Journal: Frontiers in Immunology

Article Title: NCR1 Expression Identifies Canine Natural Killer Cell Subsets with Phenotypic Similarity to Human Natural Killer Cells

doi: 10.3389/fimmu.2016.00521

Figure Lengend Snippet: Expression of NKp46 on canine PBMC using a novel anti-canine NKp46 antibody . (A) Clone 48A is specific for L-cells expressing NKp46 (open histograms: secondary antibody only, gray filled: vector only, black filled: NKp46:L-cells). (B) NKp46 is expressed on CD3 − and CD3 + lymphocytes. CD3 − /NKp46 + cells make up on average 2.3% of lymphocytes. Shown is median ± IQR ( n = 12). Representative flow plot is shown. These CD3 − /NKp46 + cells are largely negative for CD4 (median = 0%), and a subset express CD8α (median = 9.3%). They are CD5 low/− (median = 10.6%) (Solid line: negative control, Black filled: CD3 − /NKp46 + , Dotted line: CD5 expression on total lymphocytes showing two populations of CD5 + cells). Shown is median ± IQR for CD4 ( n = 7), CD5 ( n = 7), and CD8 ( n = 10). NKp46 is not expressed on CD21 + B-cells.

Article Snippet: Cells were stained with 200 ng/1,000,000 cells of mouse anti-canine NKp46 Clone 48A (isotype: IgG2a) or unconjugated TCRαβ/TCRγδ for 30 min, followed by goat anti-mouse IgG RPE (Jackson ImmunoResearch, 115-116-146) in 50% FBS/PBS, and finally with anti-canine TCRαβ/γδ (FITC/Alexa Fluor 488 when indicated; Peter Moore, UC Davis) and commercially available antibodies (Biorad) to canine CD3 (MCA1774F; clone CA17.2A12), CD8α (MCA1039A700; clone YCATE55.9), CD4 (MCA1038PECY7; clone YKIX302.9), CD21 (MCA1781A647; clone CA2.1D6), CD5 (MCA1037APC; YKIX322.3), and anti-human CD14 (MCA1568A647, MCA1568A700; TÜK4).

Techniques: Expressing, Plasmid Preparation, Negative Control

Journal: Frontiers in Immunology

Article Title: NCR1 Expression Identifies Canine Natural Killer Cell Subsets with Phenotypic Similarity to Human Natural Killer Cells

doi: 10.3389/fimmu.2016.00521

Figure Lengend Snippet: CD3 − /NKp46 + cells expand rapidly on K562 mbIL-21 feeder cells and express NK-cell associated markers . (A) CD3 − /NKp46 + cells expand on K562mbIL-21 feeder cells to make up on average 60.2% of total cells, with contaminating T-cells on average 31.8% ( n = 16, median ± IQR shown). Expanded CD3 − /NKp46 + cells are CD4 − (median = 0.08%) and are significantly decreased in percentage of cells coexpressing CD5 (median = 0.4%, p = 0.037) (open histogram = negative control, black filled = CD3 − /NKp46 + ) and increased in CD8 coexpression (median = 35%, p = 0.005, n = 7; median ± IQR shown). (B) CD3 − /NKp46 + cells express mRNA for NKp46, NKp30, Perforin, Granzyme B, NKG2D, DNAM-1, NKp80, NKp44, Ly49, and CD16 ( n = 5, all PCR reactions were conducted concurrently). (C) On average, 4.5 × 10 8 (median = 1.7e8) CD3 − /NKp46 + cells are produced at the end of 3 weeks from an average 17,774 CD3 − /NKp46 + (median = 7,000) cells at day 0 (shown is median ± IQR) representing a median 20,283-fold expansion. (D) Depletion of T-cells at day 0 significantly increases the purity of CD3 − /NKp46 + cells at day 21 (95.8 versus 40.6%) and does not hinder fold expansion (157,281 versus 64,686, n = 3).

Article Snippet: Cells were stained with 200 ng/1,000,000 cells of mouse anti-canine NKp46 Clone 48A (isotype: IgG2a) or unconjugated TCRαβ/TCRγδ for 30 min, followed by goat anti-mouse IgG RPE (Jackson ImmunoResearch, 115-116-146) in 50% FBS/PBS, and finally with anti-canine TCRαβ/γδ (FITC/Alexa Fluor 488 when indicated; Peter Moore, UC Davis) and commercially available antibodies (Biorad) to canine CD3 (MCA1774F; clone CA17.2A12), CD8α (MCA1039A700; clone YCATE55.9), CD4 (MCA1038PECY7; clone YKIX302.9), CD21 (MCA1781A647; clone CA2.1D6), CD5 (MCA1037APC; YKIX322.3), and anti-human CD14 (MCA1568A647, MCA1568A700; TÜK4).

Techniques: Negative Control, Produced

Journal: Frontiers in Immunology

Article Title: NCR1 Expression Identifies Canine Natural Killer Cell Subsets with Phenotypic Similarity to Human Natural Killer Cells

doi: 10.3389/fimmu.2016.00521

Figure Lengend Snippet: NKp46 − NK cells in canines and humans are CD16 low/− , retain NK cell phenotype and function, and NKp46 is inducible . CD3 − /CD21 − /CD14 − , NKp46 − (Null) cells are present in canine PBMC (A) as described for expanded cells in Figure A. Null cells consist of on average 5.1% of lymphocytes in PBMC, and 5.7% of expanded cells (median ± IQR shown, day 0 n = 8, day 21 n = 12). (B) The addition of null cells to fold expansion calculation decreases mean fold expansion by 50% ( n = 8, median ± IQR shown). (C) Null cells have negligible expression of CD4, CD5, and CD8 in PBMC, but increase CD8 expression with expansion similar to CD3 − /NKp46 + cells (3.1–52.4%, n = 7, median ± IQR shown). (D) NKp46 can be induced in Null cells after 5 days coculture with K562 Clone 9.mbIL-21 feeder cells, while NKp46 expression is constant in sorted CD3 − /NKp46 + cells ( n = 7). (E) Expanded Null cells express similar levels of DNAM-1, Perforin, and Granzyme B, but decreased expression of NKp46, NKG2D, NKp44, and CD16, compared to CD3 − /NKp46 + cells (mean ± SD shown). (F) Expanded Null cells have decreased cytotoxicity against Gray and melanoma-12 [ n = 9, mel-12 (in black) = 4, Gray = 6 (in blue), individual donors designated by unique symbols, median ± IQR shown], but similar cytokine secretion ( n = 3) compared to CD3 − /NKp46 + cells ( n = 7, median depicted). (G) NKp46 − NK cells are present in primary (mean = 21%) and K562 Clone 9.mbIL-21-expanded (mean = 21.7%) human NK cells ( n = 4, median ± IQR shown). (H) Spade clustering analysis of primary and mbIL-21 expanded human NK cells for CD56, NKp46, NKp30, NKp44, NKG2C, NKG2D, and NKG2A revealed that NKp46 and CD16 cluster together (outlining delineates cells that stain positive for the indicated marker) and (I) NKp46 − cells have decreased expression of CD16, NKp44, DNAM-1, and NKG2D ( n = 4, median ± IQR shown, whiskers are minimum to maximum).

Article Snippet: Cells were stained with 200 ng/1,000,000 cells of mouse anti-canine NKp46 Clone 48A (isotype: IgG2a) or unconjugated TCRαβ/TCRγδ for 30 min, followed by goat anti-mouse IgG RPE (Jackson ImmunoResearch, 115-116-146) in 50% FBS/PBS, and finally with anti-canine TCRαβ/γδ (FITC/Alexa Fluor 488 when indicated; Peter Moore, UC Davis) and commercially available antibodies (Biorad) to canine CD3 (MCA1774F; clone CA17.2A12), CD8α (MCA1039A700; clone YCATE55.9), CD4 (MCA1038PECY7; clone YKIX302.9), CD21 (MCA1781A647; clone CA2.1D6), CD5 (MCA1037APC; YKIX322.3), and anti-human CD14 (MCA1568A647, MCA1568A700; TÜK4).

Techniques: Expressing, Staining, Marker

Journal: Drug Design, Development and Therapy

Article Title:

Tranilast Inhibits Pulmonary Fibrosis by Suppressing TGFβ/SMAD2 Pathway

doi: 10.2147/dddt.s264715

Figure Lengend Snippet: Figure 4 Tranilast attenuated BLM-induced ECM production and phosphorylation of SMAD2 in mice. (A) TGFβ1 concentration in BAL fluid was evaluated by ELISA. (B) Effects of tranilast on collagen contents. Data are presented as mean ± SD in each group of 10 mice. **p < 0.05. (C) Effect of tranilast on expression of fibronectin in the murine lungs by immunohistochemistry (IHC). Bar = 200 µm. (D) The average of the percentage of fibronectin positive ratio in each of the four groups was calculated by dividing the average of each group with that of the control group. Data are presented as mean ± SD in each group of 10 mice. *p < 0.01. (E) IHC staining for phospho-SMAD2 in the murine lungs. (F) The phospho- Smad2-positive cells were counted in 10 fields at × 200 magnification. Bar = 200 µm. The average of the percentage of phospho-SMAD2-positive cells in each of the four groups was calculated by dividing the average of each group with that of the control group. Data are presented as mean ± SD in each group of 10 mice. *p < 0.01, **p < 0.05. (G) Levels of phospho- and total-SMAD2 in the whole lung lysates of mice by Western blotting analysis. (H) Fold changes were analyzed by setting the ratios of the phospho/total protein band intensities.

Article Snippet: Enzyme-Linked ImmunoSorbent Assay (ELISA) for Anti-TGFβ1 Antibody Detection TGFβ1 concentration in BAL fluid was analyzed using ELISA sandwich, specifically developed with antibodies against canine TGFβ1 (Mouse/Rat/Porcine/Canine TGFβ1, MB100B, R&D Systems, Minneapolis, USA).

Techniques: Phospho-proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemistry, Control, Western Blot