Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Interleukin-17A expression in human synovial mast cells in rheumatoid arthritis and osteoarthritis.

doi: 10.1016/j.alit.2016.04.007

Figure Lengend Snippet: Fig. 1. Expression of IL-17A in synovial MCs from OA patients and from RA patients. Representative images of IL-17Aþ MCs (tryptaseþ cells) in synovia from an OA patient (A) and an RA patient (B). Immunochemical staining of synovia with anti-tryptase mAb (green, a) and anti-IL-17A (red, b) Ab, and a merged image (yellow, c). The cells were stained with DAPI (blue). The white arrow heads indicate tryptase-IL-17A double-positive cells. Bar ¼ 10 mm. (C) As negative controls, a synovium sample from an OA patient was stained with mouse IgG1 and goat IgG. The results are representative of separate analyses using 6 patients with OA and 6 patients with RA.

Article Snippet: The following antibodies (Abs) were purchased from the indicated sources: human IgE (Calbiochem, San Diego, CA, USA); goat anti-human IL-17A polyclonal Ab (R&D Systems, Minneapolis, MN, USA); anti-human tryptase monoclonal (m) Ab (clone AA1; DakoCytomation Inc., Carpinteria, CA, USA); anti-Fc 3RIa mAb (clone CRA1; eBioscience, San Diego, CA, USA); anti-human IgE Ab (DakoCytomation Inc.); and F(ab0)2 fragments of anti-human FcgRI (clone 10.1; ID Labs Inc., London, ON, Canada).

Techniques: Expressing, Staining

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Interleukin-17A expression in human synovial mast cells in rheumatoid arthritis and osteoarthritis.

doi: 10.1016/j.alit.2016.04.007

Figure Lengend Snippet: Fig. 2. A comparison of the numbers of MCs (A), the numbers of IL-17Aþ MCs (B), the numbers of IL-17Aþ cells (C), the percentage of IL-17Aþ MCs among all the MCs (D), and the percentage of IL-17Aþ MCs among all the IL-17Aþ cells (E) in synovia from OA patients (open circles, n ¼ 6) and from RA patients (closed circles, n ¼ 6). Data are expressed as the median and inter-quartile range. N.S., not significant.

Article Snippet: The following antibodies (Abs) were purchased from the indicated sources: human IgE (Calbiochem, San Diego, CA, USA); goat anti-human IL-17A polyclonal Ab (R&D Systems, Minneapolis, MN, USA); anti-human tryptase monoclonal (m) Ab (clone AA1; DakoCytomation Inc., Carpinteria, CA, USA); anti-Fc 3RIa mAb (clone CRA1; eBioscience, San Diego, CA, USA); anti-human IgE Ab (DakoCytomation Inc.); and F(ab0)2 fragments of anti-human FcgRI (clone 10.1; ID Labs Inc., London, ON, Canada).

Techniques: Comparison

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Interleukin-17A expression in human synovial mast cells in rheumatoid arthritis and osteoarthritis.

doi: 10.1016/j.alit.2016.04.007

Figure Lengend Snippet: Fig. 3. IL-17A production from cultured synovium-derived MC following IgE- and IgG-dependent stimulation. (A) IgE-sensitized MCs from patients with OA (white bars, n ¼ 4 donors) or from patients with RA (black bars, n ¼ 4 donors) were incubated with anti-IgE for 6 h. (B) MCs from patients with OA (n ¼ 4 donors) were incubated with 1 mg/ml of F(ab0)2aFcgRI (black bars) or F(ab0)2mIgG1 (white bars) for 30 min and were then stimulated with gF(ab0)2amF(ab0)2 for 6 h. (C) MCs from patients with OA (n ¼ 6 donors) were stimulated with monomeric IgG or aggregated IgG for 6 h. (D) MCs from patients with OA (white bars, n ¼ 7 donors) and from patients with RA (black bars, n ¼ 4 donors) were stimulated with IL-33 for 6 h. (E, F) MCs from patients with OA (n ¼ 5e6 donors) were stimulated with anti-Fc

Article Snippet: The following antibodies (Abs) were purchased from the indicated sources: human IgE (Calbiochem, San Diego, CA, USA); goat anti-human IL-17A polyclonal Ab (R&D Systems, Minneapolis, MN, USA); anti-human tryptase monoclonal (m) Ab (clone AA1; DakoCytomation Inc., Carpinteria, CA, USA); anti-Fc 3RIa mAb (clone CRA1; eBioscience, San Diego, CA, USA); anti-human IgE Ab (DakoCytomation Inc.); and F(ab0)2 fragments of anti-human FcgRI (clone 10.1; ID Labs Inc., London, ON, Canada).

Techniques: Cell Culture, Derivative Assay, Incubation

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Interleukin-17A expression in human synovial mast cells in rheumatoid arthritis and osteoarthritis.

doi: 10.1016/j.alit.2016.04.007

Figure Lengend Snippet: Fig. 4. IL-17A (A) and IL-8 production (B) from cultured synovium-derived MC following various stimulations. MCs from patients with OA (n ¼ 3 donors) were stimulated with TNF- a, C5a, LPS, or IL-23 plus IL-1b for 24 h. IL-17 levels were under the detection limit in 2 donors out of 3 donors. The data of IL-17A production from one donor are shown. The data of IL-8 production are shown as the mean ± SEM. *P < 0.05.

Article Snippet: The following antibodies (Abs) were purchased from the indicated sources: human IgE (Calbiochem, San Diego, CA, USA); goat anti-human IL-17A polyclonal Ab (R&D Systems, Minneapolis, MN, USA); anti-human tryptase monoclonal (m) Ab (clone AA1; DakoCytomation Inc., Carpinteria, CA, USA); anti-Fc 3RIa mAb (clone CRA1; eBioscience, San Diego, CA, USA); anti-human IgE Ab (DakoCytomation Inc.); and F(ab0)2 fragments of anti-human FcgRI (clone 10.1; ID Labs Inc., London, ON, Canada).

Techniques: Cell Culture, Derivative Assay

Journal: Immunology letters

Article Title: A lack of confirmation with alternative assays questions the validity of IL-17A expression in human neutrophils using immunohistochemistry.

doi: 10.1016/j.imlet.2014.10.025

Figure Lengend Snippet: Fig. 1. Detection of IL-17A in human neutrophils by IHC. (A) Cyanine3 labelled IL-17A+ cells infiltrate Wolbachia-containing nodules but not Wolbachia-depleted nodules. (B) Counterstaining

Article Snippet: Immunoprecipitation was carried out using 100 g mouse anti-human IL-17A antibody (41802, R&D Systems) and 60 g goat anti-human IL-17A antibody (AF-317-NA, R&D Systems) coupled to 1 mL HiTrap NHS-activated HP columns and AKTAprime Plus low-pressure affinity chromatography system (both from GE Healthcare) using the manufacturer’s protocol.

Techniques:

Journal: Immunology letters

Article Title: A lack of confirmation with alternative assays questions the validity of IL-17A expression in human neutrophils using immunohistochemistry.

doi: 10.1016/j.imlet.2014.10.025

Figure Lengend Snippet: Fig. 2. Determination of IL-17A expression by human neutrophils. (A) Western blotting of neutrophil (PMN) cell lysate using goat anti-IL-17A antibody and mouse anti- IL-17A antibody.

Article Snippet: Immunoprecipitation was carried out using 100 g mouse anti-human IL-17A antibody (41802, R&D Systems) and 60 g goat anti-human IL-17A antibody (AF-317-NA, R&D Systems) coupled to 1 mL HiTrap NHS-activated HP columns and AKTAprime Plus low-pressure affinity chromatography system (both from GE Healthcare) using the manufacturer’s protocol.

Techniques: Expressing, Western Blot

Journal: Journal of Neuroinflammation

Article Title: RhANP attenuates endotoxin-derived cognitive dysfunction through subdiaphragmatic vagus nerve-mediated gut microbiota–brain axis

doi: 10.1186/s12974-021-02356-z

Figure Lengend Snippet: Roles of SDV in rhANP-mediated reduction of LPS-induced systemic inflammation and neuroinflammation. a Treatment schedule. Mice were intraperitoneally injected with lipopolysaccharides (LPS, 5 mg/kg). Recombinant human ANP (rhANP; 1.0 mg/kg) or 0.9% saline were intraperitoneally injected to mice at 24 h before and 10 min after LPS injection. Subdiaphragmatic vagotomy (SDV) was performed 14 days prior to LPS injection. Plasma and hippocampus were collected 24 h after LPS injection. b Body weight loss in each group (two-way ANOVA: rhANP: F 1,36 = 7.058, P = 0.0117; SDV: F 1,36 = 12.72, P = 0.0010; interaction: F 1,36 = 4.923, P = 0.0329). The plasma levels of interleukin (IL)-6 ( c ; two-way ANOVA: rhANP: F 1,36 = 13.07, P = 0.0009; SDV: F 1,36 = 15.60, P = 0.0003; interaction: F 1,36 = 20.51, P < 0.0001), IL-17A ( d ; two-way ANOVA: rhANP: F 1,36 = 6.111, P = 0.0183; SDV: F 1,36 = 7.794, P = 0.0083; interaction: F 1,36 = 6.027, P = 0.0191), interferon (IFN)-γ ( e ; two-way ANOVA: rhANP: F 1,36 = 6.460, P = 0.0155; SDV: F 1,36 = 8.447, P = 0.0062; interaction: F 1,36 = 7.836, P = 0.0082), and tumor necrosis factor (TNF)-α ( f ; two-way ANOVA: rhANP: F 1,36 = 4.828, P = 0.0345; SDV: F 1,36 = 6.217, P = 0.0174; interaction: F 1,36 = 7.883, P = 0.0080). Western blot analysis of ionized calcium-binding adapter molecule 1 (iba-1) ( g ; two-way ANOVA: rhANP: F 1,36 = 6.208, P = 0.0175; SDV: F 1,36 = 4.772, P = 0.0355; interaction: F 1,36 = 4.996, P = 0.0317), IL-6 ( h ; two-way ANOVA: rhANP: F 1,36 = 6.286, P = 0.0168; SDV: F 1,36 = 6.894, P = 0.0126; interaction: F 1,36 = 8.569, P = 0.0059), IL-17A ( i ; two-way ANOVA: rhANP: F 1,36 = 4.268, P = 0.0461; SDV: F 1,36 = 3.363, P = 0.0750; interaction: F 1,36 = 5.503, P = 0.0246), interferon (IFN)-γ ( j ; two-way ANOVA: rhANP: F 1,36 = 4.706, P = 0.0367; SDV: F 1,36 = 5.685, P = 0.0225; interaction: F 1,36 = 3.530, P = 0.0684), TNF-α ( k ; two-way ANOVA: rhANP: F 1,36 = 1.598, P = 0.2144; SDV: F 1,36 = 12.72, P = 0.0010; interaction: F 1,36 = 4.345, P = 0.0443), inducible nitric oxide synthase (iNOS) ( l ; two-way ANOVA: rhANP: F 1,36 = 4.764, P = 0.0357; SDV: F 1,36 = 2.933, P = 0.0954; interaction: F 1,36 = 5.462, P = 0.0251) and their respective β-actin in the hippocampus. Data are shown as mean ± SEM, n = 10/group. * P < 0.05, ** P < 0.01, *** P < 0.0001; N.S. not significant

Article Snippet: The membranes were blocked in 5% non-fat dried milk for 1 h at room temperature, and then incubated with the following primary antibodies: rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (iba-1; 1:1000, #016-20001: Wako Pure Chemical Industries, Ltd., Tokyo, Japan), rabbit monoclonal anti-IL-6 (1:1000, #12912S, Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-IL-17A (1:1000, #A0688, ABclonal, Wuhan, China), rabbit monoclonal anti-IFN-γ (1:1000, #A12450, ABclonal, Wuhan, China), rabbit polyclonal anti-TNF-α (1:1000, #A0277, ABclonal, Wuhan, China), rabbit monoclonal anti-inducible nitric oxide synthase (iNOS; 1:1000, #ab3523, Abcam, Cambridge, MA, USA), brain-derived neurotrophic factor (BDNF; 1:1000, #A4873, ABclonal, Wuhan, China), rabbit polyclonal phosphorylated tyrosine kinase receptor B (p-TrkB; 1:1000, #AP0423, ABclonal, Wuhan, China), rabbit polyclonal total TrkB (t-TrkB; 1:1000, #A2099, ABclonal, Wuhan, China), and mouse monoclonal anti-β-actin (1:1000, #A2319, ABclonal, Wuhan, China) overnight at 4 ℃.

Techniques: Injection, Recombinant, Western Blot, Binding Assay

Journal: Journal of Neuroinflammation

Article Title: RhANP attenuates endotoxin-derived cognitive dysfunction through subdiaphragmatic vagus nerve-mediated gut microbiota–brain axis

doi: 10.1186/s12974-021-02356-z

Figure Lengend Snippet: Effects of rhANP on plasma inflammatory cytokines after LPS-triggered endotoxemia. a Treatment schedule. Mice were intraperitoneally injected with lipopolysaccharides (LPS, 5 mg/kg) or 0.9% saline. Recombinant human ANP (rhANP; 1.0 mg/kg) or 0.9% saline were intraperitoneally injected to mice 24 h before and 10 min after LPS injection. Spleen and plasma were collected 24 h after injection of LPS or 0.9% saline. b Body weight loss in mice treated with rhANP or 0.9% saline 24 h after injection of LPS or 0.9% saline (one-way ANOVA: F 2,27 = 58.21, P < 0.0001). c Representative picture of spleen and spleen weight (one-way ANOVA: F 2,28 = 27.53, P < 0.0001). d Ratio of spleen weight/body weight (one-way ANOVA: F 2,28 = 20.17, P < 0.0001). e Plasma levels of interleukin (IL)-6 (one-way ANOVA: F 2,28 = 65.35, P < 0.0001). f Plasma levels of IL-17A (one-way ANOVA: F 2,28 = 16.82, P < 0.0001). g Plasma levels of interferon (IFN)-γ (one-way ANOVA: F 2,28 = 12.62, P < 0.0001). h Plasma levels of tumor necrosis factor (TNF)-α (one-way ANOVA: F 2,28 = 51.43, P < 0.0001). i There was a positive correlation (r = 0.797, P < 0.001) between spleen weight and plasma IL-6. j There was a positive correlation ( r = 0.629, P < 0.001) between spleen weight and plasma IL-17A. k There was a positive correlation ( r = 0.479, P = 0.006) between spleen weight and plasma IFN-γ. l Positive correlation ( r = 0.637, P < 0.001) between spleen weight and plasma TNF-α was observed. Data are shown as mean ± SEM, n = 10 or 11/group. ** P < 0.01, *** P < 0.0001

Article Snippet: The membranes were blocked in 5% non-fat dried milk for 1 h at room temperature, and then incubated with the following primary antibodies: rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (iba-1; 1:1000, #016-20001: Wako Pure Chemical Industries, Ltd., Tokyo, Japan), rabbit monoclonal anti-IL-6 (1:1000, #12912S, Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-IL-17A (1:1000, #A0688, ABclonal, Wuhan, China), rabbit monoclonal anti-IFN-γ (1:1000, #A12450, ABclonal, Wuhan, China), rabbit polyclonal anti-TNF-α (1:1000, #A0277, ABclonal, Wuhan, China), rabbit monoclonal anti-inducible nitric oxide synthase (iNOS; 1:1000, #ab3523, Abcam, Cambridge, MA, USA), brain-derived neurotrophic factor (BDNF; 1:1000, #A4873, ABclonal, Wuhan, China), rabbit polyclonal phosphorylated tyrosine kinase receptor B (p-TrkB; 1:1000, #AP0423, ABclonal, Wuhan, China), rabbit polyclonal total TrkB (t-TrkB; 1:1000, #A2099, ABclonal, Wuhan, China), and mouse monoclonal anti-β-actin (1:1000, #A2319, ABclonal, Wuhan, China) overnight at 4 ℃.

Techniques: Injection, Recombinant

Journal: Journal of Neuroinflammation

Article Title: RhANP attenuates endotoxin-derived cognitive dysfunction through subdiaphragmatic vagus nerve-mediated gut microbiota–brain axis

doi: 10.1186/s12974-021-02356-z

Figure Lengend Snippet: Effects of rhANP on the neuroinflammation and cognitive function after LPS-triggered endotoxemia. a Treatment schedule. Mice were intraperitoneally injected with lipopolysaccharides (LPS, 5 mg/kg) or 0.9% saline. Recombinant human ANP (rhANP; 1.0 mg/kg) or 0.9% saline were intraperitoneally injected to mice 24 h before and 10 min after LPS injection. The prefrontal cortex (PFC) and hippocampus were collected 24 h after injection of LPS or 0.9% saline. b , c Western blot analysis of ionized calcium-binding adapter molecule 1 (iba-1) in the prefrontal cortex (PFC) (one-way ANOVA: F 2,27 = 6.170, P = 0.0062) and hippocampus (one-way ANOVA: F 2,27 = 5.250, P = 0.0119). d , e Western blot analysis of interleukin (IL)-6 in the PFC (one-way ANOVA: F 2,27 = 5.958, P = 0.0072) and hippocampus (one-way ANOVA: F 2,27 = 17.60, P < 0.0001). f , g Western blot analysis of IL-17A in the PFC (one-way ANOVA: F 2,27 = 4.498, P = 0.0206) and hippocampus (one-way ANOVA: F 2,27 = 6.268, P = 0.0058). h , i Western blot analysis of interferon (IFN)-γ in the PFC (one-way ANOVA: F 2,27 = 11.18, P = 0.0003) and hippocampus (one-way ANOVA: F 2,27 = 7.611, P = 0.0024). j , k Western blot analysis of tumor necrosis factor (TNF)-α in the PFC (one-way ANOVA: F 2,27 = 5.530, P = 0.0097) and hippocampus (one-way ANOVA: F 2,27 = 8.550, P = 0.0013). l , m Western blot analysis of inducible nitric oxide synthase (iNOS) in the PFC (one-way ANOVA: F 2,27 = 7.328, P = 0.0029) and hippocampus (one-way ANOVA: F 2,27 = 7.597, P = 0.0024). n , o Entries in the novel arm (one-way ANOVA: F 2,27 = 31.33, P < 0.0001) and duration in the novel arm (one-way ANOVA: F 2,27 = 13.34, P < 0.0001) in the Y maze test. p Latency to eat food in the buried food test (one-way ANOVA: F 2,27 = 7.129, P = 0.0033). Data are shown as mean ± SEM, n = 10/group. * P < 0.05, ** P < 0.01, *** P < 0.0001; N.S. not significant

Article Snippet: The membranes were blocked in 5% non-fat dried milk for 1 h at room temperature, and then incubated with the following primary antibodies: rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (iba-1; 1:1000, #016-20001: Wako Pure Chemical Industries, Ltd., Tokyo, Japan), rabbit monoclonal anti-IL-6 (1:1000, #12912S, Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-IL-17A (1:1000, #A0688, ABclonal, Wuhan, China), rabbit monoclonal anti-IFN-γ (1:1000, #A12450, ABclonal, Wuhan, China), rabbit polyclonal anti-TNF-α (1:1000, #A0277, ABclonal, Wuhan, China), rabbit monoclonal anti-inducible nitric oxide synthase (iNOS; 1:1000, #ab3523, Abcam, Cambridge, MA, USA), brain-derived neurotrophic factor (BDNF; 1:1000, #A4873, ABclonal, Wuhan, China), rabbit polyclonal phosphorylated tyrosine kinase receptor B (p-TrkB; 1:1000, #AP0423, ABclonal, Wuhan, China), rabbit polyclonal total TrkB (t-TrkB; 1:1000, #A2099, ABclonal, Wuhan, China), and mouse monoclonal anti-β-actin (1:1000, #A2319, ABclonal, Wuhan, China) overnight at 4 ℃.

Techniques: Injection, Recombinant, Western Blot, Binding Assay

Journal: bioRxiv

Article Title: Recombinant outer membrane vesicles coupled with cytokines act as high-performance adjuvants against Helicobacter pylori infection in mice

doi: 10.1101/2023.06.26.546595

Figure Lengend Snippet: Construction of recombinant cytokine OMVs and their pattern diagram for immunization as adjuvant (A). HEK-293T and GES-1 cells were divided into three groups each: 100 μg of OMVs delivering cytokine IFN-γ (EGFP) was added to the first group, 100 μg of OMVs delivering cytokine IL-17A (mCherry) was added to the second, and 50 μg each of OMVs delivering cytokine IFN-γ (EGFP) or IL-17A (mCherry) was added to the third, followed by incubation in a cell culture incubator. Eukaryotic expression plasmids are highly expressed in both HEK-293T and GES-1 cells as observed by laser confocal microscopy (B). The expression of IL-17A and IFN-γ in cells by RT-qPCR (C) or quantitative ELISA (D).

Article Snippet: Monoclonal antibodies against IFN-γ, IL-17A, IL-12 (p40), IL-4, and IL-6 were added to 96-well plates (BD Biosciences, Mountain View, CA, USA).

Techniques: Recombinant, Incubation, Cell Culture, Expressing, Confocal Microscopy, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Recombinant outer membrane vesicles coupled with cytokines act as high-performance adjuvants against Helicobacter pylori infection in mice

doi: 10.1101/2023.06.26.546595

Figure Lengend Snippet: MLN cells and splenocytes of mice were isolated after 8 weeks of immunization, and the expression levels of cytokines IL-17 and IFN-γ in different groups were assessed by quantitative ELISA after immunization with UreB (A, C) or WCV (B, D) as vaccine antigens combined with recombinant OMVs or CT as adjuvants. Each group comprised nine mice, and data are expressed as mean ± SD per group. The least significant difference test was performed to determine whether the distinctions between the means of the groups were significant. P < 0.05 and P < 0.01 represent the differences between the related groups.

Article Snippet: Monoclonal antibodies against IFN-γ, IL-17A, IL-12 (p40), IL-4, and IL-6 were added to 96-well plates (BD Biosciences, Mountain View, CA, USA).

Techniques: Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Recombinant