Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) Confocal immunofluorescence microscopy images of BALF cells from an asthmatic subject showing CD163 expression by CD68 + AMΦs. The scale bar indicates 5 μm. (B) Gating strategy for identification of human CD163 + alveolar macrophages in BALF. Cellular debris was excluded using a forward light scatter/side scatter plot and doublets were excluded using width parameter on FSC and SSC properties. CD45 + cells, that co-expressed CD14 and CD68, were identified as alveolar macrophages using side scatter and CD45 bivariate plots from which lymphocytes had been excluded. A microscopic image of sorted CD45 + /CD14 + /CD68 + /CD163 + cells shows a cellular population possessing typical cellular characteristics of alveolar macrophages. (C) MFI of cell surface CD163 expression by CD45 + /CD14 + /CD68 + AMΦs in BALF from normal individuals and asthmatic subjects (n = 7, P < 0.008, paired t test). (D) A representative histogram overlay comparing cell surface CD163 expression by AMΦs from a normal individual and an asthmatic subject.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Immunofluorescence, Microscopy, Expressing

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) Intron 15–16 of the Cd163 gene was not detected by PCR of genomic DNA from Cd163 −/− mice, whereas the E. coli lacZ coding sequence from the targeting vector could only be detected in Cd163 −/− mice. Results from 2 mice are shown. (B) Cd163 mRNA was only detected by qRT-PCR in the lungs of wild type (WT), but not Cd163 −/− mice (n = 10 mice). (C) WT and Cd163 −/− mice were sensitized and challenged by daily nasal administration of HDM (25 μg) or saline, 5 days a week, for 5 weeks. End-point analysis was performed 24 h later. (D and E) Total BALF inflammatory cells (Panel D) and inflammatory cell types (Panel E) (n = 9 – 10 mice, *P < 0.05). (F) Representative lung histology from WT and Cd163 −/− mice that had been sensitized and challenged with saline or HDM were stained with hematoxylin and eosin (H & E) (x 200) or periodic acid Schiff (PAS) (x200 and x1000). (G) Serum HDM-specific IgE (n = 15 mice). (H) Quantification of MCM (n = 10 mice, *P < 0.01). ( I ) Western blots of lung proteins were reacted with antibodies against Muc5AC or β-actin. A representative blot from 6 replicate experiments is shown. (J) Relative Muc5AC expression as compared to β-actin was quantified by densitometry (n = 6, * P < 0.001). (K) Chi3L3/4 protein in BALF (n = 10 mice, *P < 0.05). Panels D, E, H and K are representative of two or three independent experiments. Panel G is pooled data from two independent experiments. Numbers of mice that were included in each experimental condition are shown.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Sequencing, Plasmid Preparation, Quantitative RT-PCR, Saline, Staining, Western Blot, Expressing

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) WT and Cd163 −/− mice were sensitized by intraperitoneal (i.p.) administration of HDM (50 μg/ml) emulsified in 2% aluminum hydroxide (ALUM) on days 1 and 4, challenged with intranasal administration of HDM (100 μg) on day 8, and end-points were analyzed on day 10. (B) BALF eosinophils (n = 10 mice, *P < 0.0001, Mann Whitney test). (C) The percentage of CD45 + /CD11c + /Siglec-F + /CD64 + /CD206 + BALF AAMs were quantified by flow cytometry (n = 10 mice, P = NS, Mann Whitney test). (D) The MFI of CD206 expression by CD45 + /CD11c + /Siglec-F + /CD64 + /CD206 + BALF AAMs was quantified by flow cytometry (n = 10 mice, P = 0.0029, Mann Whitney test). The gating strategy used to identify CD45 + /CD11c + /Siglec-F + /CD64 + BALF alveolar macrophages is presented in . Data are pooled from two independent experiments.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: MANN-WHITNEY, Flow Cytometry, Expressing

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: WT and Cd163 −/− mice were sensitized and challenged by daily nasal administration of HDM (25 μg) or saline, 5 days a week, for 5 weeks and end-points were analyzed 24 h later. (A) Cytokines in lung homogenates from HDM-challenged WT and Cd163 −/− mice (n = 18 mice). (B) Ex vivo cultures of mediastinal lymph node cells from HDM-challenged WT and Cd163 −/− mice were re-stimulated with HDM (100 μg/ml) or saline (as a control) and cytokine secretion was quantified (n = 3 – 10 mice). (C) Chemokines in BALF and lung homogenates (n = 16 – 18 mice, * P < 0.01). Data in Panels A and C are pooled from two independent experiments, while Panel B is representative of two independent experiments that showed similar results. Numbers of mice that were included in each experimental condition are shown.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Saline, Ex Vivo, Control

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) Wild type (WT) and Cd163 −/− mice were sensitized and challenged by daily intranasal administration of HDM (25 μg) or saline, 5 days a week, for 5 weeks. Mice received concurrent nasal administration of a neutralizing anti-CCL24 antibody or control immunoglobulin, 3 days a week for 5 weeks. End-point analysis was performed 24 h later. ( B ) BALF eosinophils (n = 9 – 10 mice, *P < 0.0001, HDM + control antibody vs. HDM + anti-CCL24 neutralizing antibody). ( C ) Quantification of MCM (n = 6 – 10 mice, *P < 0.0001, HDM + control antibody vs. HDM + anti-CCL24 neutralizing antibody). ( D ) Representative histologic lung sections stained with hematoxylin and eosin (x 200). (E) AMΦs were isolated from HDM-challenged WT and Cd163 −/− mice, cultured ex vivo with or without HDM (500 μg/ml) and CCL24 secretion was quantified (n = 3 – 5 mice, *P < 0.01, Cd163 −/− + HDM vs. WT + HDM). Panels B & C represent pooled data from two independent experiments, while Panel E is representative data from one of two independent experiments. Numbers of mice that were included in each experimental condition are shown.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Saline, Control, Staining, Isolation, Cell Culture, Ex Vivo

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) Bone marrow-derived dendritic cells (BMDCs) from WT and Cd163 −/− mice that had been pulsed with HDM or saline, as a control, were adoptively transferred to WT recipient mice on Day 10. All mice received nasal HDM challenges on days 23 through 26 and end-points were analyzed on day 27. ( B and C ) Total BALF inflammatory cells (Panel B) and inflammatory cell types (Panel C) (n = 9 – 16 mice). (D) CCL24 in BALF (n = 15 mice, P = NS, Mann-Whitney test). (E) CD11c + DCs isolated from MLNs of WT and Cd163 −/− mice that had been pulsed in vivo by intranasal administration of HDM (100 μg) or saline, as a control, were adoptively transferred to WT recipient mice. All mice received nasal HDM challenges on days 9 through 14 and end-points were analyzed on day 15. ( F and G ) Total BALF inflammatory cells (Panel F) and inflammatory cell types (Panel G) (n = 7 – 10 mice). ( H ) CCL24 in BALF (n = 10 mice, P = NS, Mann-Whitney test). Panels B – D and F – H represent pooled data from two independent experiments. Numbers of mice that were included in each experimental condition are shown. The same numbers of mice are shown for Panels B and C and Panels F and G.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Derivative Assay, Saline, Control, MANN-WHITNEY, Isolation, In Vivo

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) Effector splenic CD4 + T cells were isolated from wild type (WT) mice that had been sensitized by intraperitoneal (i.p.) administration of saline or HDM (100 μg/ml) emulsified in 2% aluminum hydroxide (ALUM) on days 1 and 7. On day 14, 2 × 10 6 CD4 + T cells were purified from the spleens of sensitized mice and adoptively transferred via intraperitoneal administration to recipient WT and Cd163 −/− mice. All recipient mice received intranasal HDM challenges (50 μg) on days 19, 21, 23 and 25 and endpoints were analyzed on day 27. (B) BALF eosinophils (* P < 0.001, n = 10, recipient WT vs. recipient Cd163 −/− mice). (C) Quantification of MCM. (* P < 0.001, n = 10). Data are pooled from two independent experiments. Numbers of mice that were included in each experimental condition are shown.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Isolation, Saline, Purification

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: ( A ) Proteins in D. pteronyssinus extracts that bound to immobilized recombinant human CD163 (rhCD163) were eluted, resolved by SDS-PAGE and visualized by silver staining (Lane A). Band #6 was identified as D. pteronyssinus peptidase 1 (Der p1). Also shown are proteins that eluted from the rhCD163 resin in the absence of D. pteronyssinus extracts (Lane B), proteins in D. pteronyssinus extracts that did not bind to immobilized rhCD163 (Lane C) and rhCD163 alone (Lane D). ( B ) Immobilized rhCD163 was incubated with purified Der p1 and eluted proteins were resolved by SDS-PAGE and visualized by silver staining. A protein corresponding to the molecular weight of Der p1 was eluted from immobilized rhCD163 (Lane A). Proteins that were eluted in the absence of Der p1 (Lane B), purified Der p1 proteins that did not bind to the immobilized rhCD163 resin (Lane C) and Der p1 protein alone (Lane D) are shown. ( C ) Immobilized Der p1 was incubated with rhCD163 and eluted proteins resolved by SDS-PAGE and visualized by silver staining. A protein corresponding to the molecular weight of rhCD163 was eluted from immobilized rhCD163 (Lane A). Proteins that eluted from the immobilized rhCD163 resin in the absence of rhCD163 (Lane B), rhCD163 that did not bind to immobilized Der p1 (Lane C) and rhCD163 alone (Lane D) are shown. (D) Increasing amounts of purified Der p1 were bound to plastic and incubated with 120 ng of rhCD163 with or without supplemental calcium (5 mM). The amount of rhCD163 that bound to immobilized Der p1 increased in a dose-responsive fashion (n = 3, P < 0.0001). Binding of rhCD163 to immobilized Der p1 was calcium-dependent (n = 3, * P < 0.0001, supplemental calcium vs. no supplemental calcium). (E) 250 ng of purified Der p1 was immobilized to plastic and incubated with 120 ng of rhCD163 with or without supplemental calcium (5 mM) or EGTA (5 mM). The amount of rhCD163 that bound immobilized Der p1 was quantified (n = 4, * P < 0.0001, vs. PBS alone). (F) Immobilized Der p1 was incubated with human monocyte proteins. Bound proteins were eluted and Western blots were performed using an anti-human CD163 antibody (Lane A). For comparison, human monocyte proteins were subjected to Western blotting in Lane B. (G) Immobilized Der p1 was incubated with human monocyte proteins with or without supplemental calcium (5 mM) or EGTA (5 mM). Bound proteins were eluted and Western blotting was performed using an anti-human CD163 antibody. (H) CCL24 secretion by bone marrow-derived macrophages stimulated with Der p1 (20 μg/ml) (n=6, *P < 0.05 vs. medium alone). Data are representative of two or three independent experiments.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Recombinant, SDS Page, Silver Staining, Incubation, Purification, Molecular Weight, Binding Assay, Western Blot, Comparison, Derivative Assay

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) WT and Cd163 −/− mice were sensitized by intraperitoneal injection of Der p1 or saline on days 1 and 8 and challenged with intratracheal Der p1 on day 15. End-points were analyzed 72 h later. (B). Total BALF inflammatory cells and inflammatory cell types (n = 7 – 17 mice, *P < 0.05). (C) Representative lung histologic sections from WT and Cd163 −/− mice that had been challenged with saline or Der p1 and stained with hematoxylin and eosin (H & E) (x 200) or periodic acid Schiff (PAS) (x200 and x1000). ( D ) Quantification of MCM (n = 7 – 17 mice, *P < 0.0001). 30.6 ± 0.9 airways were counted per mouse. (E) BALF CCL24 levels (n = 5 – 17 mice, * P < 0.05). ( F ) Ex vivo cultures of mediastinal lymph node cells from Der p1-challenged WT and Cd163 −/− mice were re-stimulated with Der p1 (10 μg/ml) or saline, as a control, and cytokine secretion was quantified (n = 8). Panels B – F are pooled data from two independent experiments. Numbers of mice that were included in each experimental condition are shown.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Injection, Saline, Staining, Ex Vivo, Control

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) WT mice were sensitized by intraperitoneal (i.p.) injection of Der p1 or saline, as a control, on days 1 and 7, followed by the intranasal adoptive transfer of 1 × 10 5 AMΦ’s from naive donor WT or Cd163 −/− mice on day 14. All mice received an intratracheal Der p1 (10 μg) challenge on day 17 and end-points were analyzed on day 20. (B) Gating strategy for sorting of naive murine alveolar macrophages in BALF. Cellular debris was excluded using a forward light scatter/side scatter plot and doublets were excluded using width parameter on FSC and SSC properties. CD45 + cells, that co-expressed CD11c, Siglec-F and CD64 were identified as AMΦs. A microscopic image of sorted CD45 + /CD11c + /Siglec-F + /CD64 + cells shows a cellular population possessing typical characteristics of AMΦs. ( C ) Total BALF inflammatory cells and inflammatory cell types (n = 4 – 8 mice, * P < 0.05, donor Cd163 −/− AMΦ’s vs. donor WT AMΦ’s). ( D ) BALF CCL24 levels from Der p1- challenged recipient mice. (n = 8 mice, P = 0.0016, donor Cd163 −/− AMΦ’s vs. donor WT AMΦ’s, unpaired t test). Panels C and D are representative data from one of three independent experiments. Numbers of mice that were included in each experimental condition are shown. The same numbers of mice are shown for both analyses in Panel C.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Injection, Saline, Control, Adoptive Transfer Assay

Journal: Frontiers in public health

Article Title: Circulating (1 → 3)-β-D-Glucan as an immune activation marker decreased after ART in people living with HIV.

doi: 10.3389/fpubh.2022.981339

Figure Lengend Snippet: FIGURE 4 Markers of immune activation after ART. (A,B) The percentage of PD-1 expressing on CD4+ T cells and CD8+ T cells were significantly decreased after ART (n = 39, paired Kruskal–Wallis test; paired t test). (C) the expression of HLADR+ and CD38+ within CD8+ T cells was significantly reduced (n = 39, paired Kruskal–Wallis test). (D) The levels of plasma CD163 was detected by ELISA, which decreased significantly (n = 120, paired Kruskal–Wallis test). ****p < 0.0001, **p < 0.01.

Article Snippet: Enzyme-linked immunosorbent assays (ELISAs) were performed to quantify plasma LPS (CUSABIO, Wuhan, Hubei, China), LPS binding protein (LBP, Hycultbiotech, Uden, Netherlands), soluble LPS receptor CD14 (sCD14), intestinal fatty acid binding protein (IFABP), regenerating islet-derived protein 3α (REG3α), and soluble CD163 (R&D Systems, Minneapolis, MN, USA).

Techniques: Activation Assay, Expressing, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Acta biomaterialia

Article Title: A subtype specific probe for targeted magnetic resonance imaging of M2 tumor-associated macrophages in brain tumors.

doi: 10.1016/j.actbio.2025.01.003

Figure Lengend Snippet: Fig. 6. Immunohistochemical and FACS validations of M2pep-uIONP targeting M2 TAMs. Confocal microscopic images of tumor tissues collected from mice at 24 h after i.v. injection of probes show (A) Cy7-M2pep-uIONP were highly co-localizing with CD68- and CD163-double positive M2 TAM, revealing the targeting specificity. In comparison, the non-targeted control probe Cy7-scM2pep-uIONP and Cy7-Ferumoxytol not only exhibited much less tumoral accumulation, but also were not co-localized with M2 TAM. Scale bars: 37 µm. (B) Flow cytometric zebra plots and (C) the corresponding quantification of CD68- and CD163-double positive M2 TAM targeted by FITC-M2pep-uIONP, FITC-scM2pep-uIONP, and FITC-Ferumoxytol. The gating strategy involves sorting CD163+ M2 TAM population in live cells dissociated from mouse cerebrums by gating on CD68+ pan-macrophage populations, followed by further gating to assess the FITC-labeled nanoparticles.

Article Snippet: Rabbit polyclonal anti-CD163 antibody was purchased from Boster Biological Technology (Pleasanton, CA, USA).

Techniques: Immunohistochemical staining, Injection, Comparison, Control, Labeling