Journal:

Article Title: Antisense Oligonucleotides Against Thrombospondin-1 Inhibit Activation of TGF-? in Fibrotic Renal Disease in the Rat in Vivo

doi:

Figure Lengend Snippet: Transfer of phosphorothioate ODN against TSP1 decreases activation, but not expression of TGF-β in nephritic glomeruli. In all figures treated kidneys (solid bars) were compared to nontreated control kidneys (open bars). Glomerular TGF-β1 or TGF-β2 protein (by brown immunostaining) was not changed in any group of nephritic rats (A and B). In agreement, equal levels of total TGF-β levels were determined using the PAI-1 luciferase assay. Active TGF-β levels were significantly reduced in the antisense-treated kidneys (C). Additionally, active TGF-β in nephritic glomeruli was determined by an antibody specifically recognizing the active form of TGF-β1 (D, gray cytoplasmic staining) and by an antibody specific for the phosphorylated form of the TGF-β signal-transduction molecule Smad 2/3 (H, black nuclear staining; arrowheads indicate examples for P-Smad2/3-positive nuclei). Inhibition of TSP1 expression after antisense ODN therapy but not scrambled ODN therapy was associated with a markedly decreased glomerular TGF-β activity in the left kidney as reflected by immunostaining for active TGF-β1 (E, evaluated by scoring system; F, evaluated by computerized morphometry) and by a marked reduction of glomerular cells showing positive nuclei for the TGF-β signaling molecule phospho-Smad2/3 (G). * marks significant differences (P < 0.01) of antisense groups versus the control (scrambled) group.

Article Snippet: 25 Immunostaining for matrix proteins was conducted with polyclonal antibodies to collagen I (rabbit anti-rat collagen I; Quartett, Berlin, Germany), 25 collagen IV (goat anti-human/bovine collagen IV; Southern Biotechnology Associates, Inc., Birmingham, AL), 25 active TGF-β1 (chicken anti-human active TGF-β1; R&D systems, Wiesbaden-Nordenstadt, Germany), 28 TGF-β1 (rabbit anti-human TGF-β1; Santa Cruz Biotechnology Inc., Santa Cruz, CA), 24 TGF-β2 (rabbit anti-human TGF-β2; Santa Cruz Biotechnology), 24 and a murine IgG 1 mAb against TSP1 (Dunn, Labortechnik GmbH, Asbach, Germany), 25 P-Smad2/3 (rabbit anti-human Smad2 peptide phosphorylated at Ser-433/435; Santa Cruz).

Techniques: Activation Assay, Expressing, Immunostaining, Luciferase, Staining, Transduction, Inhibition, Activity Assay

Journal:

Article Title: Therapeutic Effects of Benzoxazinorifamycin KRM-1648 Administered Alone or in Combination with a Half-Sized Secretory Leukocyte Protease Inhibitor or the Nonsteroidal Anti-Inflammatory Drug Diclofenac Sodium against Mycobacterium avium Complex Infection in Mice

doi:

Figure Lengend Snippet: Effects of 1/2 SLPI and diclofenac Na on production of TNF-α, IL-10, and TGF-β by MAC-stimulated Mφs a

Article Snippet: After rinsing of the wells with 0.1% BSA–PBS, either biotinylated rat anti-mouse TNF-α (Pharmingen) Ab, biotinylated rat anti-mouse IL-10 (Pharmingen) Ab, or chicken anti-human TGF-β Ab (R & D Systems Inc., Minneapolis, Minn.) was added as the detecting Ab and was allowed to react with the complex consisting of the corresponding cytokines and capture Abs.

Techniques: Infection

Journal: The Journal of Experimental Medicine

Article Title: CD4 + CD25 + regulatory T cells inhibit natural killer cell functions in a transforming growth factor–β–dependent manner

doi: 10.1084/jem.20051511

Figure Lengend Snippet: Selective expression of membrane-bound TGF- β on T reg cells. (A) TGF-β expression was investigated on both CD4 + CD25 high versus CD4 + CD25 low cells in flow cytometry using four-color staining with anti-CD3–APC, anti-CD4–PerCP, anti-CD25–PE, anti–TGF-β–FITC (AF-101-NA polyclonal chicken IgY), or a chicken IgY–FITC (AB-101-C) as a control antibody. (B) The specificity of the staining with the AF-101-NA polyclonal chicken IgY antibody was tested using saturating concentrations of recombinant TGF-β, which blocked the detection of membrane-bound TGF-β. (C) Expression of LAP and TGF-β–RII was also analyzed in flow cytometry on these CD3 + CD4 + T cell subsets in four-color staining.

Article Snippet: Anti–human LAP and anti–TGF-βRII antibody were obtained from R&D Systems.

Techniques: Expressing, Membrane, Flow Cytometry, Staining, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: CD4 + CD25 + regulatory T cells inhibit natural killer cell functions in a transforming growth factor–β–dependent manner

doi: 10.1084/jem.20051511

Figure Lengend Snippet: Role of membrane-bound TGF- β in T reg cell– mediated NK cell inhibition. (A) NK cells were co-cultured for 4 h with T reg cells or soluble TGF-β1 and/or a TGF-β1 blocking antibody or IgY control antibody before incubation with 51 Cr-labeled GIST 882 tumor targets. The T cell/NK cell/target ratio was 10:10:1. y axis = % specific lysis K562. The results of one representative experiment out of three are shown. Values represent means ± SEM of triplicate wells. *, P < 0.05 using Fisher's exact method. (B) Incubation of NK cells with T reg cells for 6 h before stimulation with 2 ng/ml of IL-12 for 18 h. Supernatants were collected for ELISA assaying IFN-γ levels. Values represent means ± SEM of triplicate wells. The results of one representative experiment out of three are shown. *, P < 0.05. (C, left) NK cells were cultured alone or with T reg cells at a 1:1 ratio or with 1 ng/ml of TGF-β1. After an overnight incubation, cells were analyzed by three-color staining flow cytometry (CD3–FITC, NKG2D–PE, and CD56–PC5). (right) Same as in the left panel, but comparing T reg cells with T conv or adding anti–TGF-β blocking antibody or IgY control antibody along with T reg cells.

Article Snippet: Anti–human LAP and anti–TGF-βRII antibody were obtained from R&D Systems.

Techniques: Membrane, Inhibition, Cell Culture, Blocking Assay, Incubation, Labeling, Lysis, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

Journal: Pulmonary Circulation

Article Title: Role played by Prx1-dependent extracellular matrix properties in vascular smooth muscle development in embryonic lungs

doi: 10.1086/681272

Figure Lengend Snippet: Prx1 regulates transforming growth factor (TGF)–β signaling in embryonic lungs. A, Immunohistochemical staining for TGF-β1 and TGF-β2 in lung sections derived from E15.5 (top) and D0 (bottom) Prx1WT (left) and Prx1null (right) mice. Brown indicates positive staining of antibodies. While TGF-β localization is restricted to the developing vessel area (arrows) in Prx1WT lungs, in Prx1null lungs TGF-β expression is observed in areas other than the vessel wall vicinity (arrowheads). Hematoxylin staining (blue) is used for nuclear counterstaining. Scale bar = 40 μm. B, Western blotting for TGF-β1 protein expression in E17.5 lungs from Prx1WT and Prx1null mice. C, Real-time polymerase chain reaction (PCR) for TGF-β1 messenger RNA (mRNA) expression in E17.5 lungs from Prx1WT and Prx1null mice. D, Western blotting for phosphorylated Smad2 (P-Smad2; top) and total Smad 2/3 (middle) in E17.5 lungs from Prx1WT and Prx1null mice. E, Real-time PCR for plasminogen activator inhibitor type 1 (PAI-1) mRNA expression in E17.5 lungs from Prx1WT and Prx1null mice. For Western immunoblotting (B, D), β-actin was used as a loading control. For mRNA expression by real-time PCR (C, E), target gene expression was normalized to GAPDH. Two asterisks indicate P < 0.01. WT: wild type.

Article Snippet: Ten micrograms of neutralizing antibody per milliliter against TGF-β1 (R&D Systems) was added to the medium when 10T1/2 cells were seeded in the decellularized scaffolds.

Techniques: Immunohistochemical staining, Staining, Derivative Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Targeted Gene Expression

Journal: Pulmonary Circulation

Article Title: Role played by Prx1-dependent extracellular matrix properties in vascular smooth muscle development in embryonic lungs

doi: 10.1086/681272

Figure Lengend Snippet: 10T1/2 cell differentiation to a smooth muscle phenotype is transforming growth factor (TGF)–β dependent. A, Immunostaining for α–smooth muscle actin (SMA), γ-SMA, SM22α, and smooth muscle myosin heavy chain (SMMHC; all positive reactions are green) in 10T1/2 cells, with or without TGF-β1 treatment, in the presence or absence of a TGF-β type I receptor (TGF-βRI) inhibitor (LY364947). Scale bar = 10 μm. B, α- and γ-SMA messenger RNA expression in 10T1/2 cells after stimulation with TGF-β1, with or without LY364947. Plasminogen activator inhibitor type 1 (PAI-1) was used as a positive control for canonical TGF-β signaling activation. Signals were normalized to GAPDH. C, Western immunoblotting for α- and γ-SMA expression in 10T1/2 cells after stimulation with TGF-β1, with or without a TGF-βRI inhibitor (SB431542). Phosphorylation of Smad2 (P-Smad2) was examined as a marker of canonical TGF-β signaling. GAPDH was used as a loading control.

Article Snippet: Ten micrograms of neutralizing antibody per milliliter against TGF-β1 (R&D Systems) was added to the medium when 10T1/2 cells were seeded in the decellularized scaffolds.

Techniques: Cell Differentiation, Immunostaining, RNA Expression, Positive Control, Activation Assay, Western Blot, Expressing, Marker

Journal: Pulmonary Circulation

Article Title: Role played by Prx1-dependent extracellular matrix properties in vascular smooth muscle development in embryonic lungs

doi: 10.1086/681272

Figure Lengend Snippet: Transforming growth factor (TGF)–β–dependent 10T1/2 cell differentiation tightly interacts with Prx1-dependent extracellular matrix (ECM) stiffness. A, Schema for the stiffness-controlled polyacrylamide (PAA) gel 2-D culture system. B, 10T1/2 cells were cultured on PAA gel 2-D substrates with a stiffness of either 500 (top) or 1,500 (bottom) Pa, which is parallel to the E17.5 Prx1-lung stiffness, in the presence or absence of TGF-β stimulation. Smooth muscle cell differentiation was evaluated by expression of α–smooth muscle actin (SMA; left, green), SM22α (middle, green), and smooth muscle myosin heavy chain (SMMHC; right, red). 4′,6‐Diamidino‐2‐phenylindole dihydrochloride (blue) was used for nuclear staining. Scale bar = 10 μm. C, Aspect ratio of cells cultured on 2-D PAA gels under the above-described conditions was examined to quantify cell morphology change. D–F, Real-time polymerase chain reaction results for 10T1/2 cells cultured on 2-D PAA gels of either 500- or 1,500-Pa stiffness with or without TGF-β stimulation. α-SMA (D), SM22α (E), and SMMHC (F) messenger RNA expression was examined and normalized by GAPDH or cyclophilin A (cyclo A) expression. One asterisk indicates P < 0.05, and two asterisks indicate P < 0.01. G, 3-D projection of confocal images of F-actin-stained 10T1/2 cells cultured in soft and stiff 3-D methacrylated hyaluronic acid synthetic scaffolds. F-actin is red, nucleus is blue. Scale bar = 20 μm.

Article Snippet: Ten micrograms of neutralizing antibody per milliliter against TGF-β1 (R&D Systems) was added to the medium when 10T1/2 cells were seeded in the decellularized scaffolds.

Techniques: Cell Differentiation, Cell Culture, Expressing, Staining, Real-time Polymerase Chain Reaction, RNA Expression