Journal: Journal of Virology

Article Title: Brain Large Artery Lymphocytic Inflammation and Human Immunodeficiency Virus-Related Brain Arterial Remodeling

doi: 10.1128/JVI.00081-18

Figure Lengend Snippet: Intima CD3 + /CD68 + cell phenotypes and markers of brain arterial remodeling a

Article Snippet: Briefly, anti-CD68 + antibody (primary, 1:100, catalog number M0814, from Dako Corp., Carpinteria, CA; secondary, biotinylated horse anti-mouse IgG antibody, catalog number BA-2000, from Vector Laboratories, Burlingame, CA) binding to arteries was counterstained with hematoxylin and visualized using the diaminobenzidine kit ( 10 ).

Techniques: Isolation

Journal: Toxins

Article Title: Natriuretic-like Peptide Lebetin 2 Mediates M2 Macrophage Polarization in LPS-Activated RAW264.7 Cells in an IL-10-Dependent Manner

doi: 10.3390/toxins15040298

Figure Lengend Snippet: Effect of L2 and BNP on macrophage polarization in LPS-activated RAW264.7 cells. ( a ) Representative flow cytometry plots showing the variable macrophage distribution of M1-like macrophages (CD68 + /CD206 − cells, Q1 population) and M2-like macrophages (CD68 + /CD206 + /MRC-1, Q2 population) in control and treated cells. ( b , c ) Effect of L2 on M1 and M2 macrophage subtype expression. ( d , e ) Effect of BNP on M1 and M2 macrophage subtype expression. Cells were obtained after challenging RAW264.7 cells with LPS (1 µg/mL) for 24 h followed by 48 h treatment with or without L2 or BNP (0, 0.2, 0.4 and 0.8 ng/mL) and isatin (0.1 mM), added 20 min earlier. ( f ) M2/M1 ratio in LPS-activated RAW264.7 cells. The subtypes of macrophages were identified by analyzing profiles of cell surface markers by FACS. Pro-inflammatory M1 macrophages were identified as CD68 + /CD206 ࢤ cells and M2-like macrophages assessed by double immunolabeling of CD68 and CD206/MRC-1 in control and treated cells, and the data were analyzed by BD CellQuestPro software. All results were obtained from duplicate experiments. Data are reported as mean ± SEM. *** p < 0.001 vs. corresponding control LPS group; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 vs. L2 corresponding group; †† p < 0.01 vs. group unstimulated with LPS.

Article Snippet: RAW264.7 macrophage polarization was detected after treatment with L2 and BNP by flow cytometric profiling of specific surface marker expression, including CD68 for total macrophage population quantification and CD206/MRC-1 for M2-like macrophage quantification using PE Rat Anti-Mouse CD68 and Alexa Fluor ® 647 Rat Anti-Mouse CD206 (BD BioScience, San Jose, CA, USA).

Techniques: Flow Cytometry, Expressing, Immunolabeling, Software

Journal: Toxins

Article Title: Natriuretic-like Peptide Lebetin 2 Mediates M2 Macrophage Polarization in LPS-Activated RAW264.7 Cells in an IL-10-Dependent Manner

doi: 10.3390/toxins15040298

Figure Lengend Snippet: Effect of L2 on macrophage polarization in LPS-activated RAW264.7 cells after interleukin-10 inhibition. ( a , b ) Effect of interlekin-10 (IL-10) on M1 and M2 macrophage subtype expression. Cells were obtained after challenging RAW264.7 cells with LPS for 24 h followed by 48 h treatment with or without exogenous IL-10 (0, 10 and 20 ng/mL). ( c , d ) Effect of L2 on M1 and M2 macrophage subtype expression after IL-10 inhibition. Cells were obtained after challenging RAW264.7 cells with LPS (1 µg/mL) for 24 h followed by 48 h treatment with or without L2 (0, 0.4 or 0.8 ng/mL) and IL-10 inhibitor at 10 µg/mL, added 20 min earlier. ( e ) Representative flow cytometry plots showing the variable macrophage distribution of M1-like macrophages (CD68 + /CD206 − cells, Q1 population) and M2-like macrophages (CD68 + /CD206 + /MRC-1, Q2 population) in control and treated cells. The subtypes of macrophages were identified by analyzing profiles of cell surface markers by FACS. Pro-inflammatory M1 macrophages were identified as CD68 + /CD206 − cells and M2-like macrophages assessed by double immunolabeling of CD68 and CD206/MRC-1 in control and treated cells, and the data were analyzed by BD CellQuestPro software. All results were obtained from duplicate experiments. Data are reported as mean ± SEM. *** p < 0.001 vs. corresponding control LPS group; $ p < 0.05, $$$ p < 0.001 vs. L2 corresponding group.

Article Snippet: RAW264.7 macrophage polarization was detected after treatment with L2 and BNP by flow cytometric profiling of specific surface marker expression, including CD68 for total macrophage population quantification and CD206/MRC-1 for M2-like macrophage quantification using PE Rat Anti-Mouse CD68 and Alexa Fluor ® 647 Rat Anti-Mouse CD206 (BD BioScience, San Jose, CA, USA).

Techniques: Inhibition, Expressing, Flow Cytometry, Immunolabeling, Software

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Mesenchymal Stem Cell Extracellular Vesicles Reverse Sugen/Hypoxia Pulmonary Hypertension in Rats

doi: 10.1165/rcmb.2019-0154OC

Figure Lengend Snippet: (A) Immunohistochemical staining of Cd206+ macrophages in lungs of rats treated with normoxia, (B and C) SuHx plus vehicle (B), and SuHx plus MSC-EV (C). Scale bars: 50 μm. (D–F) Average number of Cd206+ per 10× field (D), Cd68+ total rat macrophages per 10× field (E), and ratio of Cd206+/Cd68+ macrophages per 10× lens objective field of view (F) in lung sections from rats treated with Nx, SuHx plus vehicle, or SuHxEV. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Article Snippet: Lungs were kept in 4% paraformaldehyde until embedded in paraffin; sectioned in 5-μm slices; and stained with antibody against rat ACTA2 (ab5694; Abcam) to assess pulmonary vascular remodeling, antibody against rat von Willebrand factor (vWF) (ab6994; Abcam) to assess distal vessel count, and antibodies against rat Cd206 (ab64693; Abcam) or Cd68 (ED1; Bio-Rad Laboratories) for assessment of macrophage recruitment.

Techniques: Immunohistochemical staining, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: Neutrophils play a major role in the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters

doi: 10.1007/s00018-022-04643-1

Figure Lengend Snippet: Iba1 + (microglia/monocyte lineage), CD68 + (macrophages) and MPO + (neutrophils) cells presence in the olfactory epithelium before and during SARS-CoV-2 infection. Immunostaining on successive slides of the olfactory epithelium from a non-infected ( A ) or 1 dpi hamster ( B ). Only Iba1 + cells are present in the uninfected olfactory epithelium (OE) and in the lamina propria (LP). In the infected epithelium, Iba1 + cells are massively present in the OE while CD68 + and MPO cells are mostly present in the desquamated cells (red asterisk) in the lumen of the nasal cavity (white asterisk)

Article Snippet: The sections were then incubated overnight with primary antibodies directed against SARS nucleocapsid protein (1/500; mouse monoclonal; clone 1C7C7; Sigma-Aldrich), ionized calcium-binding adapter molecule 1 (Iba1) (1/500; rabbit monoclonal; clone EPR16588; Abcam), myeloperoxidase protein (MPO) (1/500; rabbit monoclonal; clone EPR20257; Abcam), CD68 (1/200; rabbit polyclonal; PA1518; Boster), cleaved caspase 3 (C3C) (1/200; rabbit polyclonal; #9661; Cell signaling), G olf (1/300; rabbit polyclonal; C-18; Santa Cruz), Olfactory Marker Protein (OMP) (1/500; goat polyclonal; 544-10001; Wako) and CK18 (1:50; mouse polyclonal; MAB3234—RGE53, Sigma-Aldrich).

Techniques: Infection, Immunostaining

Journal: Cellular and Molecular Life Sciences

Article Title: Neutrophils play a major role in the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters

doi: 10.1007/s00018-022-04643-1

Figure Lengend Snippet: CD68 + macrophage and MPO + neutrophil cells are associated with damage of the olfactory epithelium during SARS-CoV-2 infection. CD68 + ( A 1 ) and MPO + ( B 2 ) signal in the olfactory epithelium (OE, left) and lamina propria (LP, right) in either control animals (CTL) or at 1 or 2 days post-infection (dpi) (Mean normalized to control ± SEM, n = 4, * p < 0.05 (Mann–Whitney test)). Correlation between score damage and percentage of CD68 + ( A 1 ) and MPO + ( B 2 ) signal in the olfactory epithelium (left panel) and the lamina propria (right panel). Spearman test p value

Article Snippet: The sections were then incubated overnight with primary antibodies directed against SARS nucleocapsid protein (1/500; mouse monoclonal; clone 1C7C7; Sigma-Aldrich), ionized calcium-binding adapter molecule 1 (Iba1) (1/500; rabbit monoclonal; clone EPR16588; Abcam), myeloperoxidase protein (MPO) (1/500; rabbit monoclonal; clone EPR20257; Abcam), CD68 (1/200; rabbit polyclonal; PA1518; Boster), cleaved caspase 3 (C3C) (1/200; rabbit polyclonal; #9661; Cell signaling), G olf (1/300; rabbit polyclonal; C-18; Santa Cruz), Olfactory Marker Protein (OMP) (1/500; goat polyclonal; 544-10001; Wako) and CK18 (1:50; mouse polyclonal; MAB3234—RGE53, Sigma-Aldrich).

Techniques: Infection, Control, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences

Article Title: Neutrophils play a major role in the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters

doi: 10.1007/s00018-022-04643-1

Figure Lengend Snippet: Immunosuppression induced by cyclophosphamide reduces damage of the olfactory epithelium as well as OE infection area. ( A ) Expression of innate immune genes in the nasal turbinates with or without cyclophosphamide treatment at 1 and 2 days post-infection (dpi). Iba1, CD68 and Ncf2 are related to the presence of microglia/macrophages, monocytes/macrophages and neutrophils, respectively; TNFα and IL6 are two cytokines expressed during inflammation; SARS-CoV-2 N expression is related to the SARS-CoV-2 infection. Results represent the Mean ± SEM relative to vehicle-treated hamsters ( n = 4, * p < 0.05; Mann–Whitney test). Representative images of the infected olfactory epithelium immunostained for MPO (neutrophil marker) and SARS-CoV-2 N protein in ( B ) vehicle and ( C ) cyclophosphamide treated animal (olfactory epithelium (OE), lamina propria (LP)). In the vehicle condition, the lumen (white asterisk) is filled with desquamated cells (red asterisk) containing MPO signal. In the cyclophosphamide condition, MPO signal is absent and the lumen is mostly free of cellular debris . Quantification in the OE of ( D 1 ) MPO + neutrophil presence ( D 2 ) damage score ( D 3 ) SARS-CoV-2-infected area and in the lumen of the nasal cavity of ( D 4 ) desquamated cells area and ( D 5 ) percentage of SARS-CoV-2-infected area in the desquamated cells (Mean ± SEM, n = 8 areas of the nasal cavity from 4 different animals, * p < 0.05, ** p < 0.01, *** p < 0.001 (Mann–Whitney))

Article Snippet: The sections were then incubated overnight with primary antibodies directed against SARS nucleocapsid protein (1/500; mouse monoclonal; clone 1C7C7; Sigma-Aldrich), ionized calcium-binding adapter molecule 1 (Iba1) (1/500; rabbit monoclonal; clone EPR16588; Abcam), myeloperoxidase protein (MPO) (1/500; rabbit monoclonal; clone EPR20257; Abcam), CD68 (1/200; rabbit polyclonal; PA1518; Boster), cleaved caspase 3 (C3C) (1/200; rabbit polyclonal; #9661; Cell signaling), G olf (1/300; rabbit polyclonal; C-18; Santa Cruz), Olfactory Marker Protein (OMP) (1/500; goat polyclonal; 544-10001; Wako) and CK18 (1:50; mouse polyclonal; MAB3234—RGE53, Sigma-Aldrich).

Techniques: Infection, Expressing, MANN-WHITNEY, Marker

Journal: PLoS ONE

Article Title: Imaging Neuroinflammation In Vivo in a Neuropathic Pain Rat Model with Near-Infrared Fluorescence and 19 F Magnetic Resonance

doi: 10.1371/journal.pone.0090589

Figure Lengend Snippet: A , B show the affected right sciatic nerve from the CCI leg shown in stained with anti-CD68 antibody to reveal the presence of macrophages infiltrating the nerve. C , D CD68 positive cells are not present in the left leg (contralateral to surgery ) of the same CCI animal. E , F show a nerve from a separate CCI animal that also exhibits infiltration of CD68 positive cells. The boxed area is enlarged to reveal the granular cytoplasm (black arrow) of the macrophages, indicative of the presence of the nanoemulsion. Sham surgical sciatic nerve and non-surgical control sciatic nerve do not exhibit any CD68 positive cells ( G , H ) and ( I , J ). The fluorescent images A, C, G, and I were all acquired at the same sitting with the exact same image acquisition parameters. Macrophages grown in cell culture take up the nanoemulsion, exhibited as particles evident by both confocal fluorescence emissions 700–850 nm of NIR label (DiR) ( K , M ) as well as transmitted light ( L , N ).

Article Snippet: In separate immunohistochemical experiments using comparable protocols, the recovered control, sham and CCI sciatic nerves were prepared for examination using mouse anti rat CD68 antibody (MCA341R, AbD Serotech, Raleigh, NC) and Alexa fluor 488 donkey anti mouse secondary antibody (A-21202, Invitrogen, Carlsbad, CA) to assess the presence of macrophages that infiltrate the nerve.

Techniques: Staining, Control, Cell Culture, Fluorescence

Journal: Nature Medicine

Article Title: Rituximab versus tocilizumab in rheumatoid arthritis: synovial biopsy-based biomarker analysis of the phase 4 R4RA randomized trial

doi: 10.1038/s41591-022-01789-0

Figure Lengend Snippet: a , Classification into synovial pathotypes according to semiquantitative scores for CD3 + T cells, CD20 + B cells, CD68 + macrophages and CD138 + plasma cells, with representative examples from patients classified as lymphomyeloid (CD20 ≥ 2 and/or CD138≥2), diffuse-myeloid (CD68SL≥2, and CD20/CD138<2) or fibroid/pauci-immune (CD68SL/CD20/CD138<2). Right, 16-week CDAI 50% response in patients stratified by pathotype ( n = 152). Bar plots showing the proportion of CDAI 50% responders for rituximab (in blue) and tocilizumab (in yellow) within each pathotype, with corresponding exact numbers. Fisher's test, exact P values for P < 0.05. b , Approach to in silico deconvolution of synovial tissue using MCP-counter. c , MCP-counter scores for each cell type compared among CDAI 50% responders (R) and nonresponders (NR). Bar plots indicate nominal log 10 P values for tocilizumab and –log 10 P values for rituximab (two-sided Mann–Whitney test); dashed lines correspond to P = 0.05. Boxplots (right) show median and first and third quartiles, whiskers extending to the highest and lowest values. d – f , 16-week CDAI 50% response in patients stratified into B and T cell poor/rich ( d ) and macrophage/mDC poor/rich ( e ) according to median MCP-counter scores for individual cells (rich if above median, poor if below), or by combining B cell and macrophage/mDC scores from d , e ( f ). Exact P values shown when <0.05, two-sided Fisher's test comparing the proportions of responders to rituximab (in blue) and tocilizumab (in yellow). g – i , Longitudinal disease activity scores (CDAI), shown as mean ± s.d., for each month from baseline to 16 weeks for patients randomized to rituximab (in blue) or tocilizumab (in yellow) and classified as B and T cell poor/rich ( g ), macrophage/mDC poor/rich ( h ) and combined B cell/macrophage poor/rich ( i ). Comparison of CDAI between the two medications at individual time points by two-sided Mann–Whitney test, exact P values for <0.05 (adjustment for multiple comparisons by FDR). P values for the drug × time interaction term (two-way repeated-measures analysis of covariance) are shown when <0.05. c – i , n = 133 patients with baseline RNA-seq. NK, natural killer cells. mDC, myeloid dendritic cells.

Article Snippet: Briefly, tissue morphology was visualized using fluorescent antibodies CD68-AF532 (clone KP1, Novus), CD20-DL594 (clone IGEL/773, Novus) and CD3-AF647 (clone UMAB54, Origene) and Syto13 (ThermoFisher).

Techniques: In Silico, MANN-WHITNEY, Activity Assay, RNA Sequencing Assay

Journal: Nature Medicine

Article Title: Rituximab versus tocilizumab in rheumatoid arthritis: synovial biopsy-based biomarker analysis of the phase 4 R4RA randomized trial

doi: 10.1038/s41591-022-01789-0

Figure Lengend Snippet: a , Scheme showing the approach to DSP, including selection of ROIs: CD68 + lining and superficial sublining, CD20 – CD3 – deep sublining and CD3 + CD20 + lymphoid aggregates. b , MA plot showing mean expression (log 2 ) on the x axis and fold change on the y axis comparing responders and refractory patients across all ROIs. Genes significantly upregulated (FDR<0.05) in responders are shown in blue (top), and those upregulated in refractory in red (bottom); in grey, genes with FDR > 0.05; P values were calculated using a negative binomial linear model applied to count data using DESeq2 (Wald test) and were FDR adjusted n = 12 patients, six ROIs per patient. c , Example of individual genes differentially expressed in refractory (red) or responders (green). Scatterplots showing individual ROIs, boxplots showing median and first and third quartiles. FDR-adjusted P values calculated as in b are shown for differentially expressed genes between refractory and responder individuals; n = 12 patients (4 responders to rituximab, 4 responders to tocilizimab and 4 refractory). d , Examples of individual genes differentially expressed in refractory (red) or responders (green) in different ROIs. Scatterplots showing individual ROIs ( n = 12 patients, six ROIs per patient), boxplots showing median and first and third quartiles. FDR-adjusted P values calculated as in b are shown for differentially expressed genes between refractory and responder individuals. L, lining/superficial sublining; SL, deep sublining; A, lymphoid aggregates (as shown in a ).

Article Snippet: Briefly, tissue morphology was visualized using fluorescent antibodies CD68-AF532 (clone KP1, Novus), CD20-DL594 (clone IGEL/773, Novus) and CD3-AF647 (clone UMAB54, Origene) and Syto13 (ThermoFisher).

Techniques: Selection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Cirrhotic Cardiomyopathy Following Bile Duct Ligation in Rats—A Matter of Time?

doi: 10.3390/ijms24098147

Figure Lengend Snippet: Immunohistochemical staining of CD68 positive cells in rat myocardium after bile duct ligation (BDL) versus controls. Exemplary histologic images are shown for both groups at day 31 after BDL. Scale bars correspond to 250 µm length. Positive cells were counted in 10 high power fields (HPF, 40×; 2560 × 1440 pixels) and displayed as mean ± standard error of the mean (SEM). (*** P d31 < 0.001; ** P d35 = 0.002; * P d42 = 0.017, P d56 = 0.019, P group < 0.001, P time = 0.29, P group*time = 0.48). Data were analyzed using a generalized mixed model analysis corrected for multiple comparisons using the Shaffer-simulated approach. The interaction effect of group and time is expressed as group*time. p ≤ 0.05 was considered significant.

Article Snippet: Macrophages in cardiac sections were detected using a cluster of differentiation 68 (CD68) antibody (Cat. No. BM4000S, OriGene Technologies GmbH, Herford, Germany) as the primary antibody.

Techniques: Immunohistochemical staining, Staining, Ligation