Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 1. Changes of cytokine concentration in THP-1 macrophages challenged with a single LPS stimulation and a CD163 antibody. Quantification for IL-10 (A), TGF-β (B), IL-1 β (C), TNF- α (D), MCP-1 (E), and IL-6 (F) protein concentration in THP-1 macrophages with a single LPS stimulation and incubated with the anti-CD163 antibody (RM3/1) or its isotype control (Iso) for 24 h (72 h after the addition of LPS). N = 6–10 per group. *P < 0.05, vs. LPS, #P < 0.05, vs. isotype control by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Concentration Assay, Protein Concentration, Incubation, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 2. Changes of cytokine concentration in primary human macrophages challenged with a single LPS stimulation and a CD163 antibody. Quantification for IL-10 (A), TGF-β (B), IL-1 β (C), TNF-α (D), MCP-1 (E), and IL-6 (F) protein concentration in primary human macrophages with a single LPS stimulation and incubated with the anti-CD163 antibody (RM3/1) or its isotype control (Iso) for 24 h (72 h after the addition of LPS). N = 6–10 per group. #P < 0.05, vs. isotype control by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Concentration Assay, Protein Concentration, Incubation, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 3. CD163 mRNA induction in THP-1 macrophages challenged with a single LPS stimulation. Quantification for CD163 mRNA induction using Man-PEI complexed with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) in LPS-stimulated THP-1 macrophages at 24, 48, 72 and 96 h after transfection. The expression of CD163 mRNA was normalized to the respective levels of β-actin expression in each group and then calculated as fold change against the expression of the control group (Man-PEI) at each time point. N = 5–6 per group. *P < 0.05 vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Plasmid Preparation, Transfection, Expressing, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 4. CD163 protein induction in THP-1 macrophages challenged with a single LPS stimulation. Microscopic images of nuclear staining using DAPI (blue), CD163 protein (red) and mannose receptor (CD206, green) in LPS-stimulated THP-1 macrophages transfected with Man-PEI complexed with an empty vector (pEmpty), a plasmid encoding for CD163 gene (pCD163) or the Man-PEI nanoparticle alone (A). Quantification of the relative average fluorescence intensity of CD163 (B) or mannose receptor (CD206, C) in LPS-stimulated THP-1 macrophages from 48 to 96 h after transfection. The quantification of the relative average fluorescence intensity was normalized to the respective levels in the Man-PEI group, which was assigned a value equal to 1. N = 3 per group. *P < 0.05 vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Staining, Transfection, Plasmid Preparation

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 5. CD163 protein induction in THP-1 macrophages challenged with a single LPS stimulation. Representative Western blot images of CD163 and Beta-Actin proteins in LPS-stimulated THP-1 macrophages transfected with Man-PEI complexed with an empty vector (pEmp or pEmpty), a plasmid encoding for CD163 gene (pCD163) or the Man-PEI nanoparticle alone (A). Quantification of the relative expression (band density) of CD163 in LPS-stimulated THP-1 macrophages from 48 h after transfection (B). The quantification of the relative density was normalized to the respective levels in the pEmpty group, which was assigned a value equal to 1. N = 10 for pCD163 or pEmpty group. *P < 0.05 vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 6. Changes in cytokine expression in CD163-overexpressing THP-1 macrophages challenged with a single LPS stimulation. Quantification for IL-10 (A), IL-1ra (B), TGF-β (C), TNF-α (D), MCP-1 (E), IL-1β (F), and IL-6 (G) protein concentration in THP-1 macrophages with a single LPS stimulation and transfected with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) at 24, 48, 72 and 96 h after transfection. The concentration of each cytokine/chemokine was normalized to the control group (Man-PEI), which was assigned a value equal to 1. N = 4–9 per group. *P < 0.05, vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Expressing, Protein Concentration, Transfection, Plasmid Preparation, Concentration Assay, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 7. CD163 mRNA induction in THP-1 macrophages challenged with a double LPS stimulation. Quantification for CD163 mRNA using Man-PEI complexed with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) in THP-1 macrophages with a double LPS stimulation at 4 and 24 h after the second stimulus (+4 and +24, respectively). The expression of CD163 mRNA was normalized to the respective levels of β-actin expression in each group and then calculated as fold change against the expression of the control group (Man-PEI) at each time point, which was assigned a value equal to 1. N = 4–6 per group. *P < 0.05 against the pEmpty transfected group by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Plasmid Preparation, Expressing, Control, Transfection

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 8. Changes of cytokine expression in CD163-overexpressing THP-1 macrophages challenged with a double LPS stimulation. Quantification for IL-10 (A), IL-1ra (B), TGF-β (C), TNF-α (D), MCP-1 (E), IL-1β (F), and IL-6 (G) protein concentration in THP-1 macrophages with a double LPS stimulation and transfected with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) at 4 and 24 h after the second stimulus (+4 and +24, respectively). The concentration of each molecule was normalized to the control group (Man-PEI), which was assigned a value equal to 1. N = 5–13 per group. *P < 0.05, vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Expressing, Protein Concentration, Transfection, Plasmid Preparation, Concentration Assay, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 9. Changes of cytokine expression in CD163-overexpressing THP-1 macrophages challenged with a double LPS stimulation and a CD163 antibody. Quantification for IL-10 (A) and IL- 1ra (B) protein concentration in THP-1 macrophages transfected with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) and incubated with either anti- CD163 antibody (RM3/1) or its isotype control antibody at 4 (for IL-10) and 24 (for IL-1ra) h after a second LPS stimulus. The protein concentration of each group was normalized to the control group (pEmpty), which was assigned a value equal to 1. N = 5–12 per group. *P < 0.05 using Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Expressing, Protein Concentration, Transfection, Plasmid Preparation, Incubation, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 10. CD163 mRNA induction in primary human macrophages challenged with a single LPS stimulation. Quantification for CD163 mRNA induction using Man-PEI complexed with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) in primary human macrophages with a single stimulation of LPS at 48 and 96 h after transfection. The expression of CD163 mRNA was normalized to the respective levels of β-actin expression in each group, and then calculated as fold change against the expression of the pEmpty group at each time point, which was assigned a value equal to 1. N = 6–9 per group. *P < 0.05 against the pEmpty group by Student's t- test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Plasmid Preparation, Transfection, Expressing

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 11. Changes of cytokine expression in CD163-overexpressing primary human macrophages challenged with a single LPS stimulation. Quantification for IL-10 (A), IL-1ra (B), TGF-β (C), TNF-α (D), MCP-1 (E), IL-1β (F), and IL-6 (G) protein concentration in primary human macrophages with a single LPS stimulation and transfected with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) at 48 and 96 h after transfection. The concentration of each molecule was normalized to the control group (pEmpty), which was assigned a value equal to 1. N = 5–8 per group. *P < 0.05 vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Expressing, Protein Concentration, Transfection, Plasmid Preparation, Concentration Assay, Control

Journal: PLoS Pathogens

Article Title: Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function

doi: 10.1371/journal.ppat.1006206

Figure Lengend Snippet: Peripheral blood monocytes were isolated from the blood of the wild type (red), heterozygous (blue), and ΔSRCR5 (green) animals. Following cultivation in the presence of recombinant human CSF1 (rhCSF1) for seven days PMMs were analyzed by FACS. A) Co-staining with CD14-FITC and CD16-PE antibodies recognizing the native structure of the proteins (colored contour plots; red wild type, blue heterozygous, green ΔSRCR5) relative to isotype controls (grey). B) Co-staining with CD169-FITC and CD172a-PE antibodies recognizing the native structure of the proteins (colored contour plots) relative to isotype controls (grey). C) Co-staining with SWC9 (CD203a)-FITC and CD151-RPE antibodies recognizing the native structure of the proteins (colored contour plots) relative to isotype controls (grey). D) Staining against the native structure of surface expressed CD163 (colored) relative to an isotype control staining (grey).

Article Snippet: Cells were stained with antibodies targeting either mouse anti pig CD14 (AbD Serotec, MGA1273F, 1:50) and mouse anti pig CD16 (AbD Serotec, MCA2311PE, 1:200), mouse anti pig CD169 (AbD Serotec, MCA2316F, 1:50) and mouse anti pig CD172a (SoutherBiotech, 4525–09, 1:400), mouse anti human CD151 (AbD Serotec, MCA1856PE, 1:50) and mouse anti pig SWC9(CD203a) (AbD Serotec, MCA1973F, 1:50), mouse anti pig CD163 (AbD Serotec, MCA2311PE, 1:50), or mouse IgG1 or an IgG2b negative control (AbD Serotec, MCA928PE,MCA691F, or Sigma, F6397; same concentration as primary Ab).

Techniques: Isolation, Recombinant, Staining, Control

Journal: Frontiers in Genetics

Article Title: SYK Is Associated With Malignant Phenotype and Immune Checkpoints in Diffuse Glioma

doi: 10.3389/fgene.2022.899883

Figure Lengend Snippet: Expression pattern of SYK by single-cell sequencing analysis and the association between SYK expression and M2 macrophage infiltration. (A,B) SYK expression in the different cell types; (B–F) The correlation between SYK expression and CD163 and VSIG4 expression in the four cohorts. (G,H) The correlation between SYK expression and CD163 expression by IHC staining.

Article Snippet: Immunohistochemistry (IHC) was performed using a primary antibody against PD-L1 (Cell Signaling Technology Pathways, United States), CD163 (Proteintech, China), and SYK (Proteintech, China).

Techniques: Expressing, Sequencing, Immunohistochemistry

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: medRxiv

Article Title: M2 monocyte polarization in dialyzed patients is associated with increased levels of M-CSF and myeloperoxidase-associated oxidative stress: preliminary results

doi: 10.1101/2020.05.07.20094011

Figure Lengend Snippet: M1 and M2 monocytes in the blood of HD patients (n = 27) and healthy subjects (n = 23) were analyzed by flow cytometry. (A) Quantification of classical, intermediate and non-classical monocytes. Quantification of M1 monocytes assessed by the positive expression of CD86 and CCR2 (B) and of M2 monocytes assessed by the positive expression of CD206, CXCR3 and CD163 (C). Quantification of M1 (D) and M2 (E) monocytes in classical, intermediate and non-classical monocyte subsets. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: 100 μL of total blood from patients were incubated for 15 minutes at RT with PE mouse anti-human CD14 and V500 mouse anti-human CD16 antibodies (Becton Dickinson, Franklin Lakes, NJ, USA) as well as with mouse anti-human CD86-FITC and anti-human CCR2-APC monoclonal antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany) for determining the M1 polarization or with mouse anti-human CD206-FITC, anti-human CXCR3-APC and anti-human CD163-VioBlue monoclonal antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany) for the M2 polarization.

Techniques: Flow Cytometry, Expressing