Journal: International journal of molecular sciences

Article Title: CD40 Ligand-CD40 Interaction Is an Intermediary between Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy.

doi: 10.3390/ijms242115582

Figure Lengend Snippet: Figure 1. Determination of CD40 (A), CD40L (B) and TNF-α (C) levels in vitreous fluid samples. Equal volumes (15 µL) of vitreous fluid samples from patients with proliferative diabetic retinopathy (PDR; n = 12) and from nondiabetic patients with rhegmatogenous retinal detachment (RD; n = 12)

Article Snippet: Subsequently, the sections were incubated for 60 min with rabbit polyclonal anti-CD40L antibody (1:50; ab65854, Abcam, Cambridge, UK) and mouse monoclonal anti-CD40 antibody (1:1000, AMAb 90905, Atlas Antibodies, Bromma, Sweden).

Techniques:

Journal: International journal of molecular sciences

Article Title: CD40 Ligand-CD40 Interaction Is an Intermediary between Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy.

doi: 10.3390/ijms242115582

Figure Lengend Snippet: Figure 5. Immunohistochemical staining of proliferative diabetic retinopathy epiretinal fibrovascu- lar membranes. Immunohistochemical staining for CD40 showing immunoreactivity in vascular endothelial cells (arrows) (A,B) and in stromal cells (B) (black scale bar, 10 µm).

Article Snippet: Subsequently, the sections were incubated for 60 min with rabbit polyclonal anti-CD40L antibody (1:50; ab65854, Abcam, Cambridge, UK) and mouse monoclonal anti-CD40 antibody (1:1000, AMAb 90905, Atlas Antibodies, Bromma, Sweden).

Techniques: Immunohistochemical staining, Staining

Journal: International journal of molecular sciences

Article Title: CD40 Ligand-CD40 Interaction Is an Intermediary between Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy.

doi: 10.3390/ijms242115582

Figure Lengend Snippet: Figure 6. Immunohistochemical staining of proliferative diabetic retinopathy epiretinal fibrovascular membranes. Double immunohistochemical staining for CD40 (red) and CD 45 (brown) (A) or CD68 (brown) (B) demonstrating co-expression in stromal cells (arrows). No counterstain to visualize the cell nuclei was applied (black scale bar, 10 µm).

Article Snippet: Subsequently, the sections were incubated for 60 min with rabbit polyclonal anti-CD40L antibody (1:50; ab65854, Abcam, Cambridge, UK) and mouse monoclonal anti-CD40 antibody (1:1000, AMAb 90905, Atlas Antibodies, Bromma, Sweden).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: International journal of molecular sciences

Article Title: CD40 Ligand-CD40 Interaction Is an Intermediary between Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy.

doi: 10.3390/ijms242115582

Figure Lengend Snippet: Figure 7. Epiretinal fibrovascular membranes from 13 patients with PDR were studied by immuno- histochemical analysis. Significant positive correlations between the numbers of blood vessels expressing CD31 and the numbers of blood vessels expressing CD40 (A) and the numbers of stromal cells expressing CD40 (B) (Pearson’s correlation coefficient).

Article Snippet: Subsequently, the sections were incubated for 60 min with rabbit polyclonal anti-CD40L antibody (1:50; ab65854, Abcam, Cambridge, UK) and mouse monoclonal anti-CD40 antibody (1:1000, AMAb 90905, Atlas Antibodies, Bromma, Sweden).

Techniques: Expressing

Journal: International journal of molecular sciences

Article Title: CD40 Ligand-CD40 Interaction Is an Intermediary between Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy.

doi: 10.3390/ijms242115582

Figure Lengend Snippet: Figure 9. Müller cells were left untreated (C) or treated with tumor necrosis factor-α (TNF-α) for 24 h. Protein expression of CD40 (A) and CD40L (B) in the cell lysates was determined by Western blot analysis. Results are expressed as median (interquartile range) from three different experiments. (* p < 0.05 compared with the values obtained from untreated cells; Mann–Whitney test).

Article Snippet: Subsequently, the sections were incubated for 60 min with rabbit polyclonal anti-CD40L antibody (1:50; ab65854, Abcam, Cambridge, UK) and mouse monoclonal anti-CD40 antibody (1:1000, AMAb 90905, Atlas Antibodies, Bromma, Sweden).

Techniques: Expressing, Western Blot, MANN-WHITNEY

Journal: International journal of molecular sciences

Article Title: CD40 Ligand-CD40 Interaction Is an Intermediary between Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy.

doi: 10.3390/ijms242115582

Figure Lengend Snippet: Figure 10. Müller cells were left untreated or treated with soluble CD40L for 24 h. Levels of vascular endothelial growth factor (VEGF) (A), matrix metalloproteinase-9 (MMP-9) (B), monocyte chemotactic protein (MCP)-1 (C) and high-mobility group box-1 (HMGB1) (D) were quantified in the culture media by ELISA. Protein expression of CD40 (E), caspase-3 (F) and glial fibrillary acidic protein (GFAP) (G) in the cell lysates was determined by Western blot analysis. sCD40L stimulated proliferation of Müller glial cells that was almost as potent as 10 ng/mL of VEGF compared to untreated cells (H). Results are expressed as median (interquartile range) from three different experiments. (* p < 0.05 compared with the values obtained from untreated cells; Mann–Whitney test).

Article Snippet: Subsequently, the sections were incubated for 60 min with rabbit polyclonal anti-CD40L antibody (1:50; ab65854, Abcam, Cambridge, UK) and mouse monoclonal anti-CD40 antibody (1:1000, AMAb 90905, Atlas Antibodies, Bromma, Sweden).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, MANN-WHITNEY

Journal: International journal of molecular sciences

Article Title: CD40 Ligand-CD40 Interaction Is an Intermediary between Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy.

doi: 10.3390/ijms242115582

Figure Lengend Snippet: Figure 11. CD40 expression on human retinal microvascular endothelial cells (HRMECs) was evaluated through flow cytometry. Representative histogram of 4 independent experiments showing

Article Snippet: Subsequently, the sections were incubated for 60 min with rabbit polyclonal anti-CD40L antibody (1:50; ab65854, Abcam, Cambridge, UK) and mouse monoclonal anti-CD40 antibody (1:1000, AMAb 90905, Atlas Antibodies, Bromma, Sweden).

Techniques: Expressing, Flow Cytometry

Journal: International journal of molecular sciences

Article Title: CD40 Ligand-CD40 Interaction Is an Intermediary between Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy.

doi: 10.3390/ijms242115582

Figure Lengend Snippet: Figure 12. Human retinal microvascular endothelial cells were left untreated or were stimulated with tumor necrosis factor-α (TNF-α) for 24 h or TNF-α plus pre-incubation with BAY11-7085 (A), tissue inhibitor of metalloproteinase-3 (TIMP- 3) (B) or ONO-4817 (C). Levels of soluble (s) CD40 were quantified in the culture media by ELISA. Results are expressed as mean ± standard deviation from three different experiments. One-way ANOVA and independent t-test were used for comparisons between three groups and two groups, respectively. (* p < 0.05 compared with the values obtained from untreated cells. # p < 0.05 compared with TNF-α-treated cells).

Article Snippet: Subsequently, the sections were incubated for 60 min with rabbit polyclonal anti-CD40L antibody (1:50; ab65854, Abcam, Cambridge, UK) and mouse monoclonal anti-CD40 antibody (1:1000, AMAb 90905, Atlas Antibodies, Bromma, Sweden).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International journal of molecular sciences

Article Title: CD40 Ligand-CD40 Interaction Is an Intermediary between Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy.

doi: 10.3390/ijms242115582

Figure Lengend Snippet: Figure 13. Human retinal microvascular endothelial cells were left untreated (C) or were stimulated with sCD40L for 24 h. Protein expression of phospho-ERK1/2 (A), the p65 subunit of NF-κß (B), intercellular adhesion molecule-1 (ICAM-1) (C), vascular cell adhesion molecule-1 (VCAM-1) (D), vascular endothelial growth factor (VEGF) (E), CD40 (F) and caspase-3 (G) in the cell lysates was determined by Western blot analysis. Results are expressed as mean ± standard deviation from three experiments (* p < 0.05; independent t-test).

Article Snippet: Subsequently, the sections were incubated for 60 min with rabbit polyclonal anti-CD40L antibody (1:50; ab65854, Abcam, Cambridge, UK) and mouse monoclonal anti-CD40 antibody (1:1000, AMAb 90905, Atlas Antibodies, Bromma, Sweden).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: International journal of molecular sciences

Article Title: CD40 Ligand-CD40 Interaction Is an Intermediary between Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy.

doi: 10.3390/ijms242115582

Figure Lengend Snippet: Figure 15. Summary of the in vitro and in vivo experiments that documented the relevance of the CD40/CD40L pathway in proliferative diabetic retinopathy. TNF-α = tumor necrosis factor- α; VEGF = vascular endothelial growth factor; HRMEC = human retinal microvascular endothe- lial cell; ERK = extracellular-signal regulated kinase; ICAM-1 = intercellular adhesion molecule-1; VCAM-1 = vascular cell adhesion molecule-1; NF-κB = nuclear factor-Kappa B; MCP-1 = monocyte chemotactic protein-1; HMGB1 = high mobility group box-1; MMP-9 = matrix metalloproteinase-9; BRB = blood–retinal barrier; ↑= enhanced.

Article Snippet: Subsequently, the sections were incubated for 60 min with rabbit polyclonal anti-CD40L antibody (1:50; ab65854, Abcam, Cambridge, UK) and mouse monoclonal anti-CD40 antibody (1:1000, AMAb 90905, Atlas Antibodies, Bromma, Sweden).

Techniques: In Vitro, In Vivo

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cutting edge: induced expression of a RhoA-specific guanine nucleotide exchange factor, p190RhoGEF, following CD40 stimulation and WEHI 231 B cell activation.

doi: 10.4049/jimmunol.170.1.19

Figure Lengend Snippet: FIGURE 1. Induced p190RhoGEF expression after CD40 stimulation. A, Whole-cell extracts (1 mg) of the WEHI 231 B cells, which were either un- stimulated proliferating (control) or stimulated for 48 h with anti-CD40 (1 g/ml), were separated by 2D PAGE. The arrowhead indicates a protein spot that increases dramatically in its expression over the time course of CD40 stim- ulation. The gels shown are representative of two independent experiments. B, Whole-cell lysates of the mouse splenic B cells prepared as in A were separated on SDS-6% PAGE. This was followed by immunoblotting with a specific Ab against p190RhoGEF, 187. The bands shown are representative of three inde- pendent experiments. C, cDNA was prepared by PCR from the total RNA of the mouse splenic B cells that were either unstimulated (control) or stimulated for indicated times with anti-CD40 (1 g/ml). The PCR products for p190RhoGEF and -actin were separated as described in Materials and Meth- ods. The data represent three independent experiments.

Article Snippet: Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 at M cM aster U niv H lth Sci L ib on M arch 4, 2015 http://w w w .jim m unol.org/ D ow nloaded from (WEHI 231: 5 105/ml, splenic B cells: 1 106/ml) were incubated with anti-CD40 mAb (R&D Systems, Minneapolis, MN) at 1 g/ml for indicated times.

Techniques: Expressing, Control, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cutting edge: induced expression of a RhoA-specific guanine nucleotide exchange factor, p190RhoGEF, following CD40 stimulation and WEHI 231 B cell activation.

doi: 10.4049/jimmunol.170.1.19

Figure Lengend Snippet: FIGURE 2. Enhanced p190RhoGEF expression and cellular changes fol- lowing CD40 stimulation. A, The WEHI 231 B cells, either unstimulated (con- trol) or stimulated with anti-CD40 were doubly labeled for endogenous p190RhoGEF (left panels) and RhoA (middle panels) using anti-p190RhoGEF, 187 and anti-RhoA, followed by the FITC- and Texas Red-conjugated second- ary Ab, respectively. Fluorescence was visualized under a confocal microscope. The merged staining pattern is shown in the right panels. Three separate exper- iments were performed with similar results. B, Either rested (control) or CD40- stimulated WEHI 231 B cells were fixed and permeabilized. Endogenous p190RhoGEF was stained using Ab 187, followed by a FITC-conjugated sec- ondary Ab. Standard flow cytometric analyses were performed for these cells. The results are presented as a dot plot, showing changes in the size and shape (SSC-H) vs fluorescence from p190RhoGEF staining (FL1). The dot plot shown is representative of three separate experiments.

Article Snippet: Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 at M cM aster U niv H lth Sci L ib on M arch 4, 2015 http://w w w .jim m unol.org/ D ow nloaded from (WEHI 231: 5 105/ml, splenic B cells: 1 106/ml) were incubated with anti-CD40 mAb (R&D Systems, Minneapolis, MN) at 1 g/ml for indicated times.

Techniques: Expressing, Labeling, Fluorescence, Microscopy, Staining, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cutting edge: induced expression of a RhoA-specific guanine nucleotide exchange factor, p190RhoGEF, following CD40 stimulation and WEHI 231 B cell activation.

doi: 10.4049/jimmunol.170.1.19

Figure Lengend Snippet: FIGURE 3. Effects of the p190RhoGEF overexpression and RhoA activity on the changes of cell size and morphology. The WEHI 231 B cells that were transiently transfected with indicated vector(s) along with a GFP plasmid were either left unstimulated (control) or stimulated with anti-CD40 (1 g/ml) for 48 h. These cells were stained for F-actin or p190RhoGEF using rhodamine- conjugated phalloidin or Ab 187. The cells with fluorescent green were ana- lyzed under a confocal microscope. A, The changes in cell size and morphology, as well as the enhanced p190RhoGEF expression, were seen in the vector-trans- fected cells after CD40 stimulation. B and C, The cellular changes were ana- lyzed in cells transiently transfected with either the p190RhoGEF or p190RhoGEF (Y1003A) plasmid. The effect of CD40 stimulation was also de- termined in the cells that were transfected with a plasmid of p190RhoGEF (Y1003A). D and E, The cellular changes were determined in the cells that were transiently transfected with RhoA (T19N) alone or together with p190RhoGEF. The changes were also determined in the cells that were trans- fected with RhoA (T19N) after a 48-h CD40 stimulation. F, The changes in cell size and morphology were determined in cells transiently transfected with RhoA (Q63L). All of the data shown are representative of at least three inde- pendent experiments.

Article Snippet: Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 at M cM aster U niv H lth Sci L ib on M arch 4, 2015 http://w w w .jim m unol.org/ D ow nloaded from (WEHI 231: 5 105/ml, splenic B cells: 1 106/ml) were incubated with anti-CD40 mAb (R&D Systems, Minneapolis, MN) at 1 g/ml for indicated times.

Techniques: Over Expression, Activity Assay, Transfection, Plasmid Preparation, Control, Staining, Microscopy, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cutting edge: induced expression of a RhoA-specific guanine nucleotide exchange factor, p190RhoGEF, following CD40 stimulation and WEHI 231 B cell activation.

doi: 10.4049/jimmunol.170.1.19

Figure Lengend Snippet: FIGURE 4. Enhanced activation of p190RhoGEF and RhoA augments the activity of the NF-B reporter gene. The WEHI 231 B cells were transiently transfected with an empty vector, HA-tagged p190RhoGEF, and/or each form of myc-tagged RhoA (WT, Q63L, or T19N), along with reporter plasmids of NF-B luciferase and CMV--galactosidase. Luciferase light units, standard- ized to -galactosidase activity, were determined as the mean of duplicate sam- ples. Relative luciferase activities were reported as fold induction to those in the vector-transfected cells. The CD40-mediated reporter gene activity was also compared in the vector-transfected cells that were either left unstimulated (U) or stimulated with anti-CD40 for 24 h. The protein expression level of each transfected DNA is shown in the immunoblot (IB). Data are representative of three independent experiments.

Article Snippet: Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 at M cM aster U niv H lth Sci L ib on M arch 4, 2015 http://w w w .jim m unol.org/ D ow nloaded from (WEHI 231: 5 105/ml, splenic B cells: 1 106/ml) were incubated with anti-CD40 mAb (R&D Systems, Minneapolis, MN) at 1 g/ml for indicated times.

Techniques: Activation Assay, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Expressing, Western Blot

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: TGF-β insensitive dendritic cells: an efficient vaccine for murine prostate cancer

doi: 10.1007/s00262-007-0322-3

Figure Lengend Snippet:

Article Snippet: Flow cytometry Immatured DCs, tumor lysate-pulsed DCs, GFP-transduced DCs and tumor lysate-pulsed TGF-β-insensitive DCs, which were cultured for an additional 18 h, were analyzed by flow cytometry, using a panel of Abs specific for MHC class II, CD40, CD11c, CD80, and CD86 (Immunotech, Miami, FL), as previously described [ 33 ].

Techniques:

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: TGF-β insensitive dendritic cells: an efficient vaccine for murine prostate cancer

doi: 10.1007/s00262-007-0322-3

Figure Lengend Snippet: In vitro proliferation and surface co-stimulatory molecules of DCs following TGF-¦Â treatment. a Nontransduced DCs, GFP-transduced DCs and T¦ÂRIIDN-transduced DCs were incubated with TGF-¦Â1 (+) or without TGF-¦Â1 (−). Cell numbers were recorded every 3 days. In the presence of TGF-¦Â1, T¦ÂRIIDN-transduced DCs continue to proliferate and grow normally. The figures indicate average results in each group. b After freeze¨Cthawed tumor lysate and TGF-¦Â1 treatment, nontransduced DCs, GFP transduced DCs and T¦ÂRIIDN-transduced DCs were incubated with FITC-labeled antimouse CD80, CD86 antibodies (black) for 2 h at 37U+00AlãC, and then subjected to flow cytometry. Isotype-matched irrelevant FITC-conjugated antibodies (solid lines) were used as controls. Data show representative results from two independent experiments

Article Snippet: Flow cytometry Immatured DCs, tumor lysate-pulsed DCs, GFP-transduced DCs and tumor lysate-pulsed TGF-β-insensitive DCs, which were cultured for an additional 18 h, were analyzed by flow cytometry, using a panel of Abs specific for MHC class II, CD40, CD11c, CD80, and CD86 (Immunotech, Miami, FL), as previously described [ 33 ].

Techniques: In Vitro, Incubation, Labeling, Flow Cytometry