Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Comparative transcriptome analysis of human and murine choroidal neovascularization identifies fibroblast growth factor inducible-14 as phylogenetically conserved mediator of neovascular age-related macular degeneration.

doi: 10.1016/j.bbadis.2022.166340

Figure Lengend Snippet: Fig. 4. FN14 decoy receptor modulates the cytokine profile in CNV and reduces neovascularization size. (A): Experimental setup. (B): Intravitreal injection of a FN14 decoy receptor compared to PBS (control) significantly reduced CNV size. For statistical analysis, Mann–Whitney U test was applied. Color fundus (CF) and fluo rescence angiography (FA) on d7 of representative eyes of the FN14 decoy receptor (upper panel) and the PBS group (lower panel) as well as confocal microscopy (CM) images of representative lesions (COL4 in red) the size of which approximately corresponds to the mean spot size within the respective group. (C): Protein concentrations of several cytokines on d5 in the RPE of unlasered controls, CNV injected with PBS (CNV) or with FN14 decoy receptor on d1 (CNV + decoy). Data are shown as mean with SEM. For statistical analysis, paired ANOVA adjusting for total protein concentration was applied. *: p < 0.05, ns: not significant.

Article Snippet: At d1 mice received an intravitreal injection of 4 μg FN14 decoy receptor (1610-TW, R&D Systems) solved in 1 μl PBS in one eye and the same volume of PBS in the other eye, as described above.

Techniques: Injection, Control, MANN-WHITNEY, Confocal Microscopy, Protein Concentration

Journal: International Journal of Nanomedicine

Article Title: Tissue inhibitor of matrix metalloproteinases-1 loaded poly(lactic-co-glycolic acid) nanoparticles for delivery across the blood–brain barrier

doi: 10.2147/IJN.S54750

Figure Lengend Snippet: Schematic illustration of experimental plan for in vitro evaluation of NPs: The Coumarin 6 dye-loaded NPs (Dye NPs) and TIMP-1 loaded NPs (TIMP-1 NPs) without Ps80 coating and with Ps80 coating (Dye NPs + Ps80 and TIMP-1 NPs + Ps80) were evaluated in vitro. The NPs were evaluated using a rat brain endothelial cell line (RBE4) and primary rat brain capillary endothelial cells (RBCEC) co-cultured with glial cells. The NPs were evaluated for toxicity, uptake/binding and penetration. For toxicity studies, LDH assay, LY assay, and ZO1 (tight junction marker) immunocytochemistry were performed. The uptake and binding studies were done on RBCEC. For penetration studies, fluorescence spectrophotometry was used for dye-loaded NPs and ELISA for TIMP-1 NPs. Abbreviations: NPs, nanoparticles; TIMP, tissue inhibitor of matrix metalloproteinases; Ps80, polysorbate 80; LDH, lactate dehydrogenase; LY, Lucifer yellow; ELISA, enzyme-linked immunosorbent assay; immuno, immunocytochemistry; RBE4, rat brain endothelial cell line; RBCEC, rat brain capillary endothelial cells; ZO1, zona occludens 1.

Article Snippet: Recombinant mouse TIMP-1 with a 6× Histag (WBC022; R&D Systems) was used as a standard for both Western blots.

Techniques: In Vitro, Cell Culture, Binding Assay, Lactate Dehydrogenase Assay, Marker, Immunocytochemistry, Fluorescence, Spectrophotometry, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Nanomedicine

Article Title: Tissue inhibitor of matrix metalloproteinases-1 loaded poly(lactic-co-glycolic acid) nanoparticles for delivery across the blood–brain barrier

doi: 10.2147/IJN.S54750

Figure Lengend Snippet: Formulation optimization. Abbreviations: TIMP, tissue inhibitor of matrix metalloproteinases; PLGA, poly(lactic-co-glycolic acid); PDI, polydispersity index.

Article Snippet: Recombinant mouse TIMP-1 with a 6× Histag (WBC022; R&D Systems) was used as a standard for both Western blots.

Techniques: Formulation

Journal: International Journal of Nanomedicine

Article Title: Tissue inhibitor of matrix metalloproteinases-1 loaded poly(lactic-co-glycolic acid) nanoparticles for delivery across the blood–brain barrier

doi: 10.2147/IJN.S54750

Figure Lengend Snippet: ( A – C ) Characterization results of PLGA3 NPs loaded with TIMP-1. ( A ) Representative scanning electron micrograph of TIMP-1-loaded PLGA NPs. ( B ) Representative transmission electron micrograph of TIMP-1-loaded PLGA NPs; scale bar 500 nm. ( C ) Particle-size analysis by dynamic light scattering. Abbreviations: NPs, nanoparticles; TIMP, tissue inhibitor of matrix metalloproteinases; PLGA, poly(lactic-co-glycolic acid).

Article Snippet: Recombinant mouse TIMP-1 with a 6× Histag (WBC022; R&D Systems) was used as a standard for both Western blots.

Techniques: Transmission Assay, Particle Size Analysis

Journal: International Journal of Nanomedicine

Article Title: Tissue inhibitor of matrix metalloproteinases-1 loaded poly(lactic-co-glycolic acid) nanoparticles for delivery across the blood–brain barrier

doi: 10.2147/IJN.S54750

Figure Lengend Snippet: TIMP-1 release and stability: release of TIMP-1 from optimal PLGA3 NPs under in vitro conditions. Inset is representative Western blot of the protein released from the NPs, suggesting that release of TIMP-1 is stable after 48 hours. Data are means ± standard error of mean; n=3. Abbreviations: NPs, nanoparticles; TIMP, tissue inhibitor of matrix metalloproteinases; PLGA, poly(lactic-co-glycolic acid); Std, standard.

Article Snippet: Recombinant mouse TIMP-1 with a 6× Histag (WBC022; R&D Systems) was used as a standard for both Western blots.

Techniques: In Vitro, Western Blot

Journal: International Journal of Nanomedicine

Article Title: Tissue inhibitor of matrix metalloproteinases-1 loaded poly(lactic-co-glycolic acid) nanoparticles for delivery across the blood–brain barrier

doi: 10.2147/IJN.S54750

Figure Lengend Snippet: ( A – C ) Toxicity studies. ( A ) LY assay on RBCEC, showing no significant impact of NPs on endothelial cell permeability for LY compared to control (no NPs), indicating that NPs caused no significant opening of the endothelial cell monolayer. Ps80 alone and TIMP-1 alone were used as controls. ( B ) LDH assay, also showing that NPs were not toxic to the RBCEC compared to control. Ps80 alone and TIMP-1 alone were used as controls. ( C ) Representative fluorescence photomicrographs of ZO1 immunocytochemistry on RBCEC treated with TIMP-1 NPs and TIMP-1 NPs + Ps80, indicating no significant difference between the groups. Note: Values represent means ± standard error of mean of three independent experiments. Abbreviations: LY, Lucifer yellow; RBCEC, primary rat brain capillary endothelial cells; NPs, nanoparticles; TIMP, tissue inhibitor of matrix metalloproteinases; Ps80, polysorbate 80; LDH, lactate dehydrogenase; LY, Lucifer yellow.

Article Snippet: Recombinant mouse TIMP-1 with a 6× Histag (WBC022; R&D Systems) was used as a standard for both Western blots.

Techniques: Permeability, Lactate Dehydrogenase Assay, Fluorescence, Immunocytochemistry

Journal: International Journal of Nanomedicine

Article Title: Tissue inhibitor of matrix metalloproteinases-1 loaded poly(lactic-co-glycolic acid) nanoparticles for delivery across the blood–brain barrier

doi: 10.2147/IJN.S54750

Figure Lengend Snippet: In vitro blood–brain barrier penetration. Notes: (A ) Spectrophotometric assay suggests that Coumarin 6 dye-loaded NPs (Dye NPs) + Ps80 (coated with Ps80) had higher penetration, while uncoated Dye NPs did not cross the cell monolayer. Values represent means ± standard error of mean of three independent experiments, where asterisk represents P <0.05 versus group with Dye NPs + Ps80 (40 ug/mL). ( B ) For TIMP-1-loaded NPs, a TIMP-1 ELISA showed that TIMP-1 NPs + Ps80 (coated with Ps80) had much higher penetration compared to TIMP-1 NPs (uncoated NPs). Moreover, the penetration was time-dependent. After 120 minutes, there was a higher amount of TIMP-1 detected in the lower compartment compared to 30 minutes. Values represent mean ± standard error of mean of three independent experiments, where asterisks represent P <0.05 versus group with TIMP-1 NPs after 120 min of incubation and # P <0.05 versus group with TIMP-1 NPs + Ps80 after 30 minutes. Abbreviations: NPs, nanoparticles; Ps80, polysorbate 80; TIMP, tissue inhibitor of matrix metalloproteinases; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: Recombinant mouse TIMP-1 with a 6× Histag (WBC022; R&D Systems) was used as a standard for both Western blots.

Techniques: In Vitro, Spectrophotometric Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: International Journal of Nanomedicine

Article Title: Tissue inhibitor of matrix metalloproteinases-1 loaded poly(lactic-co-glycolic acid) nanoparticles for delivery across the blood–brain barrier

doi: 10.2147/IJN.S54750

Figure Lengend Snippet: In vivo blood–brain barrier (BBB) penetration. Notes: Representative fluorescence photomicrographs of immunohistochemistry on brain sections, of the specified area (marked with orange box) using a 6× Histag antibody to detect the TIMP-1-Histag fusion protein. The nucleus was labeled with DAPI. The TIMP-1 NPs + Ps80 group shows labeling indicating BBB penetration of TIMP-1 NPs + Ps80 as indicated by white arrows. Abbreviations: TIMP, tissue inhibitor of matrix metalloproteinases; DAPI, 4′,6-diamidino-2-phenylindole; NPs, nanoparticles; Ps80, polysorbate 80; PBS, phosphate-buffered saline.

Article Snippet: Recombinant mouse TIMP-1 with a 6× Histag (WBC022; R&D Systems) was used as a standard for both Western blots.

Techniques: In Vivo, Fluorescence, Immunohistochemistry, Labeling, Saline

Journal: Frontiers in Immunology

Article Title: Unique Roles for Streptococcus pneumoniae Phosphodiesterase 2 in Cyclic di-AMP Catabolism and Macrophage Responses

doi: 10.3389/fimmu.2020.00554

Figure Lengend Snippet: Phosphodiesterase 2 mutation in pneumococcus elicits a distinctive burst of IFNβ expression from macrophages. (A) Relative induction of IFNβ mRNA was measured in mouse macrophage-like RAW264.7 cell cultures infected 2 h with the indicated bacteria. Mouse IFNβ mRNA was measured and normalized to 18S rRNA using qPCR, and expressed relative to the cells infected by WT bacteria ( N = 3 experiments). (B) Relative induction of mouse CXCL10 mRNA was measured and normalized to 18S rRNA using qPCR, and expressed relative to the cells infected by WT bacteria ( N = 3 experiments). (C) Relative induction of IFNβ mRNA was measured in human THP-1 cell cultures that were matured to macrophage-like phenotypes using PMA before being infected 2 h with the indicated bacteria. Human IFNβ mRNA was measured and normalized to 18S rRNA using qPCR, and expressed relative to the cells infected by WT bacteria ( N = 6 experiments). (D) Relative induction of IFNβ mRNA was measured in mouse alveolar macrophages collected by bronchoalveolar lavage 3 h after in vivo infection of mice by intratracheal instillation with the indicated bacteria. Mouse IFNβ mRNA in these cells collected from infected lungs was measured and normalized to 18S rRNA using qPCR, and expressed relative to the cells collected from mice infected by WT bacteria ( N = 11–16 mice per group, over 4 experiments). Throughout panels, asterisk (*) indicates P < 0.05 compared to WT.

Article Snippet: Cytokine levels of IFNβ were measured in a sandwich ELISA using rat anti-mouse IFNβ mAb (ab24324; Abcam) for capture, rabbit anti-mouse IFNβ polyclonal Ab (32400-1; PBL Biomedical Laboratories) for detection, HRP-conjugated donkey anti-rabbit IgG (711-036-152; Jackson ImmunoResearch Laboratories), and recombinant mouse IFNβ standard (12400-1; PBL Biomedical Laboratories).

Techniques: Mutagenesis, Expressing, Infection, Bacteria, In Vivo

Journal: Frontiers in Immunology

Article Title: Unique Roles for Streptococcus pneumoniae Phosphodiesterase 2 in Cyclic di-AMP Catabolism and Macrophage Responses

doi: 10.3389/fimmu.2020.00554

Figure Lengend Snippet: Outcome of infection is dictated by pneumococcal phosphodiesterase 2. (A) Time-course of IFNβ protein accumulation in the supernatant was measured in mouse macrophage-like RAW264.7 cell cultures infected with the indicated bacteria. Mouse IFNβ concentration was measured using ELISA ( N = 3 experiments). (B) Time-course of cell death was measured in mouse macrophage-like RAW264.7 cell cultures infected with the indicated bacteria. Concentration of the cytosolic enzyme lactate dehydrogenase (LDH) was measured using a commercially available cytotoxicity assay kit, and expressed relative to concentrations measured after cells were completely lysed by detergent ( N = 3 experiments). Throughout panels, asterisk (*) indicates P < 0.05 compared to WT.

Article Snippet: Cytokine levels of IFNβ were measured in a sandwich ELISA using rat anti-mouse IFNβ mAb (ab24324; Abcam) for capture, rabbit anti-mouse IFNβ polyclonal Ab (32400-1; PBL Biomedical Laboratories) for detection, HRP-conjugated donkey anti-rabbit IgG (711-036-152; Jackson ImmunoResearch Laboratories), and recombinant mouse IFNβ standard (12400-1; PBL Biomedical Laboratories).

Techniques: Infection, Bacteria, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay

Journal: Frontiers in Immunology

Article Title: Unique Roles for Streptococcus pneumoniae Phosphodiesterase 2 in Cyclic di-AMP Catabolism and Macrophage Responses

doi: 10.3389/fimmu.2020.00554

Figure Lengend Snippet: The induction of IFNβ by phosphodiesterase 2 mutant pneumococci requires phagocytosis. (A) Relative induction of IFNβ mRNA was measured in mouse macrophage-like RAW264.7 cell cultures infected 2 h with the indicated bacteria in the presence or absence of cytochalasin D as a means of inhibiting actin polymerization ( N = 3 experiments). (B) Relative induction of IFNβ mRNA was measured in mouse macrophage-like RAW264.7 cell cultures infected 2 h with the indicated bacteria in the presence or absence of the indicated inhibitors pf phagosomal processing ( N = 3 experiments). For both panels, mouse IFNβ mRNA was measured and normalized to 18S rRNA using qPCR, and expressed relative to the cells infected by WT bacteria. For both panels, asterisk (*) indicates P < 0.05 compared to vehicle.

Article Snippet: Cytokine levels of IFNβ were measured in a sandwich ELISA using rat anti-mouse IFNβ mAb (ab24324; Abcam) for capture, rabbit anti-mouse IFNβ polyclonal Ab (32400-1; PBL Biomedical Laboratories) for detection, HRP-conjugated donkey anti-rabbit IgG (711-036-152; Jackson ImmunoResearch Laboratories), and recombinant mouse IFNβ standard (12400-1; PBL Biomedical Laboratories).

Techniques: Mutagenesis, Infection, Bacteria

Journal: Frontiers in Immunology

Article Title: Unique Roles for Streptococcus pneumoniae Phosphodiesterase 2 in Cyclic di-AMP Catabolism and Macrophage Responses

doi: 10.3389/fimmu.2020.00554

Figure Lengend Snippet: IFNβ expression elicited by phosphodiesterase 2 mutant pneumococci is mediated by the STING-cGAS-IRF3-IRF7 signaling pathway. (A) Relative contents of phosphorylated IRF3 and phosphorylated IRF7 protein were measured using immunoblot in cell lysates from mouse macrophage-like RAW264.7 cell cultures infected 2 h with the indicated bacteria. Relative contents of total IRF3 and IRF7 were measured for comparison, as well as GAPDH for loading control (image shown was from one experiment, representative of 2 independent experiments). (B) Relative induction of IFNβ mRNA was measured in parallel cultures of mouse macrophage-like RAW264.7 cells, one of which was deficient in STING due to gene targeting. Cultures were infected for 2 h with the indicated bacteria. Mouse IFNβ mRNA was measured and normalized to 18S rRNA using qPCR, and expressed relative to the cells infected by WT bacteria ( N = 3 experiments). Asterisk (*) indicates P < 0.05 compared to WT. (C) Relative induction of IFNβ mRNA was measured in parallel cultures of human THP-1 cell cultures that were stably transduced with lentiviruses encoding shRNA that was non-targeting (NT) or which targeted STING or cGAS. THP-1 cells were matured to macrophage-like phenotypes using PMA before being infected 2 h with the indicated bacteria. Human IFNβ mRNA was measured and normalized to 18S rRNA using qPCR, and expressed relative to the uninfected cells transduced with NT shRNA ( N = 5 experiments). Asterisk (*) indicates P < 0.05 compared to WT bacteria in cells transduced by the same shRNA, and hashtag (#) indicates P < 0.05 compared to NT shRNA-transduced cells infected by same bacteria.

Article Snippet: Cytokine levels of IFNβ were measured in a sandwich ELISA using rat anti-mouse IFNβ mAb (ab24324; Abcam) for capture, rabbit anti-mouse IFNβ polyclonal Ab (32400-1; PBL Biomedical Laboratories) for detection, HRP-conjugated donkey anti-rabbit IgG (711-036-152; Jackson ImmunoResearch Laboratories), and recombinant mouse IFNβ standard (12400-1; PBL Biomedical Laboratories).

Techniques: Expressing, Mutagenesis, Western Blot, Infection, Bacteria, Comparison, Control, Stable Transfection, Transduction, shRNA

Journal: Frontiers in Immunology

Article Title: Unique Roles for Streptococcus pneumoniae Phosphodiesterase 2 in Cyclic di-AMP Catabolism and Macrophage Responses

doi: 10.3389/fimmu.2020.00554

Figure Lengend Snippet: Dinucleotides involved in the distinctive effects of phosphodiesterase 2 mutant pneumococci. (A) Relative induction of IFNβ mRNA was measured in human THP-1 cell cultures that were matured to macrophage-like phenotypes using PMA before being stimulated 2 h with the indicated dinucleotides at a final concentration of 30 μg/ml. Human IFNβ mRNA was measured and normalized to 18S rRNA using qPCR, and expressed relative to the unstimulated cells ( N = 3 experiments). Asterisk (*) indicates P < 0.05 compared to unstimulated cells. (B) Relative induction of IFNβ mRNA was measured in mouse macrophage-like RAW264.7 cell cultures infected 2 h with the indicated bacteria that were cllected after growth in roth culture instead of on blood agar plates. Mouse IFNβ mRNA was measured and normalized to 18S rRNA using qPCR, and expressed relative to the cells infected by WT bacteria ( N = 3 experiments). (C) Relative c-di-AMP content was measured in pneumococcal cultures that were grown as used throughout experiments (except for in B ), on inverted tryptic soy agar plates containing 5% sheep’s blood and in a 5% CO 2 environment. Cyclic di-AMP was measured using a competitive ELISA ( N = 4 experiments) and normalized to the bacterial density at a given optical density (OD). Asterisk (*) indicates P < 0.05 compared to other bacterial strains. “ns” indicates not significant ( P > 0.05).

Article Snippet: Cytokine levels of IFNβ were measured in a sandwich ELISA using rat anti-mouse IFNβ mAb (ab24324; Abcam) for capture, rabbit anti-mouse IFNβ polyclonal Ab (32400-1; PBL Biomedical Laboratories) for detection, HRP-conjugated donkey anti-rabbit IgG (711-036-152; Jackson ImmunoResearch Laboratories), and recombinant mouse IFNβ standard (12400-1; PBL Biomedical Laboratories).

Techniques: Mutagenesis, Concentration Assay, Infection, Bacteria, Competitive ELISA