Journal: Biomaterials

Article Title: Macrophage Repolarization with Targeted Alginate Nanoparticles Containing IL-10 Plasmid DNA for the Treatment of Experimental Arthritis

doi: 10.1016/j.biomaterials.2015.05.028

Figure Lengend Snippet: Histogram plot (A) and mean fluorescence intensity (B) of CD163 expression in J774A.1 macrophages transfected with plasmid IL10-encapsulated control (unmodified NPs and scrambled NPs) and targeted alginate nanoparticles.

Article Snippet: Rabbit anti-CD163/M130 polyclonal antibody conjugated with Alexa Fluor 488 dye was purchased from Bioss Antibodies (Woburn, MA).

Techniques: Fluorescence, Expressing, Transfection, Plasmid Preparation

Journal: Biomaterials

Article Title: Macrophage Repolarization with Targeted Alginate Nanoparticles Containing IL-10 Plasmid DNA for the Treatment of Experimental Arthritis

doi: 10.1016/j.biomaterials.2015.05.028

Figure Lengend Snippet: Top Panel represents the agarose gel image of the PCR products for CD11b, CD86, CD163 and β-actin. The PCR product sizes are 146 bp, 99bp, 98bp, and 119 bp, respectively. β-Actin served as an internal control. Bottom Panel represents the ratio of the CD86: CD11b and CD163:CD11b to semi-quantitatively analyze the M1 and M2 percentage of total macrophages (positive for CD11b), respectively. The band intensity was calculated via NIH ImageJ software. The data is representative of macrophage population that was obtained by pooling together cells collected from hind paws of five animals per treatment group.

Article Snippet: Rabbit anti-CD163/M130 polyclonal antibody conjugated with Alexa Fluor 488 dye was purchased from Bioss Antibodies (Woburn, MA).

Techniques: Agarose Gel Electrophoresis, Software

Journal: Cell Death & Disease

Article Title: M2 macrophage-mediated interleukin-4 signalling induces myofibroblast phenotype during the progression of benign prostatic hyperplasia

doi: 10.1038/s41419-018-0744-1

Figure Lengend Snippet: a Representative CD68 immunohistochemical staining images in the early-progressed BPH, age-matched prostate and elderly BPH tissues. Scale bar, 200 μm. b Scatter plots showing macrophage clusters in the three analysed groups. The number of macrophage clusters in prostate tissues correlated positively with the stroma-to-epithelium ratio, and percent area densities of α-SMA, collagen I and fibres. c Serial histological sections showing the early-progressed BPH tissues stained for CD68, a macrophage-specific marker, and CD163, an M2 macrophage marker. The distribution of CD68 + and CD163 + cells was almost identical. Scale bar, 200 μm. d Representative immunofluorescence (IF) staining images, showing CD68 (green) and CD163 (red) expression in the infiltrating macrophages in early-progressed BPH tissues. Scale bar, 100 μm. e Quantitative RT-PCR results showing α-SMA, COL1A1 and COL3A1 expression in primary prostate fibroblast (PrPF)-early, PrPF-control and PrPF-old cells co-cultured with THP-1-derived M2 macrophages. Data are shown as relative gene expression compared to that in the respective untreated fibroblasts. f Western blots showing α-SMA and collagen I protein expression in PrPF-early, PrPF-control and PrPF-old cells co-cultured with THP-1-derived M2 macrophages. g Representative IF staining results, showing the co-expression of α-SMA (red) and collagen I (green) following a 48-h co-culture of PrPF-early, PrPF-control and PrPF-old cells with THP-1-derived M2 macrophages. The respective untreated fibroblasts were used as controls. Scale bar, 100 μm. h Solidified collagen-gel shrinkage after the seeding of PrPF-early, PrPF-control and PrPF-old cells, untreated or treated with the conditioned medium. The respective untreated fibroblast samples were used as controls, and all assays were performed in triplicate. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The primary antibodies included mouse monoclonal α-SMA (Abcam, ab7817, 1:100 dilution), CD163 (OriGene, TA506380, 1:200 dilution) and EpCAM (Cell Signaling Technology, #2929, 1:800 dilution) antibodies, and the rabbit primary antibodies included collagen I (Abcam, ab138492, 1:800 dilution), CD68 (Proteintech, 25747-1-AP, 1:400 dilution), IL4R (ImmunoWay, YT2337, 1:400 dilution), TGFβ RI (ImmunoWay, YT4627, 1:400 dilution) and vimentin (Cell Signaling Technology, #5741, 1:100 dilution) antibodies.

Techniques: Immunohistochemical staining, Staining, Marker, Immunofluorescence, Expressing, Quantitative RT-PCR, Cell Culture, Derivative Assay, Western Blot, Co-Culture Assay

Journal: Cancers

Article Title: Murlentamab, a Low Fucosylated Anti-Müllerian Hormone Type II Receptor (AMHRII) Antibody, Exhibits Anti-Tumor Activity through Tumor-Associated Macrophage Reprogrammation and T Cell Activation

doi: 10.3390/cancers13081845

Figure Lengend Snippet: Variation of pharmacodynamic markers measured in blood samples and biopsies of patients from C201 and C101 studies. ( A ) Detection of CD69 as a marker of activation of monocytes, regulatory and CD8 + T cells in blood samples of patients ( n = 20) with colorectal cancer treated with murlentamab (single agent and in combination with trifluridine/tripiracil). Data shown (boxplots) are the results from 20 patients. * p < 0.05; ** p < 0.01. p values were determined using one-way ANOVA analysis followed by Tukey’s multiple comparisons test. ( B ) Detection of ICOS (inducible co-stimulatory molecule) as a marker of lymphocyte activation in blood samples of patients with ovarian cancer treated at Gustave Roussy (Paris, France) with murlentamab in combination with carboplatin + paclitaxel ( n = 4). ( C ) Detection of CXCL9 ( n = 16) and CXCL10 ( n = 15) release in blood samples of all patients treated with murlentamab single agent in C201. ( D ) Detection of co-staining CD14/CD163 ( n = 8) and of ICOS ( n = 4) as markers of immune system regulation in FFPE (Formalin-Fixed Paraffin-Embedded) biopsies obtained from the C201 study. ( E ) Image and quantification of CD16 ( n = 5), co-staining CD16/granzyme B (GrZB) ( n = 4), and CD8 ( n = 4) as markers of immune system activation in FFPE biopsies obtained from the C201 study. ( F ) Image and quantification of CD16/granzyme B (GrZB) co-staining in FFPE biopsies obtained from the C101 study ( n = 2).

Article Snippet: CD163-PE , Miltenyi , 130-112-286 , REA812 , .

Techniques: Marker, Activation Assay, Staining, Formalin-fixed Paraffin-Embedded

Journal: Cancers

Article Title: Murlentamab, a Low Fucosylated Anti-Müllerian Hormone Type II Receptor (AMHRII) Antibody, Exhibits Anti-Tumor Activity through Tumor-Associated Macrophage Reprogrammation and T Cell Activation

doi: 10.3390/cancers13081845

Figure Lengend Snippet: Flow cytometry antibodies used.

Article Snippet: CD163-PE , Miltenyi , 130-112-286 , REA812 , .

Techniques: Flow Cytometry, In Vivo, In Vitro

Journal: Cancers

Article Title: Murlentamab, a Low Fucosylated Anti-Müllerian Hormone Type II Receptor (AMHRII) Antibody, Exhibits Anti-Tumor Activity through Tumor-Associated Macrophage Reprogrammation and T Cell Activation

doi: 10.3390/cancers13081845

Figure Lengend Snippet: Murlentamab opsonization of SKOV3-R2 + orients naïve macrophages and reprograms TAMs towards an M1-like profile. SKOV3-R2 + ovarian tumor cells were labeled with different 3C23K antibodies (3C23K-FcKO control, 3C23K-CHO normally fucosylated or murlentamab the low fucosylated form) and cultured in the presence of human monocyte-derived macrophages from healthy donors unstimulated (M0) or stimulated with M-CSF and IL-10 (TAMs). ( A ) The proportion of macrophages expressing M1/M2 membrane markers (CD32, CD64, CD80, TLR2, CD163, CD36 and CD206) was determined by flow cytometry after three days of co-culture with SKOV3-R2 + cells. ( B ) The release of cytokines (IL1β, IL12, TNFα, IL6, IFNγ, IL10) and chemokines (CCL2, CCL4, CCL5, CXCL9 and CXCL10) in the culture medium was determined by AlphaLISA after three days of co-culture with SKOV3-R2 + cells. Data shown (boxplots) are the results from three different experiments (performed with three different healthy donors). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. p values were determined using one-way ANOVA analysis followed by Tukey’s multiple comparisons test.

Article Snippet: CD163-PE , Miltenyi , 130-112-286 , REA812 , .

Techniques: Labeling, Cell Culture, Derivative Assay, Expressing, Membrane, Flow Cytometry, Co-Culture Assay

Journal: Cancers

Article Title: Murlentamab, a Low Fucosylated Anti-Müllerian Hormone Type II Receptor (AMHRII) Antibody, Exhibits Anti-Tumor Activity through Tumor-Associated Macrophage Reprogrammation and T Cell Activation

doi: 10.3390/cancers13081845

Figure Lengend Snippet: Murlentamab/pembrolizumab combination accentuates the anti-tumoral effect of murlentamab monotherapy through the enhancement of T cell activation. ( A – C ) SKOV3-R2 + ovarian tumor cells were labeled with different 3C23K antibodies (3C23K-FcKO control or murlentamab the low fucosylated form) and cultured in the presence of human monocyte-derived macrophages from healthy donors stimulated with M-CSF and IL-10 (TAMs). After 3 days of co-culture, activated T cells coming from the same healthy donor were added in the culture well for 4 more days. Pembrolizumab was added into co-culture wells everyday from day 3 to day 10. ( A ) Opsonized-SKOV3-R2 + cell number was determined by flow cytometry after one and two days of co-culture with TAMs. Data shown (mean ± SEM) are the results from three different experiments (performed with one healthy donors). ** p < 0.01 compared 3C23K-FcKO vs. Murlentamab. # p < 0.05; ## p < 0.01 compared 3C23K-FcKO + anti-PD-1 vs. Murlentamab + anti-PD-1 as determined using one-way ANOVA analysis followed by Dunnett’s multiple comparisons test. ( B , C ) The CD4 + Th1/Th2 polarization profile and the activation of T CD8 + cells were determined by flow cytometry after four days of co-culture. Data shown (mean ± SEM) are the results from three different experiments (performed with one healthy donors). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. p values were determined using one-way ANOVA analysis followed by Tukey’s multiple comparisons test. ( D , E ) 10 × 10 6 COV434-R2 + ovarian tumor cells were transplanted subcutaneously into humanized GM-CSF/IL3/IL4 hu-NOG (NOD/Shi-scid/IL2Rγ null ) mice (Taconic). After 35 days, when tumors were big enough, mice were i.p treated or not with murlentamab (5 mg/kg) +/− pembrolizumab (25 mg/kg) twice a week for 4 weeks. ( D ) Quantification of circulating CD86 + and CD163 + cells by flow cytometry from blood of tumor-bearing mice before treatment and after 24 days of treatment with murlentamab (5 mg/kg) or pembrolizumab (25 mg/kg) as single agents or murlentamab/pembrolizumab combo-therapy. Data are represented as boxplots. *** p < 0.001, **** p < 0.0001 in comparison to baseline. ( E ) In vivo tumor growth. Data are represented as mean + SEM.

Article Snippet: CD163-PE , Miltenyi , 130-112-286 , REA812 , .

Techniques: Activation Assay, Labeling, Cell Culture, Derivative Assay, Co-Culture Assay, Flow Cytometry, Comparison, In Vivo

Journal: Brain, behavior, and immunity

Article Title: Intermittent cytomegalovirus infection alters neurobiological metabolism and induces cognitive deficits in mice

doi: 10.1016/j.bbi.2023.12.033

Figure Lengend Snippet: Brain cell characterization flow panel.

Article Snippet: CD163 , AF680 , Bioss , bs-2527R-A680 , 1:400.

Techniques:

Journal: Toxicologic Pathology

Article Title: Biocompatible Solutions: Evaluating the Safety of Repeated Intra-Articular Injections of pMPCylated Liposomes for Knee Osteoarthritis Therapy in Rat Models

doi: 10.1177/01926233241271400

Figure Lengend Snippet: Antibodies used for the immunofluorescence study.

Article Snippet: Novus (CD163) , NBP3-07981 (CD163).

Techniques: Immunofluorescence, Marker

Journal: Toxicologic Pathology

Article Title: Biocompatible Solutions: Evaluating the Safety of Repeated Intra-Articular Injections of pMPCylated Liposomes for Knee Osteoarthritis Therapy in Rat Models

doi: 10.1177/01926233241271400

Figure Lengend Snippet: Immunofluorescence images of the knee joint in study 1. (A-D) These immunofluorescence (IF) images capture the right knee joint of an animal from Group 3M, which received three repeated IA injections of 15 mM CCoat. Positive cells for CD68 and CD163 cocktail are indicated by red cytoplasm, C-Maf signal is indicated by pink/purple nucleus, Pstat1 by green nuclear signal and DAPI by the blue nucleus. Autofluorescence is present within the tibia and femur, which presents as yellow color. A. The white arrows highlight the synovial membranes with IF-positive macrophages. B. IF-positive cells expressing CD68 and CD163 within the synovial membrane are observed in this image. The red cytoplasm is indicated by white arrows, while the nucleus, highlighted in blue and positive for DAPI, is marked by yellow arrows. C. These are the same cells as depicted in Figure B. The C-Maf signal is represented by the pink/purple coloration in the nucleus (white arrows), signifying the presence of M2 macrophages. D. These cells correspond to those shown in Figure B. The green nuclear signal, denoting positive P-Stat1 binding, is marked by white arrows. However, it is noteworthy that the signal is diminished both in area and intensity. (E-H) These IF images capture the right knee joint of an animal from Group 4F, which received three repeated IA injections of 30 mM CCoat. Positive cells for CD68 and CD163 cocktail are indicated by red cytoplasm, C-Maf signal is indicated by pink/purple nucleus, Pstat1 signal by green nuclear signal and DAPI by the blue nucleus. E. The white arrows point to synovial membranes with immunofluorescence-positive macrophages. F. Cells within the synovial membrane exhibit positive IF for CD68 and CD163. The red cytoplasm is indicated by white arrows, while the nucleus, highlighted in blue (yellow arrows), confirms DAPI positivity. G. These cells correspond to those in Figure F. The C-Maf signal is represented by the pink/purple coloration in the nucleus (white arrows), indicating the presence of M2 macrophages. H. These are the same cells as shown in Figure F. The green nuclear signal, indicative of positive P-Stat1 binding, is observed, but notably reduced in both area and intensity.

Article Snippet: Novus (CD163) , NBP3-07981 (CD163).

Techniques: Immunofluorescence, Expressing, Membrane, Binding Assay

Journal: Stem Cell Research & Therapy

Article Title: Human uncultured adipose-derived stromal vascular fraction shows therapeutic potential against osteoarthritis in immunodeficient rats via direct effects of transplanted M2 macrophages

doi: 10.1186/s13287-024-03946-3

Figure Lengend Snippet: A Immunohistochemical analysis of F4/80 in synovial tissue showing representative samples at 4 and 8 weeks after DMM. The sections were counterstained with hematoxylin. Scale bar = 100 μm. B F4/80 scores in each group (n = 5/group). C Immunohistochemical analysis of CD86 in synovial tissue showing representative samples at 4 and 8 weeks after DMM. The sections were counterstained with hematoxylin. Scale bar = 100 μm. D Immunohistochemical analysis of CD163 in synovial tissue showing representative samples at 4 and 8 weeks after DMM. The sections were counterstained with hematoxylin. Scale bar = 100 μm. ( E ) CD86/CD163 expression ratio (M1/M2 ratio) in each group (n = 5/group). SVF, stromal vascular fraction; ADSC, adipose-derived stromal cell; DMM, destabilization of the medial meniscus; MΦ, macrophages

Article Snippet: The samples were incubated overnight at 4 °C with a conjugated primary antibody against F4/80 (1:100; orb466169-FITC, Biorbyt), conjugated primary antibody against CD163 (1:200; BS-2527R-A594, Bioss), and primary anti-mouse antibody against hNA (1:500; ab191181, Abcam).

Techniques: Immunohistochemical staining, Expressing, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: Human uncultured adipose-derived stromal vascular fraction shows therapeutic potential against osteoarthritis in immunodeficient rats via direct effects of transplanted M2 macrophages

doi: 10.1186/s13287-024-03946-3

Figure Lengend Snippet: A Immunofluorescence analysis of F4/80, CD163, and hNA in synovial tissue of the rats at 4 weeks after DMM showing representative triple immunostaining of F4/80 (green), CD163 (red), and hNA (violet) in each group. Scale bar = 20 μm. B Comparison of the ratio of CD163- and hNA-positive cells in the F4/80-positive cells via immunofluorescence staining (n = 5/group). SVF, stromal vascular fraction; ADSC, adipose-derived stromal cell; hNA, human nuclear antigen

Article Snippet: The samples were incubated overnight at 4 °C with a conjugated primary antibody against F4/80 (1:100; orb466169-FITC, Biorbyt), conjugated primary antibody against CD163 (1:200; BS-2527R-A594, Bioss), and primary anti-mouse antibody against hNA (1:500; ab191181, Abcam).

Techniques: Immunofluorescence, Triple Immunostaining, Comparison, Staining, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: Human uncultured adipose-derived stromal vascular fraction shows therapeutic potential against osteoarthritis in immunodeficient rats via direct effects of transplanted M2 macrophages

doi: 10.1186/s13287-024-03946-3

Figure Lengend Snippet: A Flow cytometry analysis revealing the proportion of CD163-positive cells in the SVF (n = 5). B Setup of the separated pellet co-culture system. Groups including ADSC, M2Φ, and SVF were established, with each administered cell type in membrane plates and OA chondrocytes in 15 mL tubes. A control group was established with no cells in membrane plates and only OA chondrocytes in 15-mL tubes. C Gross photographs and safranin-O staining of the resulting pellets in each group. D Comparison of pellet size among each group (n = 5/group). E Analysis of TGF-β, IL-10, and MMP-13 levels in the supernatant following coculture with ADSC, M2Φ, and SVF and the chondrocyte (n = 5/group). SVF, stromal vascular fraction; M2Φ, M2 macrophages; ADSC, adipose-derived stromal cell; TGF-β, transforming growth factor-β; IL-10, interleukin-10; MMP-13, matrix metalloproteinase 13

Article Snippet: The samples were incubated overnight at 4 °C with a conjugated primary antibody against F4/80 (1:100; orb466169-FITC, Biorbyt), conjugated primary antibody against CD163 (1:200; BS-2527R-A594, Bioss), and primary anti-mouse antibody against hNA (1:500; ab191181, Abcam).

Techniques: Flow Cytometry, Co-Culture Assay, Membrane, Control, Staining, Comparison, Derivative Assay