Journal: PLoS ONE

Article Title: Myeloid Cells Expressing VEGF and Arginase-1 Following Uptake of Damaged Retinal Pigment Epithelium Suggests Potential Mechanism That Drives the Onset of Choroidal Angiogenesis in Mice

doi: 10.1371/journal.pone.0072935

Figure Lengend Snippet: ( A ) QRT-PCR analysis of time-dependent Arg-1 and Ym1 mRNA expression in RPE/choroid and retina tissues collected at indicated time points post laser induction. ( B ) RPE/choroid-lesional gene expression of Nos2 , Tnfα , Il1β , Arg-1 and Ym1 from isolated lesions at day 2 confirms localised inflammatory gene signature. ( C ) Cellular gene expression of Arg-1 and Ym1 on day 2 using CD11b MACS-isolated cells pooled from 4 RPE/choroidal or retinal tissues demonstrates early and local Arg-1 and Ym1 expression largely produced by myeloid population. Gapdh served as a normalising control in ( A – C ). Data are presented as mean ± SEM, n=3-6 for each time point. * P <0.05 vs. control; ** P <0.01 vs. control; # P <0.05 vs. non-lesion tissue on laser-treated eyes. ND, not detected.

Article Snippet: Rabbit polyclonal anti-Arg-1 (1:50) was from Santa Cruz Biotechnology.

Techniques: Quantitative RT-PCR, Expressing, Isolation, Produced

Journal: PLoS ONE

Article Title: Myeloid Cells Expressing VEGF and Arginase-1 Following Uptake of Damaged Retinal Pigment Epithelium Suggests Potential Mechanism That Drives the Onset of Choroidal Angiogenesis in Mice

doi: 10.1371/journal.pone.0072935

Figure Lengend Snippet: ( A ) Flow cytometric analysis of peripheral blood from mice following depletion of Ly6C hi CCR2 + monocyte subset using the MC-21 anti-CCR2 mAb, compared with rat IgG2b isotype control (mean ± SEM, n=3). Assessment of Ccr2 ( B ) or Arg-1 ( C ) mRNA expression normalised against Gapdh in RPE/choroid day 2 post laser in MC-21 treated mice by QRT-PCR (mean ± SEM, n=4-6). ( D ) Side view of Z-stacks of confocal images shows migration of retinal microglia (Iba1 staining) in MC-21 treated animals. Confocal images ( E ) and semi-quantitative mean fluorescence intensity (MFI) analysis using ImageJ software for Iba1 + cell accumulation ( F ) and whole lesional VEGF expression ( G ) on RPE/choroid whole-mounts taken on day 2 post laser (mean ± SEM, n≥12/group). Magnification image in (E, inset) shows macrophages co-stained for VEGF in MC-21 injected mice. ( H and I ) CNV formation on day 7 post laser was assessed by IB4 staining on RPE/choroidal whole-mounts and images were captured by confocal microscopy and analysed using a Volocity® 3D Image analysis software. Representative 3D reconstructions of neovascular complex ( H ) and quantitative data (I) show reduced CNV volume by 39% with MC-21 treatment compared with control (n=24/group). * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: Rabbit polyclonal anti-Arg-1 (1:50) was from Santa Cruz Biotechnology.

Techniques: Expressing, Quantitative RT-PCR, Migration, Staining, Fluorescence, Software, Injection, Confocal Microscopy

Journal: PLoS ONE

Article Title: Myeloid Cells Expressing VEGF and Arginase-1 Following Uptake of Damaged Retinal Pigment Epithelium Suggests Potential Mechanism That Drives the Onset of Choroidal Angiogenesis in Mice

doi: 10.1371/journal.pone.0072935

Figure Lengend Snippet: ( A ) Necrotic RPE was produced from CFDA-labelled B6-RPE07 cells (green) by heating at 95°C for 15 minutes, while RPE lysate was generated by sonication for 5 minutes on ice. Necrotic RPE cells or lysate were then added to Violet Tracer-labelled BMMΦs at different ratios (RPE to BMMΦ). After 1 hour incubation, the cells were washed, fixed and observed by confocal microscopy. Top and side view of confocal images shows engulfment of dead RPEs/debris by BMMΦs. BMMΦs were collected after 24 hours of incubation with necrotic RPE or cell lysate and RNA was extracted for QRT-PCR analysis to determine Arg-1 and Nos2 ( B ), and Vegf gene expression ( C ). 18s rRNA was used as a normalising control. ( D ) Confocal images and MFI analysis of VEGF immunocytochemistry on BMMΦs treated with necrotic RPE. ( E ) In some of experiments, after 1 hour of incubation of BMMΦs with necrotic RPE, un-engulfed dead RPE cells and its derivatives were removed and BMMΦs were further cultured for 24 hours and cell culture supernatants collected for determination of VEGF concentration using ELISA. ( F ) Human microglia cells (C13-NJ cell line) were treated with heat-induced necrotic ARPE19 cells at different ratios for 24 hours and then examined for Vegf mRNA expression. Data are presented as mean ± SEM, n≥3. * P <0.05; ** P <0.01; *** P <0.001 Arg-1 vs. Nos2 ( B ), or vs. control. NS, not significant.

Article Snippet: Rabbit polyclonal anti-Arg-1 (1:50) was from Santa Cruz Biotechnology.

Techniques: Produced, Generated, Sonication, Incubation, Confocal Microscopy, Quantitative RT-PCR, Expressing, Immunocytochemistry, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Myeloid Cells Expressing VEGF and Arginase-1 Following Uptake of Damaged Retinal Pigment Epithelium Suggests Potential Mechanism That Drives the Onset of Choroidal Angiogenesis in Mice

doi: 10.1371/journal.pone.0072935

Figure Lengend Snippet: Mice BMMΦs were treated with necrotic cells generated from B6-RPE07 cells (RPE), whole retinal tissues (Ret), retinal vascular endothelial cells (EC), YTS156 hybridoma (B cell) or bone marrow neutrophils for 24 hours. Cellular RNA was isolated and gene expression of Arg-1 ( A ), Vegf ( B ), Nos2 ( C ) and Il1β ( D ) was assessed by QRT-PCR (mean ± SEM, n≥3). 18s rRNA was utilised as a normalising control and results presented as fold change of control.

Article Snippet: Rabbit polyclonal anti-Arg-1 (1:50) was from Santa Cruz Biotechnology.

Techniques: Generated, Isolation, Expressing, Quantitative RT-PCR

Journal: Bioactive Materials

Article Title: L-arginine loading porous PEEK promotes percutaneous tissue repair through macrophage orchestration

doi: 10.1016/j.bioactmat.2024.05.025

Figure Lengend Snippet: Metabolic reprogramming effect of macrophages cultured on L-Arg loading SP . (a) Co-localized of CCR7 and iNOS of macrophages cultured on PEEK, SP, SPLA5, SPLA10 and SPLA20 surface stimulated by LPS; (b) Co-localized of CD206 and Arg-1 of macrophages cultured on PEEK, SP, SPLA5, SPLA10 and SPLA20 surface stimulated by IL-4; (c) Schematic diagram of L-Arg metabolism in different microenvironment; (d) NO content releasing from macrophages cultured on samples with different stimulations (n = 3); (e) Ornithine content producing from macrophages cultured on samples with different stimulations (n = 3). Data are expressed by mean with SD. Statistical significance was calculated by two-way ANOVA analysis and Tukey’s multiple comparison tests. *p < 0.05, **p < 0.01 and ***p < 0.001, compared with PEEK in d and e.

Article Snippet: And specific primary antibodies, rabbit polyclonal anti-F4/80 antibody (Servicebio, GB113373, 1: 5000), rabbit polyclonal anti-iNOS antibody (Servicebio, GB11119, 1: 100), rabbit polyclonal anti-Arg-1 antibody (Servicebio, GB11285, 1: 3000), and secondary antibodies, CY3-labeled goat anti-rabbit IgG (Servicebio, GB21303, 1: 300), CY5-labeled goat anti-rabbit IgG (Servicebio, GB27303, 1: 400), Alexa Fluor 488 labeled goat anti-rabbit IgG (Servicebio, GB25303, 1: 400) were chose to label macrophages, M1 type, and M2 type, respectively.

Techniques: Cell Culture, Comparison

Journal: Bioactive Materials

Article Title: L-arginine loading porous PEEK promotes percutaneous tissue repair through macrophage orchestration

doi: 10.1016/j.bioactmat.2024.05.025

Figure Lengend Snippet: Soft tissue healing. (a) Circos graph of KEGG enrichment; (b) Bubble diagram of KEGG enriched top 20 pathways of upregulated genes; (c) Bubble diagram of KEGG enriched top 20 pathways of downregulated genes; (d) Representative immunofluorescent staining images of macrophages in skin tissue implanted with samples for 14 days (F4/80 labeled macrophages with red, iNOS labeled M1 with pink, Arg-1 labeled M2 with green); (e) Representative HE staining images of skin tissue implanted with samples for 14 days (the implants are marked by dotted box); (f) Representative Masson staining images of skin tissue implanted with samples for 14 days; (g) Representative Col-III staining images of skin tissue implanted with samples for 14 days; (h) Representative CD31 staining images of skin tissue implanted with samples for 14 days.

Article Snippet: And specific primary antibodies, rabbit polyclonal anti-F4/80 antibody (Servicebio, GB113373, 1: 5000), rabbit polyclonal anti-iNOS antibody (Servicebio, GB11119, 1: 100), rabbit polyclonal anti-Arg-1 antibody (Servicebio, GB11285, 1: 3000), and secondary antibodies, CY3-labeled goat anti-rabbit IgG (Servicebio, GB21303, 1: 300), CY5-labeled goat anti-rabbit IgG (Servicebio, GB27303, 1: 400), Alexa Fluor 488 labeled goat anti-rabbit IgG (Servicebio, GB25303, 1: 400) were chose to label macrophages, M1 type, and M2 type, respectively.

Techniques: Staining, Labeling

Journal: Bioactive Materials

Article Title: L-arginine loading porous PEEK promotes percutaneous tissue repair through macrophage orchestration

doi: 10.1016/j.bioactmat.2024.05.025

Figure Lengend Snippet: The schematic illustration of the effect of L-Arg loading PEEK on both antibacterial abilities and tissue repair, including soft tissue healing and hard tissue regeneration, by macrophage orchestration via metabolic reprogramming. Under infection condition, macrophage polarizes to M1 and L-Arg is catalyzed to NO and ROS by iNOS, which play the role in sterilization. Under the tissue repair condition, macrophage will polarize to M2 and L-Arg is catalyzed to ornithine by Arg-1, which promotes the proliferation and collagen secretion of cells.

Article Snippet: And specific primary antibodies, rabbit polyclonal anti-F4/80 antibody (Servicebio, GB113373, 1: 5000), rabbit polyclonal anti-iNOS antibody (Servicebio, GB11119, 1: 100), rabbit polyclonal anti-Arg-1 antibody (Servicebio, GB11285, 1: 3000), and secondary antibodies, CY3-labeled goat anti-rabbit IgG (Servicebio, GB21303, 1: 300), CY5-labeled goat anti-rabbit IgG (Servicebio, GB27303, 1: 400), Alexa Fluor 488 labeled goat anti-rabbit IgG (Servicebio, GB25303, 1: 400) were chose to label macrophages, M1 type, and M2 type, respectively.

Techniques: Infection

Journal: Biomedicines

Article Title: Milmed Yeast Alters the LPS-Induced M1 Microglia Cells to Form M2 Anti-Inflammatory Phenotype

doi: 10.3390/biomedicines10123116

Figure Lengend Snippet: ( A ) Immunofluorescence analysis of Inducible Nitric Oxide Synthase (iNOS) expression in BV-2 cells cultured in the presence of untreated yeast grown for 24 h in different metabolic medium without LPS (on the left, first column) or after addition of Lipopolysaccharide (LPS) 1 ng/mL (second column) and ( B ) Arginase-1 (ARG-1)expression in BV-2 cells cultured in the presence of Milmed grown in different metabolic medium without LPS (on the right, first column) or after addition of LPS 1 ng/mL (second column). Quantification of the median fluorescence intensity was performed by ImageJ software and data were expressed as histograms, normalized to the number of cells for field. 4′,6-diamidino-2-phenylindole (DAPI)was used to counterstain the nuclei. Data are expressed as mean ± SD for each group ( n = 3). Statistical analysis was performed by the one-way analysis of variance (ANOVA) coupled with the Bonferroni post-test. *** p < 0.001; ### p < 0.001.

Article Snippet: For the detection of M1/M2 polarization markers, cells were incubated with rabbit polyclonal antibodies raised against iNOS (dil. 1:100—D6B65, Cell Signaling Technology, Danvers, MA, USA) or rabbit polyclonal IgG anti-Arg-1 (dil. 1:50—D4E3M, Cell Signaling Technology, Danvers, MA, USA) and subsequently with anti-rabbit Alexa Fluor 488 secondary antibodies.

Techniques: Immunofluorescence, Expressing, Cell Culture, Fluorescence, Software

Journal: Biomedicines

Article Title: Milmed Yeast Alters the LPS-Induced M1 Microglia Cells to Form M2 Anti-Inflammatory Phenotype

doi: 10.3390/biomedicines10123116

Figure Lengend Snippet: BV2 cells were pretreated with 5 × 10 2 Milmed/well grown in Yeast Extract–Peptone–Dextrose (YPD) medium or regrown in YPD medium from a dried preparation for 45 min and afterward incubated with Lipopolysaccharide (LPS) 1 ng/mL. Inducible Nitric Oxide Synthase (iNOS) and Arginase-1 (ARG-1) mRNAs expressions were evaluated by qRT-PCR at 4 h and normalized to β-actin. Data are shown as mean ± SD from three independent experiments performed in triplicate. Statistical analysis was evaluated by unpaired Student’s t test. Expression profiles were determined using the 2 −ΔΔCT method. * p < 0.05, ** p < 0.01. *** p < 0.001, **** p < 0.0001; ## p < 0.01; ### p < 0.001; #### p < 0.0001 * VS CTRL, # VS LPS.

Article Snippet: For the detection of M1/M2 polarization markers, cells were incubated with rabbit polyclonal antibodies raised against iNOS (dil. 1:100—D6B65, Cell Signaling Technology, Danvers, MA, USA) or rabbit polyclonal IgG anti-Arg-1 (dil. 1:50—D4E3M, Cell Signaling Technology, Danvers, MA, USA) and subsequently with anti-rabbit Alexa Fluor 488 secondary antibodies.

Techniques: Incubation, Quantitative RT-PCR, Expressing