Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 1. The specific induction of TNF-a from THP-1 cells and their phenotypic changes by anti-moesin pAb treatment. (A) Left panel: dose- dependent stimulation of THP-1 cells by anti-moesin pAbs. THP-1 cells were cultured for 48 h in the presence of several concentrations of anti- moesin pAbs or human IgG from healthy individuals or anti-human CD43 mAb. TNF-a concentration in the culture supernatant was measured by ELISA. *P < 0.01, **P < 0.001. The figure shows the representative results of three independent experiments. Right panel: THP-1 cells were stained with FITC-labeled anti-moesin mAb (upper panel, filled histogram), anti-CD43 mAb (bottom panel, filled histogram) or isotype antibodies (open histogram). (B) The effect of anti-moesin pAb treatment on the surface marker expression on THP-1 cells. THP-1 cells were cultured for 48 h in the presence or absence of 5 lg ml1 of anti-moesin pAbs or isotype human IgG, and the expression levels of several cell surface proteins were determined by flow cytometry. Filled histogram, untreated cells; green, cells stained with isotype control antibodies; red, human IgG-stimulated cells; blue, anti-moesin pAb-stimulated cells. The figure shows the representative results of three independent experiments.

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Marker, Expressing, Cytometry, Control

Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 2. Correlation between TNF-a secretion levels induced by anti-moesin antibodies from THP-1 cells and the expression level of moesin protein on the cell surface. (A) Moesin expression by moesin shRNA-transfected and untransfected THP-1 cells: lysate of THP-1 cells were analyzed for moesin expression level using western blotting with a mouse anti-human moesin mAb (clone 38). The figure shows representative results of three independent experiments. (B) Moesin expression levels on the surface of shRNA moesin-transfected or untransfected THP-1 cells. THP-1 cells were stained with mouse anti-moesin FITC mAb (clone 38/87) and analyzed by flow cytometry. The figure shows representative data of three independent experiments. (C) THP-1 cells transfected with shRNA moesin (clones 3 and 7) or with shRNA NC as well as untransfected cells were cultured in the presence or absence of 5 lg ml1 of anti-moesin pAbs or isotype human IgG for 48 h and levels of TNF-a in the culture supernatants were determined by ELISA. (D) THP-1 cells transfected with shRNA moesin (clone 7) were stimulated with 100 ng ml1

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Expressing, shRNA, Transfection, Western Blot, Staining, Cytometry, Clone Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 3. The anti-moesin pAb-induced activation of the ERK1/2 pathway in THP-1 cells and monocytes. (A) pAb-induced ERK1/2 phosphorylation. THP-1 cells were stimulated with anti-moesin pAbs for the indicated time and their lysates were subjected to western blotting with antibodies specific to phosphorylated ERK1/2 (phospho ERK1/2). The figure shows representative results of three independent experiments. (B) ERK1/2 activation in monocytes. The monocytes were incubated in the presence or absence of anti-moesin pAbs or IgG of healthy donors for the indicated time and the activation of ERK1/2 in cell lysates was determined as described above. The figure shows the representative results of three independent experiments using monocytes from one of the three individuals tested. Effect of an ERK1/2 inhibitor on the TNF-a secretion induced by anti-moesin pAbs in monocytic cells. THP-1 cells (C) or monocytes (D) were pre-incubated for 2 h in the presence or absence of PD98059 and then stimulated with anti-moesin pAbs for 48 h and the TNF-a levels in the supernatants were determined by ELISA and expressed as the means 6 SDs of three independent experiments.

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay

Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 4. Effect of anti-moesin pAb Fab fragments on THP-1 cells. (A) Binding of anti-moesin Fab fragments to moesin on THP-1 cells. THP-1 cells were incubated with anti-moesin pAb Fab fragments or Fab fragments of isotype human IgG followed by staining with FITC- conjugated anti-Fab fragments of human IgG. Filled histogram, cells stained with anti-moesin pAb Fab fragments; open histogram, cells stained with Fab fragments of human IgG. One representative result of three experiments is shown. (B) Effect of Fab fragments on THP-1 cells. THP-1 cells were cultured in the presence of intact anti-moesin pAbs isolated from AA patients or Fab fragments of anti-moesin pAbs or Fab fragments of IgG derived from healthy individuals for 48 h and TNF-a levels in culture supernatant were determined by ELISA. (C) Effect of Fab fragments on anti-moesin pAb TNF-a secretion. THP-1 cells were cultured in the presence of increasing concentrations of Fab fragments of anti-moesin pAbs or Fab fragments of isotype human IgG for 2 h followed by stimulation with intact anti-moesin pAbs for 48 h. The amount of TNF-a induced by anti-moesin pAbs in the absence of Fab fragments was designed as 100%. Data presented in (B and C) are the means 6 SDs of three independent experiments. *P < 0.01.

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Binding Assay, Incubation, Staining, Cell Culture, Isolation, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 5. Effect of moesin cross-linking on TNF-a secretion from THP-1 cells. (A) Binding of F(ab#)2 fragments of anti-moesin pAbs to moesin on the surface of THP-1 cells. THP-1 cells were incubated with anti-moesin pAb F(ab#)2 fragments or F(ab#)2 fragments of isotype human IgG followed by staining with FITC-conjugated anti-F(ab#)2 fragments of human IgG. Open histogram, F(ab#)2 fragment of IgG; filled histogram, F(ab#)2 fragments of anti-moesin pAbs. The figure shows a representative result of three independent experiments. (B) Effect of anti-human IgG F(ab#)2 fragments cross-linking antibodies. THP-1 cells were cultured in the presence of 5 lg ml1 of anti-moesin pAb F(ab#)2 fragments or F(ab#)2 of isotype human IgG for 30 min and thereafter 5 lg ml1 of goat anti-human IgG F(ab#)2 fragment-specific antibody was added. After 48 h of incubation, the levels of TNF-a secreted in culture supernatant were determined by ELISA. Data represent the means 6 SDs of three independent experiments. *P < 0.01, **P < 0.001. (C) ERK1/2 phosphorylation by anti-moesin pAb F(ab#)2 fragments. Lysates of THP-1 cells stimulated with anti-moesin pAb F(ab#)2 fragments or F(ab#)2 fragments of isotype human IgG in the presence of cross-linking anti-F(ab#)2 human IgG or with intact anti-moesin pAbs were analyzed by western blotting to detect phosphorylated ERK1/2. The figure shows the representative results of three independent experiments. (D) Effect of ERK1/2 inhibitor: THP-1 cells were treated for 2 h with increasing concentrations of PD98059 or with dimethyl sulfoxide followed by stimulation with cross-linked anti-moesin pAb F(ab#)2 fragments. The TNF-a level in the culture supernatant was determined by ELISA. Data represent the means 6 SDs of three independent experiments.

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Binding Assay, Incubation, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot

Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 6. The effect of anti-moesin pAbs on the activation state of moesin proteins in monocytic cells. (A) The phosphorylation of moesin. THP-1 cells were stimulated with anti-moesin pAbs for the indicated time and their lysates were subjected to western blotting with antibodies specific to phosphorylated moesin proteins (phospho moesin). (B) Phosphorylation of moesin in monocytes. The monocytes were incubated in the presence or absence of anti-moesin pAbs or IgG of healthy donors for the indicated time and the phosphorylation of moesin in cell lysates was determined as described above. (A) shows the representative results of three independent experiments while (B) shows the representative results of three independent experiments using monocytes from one of the three individuals tested.

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Incubation

Journal: Growth factors (Chur, Switzerland)

Article Title: Interleukin-3 production by basal-like breast cancer cells is associated with poor prognosis.

doi: 10.1080/08977194.2023.2297693

Figure Lengend Snippet: Figure 1. IL-3, IL-3R, IL-5 and GM-CSF expression by human breast cancer subtypes. (A) Swimmer plots representing gene expres sion of IL3+IL3RA+CSF2RB combined in breast cancer patients as a whole or separated into the molecular subtypes (luminal A, luminal B, HER2 enriched or basal-like) with number of patients, hazard ratio (HR) and the log-rank p value calculated by Cox-regression analysis; p < 0.05 considered significant. (B) Kaplan-Meier plots of the luminal A (Lum A, n = 1678), or basal-like subtype (n = 714) of breast cancer patients for gene expression of either IL3RA+CSF2RB, IL3, IL5 or CSF2 with high (blue) versus low (red) based on median expression levels, with HR and log-rank p value calculated by Cox-regression analysis; p < 0.05 consid ered significant. (C) Representative IHC analysis of IgG1 isotype control or antibodies targeting IL-3, IL-3Rα, IL-3Rβ, GM-CSF or IL-5 protein expression by human TNBC tumor tissue. DAB staining (brown) and hematoxylin counterstain (blue). White scale bar = 100µm. Yellow scale bar = 50µm.

Article Snippet: Primary antibodies (IL-3, mouse IgG1, clone 3B11, Genetex, cat#GTX84295, 1:50 dilution; IL-3Rα (CD123), mouse IgG1, clone 32703, R&D Systems, cat#MAB301, 15 mg/ml; IL-3Rβ, mouse IgG1, clone 1C1, generated by Lopez laboratory, 15 mg/ml; isotype control, mouse IgG1, BD Pharmingen, cat#555746, used at appropriately matched concentrations; GM-CSF (CSF2), mouse IgG2b, clone OTI1A3, Origene, cat#TA808006, 1:150 (6.6 mg/ml); IL-5, rabbit polyclonal, (without antigen retrieval), Origene, cat#PP1027P2, 1:500 (1 mg/ml); PECAM-1 (CD31) M-20, goat polyclonal, Santa Cruz, sc-1506, 1:1000) diluted in PBS with 3% serum and incubated overnight at 4 °C.

Techniques: Expressing, Gene Expression, Control, Staining

Journal: Rheumatology (Oxford, England)

Article Title: Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis

doi: 10.1093/rheumatology/key196

Figure Lengend Snippet: CCL3 correlates with bone resorption in vitro OCP ( n = 8 donors, four replicates/condition) differentiated into osteoclasts in medium containing anti-CCL3 or IgG1 antibody. ( A ) Anti-CCL3 exerted a concentration-dependent inhibition on osteoclastogenesis (i), reducing total tartrate-resistant acid phosphatase (TRAP)-positive cells (ii), multinucleated osteoclasts (iii) and resorption (iv). ( B ) Disks stained with TRAP and haematoxylin (day 14). M-CSF cultures lacked osteoclasts (top), M-CSF + RANKL cultures showed strong TRAP staining (bottom; scale bar = 50 µm). ( C ) Resorption pits visualized by toluidine blue (top, scale bar = 250 μm) or calcein (bottom) reduced in anti-CCL3 (8 ng/ml) cultures vs IgG1 (yellow arrows). Mean value per donor plotted. * P ≤ 0.05, ** P ≤ 0.01. OCP = osteoclast precursor cells.

Article Snippet: Cell responses were compared using media supplemented with mouse IgG1 isotype as a control (MAB002, R&D Systems, Abingdon, UK).

Techniques: In Vitro, Concentration Assay, Inhibition, Staining

Journal: Rheumatology (Oxford, England)

Article Title: Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis

doi: 10.1093/rheumatology/key196

Figure Lengend Snippet: CCL3 inhibition had no effect on resorption pit parameters Calcein-stained ivory disks were imaged by fluorescence microscopy to quantify resorption pit parameters. ( A ) Representative topographical maps of M-CSF (i), M-CSF + RANKL + IgG1 (ii), M-CSF + RANKL + anti-CCL3 (iii) disks show their naturally undulating surface and resorption pits (spherical lacuna); scale bar = 40 μm. ( B ) Measured lacuna area (i), perimeter (ii), depth (iii) and volume (iv) for anti-CCL3 vs IgG1 were unchanged (8 ng/ml). ( C ) Levels of CCL2 (i) and sIL-6R (ii) were comparable in IgG1 and anti-CCL3 (8 ng/ml) cultures (day 14). Cells from healthy human volunteers ( n = 6) were cultured, n = ≤2 disks/condition, mean ( s . e . m .) for each donor plotted.

Article Snippet: Cell responses were compared using media supplemented with mouse IgG1 isotype as a control (MAB002, R&D Systems, Abingdon, UK).

Techniques: Inhibition, Staining, Fluorescence, Microscopy, Cell Culture

Journal: Rheumatology (Oxford, England)

Article Title: Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis

doi: 10.1093/rheumatology/key196

Figure Lengend Snippet: Systemic inhibition of CCL3 reduced histological joint swelling Mice with CIA received IgG1 or anti-CCL3 on days 21, 23, 25, 27 and 28 (5 mg/kg, n = 6/group). ( A ) Arthritis progression monitored by clinical scores (i) and paw diameter (ii) saw no statistical differences (two-way analysis of variance). ( B ) Representative tartrate-resistant acid phosphatase (TRAP) and haematoxylin-stained elbow joints from IgG1 (i) and anti-CCL3 (ii). Assessment of inflammation (iii), erosion (iv) and arthritic index (v). ( C ) IgG1 wrist histology showed intense TRAP-staining (i), which reduced in anti-CCL3 (ii), with significant reductions in inflammation (iii), erosion (iv) and arthritic index (v). H = humerus, U = ulna, R = radius, C = carpal. * P ≤ 0.05, ** P ≤ 0.01, scale = 1 mm.

Article Snippet: Cell responses were compared using media supplemented with mouse IgG1 isotype as a control (MAB002, R&D Systems, Abingdon, UK).

Techniques: Inhibition, Staining

Journal: Rheumatology (Oxford, England)

Article Title: Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis

doi: 10.1093/rheumatology/key196

Figure Lengend Snippet: Bone erosions decreased after anti-CCL3 treatment during CIA Front and hind paws from CIA mice, treated with IgG1 or anti-CCL3, were processed for histology. ( A ) tartrate-resistant acid phosphatase (TRAP)-positive cells in the elbow (i) and wrist (ii) joints significantly reduced with anti-CCL3. ( B ) Radiographs of hind paws and ( C ) front paws were acquired from mice treated with IgG1 (i) or anti-CCL3 (ii) ( n = 6 mice per group). Significant reductions in erosive radiographic score for in both hind [B (iii)] and front [C (iii)] paws were quantified in mice treated with anti-CCL3 compared with IgG1 controls. * P ≤ 0.05, ** P ≤ 0.01.

Article Snippet: Cell responses were compared using media supplemented with mouse IgG1 isotype as a control (MAB002, R&D Systems, Abingdon, UK).

Techniques: