Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Immune checkpoint blockade restores HIV-specific CD4 T cell help for NK cells

doi: 10.4049/jimmunol.1701551

Figure Lengend Snippet: Stimulation of PBMCs by HIV Gag induces a CD4 T cell-dependent production of cytokines by NK cells. PBMCs were stimulated in vitro with an HIV Gag peptide pool or left unstimulated (negative control) and assessed by a delayed ICS assay, in which cells were incubated with Ag for 12–36 before addition of brefeldin and monensin for 12h and collection after 24–48h. The flow cytometry plots show IFNγ production by CD4 T cells (CD3+CD4+), B cells (CD19+), NK cells (CD56+) and monocytes (CD14+) after doublet exclusion on FSC-A/FSC-H and dead cell exclusion. ( A) Example plots for one representative donor showing IFN-γ production by CD4 T cells (CD3+CD4+), B cells (CD3-CD19+), NK cells (CD3-CD56+) and CD14+ monocytes, after doublet exclusion on FSC-A/FSC-H and dead cell exclusion after 24 or 48h of stimulation. (B) ICS frequencies of IFN-γ-producing NK cells 48h after Gag stimulation of PBMCs (n=21 untreated subjects) correlated ( C ) with HIV viral loads; and ( D ) with HIV Gag-specific CD4 T cell responses. (E ) Representative example and ( F ) summary data on 7 subjects of the impact of CD3+ or CD8+ cell depletion on IFN-γ production by NK cells. Statistical tests: Wilcoxon paired test ( BF ) and Spearman correlation ( CD ).

Article Snippet: For all samples, brefeldin-A (5ug/ml; Sigma), golgi stop (containing monensin) (0.3uL/mL BD) (and anti-CD107α (clone H4A3, PE-Cy5, BD, or BV786, BD) were added for the last 12 hours of stimulation.

Techniques: In Vitro, Negative Control, Incubation, Flow Cytometry, Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Immune checkpoint blockade restores HIV-specific CD4 T cell help for NK cells

doi: 10.4049/jimmunol.1701551

Figure Lengend Snippet: NK cell activation after HIV antigen stimulation is mainly dependent on soluble IL-2 and IL-12 secretion. ( A-D ) CD8-depleted PBMCs were stimulated for 48h with an HIV Gag peptide pool or left unstimulated, this in the presence of combinations of blocking antibodies against PD-L1, IL10Rα, IL-2 and/or IL-12. The last 12 hours were performed in the presence of Brefeldin A and monensin and anti-CD107a antibody before staining and flow cytometric acquisition. ( A ) Representative example of IFN-γ and CD107a expression by NK cells in the different Gag stimulated conditions. ( BCD ) Summary plots of the impact of IL-12 or IL-2 blockade on NK cell functions observed in the presence of combined blockade of the PD-1 and IL-10 pathways (n=14): ( B ) IFN-γ expression; ( C ) Degranulation as measured by CD107a and IFN-γ co-expression; baseline expression of CD107a precludes reliable analysis of the single positive CD107a+ cells; ( D ) TNF-α expression. Statistical tests: Non-parametric Friedman test for multiple paired comparisons with Dunn’s post tests. * p

Article Snippet: For all samples, brefeldin-A (5ug/ml; Sigma), golgi stop (containing monensin) (0.3uL/mL BD) (and anti-CD107α (clone H4A3, PE-Cy5, BD, or BV786, BD) were added for the last 12 hours of stimulation.

Techniques: Activation Assay, Blocking Assay, Staining, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Treatment With FoxP3+ Antigen-Experienced T Regulatory Cells Arrests Progressive Retinal Damage in a Spontaneous Model of Uveitis

doi: 10.3389/fimmu.2020.02071

Figure Lengend Snippet: Immunohistology and intracellular cytokine analysis (IFNγ, IL17, and IL22) of eye-draining lymph node- (DLN) and retina-infiltrating cells over the course of spontaneous EAU in dTg HEL/TCR mice. (A) Representative confocal fluorescent (a–c,e–l) and light microscopic (d) immunohistochemistry of frozen retinal sections from (P24) dTg HEL/TCR mice are shown. Sections are representative of initial retinal inflammatory changes (a–c) and advanced retinal inflammatory damage (d–l) . Infiltrating cells identified include: (a,b,f,k,l) , CD3+CD4+ double-stained T cells, CD3+CD4- single-stained, presumed double-negative (DN) T cells; (c,h) , macrophages (F4/80+); (e,i) , MHC class II+ and CD11c+ (presumed DC); and (f,k,l) , B cells. In panels (d,g,h) granulomas are shown. In panels (f,k,l) , granulomas have features of tertiary lymphoid organs (TLO). Areas of retinal HEL protein expression were identified using a polyclonal HEL-specific antibody. The slides were mounted with Hydromount TM Aqueous Non-fluorescing Mounting Media (National diagnostics, Hull, United Kingdom), and were examined with a Zeiss LSM510 confocal microscope (Carl Zeiss Meditec, Göttingen, Germany). (B) Flow cytometric analysis of intracellular cytokine expression in lymph node cell populations at different stages of EAU development. Single cell suspensions were prepared from eye-draining lymph nodes (upper panel) and retinas (lower panel), respectively ( n = 4/age group). Flow cytometric analysis of intracellular cytokine expression by three different T cell populations (1G12+CD4-, 1G12+CD4+, and 1G12-CD4+) at different time points during evolution of EAU in dTg mice was completed. Upper panel (dTg DLN): at disease onset (P21), only small numbers of antigen-specific CD4+ cells gave a positive signal for intracellular cytokines; a gradual up to fivefold increase was found by P30. Significantly fewer IFNγ-secreting antigen-specific CD4+ T cells (declining further toward P60) than IL17+ and IL22+ cells were found. In contrast, levels of IL17+ cells were sustained in the DLN through P60. IL22 expression was also sustained and increased in CD4+ but not DN HEL-specific T cells through P60. CD4+ non-antigen-specific T cells (1G12-) were low for all three cytokines over the course of the observation. Lower panel (dTg retina): Cytokine expression by cells mirrored those changes found in the DLN except at P45 when there were proportionately more non-antigen specific IL22+CD4+ T cells found in the retina. By P60 non-antigen-specific CD4+ T cells in the retina expressed similar levels of IL22 and IL17 as antigen-specific cells, which suggests some level of bystander activation. Cells had been stimulated with 50 ng/ml PMA and permeabilized with 1 μM ionomycin for 5 h in the presence of monensin (BD GolgiStop TM , BD Biosciences, Oxford, United Kingdom). The cells were surface labeled for CD4 and 1G12 (3A9 TCR) followed by intracellular cytokine labeling for IFNγ, IL17 and IL22. 1 × 10 5 events/sample were acquired on a BD LSR II flow cytometer. Data were analyzed for each specific time point using one-way ANOVA and Tukey’s Multiple Comparison post hoc Test with * p

Article Snippet: Cells were incubated for 5h in RPMI medium containing 10% FBS (both from Gibco, Fisher Scientific UK Ltd., Loughborough, United Kingdom), 50 ng/ml phorbol 12-myristate-13-acetate ( ) and 1 μM ionomycin (both from Sigma-Aldrich, St. Louis, MO, United States) for 5h in the presence of monensin (BD GolgiStopTM , BD Biosciences, Oxford, United Kingdom).

Techniques: Mouse Assay, Immunohistochemistry, Staining, Expressing, Microscopy, Activation Assay, Labeling, Flow Cytometry

Journal: Applied and Environmental Microbiology

Article Title: Redundancy in Anaerobic Digestion Microbiomes during Disturbances by the Antibiotic Monensin

doi: 10.1128/AEM.02692-17

Figure Lengend Snippet: β-Diversity of anaerobic digester microbiome samples during different periods. (A) Boxplot of weighted UniFrac distances within microbiomes from a single anaerobic digester throughout a similar operating period. Control anaerobic digester boxplot corresponds to days 203 to 306, when the slow anaerobic digester was fed only control cow manure without monensin addition; the low A anaerobic digester boxplot corresponds to days 203 to 383; the low B anaerobic digester boxplot corresponds to days 203 to 355; the fast anaerobic digester boxplot only includes samples during which the anaerobic digester was subjected to high concentrations of monensin (i.e., days 265 to 306); and the slow anaerobic digester boxplot corresponds to days 306 to 383, when the anaerobic digester was subjected to high concentrations of monensin. (B) Similarity of samples postdisturbance compared to three samples prior to disturbance (prior to P2 on days 175, 189, and 201) based on the weighted UniFrac distance metric. Similarity was calculated as one minus the average weighted UniFrac distance between the sample day and the three sample days prior to disturbance (prior to P2). Colors represent the monensin concentration gradient: dark red, high monensin concentration (1 to 5 mg · liter −1 ); pink, low monensin concentration (

Article Snippet: Seven dairy cows (Cornell University Teaching and Research Center, Dryden, NY) were split into two groups and fed the same diet, except that three cows (monensin feed-dosed cows) received a ration top-dressed with monensin (Rumensin 90 Premix [Elanco Animal Health, Indianapolis, IN] mixed with corn grain), while four cows (control cows) received no monensin (ration top-dressed with corn grain without Rumensin).

Techniques: Concentration Assay

Journal: Applied and Environmental Microbiology

Article Title: Redundancy in Anaerobic Digestion Microbiomes during Disturbances by the Antibiotic Monensin

doi: 10.1128/AEM.02692-17

Figure Lengend Snippet: β-Diversity of all anaerobic digester microbiome samples from day 175 to the end of the operating period. (A) Principal-coordinate analysis (PCoA) plot based on weighted UniFrac distance. (B) Distance-based redundancy analysis (db-RDA) plot showing the four measured parameters that best explained the variation observed in the PCoA plot. Methane, methane yield; alkalinity, bicarbonate alkalinity concentration; ammonia, total ammonium concentration; monensin, monensin concentration in the substrate. In both the PCoA and db-RDA plots, the points are colored on a gradient scale representing the duration of the operating period in days.

Article Snippet: Seven dairy cows (Cornell University Teaching and Research Center, Dryden, NY) were split into two groups and fed the same diet, except that three cows (monensin feed-dosed cows) received a ration top-dressed with monensin (Rumensin 90 Premix [Elanco Animal Health, Indianapolis, IN] mixed with corn grain), while four cows (control cows) received no monensin (ration top-dressed with corn grain without Rumensin).

Techniques: Concentration Assay

Journal: Applied and Environmental Microbiology

Article Title: Redundancy in Anaerobic Digestion Microbiomes during Disturbances by the Antibiotic Monensin

doi: 10.1128/AEM.02692-17

Figure Lengend Snippet: Heatmaps representing relative abundance of major OTUs in anaerobic digester samples during the entire sampling period for the low A anaerobic digester (A), low B anaerobic digester (B), fast anaerobic digester (C), and slow anaerobic digester (D). OTUs represented here reached at least 5% relative abundance in any one anaerobic digester sample. Lowest-level taxonomic data, as well as the OTU ID number, are provided. The sidebar color scale represents the monensin dosing rate to the anaerobic digester. For the fast digester (panel C), the black line in the red bar indicates when the monensin dose was directly increased from 1 to 5 mg · liter −1 . For the slow digester (panel D), the black lines in the red bar indicate when the monensin dose was increased (from 1 to 2 mg · liter −1 , from 2 to 3 mg · liter −1 , from 3 to 4 mg · liter −1 , and from 4 to 5 mg · liter −1 ).

Article Snippet: Seven dairy cows (Cornell University Teaching and Research Center, Dryden, NY) were split into two groups and fed the same diet, except that three cows (monensin feed-dosed cows) received a ration top-dressed with monensin (Rumensin 90 Premix [Elanco Animal Health, Indianapolis, IN] mixed with corn grain), while four cows (control cows) received no monensin (ration top-dressed with corn grain without Rumensin).

Techniques: Sampling

Journal: Applied and Environmental Microbiology

Article Title: Redundancy in Anaerobic Digestion Microbiomes during Disturbances by the Antibiotic Monensin

doi: 10.1128/AEM.02692-17

Figure Lengend Snippet: Performance parameters for the four anaerobic digesters from day 175 to the end of the operating period for specific methane yields (A), tVFA concentrations (B), total ammonium concentrations (C), and bicarbonate alkalinity concentrations (D). Points are colored on a color scale corresponding to the concentration of monensin dosed to the anaerobic digester at that time point. The different periods are marked with gray and white shading from left to right during the operating period: the first gray shading identifies P1; the first white shading identifies P2; the second gray bar identifies P3, and the second white area identifies P4 (see Table S4 in the supplemental material). The fast anaerobic digester was stopped at the end of P3 due to a loss of stability.

Article Snippet: Seven dairy cows (Cornell University Teaching and Research Center, Dryden, NY) were split into two groups and fed the same diet, except that three cows (monensin feed-dosed cows) received a ration top-dressed with monensin (Rumensin 90 Premix [Elanco Animal Health, Indianapolis, IN] mixed with corn grain), while four cows (control cows) received no monensin (ration top-dressed with corn grain without Rumensin).

Techniques: Concentration Assay

Journal: Applied and Environmental Microbiology

Article Title: Redundancy in Anaerobic Digestion Microbiomes during Disturbances by the Antibiotic Monensin

doi: 10.1128/AEM.02692-17

Figure Lengend Snippet: β-Diversity of the fast and slow anaerobic digester microbiome samples during high monensin periods (≥1 mg · liter −1 ; day 265 to 307 for fast anaerobic digester, and day 307 to 383 for slow anaerobic digester). (A) Principal-coordinate analysis (PCoA) based on weighted UniFrac distance. Time points are colored in a gradient scale corresponding to monensin concentration in the substrate, as indicated by the color sidebar. (B) Taxon biplot showing the most abundant OTUs in these samples (OTUs that reached an average relative abundance of ≥1% across the time period shown). OTUs are shown nearest to the samples that they are most abundant in. Sample time points are not shown as they are displayed in the left-hand PCoA. Point size represents average relative abundance of that OTU across all samples (minimum average relative abundance is 1.0%, maximum average relative abundance is 12.0%). Points are colored by phylum-level taxonomy. Three OTUs discussed in the text are highlighted. OTU 820298, OP11 OTU; OTU 265425, Prevotella OTU; OTU 837605, Bacteroidales OTU.

Article Snippet: Seven dairy cows (Cornell University Teaching and Research Center, Dryden, NY) were split into two groups and fed the same diet, except that three cows (monensin feed-dosed cows) received a ration top-dressed with monensin (Rumensin 90 Premix [Elanco Animal Health, Indianapolis, IN] mixed with corn grain), while four cows (control cows) received no monensin (ration top-dressed with corn grain without Rumensin).

Techniques: Concentration Assay

Journal: Journal of Virology

Article Title: Interleukin-8 and Growth-Regulated Oncogene Alpha Mediate Angiogenesis in Kaposi's Sarcoma

doi: 10.1128/JVI.76.22.11570-11583.2002

Figure Lengend Snippet: Intracellular IL-8 is present in the monocytic subpopulation of PBMC exposed to HIV-1. PBMC were treated with monensin (control) along with HIV-1 BaL , HIV-1 BRU , or TNF-α (100 ng/ml). PBMC were harvested after 6 h and analyzed for intracellular IL-8 protein by flow cytometry. (A) Lymphocyte and monocyte subpopulations were gated according to the pattern of forward scatter and side scatter. (B) Background staining in the lymphocytes and monocytes was determined by incubation with phycoerythrin-mouse IgG1 isotype control (mouse IgG-PE) and FITC-mouse IgG2a isotype control (mouse IgG-FITC). The histograms show fluorescence intensity on logarithmic scales along the x axis (FITC) and y axis (phycoerythrin). The percentage of FITC-positive cells is indicated for both the double-positive and single-positive quadrants. (C) Lymphocytes and monocytes were stained with an FITC-conjugated mouse anti-human IL-8 antibody (IL-8-FITC) and either phycoerythrin-conjugated mouse anti-human CD4 (CD4-PE) or phycoerythrin-conjugated mouse anti-human CD14 (CD14-PE), respectively. The percentage of CD4 + and CD14 + cells staining positive for IL-8 in each condition is indicated. Data shown are representative of four independent experiments.

Article Snippet: The next day, PBMC were treated for 6 h with monensin (GolgiStop; PharMingen) and incubated with virus or TNF-α as indicated in the figure legends.

Techniques: Flow Cytometry, Cytometry, Staining, Incubation, Fluorescence

Journal: PLoS ONE

Article Title: M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells

doi: 10.1371/journal.pone.0175514

Figure Lengend Snippet: In neoplastic mast cells, Kit phosphorylation at Tyr568/570, Tyr703, Tyr721, and Tyr936 occurs on the ER. ( A ) Tyrosine phosphorylation sites in human Kit. ( B - F ) Cells were treated with vehicle or ( B - D ) 5 μM M-COPA for 16 hours or ( E and F ) 250 nM monensin for 24 hours. Anti-Kit immunoprecipitates ( B and E ) and cell lysates ( C , D , and F ) were immunoblotted with anti-Kit, anti-phospho-Kit Tyr568/570 (anti-pKit Tyr568/570 ), anti-pKit Tyr703 , anti-pKit Tyr721 , and anti-pKit Tyr936 . Note that ER-localized Kit was phosphorylated at Tyr568/570, Tyr703, Tyr721 and Tyr936 in neoplastic mast cells.

Article Snippet: Monensin (Biomol, Exeter, UK), brefeldin A (Wako, Osaka, Japan), and bafilomycin A1 (Sigma) were dissolved in ethanol.

Techniques: