Journal: PLOS One

Article Title: Photobiomodulation therapy increases neural stem cell pool in aged 3xTg-AD mice

doi: 10.1371/journal.pone.0321668

Figure Lengend Snippet: A. Representative confocal images of Aβ plaques (6E10), microglia (Iba1) and CD68 showing diffuse microglial phagocytosis away from plaques in sham compared to localized phagocytosis around plaques in PBM-treated animals. B. Quantitative analysis of total and activated microglia show no overall differences in the neuroinflammatory environment surrounding Aβ plaques. (Unpaired t-test). C. Analysis of the distribution of CD68+ microglia shows a trend of higher concentration of phagocytic cells in contact with plaques, and decreasing phagocytosis away from plaques. (Two-way ANOVA).

Article Snippet: Up to six sections representing different regions of the hippocampus were immunostained with the following primary antibodies: Sox2 (Abcam), doublecortin (DCX, Santa Cruz), calretinin (CR, SWANT), β-amyloid (6E10, Biolegend), phospho-tau (pTau, AT8, Invitrogen), βIII-tubulin (Abcam), Iba1 (Synaptic Systems), and CD68 (Rockland).

Techniques: Concentration 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 , 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

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Gastric cancer-derived exosomal let-7 g-5p mediated by SERPINE1 promotes macrophage M2 polarization and gastric cancer progression.

doi: 10.1186/s13046-024-03269-4

Figure Lengend Snippet: Fig. 2 High SERPINE1 expression in GC cells promotes macrophage M2 polarization. tSNE visualization of nine single-cell clusters partitioned by unsu pervised cluster analysis, SERPINE1 expression of each single-cell, and SERPINE1 expression abundance of different single-cell clusters in the GSE134520 (A–C) and GSE167297 (D–F) datasets. (G) Flow cytometry analysis of the proportion of CD68+CD206+ macrophages in a Transwell co-culture system, with MKN45 and AGS cells overexpressing (oe_SERPINE1) or silencing SERPINE1 (shRNA#3 or sh_SERPINE1#3) in the upper chamber, and THP1 cells treated with PMA in the lower chamber. (H) Immunofluorescence staining of xenograft tumor tissues. Comparison of the proportion of M1 or M2 macrophage infiltra tion. Green indicates F4/80. Red indicates iNOS or Arg1 expression

Article Snippet: THP-1 cells were differentiated into macrophages using 150 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma) for 24 h and subsequently co-cultured with cancer-derived exosomes or GC cells in 6-well plates with 0.4-μm membranes for 72 h. Harvested macrophages were converted into single-cell suspensions, stained with Elab Fluor 488 anti-human CD68 (Mouse, 1:20, ElabScience) and APC anti-human CD206 (Mouse, 1:20, ElabScience) antibodies, and analyzed for CD68+CD206+ populations by flow cytometry (Accuri C6, BD).

Techniques: Expressing, Flow Cytometry, Co-Culture Assay, shRNA, Immunofluorescence, Staining, Comparison

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Gastric cancer-derived exosomal let-7 g-5p mediated by SERPINE1 promotes macrophage M2 polarization and gastric cancer progression.

doi: 10.1186/s13046-024-03269-4

Figure Lengend Snippet: Fig. 5 SERPINE1-mediated gastric cancer-derived exosomes facilitate the polarization of THP1 cells into M2 macrophages. (A) Schematic representation of the extraction and identification of exosomes and the induction of macrophage polarization. Transmission electron microscopy (B), nanoparticle tracking analysis (C), and western blotting (D) were used to identify the morphology, particle size, and markers of exosomes. (E) Confocal laser scanning microscopy detected Dil-labeled exosomes (red) internalized by DAPI-labeled macrophages (blue). (F–G) Immunofluorescence analysis of the proportion of CD206+ cells in THP1 cells treated with exosomes. (H–I) Flow cytometry analysis of the proportion of CD68+CD206+ cells in THP1 cells treated with exosomes. (J–K) qRT-PCR analysis of M1 markers (iNOS and TNF-α) and M2 markers (TGF-β, IL-10, and Arg-1) in THP1 cells treated with exosomes. (L–N) Transwell migration and invasion assays of GC cells (upper chamber) co-cultured with macrophages (lower chamber) ingesting exosomes

Article Snippet: THP-1 cells were differentiated into macrophages using 150 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma) for 24 h and subsequently co-cultured with cancer-derived exosomes or GC cells in 6-well plates with 0.4-μm membranes for 72 h. Harvested macrophages were converted into single-cell suspensions, stained with Elab Fluor 488 anti-human CD68 (Mouse, 1:20, ElabScience) and APC anti-human CD206 (Mouse, 1:20, ElabScience) antibodies, and analyzed for CD68+CD206+ populations by flow cytometry (Accuri C6, BD).

Techniques: Derivative Assay, Extraction, Transmission Assay, Electron Microscopy, Western Blot, Confocal Laser Scanning Microscopy, Labeling, Immunofluorescence, Flow Cytometry, Quantitative RT-PCR, Migration, Cell Culture