Journal: PLoS ONE

Article Title: Oxidation by Neutrophils-Derived HOCl Increases Immunogenicity of Proteins by Converting Them into Ligands of Several Endocytic Receptors Involved in Antigen Uptake by Dendritic Cells and Macrophages

doi: 10.1371/journal.pone.0123293

Figure Lengend Snippet: ( A ) Uptake of fluorescently-labelled proteins by CHO cells transfected with human CD36 as compared to non-transfected cells. ( B ) Binding of anti-CD36 mAb and control mouse IgA to CD36-transfected and non-transfected CHO cells, determined by cellular ELISA. ( C ) Binding of rCD36 to plate-adsorbed proteins. ( D ) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rCD36 binding to plate-adsorbed OVA-Cl. ( E ) Binding of polyclonal anti-mouse CD36 Ab and control goat IgG to BM-DC, splenic DC and PEM. A representative histogram of Ab binding to BM-DC is displayed on the left graph. ( F ) Binding of anti-mouse SR-A mAb and control rat IgG2b to BM-DC, splenic DC and PEM. A representative histogram of Ab binding to BM-DC is shown on the left graph. ( G ) Binding of SR-A present in lysates of PEM to plate-adsorbed proteins. ( H ) Effects of anti-SR-A 2F8 mAb and AcLDL on the uptake of fluorescently-labelled proteins by BM-DC. Shown are results of single experiments, each representative of at least 3 similar experiments performed (A-D, G, H) or averages +SEM from 4–6 independent experiments (E, F). The data were analysed with the unpaired (A-C, G) or one-sample (H) Student’s t-test or with ANOVA, followed by the Tukey-Kramer post-test (D). *, p < 0.05; ND, not done.

Article Snippet: Rat anti-mouse scavenger receptor A (SR-A) mAb (clone 2F8) was obtained from AbD Serotec; mouse anti-mouse CD36 mAb (CRF D-2712) from Hycult Biotech; mouse IgA isotype control mAb (M18-254), rat IgG2b isotype control mAb (A95-1), rat anti-mouse CD11b mAb (M1/70) and phycoerythrin (PE)-streptavidin conjugate from BD Biosciences; rat IgG2a isotype control mAb (54447), normal goat IgG, polyclonal goat anti-mouse CD36, anti-mouse LOX-1 (lectin-type oxidised LDL receptor-1), anti-human SREC-I (scavenger receptor expressed by endothelial cells-I) Ab and PE-conjugated rat anti-mouse LOX-1 mAb (214012) from R&D Systems; polyclonal goat anti-mouse SREC-I, anti-mouse RAGE (receptor for advanced glycation end products), anti-mouse stabilin-1 and rabbit anti-mouse-stabilin-1 Ab from Santa Cruz Biotechnology; PE-conjugated donkey anti-goat IgG Ab from SouthernBiotech; rat anti-mouse CD206/mannose receptor mAb (MR5D3) and PE-conjugated goat anti-rat IgG Ab from BioLegend; horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgA, F(ab’)2 fragments of goat anti-rat IgG and donkey anti-goat IgG Ab from Rockland.

Techniques: Transfection, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Clinical and Experimental Immunology

Article Title: Investigating the role of proinflammatory CD16 + monocytes in the pathogenesis of inflammatory bowel disease

doi: 10.1111/j.1365-2249.2010.04177.x

Figure Lengend Snippet: The peripheral blood CD14+ CD16+ monocyte population is increased in active Crohn's disease. (a) Peripheral blood mononuclear cells (PBMC) were stained for CD14, CD16 and CD36 and analysed by flow cytometry. Cells were pre-gated for high forward-scatter (FSC), side-scatter (SSC) and CD36 expression, and monocytes separated by CD14/CD16 expression. The lower panels show representative dot plots from a healthy volunteer, and Crohn's disease patients with quiescent [Crohn's disease activity index (CDAI)<150] and active (CDAI>150) inflammation. (b) The ratio of CD14+ CD16+ monocytes was increased significantly in Crohn's disease patients compared to healthy controls. *P = 0·01 (two-tailed Student's t-test with Welch's correction). (c) Further categorization of patients revealed that the CD14+ CD16+ population was increased in active Crohn's disease compared to healthy controls, but not quiescent disease or patients with ulcerative colitis. *P < 0·05 (Bonferroni's post-test following one-way analysis of variance).

Article Snippet: Slides were then incubated at 4°C overnight with the following primary antibodies: mouse anti-CD14 (BioLegend, San Diego, CA, USA), rat anti-CD16 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-CD36 (Novus Biologicals, Littleton, CO, USA).

Techniques: Staining, Flow Cytometry, Expressing, Activity Assay, Two Tailed Test

Journal: Clinical and Experimental Immunology

Article Title: Investigating the role of proinflammatory CD16 + monocytes in the pathogenesis of inflammatory bowel disease

doi: 10.1111/j.1365-2249.2010.04177.x

Figure Lengend Snippet: The number of CD14+ CD16+ monocytes is enhanced significantly in the lamina propria of Crohn's disease patients. (a) Monocytic lineage of CD14+ CD16+ cells in the colonic mucosa was confirmed by three-channel immunofluorescence microscopy for CD14 (green), CD16 (red) and CD36 (blue). No or limited CD36 staining was observed in CD14+ CD16– cells (i) and CD14– CD16+ cells (ii). In contrast, CD14+ CD16+ cells stained strongly for CD36 (iii, white pseudocolour). Colonic crypts are indicated with dashed lines. Lower panels, magnification of the boxed cells shown above. (b) Representative images of colonic mucosal sections from non-inflammatory bowel disease (IBD) control patients, non-inflamed and inflamed tissue from Crohn's disease patients, and non-inflamed ulcerative colitis samples. CD14+ CD16+ monocytes are shown in yellow pseudocolour, with nuclei in blue. (c) The number of CD14+ CD16+ cells per high-power field (HPF) was significantly increased in Crohn's disease mucosa, and enhanced further in actively inflamed tissue. Each data point represents the average of five or more HPF per patient sample. *P < 0·001 versus control, †P < 0·001 versus IBD – not inflamed [Bonferroni's post-test following one-way analysis of variance (anova)]. (d) The ratio of CD14+ CD16+ cells was increased significantly in Crohn's disease patients compared to controls. *P < 0·001 (Bonferroni's post-test following one-way anova). (e) The increase in total CD14+ CD16+ monocytes was confirmed by flow cytometry. Mononuclear cells were isolated from control and Crohn's disease colonic mucosa as described in the Materials and methods, pre-gated for forward-scatter (high), side-scatter (high) CD36+, and separated by CD14/CD16 expression. A dramatic increase of CD14+ CD16+ monocytes was seen in IBD tissue (red box). Plots are representative of three patient samples per group. (f) The tumour necrosis factor (TNF)-α mean fluorescence intensity (MFI, arbitrary units) of CD14+ CD16+ monocytes was significantly higher compared to CD14+ CD16– cells in the same field of vision. The graph shows the result from 14 HPF taken from three Crohn's disease patient samples. *P < 0·001 (two-tailed Student's t-test).

Article Snippet: Slides were then incubated at 4°C overnight with the following primary antibodies: mouse anti-CD14 (BioLegend, San Diego, CA, USA), rat anti-CD16 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-CD36 (Novus Biologicals, Littleton, CO, USA).

Techniques: Immunofluorescence, Microscopy, Staining, Flow Cytometry, Isolation, Expressing, Fluorescence, Two Tailed Test