Journal: Frontiers in Immunology

Article Title: Immunization With Bovine Herpesvirus-4-Based Vector Delivering PPRV-H Protein Protects Sheep From PPRV Challenge

doi: 10.3389/fimmu.2021.705539

Figure Lengend Snippet: Effect of BoHV-4-A-PPRV-H-ΔTK vaccination in ovine PBMC populations. PBMCs obtained from the three treatment groups [PBS (black), BoHV-4-A-ΔTK (blue), and BoHV-4-A-PPRV-H-ΔTK (red)] were stained with different monoclonal antibodies and analyzed by flow cytometry at different time points [pre-immunization (D0), pre-challenge (D42), and days 2, 4, 7, 9, 11, and 14 post-challenge (PC)]. Average percentage of (A) CD4 + cell population, (B) CD8 + cell population, (C) WC1 + (γδ T cell) population, (D) B-cell + population, (E) CD14 + cell population, (F) CD14 + CD16 + cell population, and (G) CD16 + CD14 − cell population for each sheep group were plotted. * p < 0.05 one-way ANOVA test (BoHV-4-A-PPRV-H-ΔTK vs . BoHV-4-A-ΔTK and PBS control groups). The black arrow (D42) denotes the time of virulent PPRV ICV’89 challenge in all animals.

Article Snippet: The following antibodies were used to label the different PBMC subpopulations: anti-ovine CD4 (clone 44.38), CD8 (clone 38.65), and WC1 (clone 19.19), anti-human CD14 (clone TÜK4) and CD16 (clone KD1) (all from Bio-Rad), and anti-bovine B cell marker (clone BAQ44A) (Kingfisher Biotech).

Techniques: Staining, Bioprocessing, Flow Cytometry, Control

Journal: PLoS ONE

Article Title: Pharmacological inhibition of P2RX7 ameliorates liver injury by reducing inflammation and fibrosis

doi: 10.1371/journal.pone.0234038

Figure Lengend Snippet: (A) Representative images (objective 2X) of Sirius red staining (top) and immunohistochemical staining (objective 40X) of P2RX7 (bottom) in liver tissue from a representative control and NASH-affected donor with quantification of Sirius red + area and P2RX7 + cells ( n = 5 individuals per group; n = 6–8 images per donor). Scale bar, 100 μM. Black arrows highlight P2RX7 + cells. (B) Quantification of CD45 + , CD45 + / P2RX7 + , CD14 + , CD14 + / P2RX7 + , CD68 + , CD68 + / P2RX7 + cells ( n = 5 individuals per group; n = 6–8 images per donor). (C) IL-1β, (D) caspase-1, (E) CCL2, and (F) CCL5 levels in liver tissue from control and NASH-affected liver biopsies ( n = 5 individuals per group). (G) Relative expression levels of P2RX 7, NLRP3 , AIM2 , ASC , CASP1 , IL-1β and (H) CD11b , CD45 , Tnfα , Il-6 , CCL2 , CCL5 in liver tissue from control and NASH-affected donors ( n = 5 individuals per group). n.d., for not detected. In all statistical plots, the data are shown as the mean ± SEM. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 by two-sided Student’s t-test.

Article Snippet: Anti-CD14 biotinylated antibody (1:40, #BAF383, R&D system) was visualized using streptavidin alkaline phosphatase (1:250, #SA-5100, Vector Laboratory) with wrap red.

Techniques: Staining, Immunohistochemical staining, Control, Expressing

Journal: PLoS ONE

Article Title: Pharmacological inhibition of P2RX7 ameliorates liver injury by reducing inflammation and fibrosis

doi: 10.1371/journal.pone.0234038

Figure Lengend Snippet: (A) Relative expression of P2RX 7 and NLRP3 inflammasome components NLRP3 , AIM2 , and CASP1 in liver tissue and human primary hepatocytes, KCs, HSCs, and MOs. (B) Relative expression of ACTA2 , CD14 , and CD68 in liver tissue and human primary hepatocytes, KCs, HSCs, and MOs. (C) IL-1β levels in culture media from MOs treated with LPS, ATP ± SGM-1019. In all statistical plots, the data are shown as the mean ± SEM. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 by one-way ANOVA.

Article Snippet: Anti-CD14 biotinylated antibody (1:40, #BAF383, R&D system) was visualized using streptavidin alkaline phosphatase (1:250, #SA-5100, Vector Laboratory) with wrap red.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Pharmacological inhibition of P2RX7 ameliorates liver injury by reducing inflammation and fibrosis

doi: 10.1371/journal.pone.0234038

Figure Lengend Snippet: NASH-affected livers show greater number of cells expressing P2RX7, NLRP3 inflammasome activation, IL-1β, CCL2/CCL5 and fibrosis. P2RX7 is expressed by infiltrating MOs and resident KCs in the livers of NASH-affected individuals. Pharmacological inhibition of P2RX7 in human primary CD14 + MOs and KCs block IL-1β release and differentially modulates their inflammatory response. Reduced P2RX7-dependent IL-1β secretion from MOs and KCs results in decreased hepatocyte damage, chemokine secretion, and HSCs fibrosis. P2RX7 pharmacological inhibition in a more clinically translatable animal model results in significant protection from inflammation and fibrosis.

Article Snippet: Anti-CD14 biotinylated antibody (1:40, #BAF383, R&D system) was visualized using streptavidin alkaline phosphatase (1:250, #SA-5100, Vector Laboratory) with wrap red.

Techniques: Expressing, Activation Assay, Inhibition, Blocking Assay, Animal Model

Journal: Cell reports

Article Title: The Cardiac Microenvironment Instructs Divergent Monocyte Fates and Functions in Myocarditis

doi: 10.1016/j.celrep.2019.06.007

Figure Lengend Snippet:

Article Snippet: After blocking with 1% BSA and 0.1% tween-20 in 1 × PBS, tissues were incubated with anti-CD14-biotin sheep antibodies (R & D systems) and anti-CD68 mouse antibodies (Abcam).

Techniques: Control, Northern Blot, Recombinant, Purification, Adjuvant, Lysis, Staining, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Microarray, Software

Journal: Journal of Biological Chemistry

Article Title: Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

doi: 10.1074/jbc.m109.040832

Figure Lengend Snippet: FIGURE 9. CD14 binds to solid phase lipids. A, affinity-purified preparations (2 g each) of the extracellular domains of CD14 (sCD14) and TLR4 (sTLR4) and the full-length MD-2 were analyzed by gel electrophoresis under reduc- ing and denaturing conditions. The electrophoretic gels were stained with Coomassie Blue. mol. mass st., molecular mass standards. B, phospholipids (1.25 nmol) in 20 l of ethanol were placed onto microtiter wells, and the solvent was evaporated. Nonspecific binding was blocked with 20 mM Tris buffer(pH7.4)containing0.15 MNaCl,5mMCaCl2(intheupperpanel),or2mM EGTA (in the lower panel) and 5% (w/v) bovine serum albumin (buffer A). Varying concentrations of human CD14 in buffer A were added and incu- bated at 37 °C for 1 h. The binding of CD14 to phospholipids was detected using anti-CD14 monoclonal antibody as described under “Experimental Pro- cedures.” The data shown are the means S.E. from three separate experi- ments with duplicate samples in each experiment.

Article Snippet: Mouse IgG1 isotype control, mouse monoclonal anti-human CD14 antibody, and sheep anti-human CD14 polyclonal antibody were purchased from R&D systems (Minneapolis, MN).

Techniques: Affinity Purification, Nucleic Acid Electrophoresis, Staining, Solvent, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

doi: 10.1074/jbc.m109.040832

Figure Lengend Snippet: FIGURE 10. PG Inhibits CD14 binding to solid phase LPS. A, various types of PG were coated onto microtiter plates and incubated with CD14 (1 g/ml) at 37 °C for 1 h. The binding of CD14 to PG was detected using anti-CD14 mono- clonal antibody, and the ELISA-based absorbance of CD14 bound to POPG was defined as 100%. Types of PG shown on the graph are: dilauroylphos- phatidylglycerol (DLPG), DMPG, DPPG, and 16:0/18:1 POPG. B, LPS (2 g) in 20 l of ethanol was placed onto microtiter wells, and the solvent was evaporated. After blocking the nonspecific binding with buffer A, the mix- ture of CD14 (1 g/ml) and phospholipid liposomes (20 g/ml) in buffer A, which was preincubated at 37 °C for 1 h, was added and incubated at 37 °C for 1 h. The binding of CD14 to LPS was detected using anti-CD14 mono- clonal antibody. The ELISA-based absorbance of CD14 bound to LPS was defined as 100%. The data shown are the means S.E. from three separate experiments with duplicate samples in each experiment. *, p 0.05, **, p 0.01, when compared with LPS-CD14 binding in the absence of phospholipids.

Article Snippet: Mouse IgG1 isotype control, mouse monoclonal anti-human CD14 antibody, and sheep anti-human CD14 polyclonal antibody were purchased from R&D systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Solvent, Blocking Assay, Liposomes

Journal: Journal of Biological Chemistry

Article Title: Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

doi: 10.1074/jbc.m109.040832

Figure Lengend Snippet: FIGURE11.MonoclonalantibodiesspecificfortheLPSbindingsiteinhibit CD14 interaction with POPG and PI. POPG (A) or PI (B) were coated onto microtiter plates. After blocking the nonspecific binding with buffer A, the mixture of CD14 (1 g/ml) and monoclonal antibodies or isotype control IgG (50g/ml)inbufferA,whichwaspreincubatedat37 °Cfor1h,wasaddedand incubated at 37 °C for 1 h. The binding of CD14 to phospholipids was detected using sheep anti-CD14 polyclonal antibody, and the ELISA-based absorbance of CD14 bound to phospholipid was defined as 100%. The data shown are the means S.E. from three separate experiments with duplicate samples in each experiment. *, p 0.05, when compared with CD14 binding in the absence of monoclonal antibody. mIG, mouse Ig. C, CD14 (2 g) was coated onto microtiter plates, and nonspecific binding was blocked with buffer A. Monoclonal antibodies or isotype control IgG (50 g/ml) in buffer A were added and incubated at 37 °C for 1 h. The CD14 was detected using sheep anti-CD14 polyclonal antibody, and the ELISA-based absorbance of solid phase CD14 alone was defined as 100%.

Article Snippet: Mouse IgG1 isotype control, mouse monoclonal anti-human CD14 antibody, and sheep anti-human CD14 polyclonal antibody were purchased from R&D systems (Minneapolis, MN).

Techniques: Blocking Assay, Binding Assay, Bioprocessing, Control, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cell Transplantation

Article Title: Autologous Transplantation of Amniotic Fluid-Derived Mesenchymal Stem Cells into Sheep Fetuses

doi: 10.3727/096368910x543402

Figure Lengend Snippet: Figure 2. Characterization of sheep amniotic fluid mesenchymal stem cells. The growth curve (A) demonstrates that the doubling time of both AFMSCs and GFP-FAMSCs was around 36–48 h (error bar: SD). (B) The cell size from AFMSCs and GFP-AFMSCs, which did not present statistical significance. (C) The DNA content of cells cycle by using flow cytometry analysis. The cell cycle did not showed significant difference between AFMSCs and GFP-AFMSCs. (D) The sheep AF cells stained strongly positive for MSC surface markers CD44, CD58, and CD166, confirming their mesenchymal origin. Anti-sheep CD14, CD31, and CD45 were negative compared to background staining (light gray, negative control; dark gray, anti-sheep antibodies).

Article Snippet: Mouse anti-sheep CD14, CD31, Flow Cytometry Analysis CD44, CD45, and CD58 antibodies conjugated with flu- Single cell suspensions of fetal spleen, liver, and orescein isothiocyanate (FITC) (AbD Serotec, UK), and bone marrow were prepared immediately after animal mouse anti-human CD166 conjugated with phycoery- sacrifice by straining fetal tissue through a 40-μm nylon thrin (PE) (BD Pharmingen, UK) were used for charac- mesh cell strainer (BD, UK).

Techniques: Flow Cytometry, Staining, Negative Control

Journal: Infection and Immunity

Article Title: Neisseria meningitidis Lipooligosaccharide Structure-Dependent Activation of the Macrophage CD14/Toll-Like Receptor 4 Pathway

doi: 10.1128/iai.72.1.371-380.2004

Figure Lengend Snippet: FIG. 3. TNF- induction in human macrophages by meningococcal LOS was CD14 and TLR4–MD-2 mediated. THP-1 macrophages (106/ml) were incubated with 10 or 5 g of anti-CD14 and anti-TLR4, respectively, or with anti-CD14 or anti-TLR4 in a dose-dependent manner prior to stimulation with 0.56 pmol (1 ng/ml) of meningococcal LOS from parent strain NMB (Table 1). Unstimulated cells were included as a control. Error bars represent SD from the average of four different experiments.

Article Snippet: CD14 monoclonal antibody (clone 134620) obtained from R&D Systems is produced in sheep immunized with purified, CHO cellderived, recombinant human CD14.

Techniques: Incubation, Control

Journal: Frontiers in Immunology

Article Title: Characterization of intestinal mononuclear phagocyte subsets in young ruminants at homeostasis and during Cryptosporidium parvum infection

doi: 10.3389/fimmu.2024.1379798

Figure Lengend Snippet: List of antibodies.

Article Snippet: Anti-bovine CD14 , CD14 , Mouse IgG1 , CAM36A , Bio-Rad , Sheep , Datasheet MCA6085.

Techniques:

Journal: Frontiers in Immunology

Article Title: Characterization of intestinal mononuclear phagocyte subsets in young ruminants at homeostasis and during Cryptosporidium parvum infection

doi: 10.3389/fimmu.2024.1379798

Figure Lengend Snippet: Identification of neonatal intestinal mononuclear phagocytes in the ileal Peyer’s patch of lambs and calves. Intestinal tissues from the ileal Peyer’s patch (IPP) were recovered from 10-day-old lambs ( n = 6) and calves ( n = 10). Mechanical and enzymatic dissociations were performed to obtain total isolated intestinal cells, then stained for flow cytometry. Representative gating for mononuclear phagocytes (MP) subsets are shown for the IPP of lamb (A) and calf (B) . Following exclusion of dead cells, CD11c + MHCII + cells were gated to select total MP and then four subsets were distinguished based on surface expression of CD14, CD172α, CD11b, and Cadm1: CD14 + CD172α + Cadm1 int CD11b + (subset 1, green), CD14 − CD172α − Cadm1 + CD11b − (subset 2, purple), CD14 − CD172α + Cadm1 int CD11b + (subset 3, blue), and CD14 − CD172α +/- Cadm1 − CD11b − (subset 4, yellow). Subsets 1, 2, and 3 of MP from the IPP of a 10-day-old lamb were cell sorted by flow cytometry, cytospined, and stained with hematoxylin and eosin. Pictures of the three subsets of MP were obtained by optical microscopy ( A , right panel). Pie charts represent the cell proportions in percentage of each intestinal MP subset among viable CD11c + MHCII + cells (mean ± SD) for lambs ( n = 6; A , bottom panel) and calves ( n = 10; B , bottom panel).

Article Snippet: Anti-bovine CD14 , CD14 , Mouse IgG1 , CAM36A , Bio-Rad , Sheep , Datasheet MCA6085.

Techniques: Isolation, Staining, Flow Cytometry, Expressing, Microscopy

Journal: Frontiers in Immunology

Article Title: Characterization of intestinal mononuclear phagocyte subsets in young ruminants at homeostasis and during Cryptosporidium parvum infection

doi: 10.3389/fimmu.2024.1379798

Figure Lengend Snippet: Subset proportions of intestinal mononuclear phagocytes in the lambs at different ages. Intestinal cells from the ileal Peyer’s patch were recovered from day-old ( n = 8), 10-day-old ( n = 6), and month-old lambs ( n = 5) and from ileum of 3-year-old ewes ( n = 3). Mechanical and enzymatic dissociations were performed to obtain total isolated intestinal cells, then stained for flow cytometry. Following exclusion of dead cells, CD11c + MHCII + cells were gated to select total mononuclear phagocytes (MP), and then four subsets were distinguished based on surface expression of CD14, CD172α, CD11b, and Cadm1: CD14 + CD172α + Cadm1 int CD11b + (subset 1, green), CD14 − CD172α − Cadm1 + CD11b − (subset 2, purple), CD14 − CD172α + Cadm1 int CD11b + (subset 3, blue), and CD14 − CD172α +/- Cadm1 − CD11b − (subset 4, yellow). (A) Cell proportions of ileal MP subsets according to the age of animals, expressed in percentage of MP (median ± range) with each point corresponding to one animal. (B) Pie charts of cell proportions of ileal MP subsets by age group, expressed in percentage of MP (mean ± SD). Statistical analyses were performed using Kruskal–Wallis non-parametric test followed by Dunn’s multiple comparison test to compare the medians of MP subsets between age groups; statistical significance was determined by a p -value < 0.05 (* p < 0.05, ** p < 0.01).

Article Snippet: Anti-bovine CD14 , CD14 , Mouse IgG1 , CAM36A , Bio-Rad , Sheep , Datasheet MCA6085.

Techniques: Isolation, Staining, Flow Cytometry, Expressing, Comparison

Journal: Frontiers in Immunology

Article Title: Characterization of intestinal mononuclear phagocyte subsets in young ruminants at homeostasis and during Cryptosporidium parvum infection

doi: 10.3389/fimmu.2024.1379798

Figure Lengend Snippet: Transcriptomic analyses of mononuclear phagocyte subsets in the ileal Peyer’s patch of lamb. Gene expression in the four sorted ileal mononuclear phagocyte (MP) subsets of 10-day-old lambs ( n = 5) was assessed by classical quantitative RT-PCR (D) or with the FLUIDIGM ® method (A–C) . Gene expression was defined by relative gene expression levels normalized to maximal expression across cell subsets, following normalization with three housekeeping genes ( HPRT , GAPDH , and ACTB ) and 2e -ΔCt value calculation. (A) Expression of the genes coding for the proteins used for cell sorting ( CD14 , CD172α , CADM1 , and CD11B ) in each cell subset, represented by violin plots (median and quartiles). Each point corresponds to one animal. (B) Principal component analysis (PCA) represented by its first two dimensions with corresponding variances in percentage and performed on relative gene expression values. The 66 genes displayed in (C) were included in the PCA. Each point corresponds to the data of one animal, with one point shape per individual animal. (C) MP subset-specific gene transcription represented by heatmaps of the Z -score normalized relative gene expression values for each gene analyzed in the four MP subsets, with hierarchical clustering of genes. According to their gene expression profile, subset 1 was identified as macrophages (MAC), subset 2 as type 1 conventional dendritic cells (cDC1), subset 3 as type 2 cDC (cDC2), and subset 4 as cDC no. 3. (D) Transcription of a selected set of genes characterizing DC subsets represented by the heatmap of the Z -score normalized relative gene expression values for each gene analyzed in the three DC subsets, with hierarchical clustering of genes.

Article Snippet: Anti-bovine CD14 , CD14 , Mouse IgG1 , CAM36A , Bio-Rad , Sheep , Datasheet MCA6085.

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, FACS

Journal: Frontiers in Immunology

Article Title: Characterization of intestinal mononuclear phagocyte subsets in young ruminants at homeostasis and during Cryptosporidium parvum infection

doi: 10.3389/fimmu.2024.1379798

Figure Lengend Snippet: Characterization of monocytic cells from the ileal Peyer’s patch of lamb by scRNA-sequencing. (A) Heatmap showing the top 10 differentially expressed genes (p_val_adj) in CSF1R -expressing clusters (clusters 0, 2, 3, 4, 6, 7, 8, 11, and 12), as determined by Seurat’s FindAllMarkers function. Expression levels are visualized from low expression (pink) to high expression (yellow). Complete gene lists are given in Supplementary Table 7 . (B) UMAP plots showing the expression of signature genes for macrophages [ FCGR3A ( CD16 ), FCGR1A ( CD64 ), CD14 , and CCL8 ; c0, 2, 3, 6, and 12] and of key genes characterizing the intestinal monocyte-to-macrophage differentiation [ S100A9 , CXXC5 , CLEC4E , and OVA-DOA (MHCII) ; c4 and 8] for each DC cluster. Expression levels are visualized from low expression (gray) to high expression (blue). *Genes of unknown function.

Article Snippet: Anti-bovine CD14 , CD14 , Mouse IgG1 , CAM36A , Bio-Rad , Sheep , Datasheet MCA6085.

Techniques: Sequencing, Expressing