il 15rα antagonist  (R&D Systems)

Journal: Cells

Article Title: Dual Targeting of Glioblastoma Cells with Bispecific Killer Cell Engagers Directed to EGFR and ErbB2 (HER2) Facilitates Effective Elimination by NKG2D-CAR-Engineered NK Cells

doi: 10.3390/cells13030246

Figure Lengend Snippet: Generation of NK-92 cells expressing an NKG2D-based CAR and IL-15 superagonist RD-IL15. ( A ) Lentiviral transfer plasmids encoding the NKG2D-based CAR NKAR under the control of the spleen focus-forming virus promoter (SFFV). The NKAR encompasses an immunoglobulin heavy-chain signal peptide (SP), the extracellular domain of NKG2D (amino acid residues 82-216), a flexible (G 4 S) 2 linker (L), a Myc-tag (M), a CD8α hinge region, and transmembrane and intracellular domains of CD3ζ. For co-expression of the IL-15 superagonist RD-IL15, a sequence consisting of a second signal peptide (SP), IL-15Rα sushi domain (amino acid residues 31-107), a peptide linker (L) and affinity-optimized IL-15 N72D was fused in frame to the NKAR sequence via a porcine teschovirus self-cleaving peptide (P2A). NKAR and RD-IL15 sequences are followed by an internal ribosome entry site (IRES) and enhanced green fluorescent protein (EGFP) cDNA. ( B ) The expression of EGFP (left), as well as NKG2D and the NKG2D-based CAR (right) by sorted NKAR-NK-92 and NKAR_RD-IL15-NK-92 cells was analyzed by flow cytometry, as indicated. Parental NK-92 cells were included for comparison. Unstained NK-92 cells served as a control.

Article Snippet: To examine the secretion of RD-IL15, supernatant of NKAR_RD-IL15-NK-92 cells, cultured for 3 days at an initial cell density of 1 × 10 6 cells/mL, was harvested and analyzed by sandwich ELISA with an IL-15-specific capture antibody (34593; R&D Systems, Wiesbaden, Germany), a biotin-conjugated detection antibody specific for IL-15Rα (R&D Systems), and HRP-conjugated streptavidin (Genscript, Piscataway, NJ, USA).

Techniques: Expressing, Virus, Sequencing, Flow Cytometry, Comparison

Journal: Cells

Article Title: Dual Targeting of Glioblastoma Cells with Bispecific Killer Cell Engagers Directed to EGFR and ErbB2 (HER2) Facilitates Effective Elimination by NKG2D-CAR-Engineered NK Cells

doi: 10.3390/cells13030246

Figure Lengend Snippet: Biological activity of secreted IL-15 superagonist. ( A ) RD-IL15 secreted into the culture supernatant by NKAR_RD-IL15-NK-92 cells (blue bar) during a cultivation period of 3 days was measured by sandwich ELISA using an IL-15-specific capture antibody, and biotin-conjugated IL-15Rα-specific antibody and HRP-coupled streptavidin for detection. Recombinant His-tagged RD-IL15 was used as a standard for quantification. Culture supernatant from NKAR-NK-92 cells (red bar) served as a control. Mean values ± SD are shown; n = 3. ***, p < 0.001. ( B ) Lysates of NKAR-NK-92 and NKAR_RD-IL15-NK-92 cells cultured for 24 h in medium with or without IL-2 (100 IU/mL) or IL-15 (20 ng/mL) were subjected to SDS-PAGE and immunoblotting with antibodies specific for total and phosphorylated STAT5. γ-Tubulin served as a loading control. Uncropped images of the blots are shown in . ( C ) Proliferation of NKAR-NK-92 (left) and NKAR_RD-IL15-NK-92 cells (right) grown in medium with (filled symbols) or without (open symbols) IL-2 (100 IU/mL) for 7 days. Cell numbers were determined on days 0, 1, 4, and 7. Mean values ± SD are shown; n = 3 independent experiments. ****, p < 0.0001; *, p < 0.05; ns, p > 0.05 (not significant).

Article Snippet: To examine the secretion of RD-IL15, supernatant of NKAR_RD-IL15-NK-92 cells, cultured for 3 days at an initial cell density of 1 × 10 6 cells/mL, was harvested and analyzed by sandwich ELISA with an IL-15-specific capture antibody (34593; R&D Systems, Wiesbaden, Germany), a biotin-conjugated detection antibody specific for IL-15Rα (R&D Systems), and HRP-conjugated streptavidin (Genscript, Piscataway, NJ, USA).

Techniques: Activity Assay, Sandwich ELISA, Recombinant, Cell Culture, SDS Page, Western Blot

Journal: Journal of Functional Foods

Article Title: Lycium barbarum polysaccharides improve gut microbiota composition and alleviate pulmonary inflammatory damage in allergic asthma mice by inhibiting the IL-15RA/FUT2 pathway

doi: 10.1016/j.jff.2023.105729

Figure Lengend Snippet: Fig. 2. The effects of LBP on the expression of IL-15RA and FUT2, as well as the glycosylation of lung epithelial laminarin in mice with allergic asthma. Note: A: A venn diagram was created to illustrate the intersection between the target genes of LBP obtained from the GeneCards database and the asthma-related genes identified using Phenolyzer and GeneCards. B: Coexpedia was used to analyze the co-expression relationships of the candidate target genes. C: The results of the KEGG enrichment analysis are presented, with the horizontal axis representing GeneRatio and the vertical axis representing the names of KEGG entries, along with a color gradient histogram on the right side. D: A Venn diagram was constructed to show the intersection between the target genes of LBP, downstream effect genes of IL-15RA, and significantly up-regulated genes in the GSE6858 chip. E: The gene expression heatmap for the intersecting genes obtained from D was generated using the GSE23729 gene expression data chip for allergic asthma (4 normal samples and 4 samples with allergic asthma). F: The relative IL-15RA and FUT2 mRNA expression levels in mouse lung tissues were measured using RT-qPCR in each group (n = 10). G: The relative protein expression levels of IL-15RA and FUT2 in mouse lung tissues were examined using Western blot analysis in each group (n = 10). H: Immunofluorescence staining of lectins in mouse lung tissues in each group was conducted and observed at a magnification of × 400; red fluorescence represents UEA-1 staining (n = 10). I: Flow cytometry was performed to determine the percentage of UEA-I + cells in mouse lung tissues in each group (n = 10). * P < 0.05, experiments were repeated three times.

Article Snippet: The membrane was blocked with 5% bovine serum albumin for 1 h at room temperature and probed with primary antibodies of IL-15RA (1: 1000; sc-374023, Santa Cruz); FUT2 (1: 1000; sc-100742, Santa Cruz); β-actin (1: 5000; ab6276, Abcam, Cambridge, UK).

Techniques: Expressing, Glycoproteomics, Construct, Gene Expression, Generated, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Fluorescence, Flow Cytometry

Journal: Journal of Functional Foods

Article Title: Lycium barbarum polysaccharides improve gut microbiota composition and alleviate pulmonary inflammatory damage in allergic asthma mice by inhibiting the IL-15RA/FUT2 pathway

doi: 10.1016/j.jff.2023.105729

Figure Lengend Snippet: Fig. 5. The effects of IL-15RA regulation on FUT2 expression on lung epithelial fucose glycosylation, pulmonary inflammatory injury, and intestinal microbiota composition in allergic asthma mice. Note: A: Asthma-related intestinal microbiota abundance in the gutMEGA database, with the x-axis representing the abundance of microbiota (Asthma VS Normal); B: Quantitative levels of Bifidobacterium, Faecalibacterium, and Ruminococcus in mouse feces detected by RT- qPCR; C: Relative expression levels of IL-15RA and FUT2 mRNA in mouse lung tissues in each group as detected by RT-qPCR; D: Relative protein expression levels of IL-15RA and FUT2 in mouse lung tissues in each group as detected by Western blot; E: RL values of mice in each group with varying Mch concentrations; F: Cdyn values of mice in each group with varying Mch concentrations; G: Observation of inflammatory cell infiltration and mucus secretion in mouse lung tissues in each group by H&E staining and PAS staining (×200), along with corresponding score statistics. n = 10. Black arrows indicate inflammatory cells (upper graph, dark blue) and goblet cells (lower graph, light blue); H: Measurement of TNF-α, IFN-γ, IL-1β, IL-6, and IL-2 content in mouse serum of each group by ELISA; I: Measurement of TNF-α, IFN-γ, IL-1β, IL-6, and IL-2 content in mouse BALF of each group by ELISA; J: Quantitative levels of Bifidobacterium, Faecalibacterium, and Ruminococcus in mouse feces detected by RT-qPCR; K: Detection of lectin staining results in mouse lung tissues and statistical analysis of UEA-I + cells in lung tissues (×400), with red fluorescence indicating UEA-1; L: Percentage of UEA-I + cells in mouse lung tissues detected by flow cytometry. n = 10. *P < 0.05. The experiment was repeated three times.

Article Snippet: The membrane was blocked with 5% bovine serum albumin for 1 h at room temperature and probed with primary antibodies of IL-15RA (1: 1000; sc-374023, Santa Cruz); FUT2 (1: 1000; sc-100742, Santa Cruz); β-actin (1: 5000; ab6276, Abcam, Cambridge, UK).

Techniques: Expressing, Glycoproteomics, Quantitative RT-PCR, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Fluorescence, Flow Cytometry

Journal: Journal of Functional Foods

Article Title: Lycium barbarum polysaccharides improve gut microbiota composition and alleviate pulmonary inflammatory damage in allergic asthma mice by inhibiting the IL-15RA/FUT2 pathway

doi: 10.1016/j.jff.2023.105729

Figure Lengend Snippet: Fig. 6. The effect of LBP on pulmonary epithelial fucosylation, pulmonary inflammation, and gut microbiota composition in allergic asthmatic mice. Note: A: Relative expression levels of IL-15RA and F UT2 mRNA in lung tissues of mice from different groups, determined by RT-qPCR; B: Relative protein expression levels of IL-15RA and FUT2 in mouse lung tissues from different groups, analyzed by Western blot; C: RL values of mice from different groups in response to varying concentrations of Mch; D: Cdyn values of mice from different groups in response to varying concentrations of Mch; E: H&E and PAS staining, observed at × 200 magnification, for assessing inflammatory cell infiltration and mucus secretion in mouse lung tissues from different groups, with the corresponding scoring statistics; n = 10; The black arrows indicate inflammatory cells (top images, dark blue) and goblet cells (bottom images, light blue); F: Quantification of TNF-α, IFN-γ, IL-1β, IL- 6, and IL-2 levels in mouse serum, as detected by ELISA; G: Quantification of TNF-α, IFN-γ, IL-1β, IL-6, and IL-2 levels in mouse BALF, as detected by ELISA; H: Quantitative analysis of Bifidobacterium, Faecalibacterium, and Ruminococcus in mouse feces, determined by RT-qPCR; I: Immunofluorescence staining results for lectin in mouse lung tissues, and the quantification of UEA-I + cells in lung tissues at × 400 magnification, with red fluorescence representing UEA-1; J: Flow cytometry analysis to determine the percentage of UEA-I + cells in mouse lung tissues from different groups. *P < 0.05; n = 10; Experiments were repeated three times.

Article Snippet: The membrane was blocked with 5% bovine serum albumin for 1 h at room temperature and probed with primary antibodies of IL-15RA (1: 1000; sc-374023, Santa Cruz); FUT2 (1: 1000; sc-100742, Santa Cruz); β-actin (1: 5000; ab6276, Abcam, Cambridge, UK).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Fluorescence, Flow Cytometry

Journal: Journal of Functional Foods

Article Title: Lycium barbarum polysaccharides improve gut microbiota composition and alleviate pulmonary inflammatory damage in allergic asthma mice by inhibiting the IL-15RA/FUT2 pathway

doi: 10.1016/j.jff.2023.105729

Figure Lengend Snippet: Fig. 7. The mechanism graph of the regulatory network and function of LBPs in allergic asthma. Administration of LBPs alleviates inflammatory response, enhances gut microbial community composition of bifidobacterium, fecal bacterium, and ruminococcus, and reduces pulmonary epithelial fucosylation in asthmatic mice by inhibiting the IL-15RA/FUT2 axis.

Article Snippet: The membrane was blocked with 5% bovine serum albumin for 1 h at room temperature and probed with primary antibodies of IL-15RA (1: 1000; sc-374023, Santa Cruz); FUT2 (1: 1000; sc-100742, Santa Cruz); β-actin (1: 5000; ab6276, Abcam, Cambridge, UK).

Techniques: