + R for comparing between groups of the same day (p

    Journal: Heliyon

    Article Title: Virus genes and host correlates of pathology are markedly reduced during respiratory syncytial and influenza virus co-infection in BALB/c mice

    doi: 10.1016/j.heliyon.2018.e01094

    Figure Lengend Snippet: Respiratory syncytial virus (RSV) F, G and M genes expressions across the experimental groups. Six groups of BALB/c mice were intranasally treated with 32 μl of normal saline (uninfected), RSV A2 (RSV(R)), Pr/H3N2 (IAV (I)) or co-treated simultaneously (I + R) or one after the other at 24 hours interval (IR and RI). On days 2, 4 and 7, mRNA was extracted from lungs (n = 5) for real time PCR analysis of RSV genes. (A) mRNA expression level of F gene (B) mRNA expression level of M (C) mRNA expression level of G. RSV F and G expression were markedly downregulated in I + R group by day 7 while RSV M expression was very low throughout the infection period in I + R group. CT values were normalized with GADPH, mRNA expression level was calculated using 2 −ΔΔCT method and presented as Log10 fold increase relative to uninfected controls. mRNA expressions for RSV F, M and G were not detected in IAV (single virus) infected mice. * vs Day 2 and # vs Day 4 for within group comparison while a vs RI, b vs IR and c vs I + R for comparing between groups of the same day (p

    Article Snippet: Human RSV A2 (ATCC-1540) was obtained from the National Institute for Biological Standards and Control (NIBSC), Potters Bar, Hertfordshire, EN6 3QG, WHO International Laboratory for Biological Standard, UK Official Medicine Control Laboratory, and was propagated on Hep-2 cell line.

    Techniques: Mouse Assay, Real-time Polymerase Chain Reaction, Expressing, Infection

    a , Hierarchical clustering of F. prausnitzii strains and type strains of other species of the family Ruminococcaceae based on the gene orthologues content. Orthologous protein products were grouped using OrthoMCL. Clustering performed with Euclidean distances using Ward.D2 algorithm. F. prausnitzii clade highlighted in red. Clustering identified 245 non-paralogous single copy genes constituting the core genome of the family. b , Maximum-likelihood phylogenetic tree of the family Ruminococcaceae based on concatenated alignments of 245 highly conserved proteins. Phylogeny inference done with PROTGAMMABLOSUM62 model, 100 bootstrap replicates. Phylogroups I, IIa, and IIb highlighted in red, purple and light blue respectively

    Journal: BMC Genomics

    Article Title: Comparative analysis of Faecalibacterium prausnitzii genomes shows a high level of genome plasticity and warrants separation into new species-level taxa

    doi: 10.1186/s12864-018-5313-6

    Figure Lengend Snippet: a , Hierarchical clustering of F. prausnitzii strains and type strains of other species of the family Ruminococcaceae based on the gene orthologues content. Orthologous protein products were grouped using OrthoMCL. Clustering performed with Euclidean distances using Ward.D2 algorithm. F. prausnitzii clade highlighted in red. Clustering identified 245 non-paralogous single copy genes constituting the core genome of the family. b , Maximum-likelihood phylogenetic tree of the family Ruminococcaceae based on concatenated alignments of 245 highly conserved proteins. Phylogeny inference done with PROTGAMMABLOSUM62 model, 100 bootstrap replicates. Phylogroups I, IIa, and IIb highlighted in red, purple and light blue respectively

    Article Snippet: Prophage induction For prophage induction, overnight both cultures of 9 F. prausnitzii strains (A2–165, ATCC 27766, ATCC 27768, 942/18–1, 942/30–2, 942/32–1, 922/41–1, 923/51–1, 923/61–1) as well as Subdoligranulum /Gemmiger sp. strain 924/74 were inoculated into 10 ml of fresh clarified M2GSC broth at 1:100 ratio and incubated anaerobically at 37 °C.

    Techniques:

    a , PCoA ordination based on composition of gene orthologues reveals two distinct F. prausnitzii genomogroups. Ordination performed with Euclidean distances. b , The 20 COG categories showing differential abundance between core and accessory genome in F. prausnitzii . COG categories on the right are enriched in the accessory part of species pangenome ( p lt; 0.05 in Wilcoxon test)

    Journal: BMC Genomics

    Article Title: Comparative analysis of Faecalibacterium prausnitzii genomes shows a high level of genome plasticity and warrants separation into new species-level taxa

    doi: 10.1186/s12864-018-5313-6

    Figure Lengend Snippet: a , PCoA ordination based on composition of gene orthologues reveals two distinct F. prausnitzii genomogroups. Ordination performed with Euclidean distances. b , The 20 COG categories showing differential abundance between core and accessory genome in F. prausnitzii . COG categories on the right are enriched in the accessory part of species pangenome ( p lt; 0.05 in Wilcoxon test)

    Article Snippet: Prophage induction For prophage induction, overnight both cultures of 9 F. prausnitzii strains (A2–165, ATCC 27766, ATCC 27768, 942/18–1, 942/30–2, 942/32–1, 922/41–1, 923/51–1, 923/61–1) as well as Subdoligranulum /Gemmiger sp. strain 924/74 were inoculated into 10 ml of fresh clarified M2GSC broth at 1:100 ratio and incubated anaerobically at 37 °C.

    Techniques:

    A heatmap of gene orthologues differentially abundant between the two F. prausnitzii genomogroups ( p lt; 0.05 in Wilcoxon test). Dendrogram on top reflect hierarchical clustering using Ward.D2 algorithm. Only groups with positive COG annotation are shown, see annotation bar on the left and the relevant legend inset. Orange colour in heatmap corresponds to single copy orthologues, other colours used for orthologous groups with multiple member per genome (see colour code on the right). An expanded version of this heatmap is given in Additional file 8 : Figure S5, where all orthologous groups are shown, regardless of COG annotation availability

    Journal: BMC Genomics

    Article Title: Comparative analysis of Faecalibacterium prausnitzii genomes shows a high level of genome plasticity and warrants separation into new species-level taxa

    doi: 10.1186/s12864-018-5313-6

    Figure Lengend Snippet: A heatmap of gene orthologues differentially abundant between the two F. prausnitzii genomogroups ( p lt; 0.05 in Wilcoxon test). Dendrogram on top reflect hierarchical clustering using Ward.D2 algorithm. Only groups with positive COG annotation are shown, see annotation bar on the left and the relevant legend inset. Orange colour in heatmap corresponds to single copy orthologues, other colours used for orthologous groups with multiple member per genome (see colour code on the right). An expanded version of this heatmap is given in Additional file 8 : Figure S5, where all orthologous groups are shown, regardless of COG annotation availability

    Article Snippet: Prophage induction For prophage induction, overnight both cultures of 9 F. prausnitzii strains (A2–165, ATCC 27766, ATCC 27768, 942/18–1, 942/30–2, 942/32–1, 922/41–1, 923/51–1, 923/61–1) as well as Subdoligranulum /Gemmiger sp. strain 924/74 were inoculated into 10 ml of fresh clarified M2GSC broth at 1:100 ratio and incubated anaerobically at 37 °C.

    Techniques:

    Circular map of complete 2.97 Mbp chromosome of F. prausnitzii APC918/95b, representative for phylogroup I. Innermost circle (green and purple), GC skew; circle 2 (black), G + C content; circle 3 (grey), predicted prophage remnants; circles 4 and 5 (dark red and blue), ORFs located on + and - DNA strands respectively; circle 6 (green), genes specific for genomogroup I; circle 7 (orange), tRNA and rRNA genes; circles 8–11 (pink), homologous genomic segments of gt; 1000 nt from other representatives of genomogroup I (APC924/119, APC923/51–1, ATCC 27768 T , CNCM 4573,); circles 12–16 (light blue), homologous genomic segments of gt; 1000 nt (aligned by Mauve) from representatives of genomogroup II (APC942/30–2, A2–165, APC922/41–1, APC923/61–1, KLE1255); circle 17, genes annotated as integrase, recombinase, replication initiator protein, mobilization protein, transposase

    Journal: BMC Genomics

    Article Title: Comparative analysis of Faecalibacterium prausnitzii genomes shows a high level of genome plasticity and warrants separation into new species-level taxa

    doi: 10.1186/s12864-018-5313-6

    Figure Lengend Snippet: Circular map of complete 2.97 Mbp chromosome of F. prausnitzii APC918/95b, representative for phylogroup I. Innermost circle (green and purple), GC skew; circle 2 (black), G + C content; circle 3 (grey), predicted prophage remnants; circles 4 and 5 (dark red and blue), ORFs located on + and - DNA strands respectively; circle 6 (green), genes specific for genomogroup I; circle 7 (orange), tRNA and rRNA genes; circles 8–11 (pink), homologous genomic segments of gt; 1000 nt from other representatives of genomogroup I (APC924/119, APC923/51–1, ATCC 27768 T , CNCM 4573,); circles 12–16 (light blue), homologous genomic segments of gt; 1000 nt (aligned by Mauve) from representatives of genomogroup II (APC942/30–2, A2–165, APC922/41–1, APC923/61–1, KLE1255); circle 17, genes annotated as integrase, recombinase, replication initiator protein, mobilization protein, transposase

    Article Snippet: Prophage induction For prophage induction, overnight both cultures of 9 F. prausnitzii strains (A2–165, ATCC 27766, ATCC 27768, 942/18–1, 942/30–2, 942/32–1, 922/41–1, 923/51–1, 923/61–1) as well as Subdoligranulum /Gemmiger sp. strain 924/74 were inoculated into 10 ml of fresh clarified M2GSC broth at 1:100 ratio and incubated anaerobically at 37 °C.

    Techniques:

    Circular map of complete 2.83 Mbp chromosome of F. prausnitzii APC942/30–2, representative for phylogroup II. Innermost circle (green and purple), GC skew; circle 2 (black), G + C content; circle 3 (grey), predicted prophage remnants; circles 4 and 5 (dark red and blue), ORFs located on + and - DNA strands respectively; circle 6 (green), genes specific for genomogroup I; circle 7 (orange), tRNA and rRNA genes; circles 8–11 (light blue), homologous genomic segments of gt; 1000 nt from other representatives of genomogroup II (A2–165, APC922/41–1, APC923/61–1, KLE1255); circles 12–16 (pink), homologous genomic segments of gt; 1000 nt (aligned by Mauve) from representatives of genomogroup I (APC918/95b, APC924/119, APC923/51–1, ATCC 27768 T , CNCM 4573); circle 17, genes annotated as integrase, recombinase, replication initiator protein, mobilization protein, transposase

    Journal: BMC Genomics

    Article Title: Comparative analysis of Faecalibacterium prausnitzii genomes shows a high level of genome plasticity and warrants separation into new species-level taxa

    doi: 10.1186/s12864-018-5313-6

    Figure Lengend Snippet: Circular map of complete 2.83 Mbp chromosome of F. prausnitzii APC942/30–2, representative for phylogroup II. Innermost circle (green and purple), GC skew; circle 2 (black), G + C content; circle 3 (grey), predicted prophage remnants; circles 4 and 5 (dark red and blue), ORFs located on + and - DNA strands respectively; circle 6 (green), genes specific for genomogroup I; circle 7 (orange), tRNA and rRNA genes; circles 8–11 (light blue), homologous genomic segments of gt; 1000 nt from other representatives of genomogroup II (A2–165, APC922/41–1, APC923/61–1, KLE1255); circles 12–16 (pink), homologous genomic segments of gt; 1000 nt (aligned by Mauve) from representatives of genomogroup I (APC918/95b, APC924/119, APC923/51–1, ATCC 27768 T , CNCM 4573); circle 17, genes annotated as integrase, recombinase, replication initiator protein, mobilization protein, transposase

    Article Snippet: Prophage induction For prophage induction, overnight both cultures of 9 F. prausnitzii strains (A2–165, ATCC 27766, ATCC 27768, 942/18–1, 942/30–2, 942/32–1, 922/41–1, 923/51–1, 923/61–1) as well as Subdoligranulum /Gemmiger sp. strain 924/74 were inoculated into 10 ml of fresh clarified M2GSC broth at 1:100 ratio and incubated anaerobically at 37 °C.

    Techniques:

    BLASTn-based average nucleotide identity (ANIb) between available 31 complete and draft genomes of F. prausnitzii . Dendrogram on top built by hierarchical clustering using Ward.D2 algorithm

    Journal: BMC Genomics

    Article Title: Comparative analysis of Faecalibacterium prausnitzii genomes shows a high level of genome plasticity and warrants separation into new species-level taxa

    doi: 10.1186/s12864-018-5313-6

    Figure Lengend Snippet: BLASTn-based average nucleotide identity (ANIb) between available 31 complete and draft genomes of F. prausnitzii . Dendrogram on top built by hierarchical clustering using Ward.D2 algorithm

    Article Snippet: Prophage induction For prophage induction, overnight both cultures of 9 F. prausnitzii strains (A2–165, ATCC 27766, ATCC 27768, 942/18–1, 942/30–2, 942/32–1, 922/41–1, 923/51–1, 923/61–1) as well as Subdoligranulum /Gemmiger sp. strain 924/74 were inoculated into 10 ml of fresh clarified M2GSC broth at 1:100 ratio and incubated anaerobically at 37 °C.

    Techniques:

    Mauve alignment of four representative complete genomes within each of the four species of human gut-associated bacteria: F. prausnitzii , Clostridiodes difficile , Bacteroides fragilis and Escherichia coli . Blocks of the same colour correspond to Locally Collinear Blocks (LCBs); +, positive DNA strand; −, negative DNA strand

    Journal: BMC Genomics

    Article Title: Comparative analysis of Faecalibacterium prausnitzii genomes shows a high level of genome plasticity and warrants separation into new species-level taxa

    doi: 10.1186/s12864-018-5313-6

    Figure Lengend Snippet: Mauve alignment of four representative complete genomes within each of the four species of human gut-associated bacteria: F. prausnitzii , Clostridiodes difficile , Bacteroides fragilis and Escherichia coli . Blocks of the same colour correspond to Locally Collinear Blocks (LCBs); +, positive DNA strand; −, negative DNA strand

    Article Snippet: Prophage induction For prophage induction, overnight both cultures of 9 F. prausnitzii strains (A2–165, ATCC 27766, ATCC 27768, 942/18–1, 942/30–2, 942/32–1, 922/41–1, 923/51–1, 923/61–1) as well as Subdoligranulum /Gemmiger sp. strain 924/74 were inoculated into 10 ml of fresh clarified M2GSC broth at 1:100 ratio and incubated anaerobically at 37 °C.

    Techniques: