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: BMC Immunology

Article Title: Methylprednisolone inhibits interleukin-17 and interferon-gamma expression by both naive and primed T cells

doi: 10.1186/1471-2172-9-47

Figure Lengend Snippet: The effect of MP on IL-17 and IFN-γ gene expression and production in LNC . LNC isolated from DA rats (A, C-H) or AO rats (B) were stimulated with ConA (2.5 μg/ml) for 48 hours (A, B, E, F, H) or for various time periods (C, D) in the presence or absence of various MP concentrations (A, B), or with 10 ng/ml of MP (C-H). Subsequently, culture supernatants were collected for ELISA (A, B, C, D) or cells were collected for CFSE FACS analysis (H) or RNA isolation (E, F, G). Alternatively, cells were grown for additional 16 hours in the presence of 3 H-thymidine for proliferation assay (A). Results are presented as % of values obtained in corresponding cultures without MP – control (A, B) or as concentrations (C, D), or as 2 -dCt (E, F, G). CFSE staining profiles are: dark grey shadow – ConA, light grey line – ConA+MP, while the numbers in the table represent % of cells in corresponding phase. *p < 0.05 represents statistically significant difference to the corresponding culture without MP.

Article Snippet: Cell-free supernatants were frozen until analyzed by the protocol recommended by the manufacturers of the ELISA kits (OptEIA Mouse IL-17 Set, BD Biosciences; rat IFN-γ and rat IL-6 ELISA DuoSets, R&D systems, Minneapolis, MN).

Techniques: Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Staining

Journal: BMC Immunology

Article Title: Methylprednisolone inhibits interleukin-17 and interferon-gamma expression by both naive and primed T cells

doi: 10.1186/1471-2172-9-47

Figure Lengend Snippet: The effect of MP on MBP-specific production of IL-17 and IFN-γ . DLNC isolated from 4 rats 6 days after immunization with SCH-CFA, or SCC isolated from 3 rats 10 days after immunization with SCH-CFA (clinical score – 1.5) were stimulated with MBP (10 μg/ml) and cultivated in the presence or absence of MP (10 ng/ml) for 72 hours. *p < 0.05 represents statistically significant difference to the corresponding culture without MP.

Article Snippet: Cell-free supernatants were frozen until analyzed by the protocol recommended by the manufacturers of the ELISA kits (OptEIA Mouse IL-17 Set, BD Biosciences; rat IFN-γ and rat IL-6 ELISA DuoSets, R&D systems, Minneapolis, MN).

Techniques: Isolation

Journal: BMC Immunology

Article Title: Methylprednisolone inhibits interleukin-17 and interferon-gamma expression by both naive and primed T cells

doi: 10.1186/1471-2172-9-47

Figure Lengend Snippet: The effect of MP on IL-17 and IFN-γ production in T lymphocytes . CD3 + cells and LNC were stimulated with anti-CD3 antibody and anti-CD28 antibody (both at 1 μg/ml) in the absence or presence of MP (10 ng/ml) for 24 (A, C) and 48 (B, D) hours, and cell culture supernatants analyzed for IL-17 (A, B) and IFN-γ (C, D). CD3 - cells were stimulated with LPS (1 μg/ml) for 48 hours and cell culture supernatants analyzed for IL-6 (E). Results are presented as % of values obtained in the corresponding cultures without MP. *p < 0.05 represents statistically significant difference to the corresponding culture without MP.

Article Snippet: Cell-free supernatants were frozen until analyzed by the protocol recommended by the manufacturers of the ELISA kits (OptEIA Mouse IL-17 Set, BD Biosciences; rat IFN-γ and rat IL-6 ELISA DuoSets, R&D systems, Minneapolis, MN).

Techniques: Cell Culture

Journal: BMC Immunology

Article Title: Methylprednisolone inhibits interleukin-17 and interferon-gamma expression by both naive and primed T cells

doi: 10.1186/1471-2172-9-47

Figure Lengend Snippet: The effect of IFN-γ addition or neutralization on the inhibition of IL-17 production by MP . LNC were stimulated with ConA (2.5 μg/ml) and incubated with or without MP (10 ng/ml) and/or IFN-γ (IFN – 100 ng/ml), and/or anti-IFN-γ-neutralizing antibody (a-IFN – 1 μg/ml) or isotype control antibody (a-Iso – 1 μg/ml) for 24 hours. Subsequently, culture supernatants were collected for ELISA and IL-17 (A) and IFN-γ(B) production was determined. *p < 0.05 represents statistically significant difference to control cultures (medium).

Article Snippet: Cell-free supernatants were frozen until analyzed by the protocol recommended by the manufacturers of the ELISA kits (OptEIA Mouse IL-17 Set, BD Biosciences; rat IFN-γ and rat IL-6 ELISA DuoSets, R&D systems, Minneapolis, MN).

Techniques: Neutralization, Inhibition, Incubation, Enzyme-linked Immunosorbent Assay