Journal: Cellular & molecular immunology

Article Title: A novel cytokine consisting of the p40 and EBI3 subunits suppresses experimental autoimmune arthritis via reciprocal regulation of Th17 and Treg cells.

doi: 10.1038/s41423-021-00798-2

Figure Lengend Snippet: Fig. 2 In vivo therapeutic effect of p40-EBI3 on the development of autoimmune arthritis. One week after immunization with type II collagen (CII), mice with CII-induced arthritis (CIA) were administered 100 μg of p40-EBI3 vector or FLAG vector (mock) in the muscles of both thighs by electroporation; they then received the p40-EBI3 vector or FLAG vector intravenously by hydrodynamic injection at weekly intervals until 10 weeks after CII immunization. A Representative results from one of three independent experiments are shown for the clinical scores (left) and incidences of arthritis (right) for each treatment group over time. B The concentrations of CII-specific IgG, IgG1, and IgG2a in sera from mice in each treatment group at 10 weeks after CII immunization were determined by ELISA. Bars show the mean ± SD of 10 mice per group. C At 10 weeks after the initial CII immunization, tissue sections were obtained from the ankle joints of mice with CIA and stained with hematoxylin and eosin (H&E) or antibodies specific for IL-1β, TNF-α, IL-17, IL-6, IFN-γ, HIF-1α, VEGF, RANKL, RANK, or TRAP. Representative images of antibody-positive cells (stained brown) are shown (top, scale bar = 100 μm); the results are depicted as the number of antibody- positive cells (mean ± SD) for five mice per group (bottom) (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Serially diluted serum samples were applied and incubated at room temperature for 1 h. The plates were washed, and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, IgG1, or IgG2a (Bethyl Laboratories, Montgomery, TX, USA) was added.

Techniques: In Vivo, Plasmid Preparation, Muscles, Electroporation, Injection, Enzyme-linked Immunosorbent Assay, Staining

Journal: Cellular & molecular immunology

Article Title: A novel cytokine consisting of the p40 and EBI3 subunits suppresses experimental autoimmune arthritis via reciprocal regulation of Th17 and Treg cells.

doi: 10.1038/s41423-021-00798-2

Figure Lengend Snippet: Fig. 3 Attenuation of arthritis via regulation of the phenotype of immune cells in p40-EBI3 transgenic C57BL/6 mice achieved by microinjection of the transgene. Collagen-induced arthritis (CIA) was induced in WT or p40-EBI3 transgenic C57BL/6 mice as described in the Methods section. A Representative results from one of three independent experiments are shown for the clinical scores (left) and incidences of arthritis (right) for each treatment group over time. B The concentrations of total IgG in sera from mice in each group at 12 weeks after CII immunization were determined by ELISA. C Spleens were obtained from mice in each group at 12 weeks after CII immunization and analyzed by flow cytometry for B220+GL-7+ germinal center B cells and B220−CD138+ plasma cells. Representative flow cytometry plots from one of three independent experiments are shown (left); the results are depicted as the mean ± SD of three mice per group (right). D Mice were sacrificed at 12 weeks after CIA induction; the populations of IFN-γ+, IL-4+, IL-17+, and CD25+Foxp3+ cells among splenic CD4+ T cells were analyzed by flow cytometry. Representative flow cytometry plots from one of three independent experiments are shown (left); the results are depicted as the mean ± SD of three mice per group (right) (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Serially diluted serum samples were applied and incubated at room temperature for 1 h. The plates were washed, and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, IgG1, or IgG2a (Bethyl Laboratories, Montgomery, TX, USA) was added.

Techniques: Transgenic Assay, Microinjection, Enzyme-linked Immunosorbent Assay, Cytometry, Clinical Proteomics

Journal: Cellular & molecular immunology

Article Title: A novel cytokine consisting of the p40 and EBI3 subunits suppresses experimental autoimmune arthritis via reciprocal regulation of Th17 and Treg cells.

doi: 10.1038/s41423-021-00798-2

Figure Lengend Snippet: Fig. 6 In vivo antiosteoclastogenic and anti-inflammatory properties of the p40-EBI3-Fc protein with respect to the development of collagen- induced arthritis (CIA). Mice with CIA received the p40-EBI3-Fc protein (2.5 mg/kg) or vehicle intraperitoneally daily after induction of CIA for 9 weeks. A Representative results from one of three independent experiments are shown for the clinical scores (left) and incidences of arthritis (right) for each treatment group over time. B At 63 days after initial CII immunization, tissues were obtained from the ankle joints of mice with CIA and stained with hematoxylin and eosin (H&E; original magnification, ×40), safranin O (original magnification, ×200), toluidine blue (original magnification, ×200), or tartrate-resistant acid phosphatase (TRAP; original magnification, ×200) to examine arthritis severity. C Isolated splenic CD4+ T cells from each group of mice were cultured for 3 days. T cell proliferation was determined by a [3H] thymidine incorporation assay. D The concentrations of CII-specific IgG, IgG1, and IgG2a in sera from mice in each group were determined by ELISA. Bars show the mean ± SD of four mice per group. E Tissue sections generated from the ankle joints of mice with CIA treated with or without p40- EBI3-Fc were stained with antibodies against TNFα, IL-1β, IL-6, RANKL, VEGF, and HIF-1α or with an isotype control. Cells stained with each antibody are shown in brown (upper). The numbers of positive cells were counted in high-power fields (magnification, ×400) with the aid of Adobe Photoshop software and averaged for three randomly selected fields per tissue section. Bars show the mean ± SD of three mice per group (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Serially diluted serum samples were applied and incubated at room temperature for 1 h. The plates were washed, and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, IgG1, or IgG2a (Bethyl Laboratories, Montgomery, TX, USA) was added.

Techniques: In Vivo, Staining, Isolation, Cell Culture, Thymidine Incorporation Assay, Enzyme-linked Immunosorbent Assay, Generated, Control, Software

Journal: Journal of Translational Medicine

Article Title: TSLP promoting B cell proliferation and polarizing follicular helper T cell as a therapeutic target in IgG4-related disease

doi: 10.1186/s12967-022-03606-1

Figure Lengend Snippet: The expression of TSLP and its receptors in IgG4-RD. A Plasma levels of TSLP in HC, treatment naïve IgG4-RD patients and IgG4-RD patients with disease remission measured by Elisa. B Representative immunohistochemical staining of TSLP in LG from patients with SS (left) and SMG from patients with IgG4-RD (right). C The expression levels of TSLP in SMG estimated by semiquantitative analysis. D Trible staining for CD11c (green), CD4 (green), CD19 (green), CRLF2 (TSLPR, red) and DAPI (blue) in SMG from representative patients with IgG4-RD. E Percentages of TSLPR and IL-7Ra in CD4+, CD8+, CD19+, and Lin-CD11c+HLA-DR+ cells respectively. F Correlations between serum levels of IgG, IgE, IgG1, IgG4, RI and plasma level of TSLP. HC healthy controls, IgG4-RD IgG4-related disease, LG labial gland, SMG submandibular gland, SS primary Sjogren’s syndrome, RI responder index. *p < 0.05; ***p < 0.001; ns = not significant

Article Snippet: Antibodies in B cell culture supernatant was quantified by IgG Elisa kit (CUSABIO), IgG4 Elisa kit (CUSABIO) and IgE Elisa kit (Bethyl Laboratories).

Techniques: Expressing, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining

Journal: Journal of Translational Medicine

Article Title: TSLP promoting B cell proliferation and polarizing follicular helper T cell as a therapeutic target in IgG4-related disease

doi: 10.1186/s12967-022-03606-1

Figure Lengend Snippet: The effect of TSLP on B cells in IgG4-RD. CD19+ B cells from IgG4-RD patients were stimulated with IL-4, anti-IgM, CD40L, with or without TSLP. Proliferation quantified by Ki-67 ( A , B ), apoptosis measured by 7-AAD and Annexin-V ( C , D ), and B cell subsets ( E , F ) were assessed on 5 days. Supernatant IgG and IgG4 levels on 7 days were quantified by ELISA ( G ). *p < 0.05

Article Snippet: Antibodies in B cell culture supernatant was quantified by IgG Elisa kit (CUSABIO), IgG4 Elisa kit (CUSABIO) and IgE Elisa kit (Bethyl Laboratories).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Veterinary Internal Medicine

Article Title: Investigation of the presence of specific neural antibodies in dogs with epilepsy or dyskinesia using murine and human assays

doi: 10.1111/jvim.16744

Figure Lengend Snippet: Canine anti‐IgLON5 antibodies. Immunofluorescence studies: Diluted serum (1:20) from a dog from the epilepsy/dyskinesia group (Golden Retriever, female, 3 years) is incubated with IgLON5‐transfected human embryonic kidney (HEK) cells. Binding of antibodies to cell surfaces is visualized by two different secondary anti‐canine antibodies coupled with immunofluorescence dyes and subsequently overlaid. The results demonstrate that there are canine anti‐IgLON5 IgG antibodies. Endpoint titration gave a titer of 1:40 (not shown). (A) anti‐canine IgG heavy and light chain (a sensitive secondary antibody), coupled with the red Alexa 594 dye. (B) overlay; yellow: double‐stainings. (C) anti‐canine IgG Fc fragment (the more specific secondary antibody), coupled with the green Alexa 488 dye. Bar: 25 μm.

Article Snippet: Secondary AB was a polyclonal rabbit anti‐dog immunoglobulin‐G (IgG) AB directed against canine IgG heavy and light chains (catalog no. 304‐065‐003; Jackson ImmunoResearch/Dianova) conjugated with red Alexa Fluor 594 used at a dilution of 1:100 and incubated for 30 minutes at room temperature; nuclei were counterstained with Hoechst 33342, 1:10 000.

Techniques: Immunofluorescence, Incubation, Transfection, Binding Assay, Titration