Journal: Annals of Dermatology

Article Title: Hyaluronan Oligosaccharides Improve Rosacea-Like Phenotype through Anti-Inflammatory and Epidermal Barrier-Improving Effects

doi: 10.5021/ad.2020.32.3.189

Figure Lengend Snippet: Treatment with hyaluronan oligosaccharides (oligo-HAs) reduced inflammatory cell infiltration, interleukin-17 (IL-17), and kallikrein 5 (KLK5) expressions. (A) Hematoxylin and eosin-stained tissue sections of LL-37 induced rosacea-like mice showed marked inflammatory cellular infiltration. Further treatments with oligo-HAs reduced cellular infiltration (H&E, ×100). (B) Majority of infiltrated cells were CD4+ T cells and infiltration of CD4+ T cells decreased in oligo-HAs treated group (CD4, ×100). (C) IL-17A expression was markedly increased in the epidermis of LL-37 induced rosacea-like mice. Oligo-HAs decreased the IL-17A expression regardless of the injection method (IL-17A, ×200). (D) Expression of KLK5 was increased in LL-37 induced rosacea-like mice (arrow head). Further treatment with oligo-HAs reduced KLK5 expression (KLK5, ×400). i.d.: intradermally, i.p.: intraperitoneally.

Article Snippet: To detect CD4, IL-17A, KLK5, filaggrin, and CD44 in histological specimens from mice, primary antibodies including a monoclonal mouse anti-CD4 antibody (BD Biosciences, Franklin Lakes, NJ, USA), monoclonal rat anti-mouse IL-17A antibody (Covalab, Villeurbanne, France), polyclonal rabbit anti-KLK5 antibody (Abcam, Cambridge, UK), polyclonal mouse anti-profilaggrin antibody (Covance, Princeton, NJ, USA), and monoclonal rat anti-human/mice CD44 antibody (eBioscience, San Diego, CA, USA) were used.

Techniques: Staining, Expressing, Injection

Journal: Journal of Inflammation (London, England)

Article Title: Comparative effects of the herbal constituent parthenolide (Feverfew) on lipopolysaccharide-induced inflammatory gene expression in murine spleen and liver

doi: 10.1186/1476-9255-2-6

Figure Lengend Snippet: IL-6 protein production in sera following parthenolide and LPS co-treatment. Female B6C3F1 mice were co-treated with parthenolide (5 mg/kg, i.p.) or 50 μl DMSO and LPS (1 mg/kg, i.p.) or 100 μl water. After 90 minutes, blood was collected and serum analyzed for IL-6 by ELISA. The letter (a) indicates a significant difference compared to vehicle and parthenolide controls; (b) indicates a significant difference compared to LPS. Data are mean ± SEM (n = 16, controls n = 4), and is a combination of 4 separate experiments.

Article Snippet: IL-6 analysis was performed using purified and biotin-conjugated rat anti-mouse IL-6 antibodies from PharMingen (San Diego, CA) as described previously [ ].

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation (London, England)

Article Title: Comparative effects of the herbal constituent parthenolide (Feverfew) on lipopolysaccharide-induced inflammatory gene expression in murine spleen and liver

doi: 10.1186/1476-9255-2-6

Figure Lengend Snippet: IL-6 mRNA expression levels in spleen and liver following parthenolide and LPS co-treatment. Female B6C3F1 mice were co-treated with parthenolide (5 mg/kg, i.p.) or 50 μl DMSO and LPS (1 mg/kg, i.p.) or 100 μl water. Spleen and liver were collected after 90 minutes and total RNA was extracted and subjected to real-time, one-step RT-PCR using TaqMan primers and probes. IL-6 mRNA levels were normalized using 18S rRNA and related to spleen control values. (a) indicates a significant difference compared to vehicle and parthenolide controls; (b) indicates a significant difference compared to LPS. Data are mean ± SEM (n = 16, controls n = 4), and is a combination of 4 separate experiments.

Article Snippet: IL-6 analysis was performed using purified and biotin-conjugated rat anti-mouse IL-6 antibodies from PharMingen (San Diego, CA) as described previously [ ].

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Molecules

Article Title: Enhanced Anti-Allergic Activity of Milk Casein Phosphopeptide by Additional Phosphorylation in Ovalbumin-Sensitized Mice

doi: 10.3390/molecules24040738

Figure Lengend Snippet: Effects of oral feeding with differently phosphorylated CPP-III on cytokine production and gene expression of OVA-sensitized mice. OVA-sensitized mice were fed differently phosphorylated CPP-III for a period of 6 weeks, and the collected spleen (SP) cells were incubated with PBS (unstimulated) for normal group and stimulated with 50 μg/mL (final concentration) of OVA for the control, N-CPP, P-CPP, and D-CPP groups. The levels of IFN-γ ( A ) and IL-4 ( B ) in the culture supernatant after incubation for 72 h were determined by sandwich ELISA. Relative mRNA expression levels of IL-4 and GATA-3 ( C ) were determined by qPCR ( C ). Data represent the mean ± SE of individual mice in the group. * p < 0.05; ** p < 0.01 vs sham group.

Article Snippet: The levels of cytokines secreted into the murine SP cell culture supernatants were measured by sandwich ELISA using the following HRP-conjugated antibodies and hydrogen peroxide with O -phenylenediamine as the substrate: rat anti-mouse IL-4 and IFN-γ antibodies, biotinylated rat anti-mouse IL-4 and IFN-γ antibodies, and streptavidin-HRP conjugate (all purchased from Peprotech, Rocky Hill, NJ, USA).

Techniques: Expressing, Incubation, Concentration Assay, Sandwich ELISA

Journal: PLoS ONE

Article Title: NK-4 exerts selective regulatory effects on the activation and function of allergy-related Th2 cells

doi: 10.1371/journal.pone.0199666

Figure Lengend Snippet: Th1 clone #4 (A and B) and Th2 clone D10.G4.1 cells (C to E) (2.5 x 10 4 cells/well) were stimulated with immobilized anti-CD3ε mAb (8 μg/ml) in the presence or absence (control) of varying concentrations of NK-4 for 48 h at 37°C in 96-well plates. Concentrations of IFN-γ (A), IL-4 (C) and IL-5 (D) in culture supernatants were measured by ELISA. Cell numbers of #4 (B) and D10.G4.1 (E) were determined by cell counting kit-8. Results are the means ± S.D. of triplicate cultures. Results are representative of three independent experiments with similar results. * p < 0.05, ** p < 0.01 compared with control cultures.

Article Snippet: The Abs for coating the plates and the biotinylated secondary mAbs were as follows: for IL-4, rat mAb anti-mouse IL-4 (BVD4-1D11, BD Biosciences, San Diego, CA) and biotinylated mAb anti-mouse IL-4 (BVD6-24G2, BD Biosciences); for IL-5, rat mAb anti-mouse/human IL-5 (TRFK5, BD Biosciences) and biotinylated mAb anti-mouse IL-5 (TRFK4, BD Biosciences); for IFN-γ, polyclonal rabbit anti-mouse IFN-γ Ab (prepared in our laboratory) and biotinylated rat mAb anti-mouse IFN-γ (XMG1.2, BD Pharmingen).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Counting

Journal: PLoS ONE

Article Title: NK-4 exerts selective regulatory effects on the activation and function of allergy-related Th2 cells

doi: 10.1371/journal.pone.0199666

Figure Lengend Snippet: Th1 clone #4 (2.5 x 10 4 cells/well) were stimulated with OVA (200 μg/ml) and MMC-treated BALB/c mouse spleen cells (1.2 x 10 6 cells/well) in the presence or absence (control) of varying concentrations of NK-4 for 48 h at 37°C in 96-well plates (A). D10.G4.1 cells (2.5 x 10 4 cells/well) were stimulated with MMC-treated C57BL/6 mouse spleen cells (1.2 x 10 6 cells/well) (B and C). Concentrations of IFN-γ (A), IL-4 (B) and IL-5 (C) in culture supernatants were measured by ELISA. Results are the means ± S.D. of triplicate cultures. Results are representative of three independent experiments with similar results. ** p < 0.01 compared with control cultures.

Article Snippet: The Abs for coating the plates and the biotinylated secondary mAbs were as follows: for IL-4, rat mAb anti-mouse IL-4 (BVD4-1D11, BD Biosciences, San Diego, CA) and biotinylated mAb anti-mouse IL-4 (BVD6-24G2, BD Biosciences); for IL-5, rat mAb anti-mouse/human IL-5 (TRFK5, BD Biosciences) and biotinylated mAb anti-mouse IL-5 (TRFK4, BD Biosciences); for IFN-γ, polyclonal rabbit anti-mouse IFN-γ Ab (prepared in our laboratory) and biotinylated rat mAb anti-mouse IFN-γ (XMG1.2, BD Pharmingen).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: Murine astrocytes produce IL-24 and are susceptible to the immunosuppressive effects of this cytokine

doi: 10.1186/s12974-019-1444-1

Figure Lengend Snippet: IL-24 augments the expression of suppressive cytokine signaling components by murine astrocytes and limits inflammatory cytokine release by these cells. a Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 30 min and the presence of phosphorylated STAT3 and STAT1 was determined by immunoblot analysis. Expression of β-actin is shown as a loading control and these immunoblots are representative of two separate experiments. b Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 2 or 4 h, and SOCS3 mRNA expression was determined by semi-quantitative RT-PCR. Expression of the housekeeping gene product GAPDH is shown, and relative SOCS3 expression was determined by densitometric analysis and normalized to untreated cells. Data is expressed as the mean ± the SEM of three independent experiments, and an asterisk indicates a statistically significant difference from unchallenged cells at each time point ( p < 0.05). c Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 8 h prior to immunoblot analysis for SOCS3 protein expression. Expression of the housekeeping gene β-actin is shown and relative SOCS3 protein expression was determined by densitometric analysis normalized to untreated cells. Asterisks indicate statistically significant differences from unchallenged cells (p < 0.05). d Astrocytes were untreated or treated with IL-24 (0.5, 3, 10, 30, or 100 ng/mL) for 4 h prior to challenge with bacterial LPS (5 ng/mL) or vehicle control for 12 h, and IL-6 secretion was determined by specific capture ELISA. Asterisks indicate a statistically significant difference ( p < 0.05) from similarly challenged cells in the absence of IL-24 ( n = 3). e Primary astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 4 h prior to being uninfected or infected with Nm for 48 h before cell viability analysis via MTS assay. Data is presented as the mean absorbance ± SEM for three experiments. 0.1% Triton X-100 was used as a positive control, and an asterisk indicates a statistically significant difference from unchallenged cells in the absence of IL-24 ( p < 0.05)

Article Snippet: Commercially available Duoset® ELISA kits were used to measure IL-10 and TNF-α secretion (R&D Systems), while murine IL-6 secretion was measured using a rat anti-mouse IL-6 capture antibody (Clone MP5-20F3) and a biotinylated rat anti-mouse IL-6 detection antibody (Clone MP5-C2311) (BD Biosciences).

Techniques: Expressing, Recombinant, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Infection, MTS Assay, Positive Control