Journal: Journal of Reproduction & Infertility

Article Title: Investigating the Effect of Crocus sativus (Saffron) Petal Hydro-alcoholic Extract on Ovarian Follicle, Inflammatory Markers, and Antioxidant Enzymes in Mice Model of Polycystic Ovary Syndrome

doi: 10.18502/jri.v23i1.8447

Figure Lengend Snippet: The plasma level(s) of TNF-α, IL6, IL1ß, IL18, and CRP

Article Snippet: Levels of IL-6, TNF-α, IL-1ß, IL-18, and CRP in all serum samples were determined by special ELISA kits (Accu-Bind, Monobind Inc., USA) ( ).

Techniques:

Journal: Journal of Reproduction & Infertility

Article Title: Investigating the Effect of Crocus sativus (Saffron) Petal Hydro-alcoholic Extract on Ovarian Follicle, Inflammatory Markers, and Antioxidant Enzymes in Mice Model of Polycystic Ovary Syndrome

doi: 10.18502/jri.v23i1.8447

Figure Lengend Snippet: The plasma level(s) of TNF-α, IL6, IL1ß, IL18, and CRP

Article Snippet: Levels of IL-6, TNF-α, IL-1ß, IL-18, and CRP in all serum samples were determined by special ELISA kits (Accu-Bind, Monobind Inc., USA) ( ).

Techniques:

Journal: Shock (Augusta, Ga.)

Article Title: The Protective Effect of a Short Peptide Derived from Cold-inducible RNA-binding Protein in Renal Ischemia-Reperfusion Injury

doi: 10.1097/SHK.0000000000000988

Figure Lengend Snippet: Serum was collected 24 h after RIR and used to measure (A) TNF-α, (B) IL-6, (C) IL-1β using ELISA. Data are expressed as mean ± SEM (sham: n = 4 mice; RIR + vehicle: n = 5 mice; RIR + vehicle: n = 5 mice) and compared by one-way analysis of variance and Student-Newman-Keuls method. *P < 0.05 versus sham; #P < 0.05 versus vehicle. RIR, renal ischemia-reperfusion; SEM, standard error of the mean; TNF-α, tumor necrosis factor-α; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: Serum TNF-α, IL-6, and IL-1β were determined with an ELISA kit, specific to mouse TNF-α, IL-6, and IL-1β (BD Biosciences, San Diego, CA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Helicobacter pylori metabolites exacerbate gastritis through C-type lectin receptors

doi: 10.1084/jem.20200815

Figure Lengend Snippet: αCAG induces innate and acquired immune responses as potent as TDM. (A) Reporter cells expressing Mincle + FcRγ were stimulated with αCG, αCAG C14:0, or TDM (0.003, 0.03, and 0.3 nmol/well) for 20 h and analyzed for GFP expression. (B) Binding of Mincle-Ig to plate-coated αCG, αCAG C14:0, or TDM (0.001, 0.01 and 0.1 nmol/well). Bound proteins were detected with anti-human IgG-HRP. (C) WT and Clec4e −/− BMDCs were stimulated with αCAG C14:0 (0.001, 0.01, 0.1, and 1 nmol/well) or LPS for 1 d and cytokine production was quantified. (D) WT and Clec4e −/− BMDCs were stimulated with αCAG C14:0 for 1 d and analyzed for the surface expression of CD80 and CD40. (E) IFN-γ–primed BMDCs were stimulated with αCAG C14:0 for 1 d and analyzed for the expression of intracellular NOS2. SSC, side scatter. (F) Reporter cells expressing human Mincle + FcRγ were stimulated with αCG, αCAG C14:0, or TDM (0.003, 0.03, and 0.3 nmol/well) and analyzed for GFP expression. (G) hMoDCs were stimulated with αCG, αCAG C14:0 or TDM (0.04, 0.1, and 0.3 nmol/well) for 1 d, and IL-8 production was quantified. (H) BMDCs were stimulated with 0.1 nmol/well of αCAG C14:0, and co-cultured with OT-II CD4 + T cells in the presence of OVA 323–339 peptide for 3 d. Cytokine concentrations were measured by ELISA. (I) Mice were immunized with OVA + αCAG C14:0 and challenged with heat-aggregated OVA at 7 d after immunization. At day 7 after OVA challenge, B cell–depleted inguinal LN cells were stimulated with indicated amounts of OVA for 4 d, and IFN-γ production was quantified. The results of the statistical analysis show the difference between WT and Clec4e −/− mice. Data are presented as the mean ± SD of triplicate (B, C, G, and H) or duplicate (A, F, and I) assays and representative of three (A, B, F, and H), five (C and D), two (E and I), or four (G) independent experiments. An unpaired two-tailed Student’s t test was used for the statistical analyses. *, P < 0.05; **, P < 0.01.

Article Snippet: The ELISA kits for TNF, IL-6, and IFN-γ were from BD Biosciences.

Techniques: Expressing, Binding Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Helicobacter pylori metabolites exacerbate gastritis through C-type lectin receptors

doi: 10.1084/jem.20200815

Figure Lengend Snippet: Pathogenic role of Mincle in H. pylori –induced gastritis. (A and B) WT or Clec4e −/− mice were orally administered with 3 × 10 8 CFU of H. pylori SS1 three times in 2 d. At 6 wk after infection, cells from gastric LNs (A) or Peyer’s patches (B) were stimulated with indicated concentrations of H. pylori lysates (Ag) in the presence of BMDCs for 4 d. Uninfected WT mice were used as control. The concentrations of IFN-γ (left panel) and IL-17 (right panel) in the supernatant were determined by ELISA. (C) The number of bacteria recovered from the stomachs of WT and Clec4e −/− mice at 8 wk after H. pylori infection. Bacterial numbers were determined by counting the number of colonies on H. pylori selective agar plates. (D) H&E (HE) staining and immunohistochemical staining of anti-CD3 and F4/80 of stomach sections from uninfected and H. pylori –infected mice after 8 wk. Scale bar, 100 µm. (E) The numbers of Ly6G + CD11b + or F4/80 + CD11b + cells in gastric MNC from mice at 6 wk after infection. (F) Heat map of differentially expressed genes based on RNA-sequencing analysis using RNA extracted from the stomachs of H. pylori –infected WT or Clec4e −/− mice ( n = 3 in each group) after 8 wk. (G–I) H. pylori –infected mice were injected with anti-Mincle mAb or rat IgG as a control Ab (cont. Ab). After 8 wk, single-cell suspensions of mesenteric LN (G), spleen (H), or splenic CD4 + T cells (I; in the presence of BMDCs) were stimulated with H. pylori lysates (2, 20, and 200 µg/ml) for 4 d. Uninfected WT mice were used as control. The concentrations of IFN-γ in the supernatant were determined by ELISA. (J) The frequency of Ly6G + CD11b + cells in gastric MNCs was analyzed at 6 wk after infection with anti-Mincle mAb or cont. Ab treatment. Bars indicate the average number. (K) Bacterial CFU in the stomach of infected mice was determined by counting the number of colonies on H. pylori selective agar plates. Littermates (A–F) or C57BL/6J mice obtained from CLEA Japan (G–K) were used as WT mice. Data are presented as the mean ± SD of triplicate assays (A, B, and G–I) from three (WT vs. Clec4e −/− ) or two (Control Ab vs. Anti-Mincle) independent experiments (at least six infected mice in each group) with similar results. An unpaired two-tailed Student’s t test was used for the statistical analyses. *, P < 0.05; **, P < 0.01.

Article Snippet: The ELISA kits for TNF, IL-6, and IFN-γ were from BD Biosciences.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining, RNA Sequencing Assay, Injection, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Helicobacter pylori metabolites exacerbate gastritis through C-type lectin receptors

doi: 10.1084/jem.20200815

Figure Lengend Snippet: Cellular immunity during H. pylori infection. (A) Gating strategy for analyzing gastric MNCs by flow cytometry. Numbers indicate the percentages of cells in each gate. PI, propidium iodide; SSC, side scatter; FSC, forward scatter. (B) Gastric MNCs from uninfected and H. pylori –infected WT or Clec4e −/− mice were stained with anti-Ly6G, F4/80, CD11b, TCRβ and B220 at 6 wk after infection. Numbers indicate the percentages of cells in each gate. (C) Metagenome analysis of gastric mucosal swabs from uninfected WT mice, H. pylori –infected WT and Clec4e −/− mice ( n = 3 in each group) after 12 wk. (D) H. pylori –infected WT mice were injected with anti-Mincle mAb or rat IgG as a control Ab (cont. Ab). At 8 wk after infection, Peyer’s patch cells were collected and cultured for 4 d in the absence of exogenous H. pylori lysates. Uninfected WT mice were used as control. The concentrations of IFN-γ and IL-17 in the supernatants were determined by ELISA. (E) H&E (HE) staining and immunohistochemical staining with anti-CD3 of stomach sections from uninfected and H. pylori –infected mice treated with anti-Mincle mAb or control Ab. Scale bar, 100 µm. (F) Mouse invariant NKT hybridoma cells (DN32.D3) expressing CD1d were incubated with indicated amounts of αCG, αCAG, αCPG, or α-GalCer for 1 d. Each lipid was dissolved in DMSO. The expressions of CD69 were analyzed by flow cytometry. (G) The number of bacteria recovered from the stomachs of WT and Traj18 −/− mice at 8 wk after H. pylori infection. Bacterial numbers were determined by counting the number of colonies on H. pylori selective agar plates. (H) H&E staining of stomach sections from H. pylori –infected WT and Traj18 −/− mice after 8 wk. Scale bar, 100 µm. Data are presented as the mean ± SD of triplicate assays (D and F) from two independent experiments (at least six infected mice in each group) with similar results. An unpaired two-tailed Student’s t test was used for the statistical analyses. *, P < 0.05; **, P < 0.01.

Article Snippet: The ELISA kits for TNF, IL-6, and IFN-γ were from BD Biosciences.

Techniques: Infection, Flow Cytometry, Staining, Injection, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Expressing, Incubation, Two Tailed Test

Journal: Advanced Science

Article Title: Extracellular Vesicles Derived from Ligament Tissue Transport Interleukin‐17A to Mediate Ligament‐To‐Bone Crosstalk in Ankylosing Spondylitis

doi: 10.1002/advs.202406876

Figure Lengend Snippet: Pathological ossification and inflammatory activation in non‐ossified ligaments from patients with AS. A). µCT images show the 3D reconstruction and spinal ligament tissue from patients with AS. The red pentagram indicates uncalcified ligament. B). SOFG and HE staining of the AS patient ligament sections showing osteogenic differentiation and disorganized arrangement of collagen fibers. C,D). Representative immunohistochemistry and immunofluorescence staining images showing over‐expression of RUNX2, CD68, and PDGFRα in AS spinal ligament tissue. E). Heatmap of differentially upregulated genes, regulation of osteoblast differentiation (GO:0045667), and cytokine‐mediated signaling pathway (GO:0019221). F). Quantitative analysis of the immunofluorescence area occupied by stained regions in the different groups in (D). G). Concentration of CXCL12 in AS ligament tissue measured using ELISA. H). Pearson's correlation analysis of PDGFRα expression and the expression of markers of AS‐related osteoblast differentiation (RUNX2 and CXCL12) and inflammation (CD68) based on the ELISA and immunofluorescence staining results. I). Left: experimental scheme of AS or HD ligament tissue co‐culture with AS‐MSCs. Middle: ARS staining of AS‐MSCs after 21 days of osteogenic induction. Right: quantitative analysis of the ARS staining (absorption, 562 nm). n = 5 per group. J). Experimental scheme of AS or HD ligament tissue co‐culture with AS‐BMMCs. Concentration of inflammation factors after co‐culturing AS or HD ligament tissue with AS‐BMMCs measured using ELISA. IL, interspinous ligament; SL, supraspinous ligament; SP, spinous process; HE, hematoxylin‐eosin staining; SOFG, Safranin O–Fast Green staining.

Article Snippet: The following ELISA kits were used according to the manufacturer's protocol: CXCL12 (Solarbio, China), TNF‐α (Solarbio, China), IL‐1β (Solarbio, China), IL‐6 (Solarbio, China), and MMP14 (Thermo Scientific, USA).

Techniques: Activation Assay, Staining, Immunohistochemistry, Immunofluorescence, Over Expression, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Co-Culture Assay

Journal: Advanced Science

Article Title: Extracellular Vesicles Derived from Ligament Tissue Transport Interleukin‐17A to Mediate Ligament‐To‐Bone Crosstalk in Ankylosing Spondylitis

doi: 10.1002/advs.202406876

Figure Lengend Snippet: AS‐LTEVs facilitate inflammation activation by delivering IL‐17A in vivo. A). Diagrammatic representation of experimental strategies for macrophage polarization. B). Representative flow cytometry results of M1 macrophages after treatment with PBS, HD‐LTEVs, AS‐LTEVs, AS‐LTEVs with secukinumab, AS‐LTEVs with secukinumab, and Dynasore. Percentages indicate the proportion of M1 macrophages. n = 3 per group. C). Representative flow cytometry results of DCFH‐DA after treatment with PBS, HD‐LTEVs, AS‐LTEVs, AS‐LTEVs with secukinumab, AS‐LTEVs with secukinumab, and Dynasore. The right panel shows the quantitative analysis of the positive cell rate of ROS in the different groups. n = 4 per group. D–F). ELISA‐based measurement of TNF‐α, IL‐10, and IL‐1β levels in supernatants prepared from the culture medium after each of the different treatments in (B). n = 5 per group. G). Schematic diagram of the experimental schedule for the CAIA mouse model. H). µCT scans showing pathological new bone formation in hind paws in different CAIA model mice groups. I). HE staining of CAIA model mice paws and ankle joints sections showing articular destruction and inflammatory cell infiltration. J). Quantitative analysis of structural parameters of new bone by µCT analysis. n = 5 per group. K–M). Concentration of IL‐17A, TNF‐α, and IL‐6 in the serum of CAIA model mice. N. Therapeutic efficacy of the various substances assessed in terms of changes in the main symptoms of CAIA: arthritis clinical score, paw thickness, paw temperature, and pain‐associated behavior analysis using the von Frey filament test. n = 5 per group. DCFH‐DA, dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species.

Article Snippet: The following ELISA kits were used according to the manufacturer's protocol: CXCL12 (Solarbio, China), TNF‐α (Solarbio, China), IL‐1β (Solarbio, China), IL‐6 (Solarbio, China), and MMP14 (Thermo Scientific, USA).

Techniques: Activation Assay, In Vivo, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Concentration Assay, Drug discovery