Journal: Oncoimmunology

Article Title: Reduction of CD11b + myeloid suppressive cells augments anti-neuroblastoma immune response induced by the anti-GD 2 antibody ch14.18/CHO

doi: 10.1080/2162402X.2020.1836768

Figure Lengend Snippet: Evaluation of tumor infiltrating CD11b + cells. (a-d) Representative immunohistochemical images of tumor infiltrating leukocytes (a) and CD11b + cells (c) and respective negative controls (b and (d). Primary tumors obtained from A/J mice inoculated subcutaneously with NXS2-HGW NB cells were stained with either anti-CD45- (A) or anti-CD11b Ab (C) for detection of leukocytes and CD11 + cells, respectively. Magnification of 100 ×. (e) Gating strategy of flow cytometric analysis of CD11b + cell subsets (GD 2 − /CD45 + /CD11b + ). A dot plot of GD 2 and CD45 expression showing a living cell fraction (left) was used to define a GD 2 − /CD45 + cell population that was next characterized in terms of CD11b expression (open black curve) using a histogram (right). Isotype Ab served as a negative control (filled gray curve). (f) Quantitative analysis of leukocytes and CD11b + cells infiltrating primary tumors in untreated mice (0.9% NaCl; white columns). Leukocytes were calculated as a percent of the viable GD 2 -negative CD45-positive cells relative to all viable cells detected in primary tumor tissue. Two subsets of CD11b + (CD11b + cells (CD11b + ) and cells showing high expression of CD11b (CD11b high )) were calculated as a percent of viable GD 2 − /CD45 + /CD11b + cells relative to all viable leukocytes detected in tumor tissue

Article Snippet: Mice were untreated (0.9% NaCl, i.p.) or treated with ch14.18/CHO alone (provided by Polymun Scientific, Klosterneuburg, Austria), 15 mg/kg, i.p., days 4, 5, 6, 7, and 8 after tumor cell injection), anti-CD11b Ab alone (Bio X Cell, West Lebanon, NH, USA, clone M1/70, 25 mg/kg, i.p., days 4, 7, 11, 14, and 18), 5-FU alone (Merck, Darmstadt, Germany, 50 mg/kg, i.p., days 4, 11, and 18), or ch14.18/CHO and 5-FU.

Techniques: Immunohistochemical staining, Staining, Expressing, Negative Control

Journal: Oncoimmunology

Article Title: Reduction of CD11b + myeloid suppressive cells augments anti-neuroblastoma immune response induced by the anti-GD 2 antibody ch14.18/CHO

doi: 10.1080/2162402X.2020.1836768

Figure Lengend Snippet: Antitumor effects of the ch14.18/CHO treatment in combination with reduction of suppressive myeloid cells in vivo . (a) Schematic overview of the treatment protocol. Four days after tumor cell injection, mice received either ch14.18/CHO or anti-CD11b Ab or 5-FU or combination of ch14.18/CHO and 5-FU. (b) Analysis of tumor growth in mice treated with either anti-CD11b Ab (open squares) or 5-FU (open circles). Controls received 0.9% NaCl (closed squares). Data are shown as mean values ± SEM. Statistical analysis of differences between anti-CD11b and control, 5-FU and control as well anti-CD11b and 5-FU groups was performed with one-tailed t-test: * P < .05 for 0.9% NaCl vs. anti-CD11b; § P < .05, §§ P < .01, §§§ P < .001 for 0.9% NaCl vs. 5-FU; # P < .05, ## P < .01 for anti-CD11b vs. 5-FU. (c) Analysis of tumor infiltrating effector cells of mice treated with anti-CD11b Ab (anti-CD11b, black columns) or 5-FU (5-FU, gray columns) in comparison with the untreated controls (0.9% NaCl, white columns). Leukocytes shown in % of all viable CD45 + /GD2 − cells and CD11b + cells shown in % of all viable CD45 + /CD11b + /GD2 − cells were assessed in tumor tissue using flow cytometry analysis. To show impact of anti-CD11b and 5-FU treatments on effector cell count, t-test was used. *** P < .001 vs. 0.9% NaCl. (d) Analysis of tumor growth in mice treated with either ch14.18/CHO alone (gray circles) or ch14.18/CHO in combination with 5-FU (closed circles). Controls received 0.9% NaCl (closed squares). Data are shown as mean values ± SEM. Statistical analysis of differences between ch14.18/CHO and control, combined treatment (ch14.18/CHO + 5-FU) and control as well as combined treatment (ch14.18/CHO + 5-FU) and 5-FU groups was performed with one-tailed t -test: * P < .05, * P < .01, * P < .001 for 0.9% NaCl vs. ch14.18/CHO group; # P < .05 for ch14.18/CHO vs. combined treatment group (ch14.18/CHO + 5-FU); § P < .05, §§ P < .01, §§§ P < .001 for 0.9% NaCl vs. combined treatment group (ch14.18/CHO + 5-FU)

Article Snippet: Mice were untreated (0.9% NaCl, i.p.) or treated with ch14.18/CHO alone (provided by Polymun Scientific, Klosterneuburg, Austria), 15 mg/kg, i.p., days 4, 5, 6, 7, and 8 after tumor cell injection), anti-CD11b Ab alone (Bio X Cell, West Lebanon, NH, USA, clone M1/70, 25 mg/kg, i.p., days 4, 7, 11, 14, and 18), 5-FU alone (Merck, Darmstadt, Germany, 50 mg/kg, i.p., days 4, 11, and 18), or ch14.18/CHO and 5-FU.

Techniques: In Vivo, Injection, Control, One-tailed Test, Comparison, Flow Cytometry, Cell Counting

Journal: Oncoimmunology

Article Title: Reduction of CD11b + myeloid suppressive cells augments anti-neuroblastoma immune response induced by the anti-GD 2 antibody ch14.18/CHO

doi: 10.1080/2162402X.2020.1836768

Figure Lengend Snippet: Impact of the ch14.18/CHO treatment and reduction of suppressive myeloid cells on survival and gene expression. The GD 2 expressing murine NB cells NXS2-HGW were injected subcutaneously into A/J mice followed by the treatment with the chimeric anti-GD 2 Ab ch14.18/CHO, anti-CD11b Ab, 5-FU, or combination of ch14.18/CHO and 5-FU. (a) Comparison of OS probabilities of mice treated with either ch14.18/CHO alone (black dashed line) or 5-FU alone (gray dashed line) or ch14.18/CHO in combination with 5-FU (black solid line) with control mice (0.9% NaCl, gray solid line). Death ahead of schedule and a tumor volume of 750 mm 3 were defined as event. LogRank test. * P < .05 vs. ch14.18/CHO, *** P < .001 vs. 5-FU, $$$ P < .001 vs. 0.9% NaCl, # P < .05 vs. 0.9% NaCl. (b) Comparison of mRNA expression levels of suppressive myeloid cell-associated and modulating genes between primary tumor tissue and NB cells NXS2-HGW used for tumor establishment. Tumor samples were collected from tumor bearing mice treated with ch14.18/CHO, anti-CD11b, 5-FU, or ch14.18/CHO in combination with 5-FU as well as control mice. Expression levels of Arg1, IL-1β, IL-10, CCL2, NOS2, IFN-γ, M-CSFr, IL-6 r, IL-8, IDO, GM-CSF, IL-4, M-CSF, IL-6, TGF-β1, and VEGF-A were determined using RT-PCR analysis relative to the internal control GAPDH and are represented as colors. The color spectrum between light- and dark gray was used to represent the different levels of expression with light gray for low- and dark gray for high expression of the respective gene. Statistical analysis of expression level differences between anti-CD11b and control, 5-FU and control, anti-CD11b and 5-FU as well as ch14.18/CHO and ch14.18/CHO in combination with 5-FU groups was performed with one-tailed t -test: * P < .05 vs. 0.9% NaCl, # P < .05 vs. anti-CD11b

Article Snippet: Mice were untreated (0.9% NaCl, i.p.) or treated with ch14.18/CHO alone (provided by Polymun Scientific, Klosterneuburg, Austria), 15 mg/kg, i.p., days 4, 5, 6, 7, and 8 after tumor cell injection), anti-CD11b Ab alone (Bio X Cell, West Lebanon, NH, USA, clone M1/70, 25 mg/kg, i.p., days 4, 7, 11, 14, and 18), 5-FU alone (Merck, Darmstadt, Germany, 50 mg/kg, i.p., days 4, 11, and 18), or ch14.18/CHO and 5-FU.

Techniques: Gene Expression, Expressing, Injection, Comparison, Control, Reverse Transcription Polymerase Chain Reaction, One-tailed Test

Journal: Oncoimmunology

Article Title: Reduction of CD11b + myeloid suppressive cells augments anti-neuroblastoma immune response induced by the anti-GD 2 antibody ch14.18/CHO

doi: 10.1080/2162402X.2020.1836768

Figure Lengend Snippet: PCR conditions and primer sequences of CD11b+ myeloid cell-associated and modulating genes. To investigate mRNA expression of CD11b+ myeloid cell-associated and modulating genes in tumor tissue and the neuroblastoma cells NXS2-HGW, RT-PCR analysis was used

Article Snippet: Mice were untreated (0.9% NaCl, i.p.) or treated with ch14.18/CHO alone (provided by Polymun Scientific, Klosterneuburg, Austria), 15 mg/kg, i.p., days 4, 5, 6, 7, and 8 after tumor cell injection), anti-CD11b Ab alone (Bio X Cell, West Lebanon, NH, USA, clone M1/70, 25 mg/kg, i.p., days 4, 7, 11, 14, and 18), 5-FU alone (Merck, Darmstadt, Germany, 50 mg/kg, i.p., days 4, 11, and 18), or ch14.18/CHO and 5-FU.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: CD11b is required for stretch-mediated changes in macrophage activation. (A) Representative Western blot of CD11b and GAPDH for unstimulated, IFNγ/LPS, and IL4/IL13 stimulated macrophages exposed to 0% and 20% static and cyclic stretch (left). Quantification of average across three independent experiments for CD11b expression (right). Values were normalized to GAPDH and made relative to 0% stretch and unstimulated condition. (B) Representative immunofluorescence images (top) and quantification of relative Itgam gene expression in unstimulated macrophages treated with non-target (siControl) or CD11b (siCD11b) siRNA. Data relative to siControl condition. (C) Secretion of TNFα, IL6, and MCP1 for unstimulated and IFNγ/LPS stimulated macrophages treated with siControl or siCD11b and exposed to either 0% control, 20% static, or 20% cyclic stretch. Data normalized to a siControl and IFNγ/LPS treated internal control exposed to 0% stretch within each biological replicate. (D) Relative Arg1 gene expression in IL4/IL13 stimulated and siControl or siCD11b treated BMDMs exposed to 0% control, 20% static, or 20% cyclic stretch. Data relative to 0% siControl condition. Error bars indicate standard deviation of the mean for three separate experiments and * p < 0.05 when compared to the corresponding 0% stretch condition as determined by Student’s t-test (A, C) or paired t-test (B, D) .

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Activation Assay, Western Blot, Expressing, Immunofluorescence, Gene Expression, Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: Modulation of CD11b by adhesion time regulates stretch-mediated macrophage inflammatory responses. (A) Phase contrast images (top) of macrophages following 4 hrs (left) and 24 hrs (right) of adhesion prior to stimulation and stretch. Fluorescence images (bottom) of macrophages labelled for CD11b (red), actin (green), and nuclei (blue) following 4 hrs (left) and 24 hrs (right) of culture. (B) Averaged relative median fluorescence intensity across three independent experiments of CD11b as measured by flow cytometry. Values normalized to 4 hrs adhesion condition. (C) Secretion of TNFα for unstimulated and IFNγ/LPS stimulated macrophages exposed to 0% and 20% cyclic stretch after 4 hrs (left) and 24 hrs (right) of adhesion. Values are normalized to a 0% stretch IFNγ/LPS internal control within each biological replicate. (D) Representative Western blots (left) and corresponding quantification for ARG1 (right) for unstimulated, IFNγ/LPS, and IL4/IL13 stimulated macrophages allowed to adhere for 4 hrs prior to stimulation and stretch. Values were normalized to GAPDH and made relative to IL4/IL13 stimulated and 0% stretch conditions, respectively. Error bars indicate standard deviation of the mean for three separate experiments and * p < 0.05 when compared to the corresponding 0% stretch condition as determined by Student’s t-test (C) or paired t-test (B, D) .

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Fluorescence, Flow Cytometry, Control, Western Blot, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: Crosstalk between Piezo1 and CD11b mediates macrophage response to stretch. (A) Representative immunofluorescence images (left) and quantification of mean Piezo1-tdT intensity in unstimulated, IFNγ/LPS, and IL4/IL13 treated macrophages exposed to 0% control, 20% static, or 20% cyclic stretch. Data normalized to the 0% control condition. (B) Secretion of TNFα in IFNγ/LPS stimulated macrophages treated with siControl or siPiezo1 and exposed to either 0% or 20% cyclic stretch. Data normalized to a siControl treated internal control exposed to 0% stretch within each biological replicate. (C) Secretion of TNFα in IFNγ/LPS stimulated macrophages treated with DMSO or Yoda1 and exposed to either 0% or 20% cyclic stretch. Data normalized to a DMSO treated internal control exposed to 0% stretch within each biological replicate. (D) Relative Piezo1 gene expression in unstimulated macrophages treated with non-target (siControl) or CD11b (siCD11b) siRNA. Gene expression is normalized to the siControl treated condition. (E) Relative Itgam, Itgb1, Itgb2 , and Itgb3 gene expression in unstimulated and siControl or siPiezo1 treated macrophages. Gene expression is normalized to the siControl treated condition. Error bars indicate standard deviation of the mean for three separate experiments and * p < 0.05 when compared to the corresponding 0% stretch condition as determined by Student’s t-test.

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Immunofluorescence, Control, Gene Expression, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: Stretch-induced changes in macrophage activation require modulation of actin. (A) Representative images of F-actin in unstimulated, IFNγ/LPS, and IL4/IL13 stimulated macrophages exposed to 0%, 20% static, and 20% cyclic stretch. Quantification of mean F-actin fluorescence intensity across three independent experiments (right). Data normalized to the unstimulated and 0% stretch control. (B) Representative images of F-actin in unstimulated, IFNγ/LPS, and IL4/IL13 stimulated macrophages exposed to siControl or CD11b siRNA. Quantification of mean F-actin fluorescence intensity across three independent experiments (right). Values normalized to unstimulated and siControl condition. (C) Secretion of TNFα in IFNγ/LPS stimulated macrophages treated with DMSO or CytoD and exposed to 0% and 20% static or cyclic strains. Values are normalized to a DMSO, 0% stretch, and IFNγ/LPS stimulated internal control within each biological replicate. (D) Representative Western blot (left) and quantification of ARG1 expression in IL4/IL13 stimulated macrophages treated with DMSO or CytoD and exposed to 0% and 20% static or cyclic strains. Expression is relative to GAPDH. Error bars indicate standard deviation of the mean for three separate experiments and * p < 0.05 when compared to the corresponding 0% stretch condition as determined by paired t-test (A, B) and Student’s t-test (C, D) .

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Activation Assay, Fluorescence, Control, Western Blot, Expressing, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: Summary of stretch mediated changes in macrophage function.

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Expressing

Journal: Journal of Lipid Research

Article Title: Hepatoprotective drug screening identifies daclatasvir, a promising therapeutic candidate for MASLD by targeting PLIN2

doi: 10.1016/j.jlr.2025.100835

Figure Lengend Snippet: Daclatasvir treatment ameliorates liver damage and fibrosis in MCD diet-induced MASH mouse model. A: Schematic illustration of the experimental workflow for the daclatasvir treatment strategy in a methionine-choline deficient (MCD) diet-induced MASH mouse model. B: Liver weight, LW/BW, and LW/BMI were recorded for MCD diet-fed mice after 3 weeks of treatment with either vehicle or daclatasvir. C: Representative images of H&E, PSR, and CD11b staining of liver samples from vehicle or daclatasvir treated mice at 4 weeks of MCD diet administration. Scale bar, 200 μm for H&E and PSR; 100 μm for CD11b. D: Quantitative results for PSR and CD11b staining shown in (C). E: Enzyme activities of serum hepatic transaminases in MCD diet-fed mice after 3 weeks of treatment with either vehicle or daclatasvir. F–H: The mRNA expression of genes associated with lipid catabolism (F), inflammation (G), and fibrosis (H) in liver tissues from MCD diet-induced MASH mice following 3 weeks of daclatasvir or vehicle treatment. For (B–H), n = 6 mice per group. Data are presented as mean ± SD; ∗ P < 0.05, ∗∗ P < 0.01; Student’s t test for statistics.

Article Snippet: The sections were incubated with a rabbit anti-CD11b primary antibody (BM3925, Boster) overnight at 4°C, followed by incubation with a goat anti-rabbit fluorophore-conjugated secondary antibody (A-11036, Invitrogen).

Techniques: Staining, Expressing

Journal: The EMBO Journal

Article Title: A G1‐like state allows HIV ‐1 to bypass SAMHD 1 restriction in macrophages

doi: 10.15252/embj.201696025

Figure Lengend Snippet: A–D Peritoneal macrophages from 6‐ to 10‐week‐old C57BL/6 mice were stained for the macrophage markers (A) F4/80, (B) CD11b, (C) MCM2 or (D) active DNA synthesis (EdU) 2 h after isolation. Scale bars: 20 μm. E–K Peritoneal macrophages were isolated from 6‐ to 10‐week‐old WT or SAMHD1 −/− (KO) mice. Cells were infected with VSV‐G‐pseudotyped HIV‐1 GFP for 48 h and analysed for (G) infection, (H) MCM2 expression, (I) EdU incorporation (EdU was added at the time of infection), and co‐localisation between infection and (J) MCM2 protein or (K) active DNA synthesis (by EdU incorporation). (E, F) Random microscopic fields and plot profiles show an example of immunofluorescence intensity along infected cells. Scale bars: 10 μm. On average, 10 4 cells in each experiment were recorded and analysed using Hermes WiScan cell‐imaging system and ImageJ ( n ≥ 2, mean ± s.e.m.; (ns) non‐significant; **P ‐value ≤ 0.01, unpaired t ‐test).

Article Snippet: Antibodies used were as follows: anti‐cdc2 (Cell Signaling Technology, Beverly, MA, USA); anti‐CDK2 (H‐298, Santa Cruz Biotechnology); anti‐pCDK2(Thr160) (Bioss Inc., MA, USA); anti‐CDK4 (DCS156, Cell Signaling Technology); anti‐CDK6 (B‐10, Santa Cruz Biotechnology); anti‐SAMHD1 (ab67820, Abcam, UK), beta‐actin (ab6276, abcam, UK); mouse anti‐MCM2 (BM‐28, BD Biosciences, UK); and rabbit anti‐MCM2 (SP85) from Sigma; pSAMHD1 (a kind gift from M. Benkirane) and ProSci (Poway, CA, USA); anti‐Geminin (NCL‐L‐Geminin, Leica); anti‐mouse F4/80 and CD11b (kind gift from S. Yona, UCL); anti‐human CD68, CD14, CD163, CD80, CD86, CD40 (kind gift from M. Noursadeghi).

Techniques: Staining, DNA Synthesis, Isolation, Infection, Expressing, Immunofluorescence, Imaging

Journal: Cancers

Article Title: Overexpression of Murine Rnaset2 in a Colon Syngeneic Mouse Carcinoma Model Leads to Rebalance of Intra-Tumor M1/M2 Macrophage Ratio, Activation of T Cells, Delayed Tumor Growth, and Rejection

doi: 10.3390/cancers12030717

Figure Lengend Snippet: Quantification of different key markers of tumor infiltrating immune cells, as follows: CD86 + M1 and CD206 + M2 (panel A ), Gr-1 (GR) for granulocytes and CD11b + Gr-1 + for MDSCs (panel B ), CD31 for vessels (panel C ), and CD4 and CD8 for T cells (panel D ) per HMMF in BALB/c mice injected with C51 P, C51 E, and C51 FL Rnaset2 cell lines ( N = six animals in group). The mean ± SE are indicated. Immunohistochemistry and immunofluorescence analyses of tumor sections from BALB/c mice injected with C51 P, C51 E, and C51 FL Rnaset2 cell lines at indicated time points are shown. An ANOVA statistical analysis was performed assuming p < 0.05 as a threshold value. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Cryostat sections (6 μm thick) were air-dried and fixed in cold acetone for 10 min. Immunostaining was performed using a previously described procedure [ ] and the following primary mAbs: anti-CD4 and anti-CD8 T lymphocytes (DITTA), anti-CD31 (clone MEC 13.3, kindly provided by A. Mantovani, Humanitas Institute, Milan), anti-granulocyte Gr-1 (clone RB6-8C5), anti-myeloid cells CD11b (clone M1/70) from ImmunoKontact (Oxford, UK); anti-M1-type CD86 (clone PO.3) and anti-M2-type CD206 (clone MR5D3), both from AbD Serotec (Oxford, UK).

Techniques: Injection, Immunohistochemistry, Immunofluorescence

Journal: Cancers

Article Title: Overexpression of Murine Rnaset2 in a Colon Syngeneic Mouse Carcinoma Model Leads to Rebalance of Intra-Tumor M1/M2 Macrophage Ratio, Activation of T Cells, Delayed Tumor Growth, and Rejection

doi: 10.3390/cancers12030717

Figure Lengend Snippet: Cell cytometric flow assessment in spleens from groups of BALB/c mice ( N = three animals in group) at one month after tumor injection, as follows, i.e., receiving C51 P, C51 E tumor cells lines and C51 challenge in rejecting and tumor-free C51 FL Rnaset2 mice. The cytometric flow analysis has been conducted after in vitro tumor digestion to quantify: Percentage of tumor cell infiltration of F4/80 + mature macrophages (panel A ); percentage of TNFα-secreting CD11b + macrophages by intracellular staining, following in vitro 4 h LPS stimulation (panel B ); percentage of CD4 + and CD8 + T cells (panel C ); and percentage of IFNγ-producing CD4 + and CD8 + T cells by intracellular staining, following in vitro 4 h PMA plus ionomycin stimulation (panel D ). An ANOVA statistical analysis was performed assuming p < 0.05 as a threshold value. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Cryostat sections (6 μm thick) were air-dried and fixed in cold acetone for 10 min. Immunostaining was performed using a previously described procedure [ ] and the following primary mAbs: anti-CD4 and anti-CD8 T lymphocytes (DITTA), anti-CD31 (clone MEC 13.3, kindly provided by A. Mantovani, Humanitas Institute, Milan), anti-granulocyte Gr-1 (clone RB6-8C5), anti-myeloid cells CD11b (clone M1/70) from ImmunoKontact (Oxford, UK); anti-M1-type CD86 (clone PO.3) and anti-M2-type CD206 (clone MR5D3), both from AbD Serotec (Oxford, UK).

Techniques: Injection, In Vitro, Staining