Journal: Central-European journal of immunology

Article Title: Targeting the serum marker interleukin 9 improves the underlying characterization and immune homeostasis in rheumatoid arthritis.

doi: 10.5114/ceji.2024.141695

Figure Lengend Snippet: Fig. 3. Interleukin 9 affected the severity of arthritis in CIA rats. A) ELISA for the detection of IL-9 levels. B) Rat arthritis score. C) Analysis of cartilage damage by saffron O-solid green staining. The descriptive statistics are pre- sented as mean ±SD. *p < 0.05 vs. the CIA group, n = 3

Article Snippet: We measured the levels of serum markers using ELISA with a specialized commercial human IL-9 ELISA Kit (CSB-E04642h, Cusabio, Wuhan, China), human Gal1 ELISA Kit (CSB-EL012882h, Cusabio), human anti-CII ELISA Kit (HB3371-Hu, Shanghai Hengyuan Biotechnology Co., Ltd, Shanghai, China), human sSR-A ELISA Kit (HB3370-Hu, Shanghai Hengyuan Biotechnology Co., Ltd), human anti-Sa ELISA Kit (ml382511V), human RF ELISA Kit (ml060678V), human ACPA ELISA Kit (ml233305V), rat IL-9 ELISA Kit (CSB-E07294r, Cusabio), rat Gal1 ELISA Kit (JL25762, Shanghai Jianglai Biotechnology Co., Ltd, Shanghai, China), rat anti-CII ELISA Kit (HB1364-Ra, Shanghai Hengyuan Biotechnology Co., Ltd), rat sSR-A ELISA Kit (HB1363-Ra, Shanghai Hengyuan Biotechnology Co., Ltd), rat anti-Sa ELISA Kit (ml202241V), rat RF ELISA Kit (ml059541V), and rat ACPA ELISA Kit (ml201458V).

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal: Respiratory Research

Article Title: MiR-493-5p inhibits Th9 cell differentiation in allergic asthma by targeting FOXO1

doi: 10.1186/s12931-022-02207-2

Figure Lengend Snippet: Down regulation of miR-493-5p and higher proportion of Th9 cells in murine asthma models. OVA was used to induce allergic airway inflammation in mice. The mice were euthanasia by dislocation of the cervical spine after the measurement of airway hyperreactivity, and then their lung tissues and BALF were collected (n = 8 for each group). A The IL-9 levels in lung tissue and BALF were measured by ELISA, and IL-9 level in lung tissue and BALF of asthma groups were significantly higher than the control groups. B The miR-493-5p expression in lung tissue and BALF were detected by RT-PCR, and the miR-493-5p expression were both obviously diminished in asthma groups compared with control groups. C MiR-493-5p, FOXO1, IRF4 and IL-9mRNA expression in lung tissues were detected by RT-PCR, and IL-9, FOXO1, IRF4 expression were all significantly upregulated in lung tissues from asthmatic mice. D , E The proportion of CD4 + T cells secreting IL-9 in PBMCs were analysed by flow cytometry, the results revealed that the higher proportion of CD4 + T cells secreting IL-9 in the PBMCs of the asthma mice . ** P < 0.01, compared to control group

Article Snippet: All samples were preserved at − 80 °C for subsequent assay of IL-9 by ELISA (Elabscience, E-EL-M0724c) according to the instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry

Journal: Respiratory Research

Article Title: MiR-493-5p inhibits Th9 cell differentiation in allergic asthma by targeting FOXO1

doi: 10.1186/s12931-022-02207-2

Figure Lengend Snippet: MiR-493-5p negatively regulated the differentiation of Th9 cells in vitro. The CD4 + T cells were separated from widetype mice by density gradient centrifugation and magnetic beads. To investigate the effect of miR-493-5p on Th9 cells differentiation, both CD4 + T cells which were transfected with miR-493-5p mimic or treated with the inhibitor and their negative control (NC) were under the condition of inducing Th9 cell differentiation. A – C The mRNA level of IL-9, IRF4 and FOXO1 was detected by RT-qPCR. The data showed that the mRNA of FOXO1, IL-9 and IRF4 were downregulated in CD4 + T cells treated with miR-493-5p mimic and upregulated in CD4 + T cells treated with miR-493-5p inhibitor. D Groups. E – H The protein production of IL-9, IRF4 and FOXO1 was detected by western blot. The data showed that protein of FOXO1, IL-9 and IRF4 were downregulated in CD4 + T cells treated with miR-493-5p mimic and upregulated in CD4 + T cells treated with miR-493-5p inhibitor. I , J The proportion of CD4 + Th9 cells was analysed by flow cytometry and K IL-9 secretion in cell supernatant was measured by ELISA, and the data were all consistent with the proportion of Th9 cells. Data are from three experiments (mean and SD of three independent replicates). ** P < 0.01 compared to A group, # P < 0.05 compared to B group, ## P < 0.01 compared to B group

Article Snippet: All samples were preserved at − 80 °C for subsequent assay of IL-9 by ELISA (Elabscience, E-EL-M0724c) according to the instructions.

Techniques: In Vitro, Gradient Centrifugation, Magnetic Beads, Transfection, Negative Control, Cell Differentiation, Quantitative RT-PCR, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Toll like Receptor 2 engagement on CD4 + T cells promotes TH9 differentiation and function

doi: 10.1002/eji.201646846

Figure Lengend Snippet: TLR2, but no other TLR ligands increase TGF-β and IL-4 driven IL-9 cytokine secretion. Naive CD4+ T cells were activated with anti-CD3 and anti-CD28 mAbs in the presence of different TLR ligands (1μg/ml) under non-polarizing and TH9 polarizing conditions for 48 h. IL-9 (A) and IFN-γ (B) ELISA were measured by culture supernatants. Means ± SD of three independent experiments are shown. * p < 0.05, ** p < 0.01, NS denotes non-significant.

Article Snippet: IFN-γ was measured by sandwich ELISA (R&D Systems; MIF00) and IL-9 ELISA was done using the DuoSet ELISA kit (R&D Systems; DY409) by following manufactured protocol.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Toll like Receptor 2 engagement on CD4 + T cells promotes TH9 differentiation and function

doi: 10.1002/eji.201646846

Figure Lengend Snippet: TLR2 engagement on Antigen85B specific CD4+ T cells increases TH9 differentiation driven by TGF-β and IL-4. Naive Ag85B transgenic CD4+ T cells were co-incubated with TLR2 KO BMDM pulsed with Ag85B peptide (1μg/ml) and co-stimulated with or without TLR2 ligand (P3CSK4) under non-polarizing and TH9 polarizing conditions for 48 h. (A) CD4+ T cells were permeabilized and labeled with mAbs to IL-9 and IFN-γ and percentages of IL-9+ or IFN-γ+ cells were determined by flow cytometry. (B, C) IL-9 and IFN-γ were measured in culture supernatants by ELISA. Means ± SD of five independent experiments are shown. * p < 0.05, ** p < 0.01. NS denote non- significant.

Article Snippet: IFN-γ was measured by sandwich ELISA (R&D Systems; MIF00) and IL-9 ELISA was done using the DuoSet ELISA kit (R&D Systems; DY409) by following manufactured protocol.

Techniques: Transgenic Assay, Incubation, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Toll like Receptor 2 engagement on CD4 + T cells promotes TH9 differentiation and function

doi: 10.1002/eji.201646846

Figure Lengend Snippet: TLR2 engagement on human CD4+ T cells enhances IL9 mRNA and protein expression driven by polyclonal activation and TGF-β and IL-4. Naïve CD4+ T cells from three human donors were stimulated with anti-CD3 (10ug/ml) and anti-CD28 mAbs (1ug/ml) and exogenous TGF-β (5ng/ml) and IL-4 (10ng/ml), with or without P3CSK4 (2μg/ml) for 48h. (A) Relative expression of Il9 mRNA under non-polarizing and polarizing conditions with or without P3CSK4 (n=3) is shown. (B) IL-9 cytokine in culture supernatants were determined by ELISA. Means ± SD of three technical replicates for each donor are shown.

Article Snippet: IFN-γ was measured by sandwich ELISA (R&D Systems; MIF00) and IL-9 ELISA was done using the DuoSet ELISA kit (R&D Systems; DY409) by following manufactured protocol.

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Critical role of IL-33 receptor ST2 in experimental cerebral malaria development.

doi: 10.1002/eji.201445206

Figure Lengend Snippet: Figure 6. ST2 deficiency does not affect lung or spleen inflammation after PbA sporozoite infection. WT- or ST2-deficient mice were infected with 1000 PbA sporozoites. (A) Lung inflammation with alveolar septae thickening with mononuclear cell infiltration, hemorrhage in alveoli, and interstitial edema on day 9 post infection in WT or ST2−/−mice; H&E staining. Magnification 20×; scale bar = 100 μm. Images are representative of two independent experiments. (B) Semiquantitative score of lung pathology, expressed in arbitrary unit (AUs). The bar graphs show the mean ± SEM of n = 9 mice per group and are pooled from two independent experiments. (C–F) PbA-induced spleen inflammation in WT or ST2−/−mice. The absolute numbers of CD8+ (C) and CD4+ (D) T cells, respectively, CD8+CD69+ and CD4+CD69+ T cells, CD8+CXCR3+ and CD4+CXCR3+ T cells per spleen are shown. IFN-γ (E) and IL-10 (F) levels were measured by ELISA in spleen homogenates on days 0 and 9 post infection. (C–F) Data are expressed as mean ± SEM of n = 5–6 mice per condition and are representative of two independent experiments; *p < 0.05; statistical analysis with Kruskal–Wallis test followed by Dunn’s comparison test.

Article Snippet: After centrifugation IL-33, IL-10, and IFN-γ levels were determined in supernatants using ELISA assay kits (Mouse DuoSet, R&D Systems, Minneapolis, USA).

Techniques: Infection, Staining, Enzyme-linked Immunosorbent Assay, Comparison