Journal: BMB Reports

Article Title: Effects of α-lipoic acid on LPS-induced neuroinflammation and NLRP3 inflammasome activation through the regulation of BV-2 microglial cells activation

doi: 10.5483/BMBRep.2019.52.10.026

Figure Lengend Snippet: α-LA inhibits the expression of pro-inflammatory cytokines in LPS-treated BV-2 microglial cells. (A) Effects of α-LA on cell viability. BV-2 microglial cells were incubated with LPS (1 μg/ml) for 30 min followed by treatment with the indicated concentrations of α-LA for 24 h. Thereafter, cell viability was assessed through the MTT assay. (B and C) BV-2 microglial cells were treated with LPS (1 μg/ml) for 30 min followed by treatment with the indicated concentrations of α-LA at the suggested times. The cell-free conditioned culture medium was collected and analyzed with ELISA for TNF-α, IL-6. Data from three independent experiments are presented as means ± S.D. *≤ 0.05, **< 0.01, ***< 0.001 and are related to both LPS-induced cells and α-LA treated cells.

Article Snippet: Both TNF-α and IL-6 were quantitatively measured through an enzyme-linked immunosorbent assay (ELISA) using the mouse TNF-α and IL-6 DuoSet ELISA kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Incubation, MTT Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: The negative cofactor 2 complex is a key regulator of drug resistance in Aspergillus fumigatus

doi: 10.1038/s41467-019-14191-1

Figure Lengend Snippet: a , b A549 epithelial cells or macrophages were infected with the wild-type (WT), the nctA null mutant ( ∆nctA ) or the nctA reconstituted isolate ( nctA rec ) for 24 h. Cytotoxicity of each mutant was evaluated by measuring the release of lactate dehydrogenase (LDH) activity into the culture medium. The data represent five different infection challenges with triplicate LDH activity measurements. Data are shown in fold change of LDH activity relative to the wild-type-infected cells. The error bars mean the standard error of the mean (SEM), and P- values were calculated by Kruskal–Wallis test with Dunn’s correction: * P < 0.0180; ** P < 0.0056 (for a , A549 cells). ** P < 0.0032; *** P < 0.0007 (for b , macrophages). c – f Granulocyte macrophage colony-stimulating factor-induced bone marrow-derived dendritic cells (gm-csf BMDCs) were infected with the A. fumigatus strains with the multiplicity of infection (MOI) = 5:1. Proinflammatory cytokines were quantified by ELISA. Data represent three biological replicates ( c – e ) or five biological replicates ( f ) with ± SEM. P- values were calculated by ANOVA with Tukey’s correction: *** P < 0.002; **** P < 0.0001. g , h Activation of dendritic cells measured by CD40 and CD80 markers by flow cytometry. Data represent three biological replicates with ±SEM. P -values were calculated by ANOVA: *** P < 0.0002; **** P < 0.0001 (for g ). *** P < 0.0004; **** P < 0.0001 (for h ). Source data are provided as a Source Data file.

Article Snippet: The concentration of IL-8 and IL-6 were determined in A549 epithelial cells co-cultured with A. fumigatus strains by using the Human IL-8/CXCL8 and IL-6 DuoSet ELISA according to the manufacturer’s instructions (R&D systems).

Techniques: Infection, Mutagenesis, Activity Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Flow Cytometry

Journal: ALTEX

Article Title: Optimization of the monocyte activation test for evaluating pyrogenicity of tick-borne encephalitis virus vaccine

doi: 10.14573/altex.2002252

Figure Lengend Snippet: Fig. 3: Recovery of RSE at different doses of Encepur vaccine and excipient matrix Peripheral blood mononuclear cells (PBMC) were stimulated with nine different doses of Encepur vaccine or excipient solution spiked-in with a fixed dose of RSE (0.2 EU/mL) or R848 (0.3 µg/mL). Recovery of RSE or R-848 was measured by IL-6 release in terms of equivalent of endotoxin unit per mL (eEU/mL) or equivalent of R848 µg per mL (eµg/mL), respectively, by ELISA. The range of valid eEU/mL or eµg/ mL values (50-200% of the spiked dose of RSE or R848) is indicated by red dashed lines. Dots represent single recovery values obtained from 5 different donors for the spike of RSE in the Encepur vaccine (A) or in the excipient matrix (B) and 4 different donors when R848 was spiked-in into Encepur vaccine (C) or excipient matrix (D).

Article Snippet: Subsequently, a modified version of the Duoset IL-6 ELISA kit from R&D Systems (Minneapolis, MN, USA) was used to measure IL-6 released in the MAT assay.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: ALTEX

Article Title: Optimization of the monocyte activation test for evaluating pyrogenicity of tick-borne encephalitis virus vaccine

doi: 10.14573/altex.2002252

Figure Lengend Snippet: Fig. 5: Application of semi- quantitative Method B calculation to Encepur-optimized MAT assay Peripheral blood mononuclear cells (PBMC) were stimulated with RSE (R1 = 0.025, R2 = 0.05, R3 = 0.1, R4 =0.2 and R5 = 0.4 EU/mL) or Encepur vaccine (1:100, 1:200 and 1:400) according to the plate layout presented in Scheme 31, and optical density (OD) values were measured by IL-6 ELISA. Results obtained from an experimental session performed by two different analysts on PBMC of the same donor are shown. Squares indicate OD obtained by stimulation with Encepur alone, while triangles represent OD values from PBMC treated with vaccine spiked-in with 0.1 (A) or 0.2 (B) EU/mL RSE. Numbers close to triangles indicate the percentage of RSE recovery. The threshold of pyrogenic dose was fixed equal to the assay limit of detection (LOD) as described in Ph. Eur. (A) or equal to the assay sensitivity (AS) (B) in the proposed modified version of Method B. One representative experiment out of three experiments yielding similar results is shown.

Article Snippet: Subsequently, a modified version of the Duoset IL-6 ELISA kit from R&D Systems (Minneapolis, MN, USA) was used to measure IL-6 released in the MAT assay.

Techniques: Enzyme-linked Immunosorbent Assay, Modification

Journal: Science translational medicine

Article Title: Oral delivery of liquid mRNA therapeutics by engineered capsule for treatment of preclinical intestinal disease

doi: 10.1126/scitranslmed.adu1493

Figure Lengend Snippet: ( A ) Enzyme-linked immunosorbent assay (ELISA) analysis of IL-10 concentration in supernatant from Caco-2, MC-38 and Raw 264.7 cells treated with various amounts of PBS, IL-10 -mRNA or IL-10 -mRNA NPs for 24 h. ( B ) Western blot analysis of IL-10 expression in MC-38 cells treated with PBS, IL-10 -mRNA, blank NPs or IL-10 -mRNA NPs for 12 h. IL-10 -mRNA 750 ng/mL. ( C ) Confocal microscopy images of immunofluorescence staining of IL-10 expression in MC-38 cells treated with free IL-10 -mRNA or IL-10 -mRNA NPs. Hoechst (blue) was used to stain the cell nuclei. An Alexa Fluor 647-labeled antibody was used to stain IL-10. ( D ) Schematic illustration of how IL-10 -mRNA NPs directly transfect macrophages, inhibiting the polarization of macrophages to proinflammatory M1 phenotype in the presence of lipopolysaccharide (LPS), an inflammatory stimulator. ( E ) ELISA analysis of anti-inflammatory cytokine IL-10 and proinflammatory cytokine TNF-α and IL-6 in supernatant from RAW 264.7 cells treated with PBS or IL-10 -mRNA NPs (w/wo LPS stimulation) following procedures illustrated in (D). ( F ) Schematic illustration of how IL-10 -mRNA NPs first transfect intestinal epithelial cells, then indirectly induce the repolarization of macrophages from proinflammatory M1 phenotype to anti-proinflammatory M2 phenotype. ( G ) ELISA analysis of IL-10 and TNF-α and IL-6 in supernatant from RAW 264.7 cells treated with supernatant from Caco-2 cells incubated with PBS or IL-10 -mRNA NPs following procedures illustrated in (F). ( H ) Ex vivo images of excised rat gastrointestinal (GI) tract at various time points from 15 min to 6 h post-administration. Rats were orally administered six Cy5-mRNA-RNACaps (L100-55 coated). The left panel shows enlarged images of RNACaps at 1 h and 2 h (images 1–4). Cy5-mRNA: 50 μg per rat. Color scale, 0-255 gray value. ( I ) Confocal microscopy images of intestine sections from rats treated with Cy5-mRNA-RNACaps (purple) for 4 h. Hoechst (blue) was used to stain the cell nuclei. Scale bar, 100 μm. ( J ) Ex vivo images of excised intestines at 6 h post-administration. Rats were orally administered six Cy5-mRNA-RNACaps (L100 coated). Color scale, 0-255 gray value. Data are presented as mean ± S.D. Dots represent individual sample replicates. Statistical significance was evaluated by one-way ANOVA with Tukey’s post hoc analysis in ( E ) and ( G ). ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data in (A, B, C, E, G, H, I, J) are representative of n = 3 independent experiments. All the schematic illustrations were created using Adobe Illustrator.

Article Snippet: Rat TNF-α ELISA (438204, Biolegend), Rat IL-1 beta/IL-1F2 ELISA (DY501-05, R&D Systems), Rat IL-6 ELISA (DY506-05, R&D Systems), Rat IL-17A ELISA (437904, Biolegend), Rat JE/MCP-1/CCL2 ELISA (DY3144-05, R&D Systems).

Techniques: In Vitro, Ex Vivo, Imaging, In Vivo, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, Expressing, Confocal Microscopy, Immunofluorescence, Staining, Labeling, Incubation

Journal: Science translational medicine

Article Title: Oral delivery of liquid mRNA therapeutics by engineered capsule for treatment of preclinical intestinal disease

doi: 10.1126/scitranslmed.adu1493

Figure Lengend Snippet: ( A ) Experimental timeline for oral administration of IL-10 -mRNA-RNACaps to acute colitis rat models. IL-10 -mRNA: 25 μg in 3 RNACaps per rat. Acute colitis was induced in rats by providing free access to drinking water supplemented with 6.0% (w/w) dextran sulfate sodium (DSS) for 8 days. Rats were treated with RNACaps on day 2, 5 and 8. ( B and C ) Relative body weight (B) and disease activity index (DAI) (C) of healthy (plain water-treated), DSS-treated or DSS plus IL-10 -mRNA-RNACap-treated rats were monitored daily. ( D ) Quantification of colon length on day 8. ( E to J) Colon tissue protein expression of IL-10 (E), tumor necrosis factor-alpha (TNF-α) (F), interleukin-1β (IL-1β) (G), IL-6 (H), IL-17A (I) and monocyte chemoattractant protein-1 (MCP-1) (J) by ELISA. ( K to P ) Quantification of protein expression in blood of IL-10 (K), TNF-α (L), IL-1β (M), IL-6 (N), IL-17A (O) and MCP-1 (P) by ELISA. ( Q ) H&E staining images of the colon tissue sections. Dashed box indicates inset. Scale bars, 500 μm and 400 μm. Data are presented as mean ± S.D. Dots represent individual sample replicates. Statistical significance was evaluated by one-way ANOVA with Tukey’s post hoc analysis in (B to P). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. n = 5 animals per group for all panels. All the schematic illustrations were created using Adobe Illustrator.

Article Snippet: Rat TNF-α ELISA (438204, Biolegend), Rat IL-1 beta/IL-1F2 ELISA (DY501-05, R&D Systems), Rat IL-6 ELISA (DY506-05, R&D Systems), Rat IL-17A ELISA (437904, Biolegend), Rat JE/MCP-1/CCL2 ELISA (DY3144-05, R&D Systems).

Techniques: Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: Science translational medicine

Article Title: Oral delivery of liquid mRNA therapeutics by engineered capsule for treatment of preclinical intestinal disease

doi: 10.1126/scitranslmed.adu1493

Figure Lengend Snippet: ( A ) Experimental timeline for the oral administration of IL-10 -mRNA-RNACaps in acute colitis rat models. Rats were given free access to drinking water supplemented with 8.0% (w/w) DSS for 10 days to induce colitis. Afterward, plain water was provided, and rats were treated with RNACaps ( IL-10 -mRNA: 25 μg in 3 RNACaps per rat.) on days 11, 14 and 17 or sulfasalazine (SSZ, standard therapy, 100 mg/kg/day) daily. ( B and C ) Relative body weight (B) and DAI (C) of healthy (plain water-treated), DSS-treated, DSS plus IL-10 -mRNA-RNACap-treated or DSS plus SSZ-treated rats were monitored daily. ( D ) Quantification of colon length on day 17. ( E to J ) Quantification tissue protein expression of IL-10 (E), TNF-α (F), IL-1β (G), IL-6 (H), IL-17A (I) and MCP-1 (J) by ELISA. ( K to P ) Quantification of protein expression in blood of IL-10 (K), TNF-α (L), IL-1β (M), IL-6 (N), IL-17A (O) and MCP-1 (P) by ELISA. ( Q ) H&E staining images of the corresponding colon tissue sections. Dashed box indicates inset. Scale bars, 100 μm and 400 μm. Data are presented as mean ± S.D. Dots represent individual sample replicates. Statistical significance was evaluated by one-way ANOVA with Tukey’s post hoc analysis in (B to P). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. n = 5 animals per group for all panels. All the schematic illustrations were created using Adobe Illustrator.

Article Snippet: Rat TNF-α ELISA (438204, Biolegend), Rat IL-1 beta/IL-1F2 ELISA (DY501-05, R&D Systems), Rat IL-6 ELISA (DY506-05, R&D Systems), Rat IL-17A ELISA (437904, Biolegend), Rat JE/MCP-1/CCL2 ELISA (DY3144-05, R&D Systems).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: Science translational medicine

Article Title: Oral delivery of liquid mRNA therapeutics by engineered capsule for treatment of preclinical intestinal disease

doi: 10.1126/scitranslmed.adu1493

Figure Lengend Snippet: ( A ) Experimental timeline for oral administration of IL-10 -mRNA-RNACaps to rats for safety assessment. IL-10 -mRNA: 25 μg in 3 RNACaps per rat. ( B and C ) Analysis of blood chemistry, including aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) and complete blood count analysis, including white blood cell count (WBC), neutrophil (NE) (B), lymphocyte (LY), red blood cell count (RBC), hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelets (PLT) (C). ( D ) The rats were euthanized at the end of the study, and the indicated organs were sectioned and stained with H&E. Scale bars, 100 μm and 400 μm. ( E and F ) Cytokine concentration in the serum 6 h following oral administration of Fluc -mRNA-RNACap ( Fluc -mRNA: 25 μg in 3 RNACaps per rat) to healthy rats were measured using ELISA and Luminex. Cytokines measured include IL-1ra, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, and IL-10 (E) or IL-12p70, IL-13, IL-17A, IL-18, IFN-γ, TNF-α, GM-CSF, and VEGF (F). Data are presented as mean ± S.D. Dots represent individual sample replicates. Statistical significance was evaluated by unpaired two-tailed Student’s t-test in (B, C, E, F). * P < 0.05, *** P < 0.001. n = 3 animals per group for all panels. All the schematic illustrations were created using Adobe Illustrator.

Article Snippet: Rat TNF-α ELISA (438204, Biolegend), Rat IL-1 beta/IL-1F2 ELISA (DY501-05, R&D Systems), Rat IL-6 ELISA (DY506-05, R&D Systems), Rat IL-17A ELISA (437904, Biolegend), Rat JE/MCP-1/CCL2 ELISA (DY3144-05, R&D Systems).

Techniques: In Vivo, Cell Characterization, Concentration Assay, Staining, Enzyme-linked Immunosorbent Assay, Luminex, Two Tailed Test

Journal: Frontiers in Veterinary Science

Article Title: The immunomodulation–immunogenicity balance of equine Mesenchymal Stem Cells (MSCs) is differentially affected by the immune cell response depending on inflammatory licensing and major histocompatibility complex (MHC) compatibility

doi: 10.3389/fvets.2022.957153

Figure Lengend Snippet: Primers used for gene expression analysis by RT-qPCR.

Article Snippet: For IL6 analysis (Equine IL-6 DuoSet ELISA, R&D Systems, REF: DY1886), baseline and control supernatants were diluted 1:1 in the reagent diluent, and supernatants from co-cultures were not diluted.

Techniques: Gene Expression, Sequencing, Amplification

Journal: Frontiers in Veterinary Science

Article Title: The immunomodulation–immunogenicity balance of equine Mesenchymal Stem Cells (MSCs) is differentially affected by the immune cell response depending on inflammatory licensing and major histocompatibility complex (MHC) compatibility

doi: 10.3389/fvets.2022.957153

Figure Lengend Snippet: Interleukin 6 (IL6) gene expression and secretion by equine mesenchymal stem cells (MSCs) in the different scenarios. (A) IL6 mRNA relative expression and (C) IL6 secretion (pg/mL) before (baseline; orange bars) and after equine MSC-naive (light blue bars) and MSC-primed (dark blue bars) were exposed in vitro to phytohemagglutinin (PHA)-activated peripheral blood lymphocytes (PBLs) (immunosuppression assays). (B) IL6 mRNA relative expression and (D) IL6 secretion before (baseline; orange bars) and after MSC-naive (light green bars) and MSC-primed (dark green bars) were exposed in vitro to resting PBLs [modified one-way mixed lymphocyte reaction (MLR) assays]. Co-cultures of MSCs and PBLs were autologous ( n = 3), allogeneic, matched ( n = 8), or mismatched ( n = 7) for the major histocompatibility complex. Changes in gene expression are represented as mean ± S.E.M of the relative mRNA expression, using baseline MSC-naive as reference sample (light orange bar, value 1). Concentration of IL6 in the supernatant from the different conditions is represented as mean ± S.E.M (pg/mL). Significant differences of each condition compared with the baseline MSC-naive (light orange bar) are represented by hashes (#) above the corresponding bar ( # p < 0.05; #### p < 0.0001). Significant differences compared with the baseline MSC-primed (dark orange bar) are represented by a cross (+) above the corresponding bar ( + p < 0.05; ++ p < 0.01). Significant differences between experimental conditions are represented by a squared line with an asterisk ( * p < 0.05; *** p < 0.001).

Article Snippet: For IL6 analysis (Equine IL-6 DuoSet ELISA, R&D Systems, REF: DY1886), baseline and control supernatants were diluted 1:1 in the reagent diluent, and supernatants from co-cultures were not diluted.

Techniques: Gene Expression, Expressing, In Vitro, Modification, Immunopeptidomics, Concentration Assay