Journal: The Journal of allergy and clinical immunology

Article Title: Activin A and TGF-β promote T H 9 cell–mediated pulmonary allergic pathology

doi: 10.1016/j.jaci.2011.12.965

Figure Lengend Snippet: Induction of TH9 differentiation in vitro by activin A. A, TH9 cells generated in the presence of IL-4 alone or in association with TGF-β1, activin A, and/or IL-25. B, Percentage of TH9 cells plotted in a histogram. C, Phenotype of TH9 cells differentiated in the presence of IL-4 with either TGF-β1 or activin A. D, IL-9 secretion from activin A–derived TH9 cells after 4 days in culture in the presence of 0.1 ng/mL of TGF-β1. Data shown represent means ± SEMs.

Article Snippet: In blocking experiments, mice received 20 μg of neutralizing antibody to murine activin A (R&D Systems, Abingdon, UK) and/or anti-TGF-β (5 mg/kg, clone 1D11 pan neutralizing TGF-β1-3; Genzyme Corporation, Cambridge, Mass) or isotype control intraperitoneally.

Techniques: In Vitro, Generated, Derivative Assay

Journal: The Journal of allergy and clinical immunology

Article Title: Activin A and TGF-β promote T H 9 cell–mediated pulmonary allergic pathology

doi: 10.1016/j.jaci.2011.12.965

Figure Lengend Snippet: Acute blockade of TGF-β and activin A inhibits TH9 differentiation. A, Schematic of experimental protocol. B, TH9 cells recovered from the lung after treatment with either anti–TGF-β and/or anti–activin A in mice challenged with either PBS or HDM. Total cells (C), lung eosinophils (D), and TH2 cells (E) recovered from the lung. Serum mMCP-1 levels (F) and intraepithelial mast cells (G) per lung section. IL-9 (H) and IL-13 (I) levels in supernatants from lymph node cell cultures. J, IL-25 levels in the lungs. Data shown represent means ± SEMs. *P < .05. mMCP-1, Mouse mast cell protease-1.

Article Snippet: In blocking experiments, mice received 20 μg of neutralizing antibody to murine activin A (R&D Systems, Abingdon, UK) and/or anti-TGF-β (5 mg/kg, clone 1D11 pan neutralizing TGF-β1-3; Genzyme Corporation, Cambridge, Mass) or isotype control intraperitoneally.

Techniques:

Journal: The Journal of allergy and clinical immunology

Article Title: Activin A and TGF-β promote T H 9 cell–mediated pulmonary allergic pathology

doi: 10.1016/j.jaci.2011.12.965

Figure Lengend Snippet: Chronic blockade of TGF-β and activin A reduces airway remodeling. A, Schematic of experimental protocol. B, Peribronchial and perivascular cellular infiltrate (H&E), purple-colored mucin-containing cells in the epithelium (PAS), and perivascular and peribronchiolar collagen (sirrius red) and brown stained intraepithelial mast cells. Original magnification ×40. Scale bar = 50 μm. C, Quantification of mucus positive cells. D, Intraepithelial mast cells per lung section. E, Serum mMCP-1 levels. F, Total lung collagen. G, Total cells recovered from BAL and lung. H, IL-25 levels in the lung. I, Airway resistance (RI) following 3-week HDM challenge. Data shown represent means ± SEMs. *P < .05. BAL, Bronchoalveolar lavage; MCPT7, mouse tryptase beta 1; mMCP-1, mouse mast cell protease-1; PAS, periodic acid-Schiff.

Article Snippet: In blocking experiments, mice received 20 μg of neutralizing antibody to murine activin A (R&D Systems, Abingdon, UK) and/or anti-TGF-β (5 mg/kg, clone 1D11 pan neutralizing TGF-β1-3; Genzyme Corporation, Cambridge, Mass) or isotype control intraperitoneally.

Techniques: Staining

Journal:

Article Title: Glucose Stimulation of Transforming Growth Factor-? Bioactivity in Mesangial Cells Is Mediated by Thrombospondin-1

doi:

Figure Lengend Snippet: Time-dependent activation of TGF-β and stimulation of TSP-1 expression in media conditioned by mesangial cells in the presence of high glucose levels. RMCs were treated as indicated in Materials and Methods. Conditioned media were harvested on days 1 and 2 and analyzed for levels of active (A) or total (B) TGF-β in samples by the NRK soft agar assay. Results are expressed as the mean number of colonies from triplicate determinations ±SD. Results are representative of three separate experiments. The baseline colony formation in this assay in wells containing 2.5 ng/ml EGF without conditioned media was 12 ± 2 colonies. *, For active TGF-β in RMC cultured with 30 mmol/L versus 5 mmol/L, P < 0.001. *, For total TGF-β in RMC cultured with 30 mmol/L versus 5 mmol/L, P < 0.005. The Student’s t-test was used to analyze data. C: Conditioned media were harvested on days 1 and 2, and a 60-μl aliquot of 1 ml of conditioned medium loaded in each well was subjected to 8% SDS-PAGE under reducing conditions, transferred to nitrocellulose, and analyzed by Western blotting with mouse 133 monoclonal antibody to thrombospondin as indicated in Materials and Methods. Lane 1, conditioned media harvested on day 1 from RMC cultured with 5 mmol/L of glucose; lane 2, conditioned media harvested on day 1 from RMC cultured with 30 mmol/L of glucose; lane 3, conditioned media harvested on day 2 from RMC cultured with 5 mmol/L of glucose; and lane 4, conditioned media harvested on day 2 from RMC cultured with 30 mmol/L of glucose. Immunoblots were analyzed by scanning densitometry and quantified by one-dimensional gel analysis (One-Dscan version 1.31; Scanalytics). Results are representative of three separate experiments.

Article Snippet: Monoclonal anti-TGF-β1-3 antibody was purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Expressing, Soft Agar Assay, Cell Culture, SDS Page, Western Blot

Journal:

Article Title: Glucose Stimulation of Transforming Growth Factor-? Bioactivity in Mesangial Cells Is Mediated by Thrombospondin-1

doi:

Figure Lengend Snippet: Anti-TSP antibody and antagonist peptides (GGWSHW and LSKL) block TSP-mediated activation of latent TGF-β secreted by RMCs under high glucose conditions. RMCs were made quiescent for 48 hours in serum-free media. Cells were then stimulated for 48 hours with either 5 mmol/L or 30 mmol/L of glucose in the presence of TSP antagonist peptides (LSKL at 1 μmol/L, GGWSHW at 10 μmol/L), control peptides (SLLK at 1 μmol/L, GGYSHW at 10 μmol/L), or aprotinin (200 μg/ml). Aliquots of conditioned media (0.4 ml) were assayed for either (A) active or (B) total TGF-β levels using the NRK soft agar assay. The dashed line represents the baseline colony formation in this assay in wells containing only 2.5 ng/ml EGF without conditioned media (12 ± 2 colonies). Results are expressed as the mean number of colonies from triplicate determinations ±SD. One-way analysis of variance analysis was used to determine significance: *, P < 0.05 for 30 mmol/L of glucose control versus LSKL peptide or GGWSHW peptide in 30 mmol/L of glucose. Results for samples treated with either LSKL or GGWSHW peptides in 30 mmol/L of glucose were not statistically different from control samples in 5 mmol/L of glucose. Results are representative of three separate experiments. C: RMC stimulated with 5 mmol/L or 30 mmol/L of glucose were treated with Fab fragments (24 μg/ml) purified from 133 anti-TSP1 antibody (α-TSP). Results are expressed as the mean number of colonies from triplicate determinations ±SD. *, P < 0.01 for 30 mmol/L of glucose control versus Fab fragments in 30 mmol/L of glucose. D: Media from RMC grown in 5 or 30 mmol/L of glucose were treated with either 15 μg/ml anti-TGF-β1-3 antibody (α-TGF) or 15 μg/ml nonimmune IgG (NI) before addition to the NRK assay. Results are the means of triplicate determinations.

Article Snippet: Monoclonal anti-TGF-β1-3 antibody was purchased from R&D Systems (Minneapolis, MN).

Techniques: Blocking Assay, Activation Assay, Soft Agar Assay, Purification

Journal:

Article Title: Transforming Growth Factor-?-induced Mobilization of Actin Cytoskeleton Requires Signaling by Small GTPases Cdc42 and RhoA

doi: 10.1091/mbc.01-08-0398

Figure Lengend Snippet: TGF-β1–induced actin reorganization and membrane ruffling in RBL-2H3 cells. (A) RBL-2H3 cells were serum-starved for 12 h and treated with 25 ng/ml TGF-β1 for 5, 15, 30, and 60 min. FcεRI activation was induced by addition of the cross-linking antibody BC4. (B) Monoclonal mouse anti-TGF-β1-3 neutralizing antibody was added to RBL-2H3 cells at a concentration of 25 μg/ml 1 h before stimulation with 25 ng/ml TGF-β1 for 15 min. Filamentous actin was visualized by TRITC-labeled phalloidin. Bar, 20 μm.

Article Snippet: Mouse monoclonal anti-FcεRI antibody (clone BC4) was a generous gift from Dr. R. Siraganian (National Institutes of Health, Bethesda, MD), mouse monoclonal anti-TGF-β1-3 neutralizing antibody was from Genzyme (Cambridge, MA), mouse monoclonal anti-myc (9E10) was from Santa Cruz Biotechnology (Santa Cruz, CA), fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibodies were from DAKO (Glostrup, Denmark), and mouse monoclonal anti-Flag M5 was from Sigma Chemical (St. Louis, MO).

Techniques: Activation Assay, Concentration Assay, Labeling

Journal: PLoS ONE

Article Title: Aspergillus terreus Accessory Conidia Are Unique in Surface Architecture, Cell Wall Composition and Germination Kinetics

doi: 10.1371/journal.pone.0007673

Figure Lengend Snippet: Staining for β1-3-glucan surface exposure on A. fumigatus conidia (A), A. terreus phialidic conidia (B) and A. terreus accessory conidia (C) was performed and evaluated by fluorescence microscopy. Arrows denote the β1-3-glucan staining on accessory conidia.

Article Snippet: Monoclonal mouse IgG antibody to β1-3-glucan (Biosupplies Australia, Parkville, Australia) was added at 1∶20 concentration and conidia were incubated on ice for 45 minutes.

Techniques: Staining, Fluorescence, Microscopy