Journal: International journal of molecular sciences

Article Title: Inhibitory Effects of Extracellular Vesicles from iPS-Cell-Derived Mesenchymal Stem Cells on the Onset of Sialadenitis in Sjögren's Syndrome Are Mediated by Immunomodulatory Splenocytes and Improved by Inhibiting miR-125b.

doi: 10.3390/ijms24065258

Figure Lengend Snippet: Figure 3. Effects of iEVs on splenic Th17 cells. Four-month-old female NOD.B10.H2b mice were IV injected with PBS, PD15 iEVs, or PD45 iEVs twice a week for two weeks. Splenocytes were collected at two weeks after last injection. (A,B) Percentages of IL17+ cells in splenic CD4+ T cells were examined with flow cytometry. (C) The mRNA levels of Th17 markers (IL17a, IL21, and Rorc) and negative regulator IL1rn in spleen were examined with qRT-PCR. N = 5, ns: not significant, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Article Snippet: These cells were stained with fluorescent-labeled antibodies against F4/80 (BD Pharmingen 123116, 123107, San Diego, CA, USA), CD3 (BioLegend 100236, San Diego, CA, USA), CD19 (BioLegend 152409), CD38 (BioLegend 102707), CD206 (BioLegend 141706), CD4 (BioLegend 100408), IL17a (130-112-009, Miltenyi Biotec, San Diego, CA, USA), or the corresponding isotype controls (BioLegend 400608, 400511, 400612, 400207, 1:100).

Techniques: Injection, Cytometry, Quantitative RT-PCR

Journal: International journal of molecular sciences

Article Title: Inhibitory Effects of Extracellular Vesicles from iPS-Cell-Derived Mesenchymal Stem Cells on the Onset of Sialadenitis in Sjögren's Syndrome Are Mediated by Immunomodulatory Splenocytes and Improved by Inhibiting miR-125b.

doi: 10.3390/ijms24065258

Figure Lengend Snippet: Figure 6. Effects of inhibiting miR-125b in aging iEVs on the polarization of splenic macrophages. Four-month-old female NOD.B10.H2b mice were IV injected with PBS, Ctrl EVs, or 125KD EVs twice a week for two weeks. Splenocytes were collected at two weeks after last injection. (A,B) Percentages of IL17+ cells in CD4+ splenocytes were examined with flow cytometry. (C) The mRNA levels of Th17 markers (IL17a, IL21, and Rorc) and negative regulator IL1rn in spleen were examined with qRT-PCR. N = 5, ns: not significant, *: p < 0.05, **: p < 0.01, ****: p < 0.0001.

Article Snippet: These cells were stained with fluorescent-labeled antibodies against F4/80 (BD Pharmingen 123116, 123107, San Diego, CA, USA), CD3 (BioLegend 100236, San Diego, CA, USA), CD19 (BioLegend 152409), CD38 (BioLegend 102707), CD206 (BioLegend 141706), CD4 (BioLegend 100408), IL17a (130-112-009, Miltenyi Biotec, San Diego, CA, USA), or the corresponding isotype controls (BioLegend 400608, 400511, 400612, 400207, 1:100).

Techniques: Injection, Cytometry, Quantitative RT-PCR

Journal: Acta pharmacologica Sinica

Article Title: Pirfenidone ameliorates silica-induced lung inflammation and fibrosis in mice by inhibiting the secretion of interleukin-17A.

doi: 10.1038/s41401-021-00706-4

Figure Lengend Snippet: Fig. 3 Pirfenidone inhibited the secretion of IL-17A in early-stage silicosis models. ELISA was used to detect the protein levels of IL-17A in serum (a) and BALF (b). The mRNA and protein levels of IL-17A in lung tissues from different groups were measured by qPCR (c), immunohistochemistry (d), and Western blot analysis (e). All data were presented as mean ± SD; n = 10 for each group; ***P < 0.001, ****P < 0.0001, ns (no statistical significance).

Article Snippet: IL-17A neutralization By intraperitoneal injection, 100 μg of anti-mouse IL-17A antibody (M00421-2, BOSTER, Wuhan, China) or isotype control IgG (ab18443, abcam, Cambridge, UK) was given to silica-exposed mice weekly for 4 weeks, starting 1 day after silica exposure.

Techniques: Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Western Blot

Journal: Acta pharmacologica Sinica

Article Title: Pirfenidone ameliorates silica-induced lung inflammation and fibrosis in mice by inhibiting the secretion of interleukin-17A.

doi: 10.1038/s41401-021-00706-4

Figure Lengend Snippet: Fig. 6 Pirfenidone diminished the production of IL-17A in advanced silicosis models. ELISA was performed to measure the protein expression of IL-17A in serum (a) and BALF (b) from different groups. The mRNA and protein levels of IL-17A in lung tissues were measured by qPCR (c), immunohistochemistry (d) and Western blot analysis (e), respectively. All data were presented as mean ± SD; n = 10 for each group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: IL-17A neutralization By intraperitoneal injection, 100 μg of anti-mouse IL-17A antibody (M00421-2, BOSTER, Wuhan, China) or isotype control IgG (ab18443, abcam, Cambridge, UK) was given to silica-exposed mice weekly for 4 weeks, starting 1 day after silica exposure.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemistry, Western Blot

Journal: Cellular and Molecular Immunology

Article Title: Characterization and allergic role of IL-33-induced neutrophil polarization

doi: 10.1038/cmi.2017.163

Figure Lengend Snippet: Primers used for real-time PCR analysis

Article Snippet: Fluorescein isothiocyanate-conjugated anti-Ly6G (1A8), PE-conjugated anti-IL-1R2 (4E2), PE-conjugated anti-mSiglec F (E50–2440) were purchased from BD Biosciences Pharmingen (San Diego, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Cellular and Molecular Immunology

Article Title: Characterization and allergic role of IL-33-induced neutrophil polarization

doi: 10.1038/cmi.2017.163

Figure Lengend Snippet: IL-33-induced neutrophils express unique cytokine and chemokine receptors compared with N0- or LPS-treated neutrophils. (a) Expression profiling of lineage-restricted receptors of cytokines and chemokines in N0, N(LPS) and N(IL-33) cells. Genes with predominant expression levels in one cell type (at least twofold change compared with other cell types) were organized to draw the plot. Colors represent genes above (red) or below (blue) the second highest expression in three cell types. (b) Freshly isolated bone marrow neutrophils were stimulated with LPS and IL-33 for the indicated times. The levels of cytokine and chemokine receptors in N(IL-33) cells, including IL-13Rα1, CXCR1, CCR1, IL-1R2 and CXCR2, were determined by real-time PCR. (c) Flow cytometric analysis of IL-1R2 expression on the surface of N0, N(LPS) and N(IL-33) cells. (d) The average percentages of IL-1R2+ cells in different neutrophil subpopulations were summarized. Data were shown as mean±s.d. (n=3). *P<0.05, **P<0.01 and ***P<0.001 compared with control or LPS-treated cells. CCR, C-C motif chemokine receptor; CXCR, C-X-C motif chemokine receptor; IL-1R, interleukin-1 receptor; LPS, lipopolysaccharide.

Article Snippet: Fluorescein isothiocyanate-conjugated anti-Ly6G (1A8), PE-conjugated anti-IL-1R2 (4E2), PE-conjugated anti-mSiglec F (E50–2440) were purchased from BD Biosciences Pharmingen (San Diego, CA, USA).

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Immunology

Article Title: Characterization and allergic role of IL-33-induced neutrophil polarization

doi: 10.1038/cmi.2017.163

Figure Lengend Snippet: The specific phenotype of IL-33-treated neutrophils is mediated by JNK and NF-κB signaling pathways. (a) Neutrophils freshly isolated from bone marrow were treated with IL-33 for 15, 30 and 120 min, respectively. The activities of JNK, p38, STAT1, Erk and p65 in cytoplasm were detected by western blotting. (b) The c-jun and p65 protein levels in nucleus of neutrophils after IL-33 treatment for 1 and 2 h, respectively, were determined by western blotting. (c and d) Neutrophils were pretreated with the indicated concentrations of JNK inhibitor (SP600125) and NF-κB inhibitor for 30 min and then stimulated with IL-33 for 24 h. The levels of IL-9, CCL2, CCL7, CCL12, IL-1R2 and IL-13Ra1 expression were detected by real-time PCR. (e and f) The concentrations of IL-9, IL-4, IL-5, IL-13, CCL2 and CCL7 in the supernatant of neutrophils pretreated with either SP600125 or NF-κB inhibitor and then cultured with IL-33 for 24 h were determined by ELISA assay kits. Assays were performed more than three times. Data are shown as mean±s.d. (n=3). *P<0.05, **P<0.01 and ***P<0.001 compared with control. CCL, C-C motif chemokine ligand; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal–regulated kinase; IL, interleukin; JNK, c-Jun N-terminal kinase; NF, nuclear factor; STAT, signal transducer and activator of transcription factor.

Article Snippet: Fluorescein isothiocyanate-conjugated anti-Ly6G (1A8), PE-conjugated anti-IL-1R2 (4E2), PE-conjugated anti-mSiglec F (E50–2440) were purchased from BD Biosciences Pharmingen (San Diego, CA, USA).

Techniques: Isolation, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Immunology

Article Title: Characterization and allergic role of IL-33-induced neutrophil polarization

doi: 10.1038/cmi.2017.163

Figure Lengend Snippet: N(IL-33) neutrophils promote OVA-induced allergic airway inflammation in vivo. OVA-presensitized WT and IL-33-TG mice were challenged with aerosolized OVA daily for 3 days as described in the Materials and methods section. (a) IL-9+ and IL-1R2+ neutrophils in BAL fluid of wild-type asthma mice were detected by flow cytometry. (b) H&E staining of lung tissues from control or OVA-induced asthma WT and IL-33-TG mice is presented. (c) Ratio of wet/dry lung weights of WT and IL-33-TG mice after OVA challenge was measured as described in the Materials and methods section. (d) Cell numbers in BAL fluid of WT and IL-33-TG mice after OVA challenge. (e) Flow cytometric analysis of infiltrated cells such as eosinophils in BAL fluid of WT and IL-33-TG mice after OVA challenge. (f) Proportion of neutrophils in BAL fluid of WT and IL-33-TG mice after OVA challenge is presented. (g) The mRNA expression levels of IL-4, IL-5, IL-9, IL-13, CCL2 and CCL7 in lung tissues of WT and IL-33-TG mice after OVA challenge were detected by real-time PCR. (h) Wild-type neutrophils were stimulated with IL-33, and N0 or N(IL-33) cells were transferred into OVA-presensitized syngeneic wild-type recipients before challenged with aerosolized OVA for 3 days as described in the Materials and methods section. H&E staining of lung tissues from mice treated as described. (i) Cell numbers in BAL fluid of OVA-induced asthma mice transferred with N0 and N(IL-33) cells. (j) The infiltrated cells in BAL fluid of asthma mice treated with N0 and N(IL-33) cells were detected by flow cytometry. Eosinophil numbers in BAL fluid were presented. (k and l) Quantitative PCR analysis of IL-4, IL-5, IL-9, IL-13, CCL2 and CCL7 expression levels in lung tissues of asthma mice treated with N0 and N(IL-33) cells. Data are expressed as mean±s.d. (n=3–5) and are one representative of the three independent experiments with similar results. *P<0.05, **P<0.01 and ***P<0.001 compared with control mice. BAL, bronchoalveolar lavage; CCL, C-C motif chemokine ligand; H&E, hematoylin and eosin; IL, interleukin; OVA, ovalbumin; WT, wild type.

Article Snippet: Fluorescein isothiocyanate-conjugated anti-Ly6G (1A8), PE-conjugated anti-IL-1R2 (4E2), PE-conjugated anti-mSiglec F (E50–2440) were purchased from BD Biosciences Pharmingen (San Diego, CA, USA).

Techniques: In Vivo, Flow Cytometry, Staining, Expressing, Real-time Polymerase Chain Reaction

Journal: mAbs

Article Title: Highly potent anti-CD20-RLI immunocytokine targeting established human B lymphoma in SCID mouse

doi: 10.4161/mabs.28699

Figure Lengend Snippet: Figure 2. Production and purification of the anti-CD20-RLI ICK. (A) ICK construction, PL: Peptidic Linker within RLI fusion protein.33(B) SDS-PAGE under non reducing (left panel) and reducing (middle panel) conditions (Lane 1: 5µg of RTX, line 2: 5µg of anti-CD20-RLI); western blot analysis using anti-IgG Ab (right panel, line 1: 0.1µg of anti-CD20-RLI) or anti-IL-15 Ab (right panel, line 2: 0.1µg of anti-CD20-RLI). kDa: kilo Dalton; Mw: Molecular weight. (C) gel-filtration analysis of RTX (left panel) or affinity-purified anti-CD20-RLI (right panel) revealing mainly monomer.

Article Snippet: 33 Control human isotype IgG was purchased from Santa-Cruz Biotechnology, peroxidase-conjugated polyclonal goat anti-human IgG (H+L) (109–036–003) from Jackson ImmunoResearch, anti-human IgG (H+L) (UP892370) from Interchim, mouse anti-human IL-15 mAb (B-E29) from Gen-Probe and RTX, an anti-CD20 mAb from Roche.

Techniques: Purification, SDS Page, Western Blot, Molecular Weight, Filtration, Affinity Purification

Journal: mAbs

Article Title: Highly potent anti-CD20-RLI immunocytokine targeting established human B lymphoma in SCID mouse

doi: 10.4161/mabs.28699

Figure Lengend Snippet: Figure 4. Cytokine-dependent functional effects of anti-CD20-RLI. (A) Kit225 or (B) 32Dβ cell proliferation induced by increasing concentrations of human IL-15 (▲), RLI (large-black-square) or anti-CD20-RLI (black-square) was assessed by Alamar blue reduction assay. (C) Phosphorylation of STAT5 was evaluated in 32Dβ cells stimulated during 30min by increasing concentrations of human IL-15 (▲), RLI (large-black-square) or anti-CD20-RLI (black-square). Data are means ± SEM of three experiments.

Article Snippet: 33 Control human isotype IgG was purchased from Santa-Cruz Biotechnology, peroxidase-conjugated polyclonal goat anti-human IgG (H+L) (109–036–003) from Jackson ImmunoResearch, anti-human IgG (H+L) (UP892370) from Interchim, mouse anti-human IL-15 mAb (B-E29) from Gen-Probe and RTX, an anti-CD20 mAb from Roche.

Techniques: Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: Immunomodulatory Activity of Dietary Fiber: Arabinoxylan and Mixed-Linked Beta-Glucan Isolated from Barley Show Modest Activities in Vitro

doi: 10.3390/ijms12010570

Figure Lengend Snippet: IL-8 secretion from Caco-2 ( A ) and HT-29 ( B ) cells in response to treatment with fiber fractions extracted from the barley variety Tyra, zymosan and PMII (all 1 mg/mL). Cells were incubated with fiber of the respective fiber fractions in cell culture medium for 24 hours before IL-8 secretion was measured. Each bar represents the average ± SD of one representative experiment from a total of three independent experiments. * p < 0.05.

Article Snippet: Monoclonal mouse anti-human IL-8 antibody (BD Bioscience Pharmingen, San Diego, CA) suspended in coating buffer (0.1 M Carbonate/Bicarbonate buffer pH 9.6) was added to MaxiSorp TM ELISA plates (Nunc, Roskilde, Denmark) and incubated over night at 4 °C.

Techniques: Incubation, Cell Culture