Journal: Nature Communications

Article Title: Augmentation of vaccine-induced humoral and cellular immunity by a physical radiofrequency adjuvant

doi: 10.1038/s41467-018-06151-y

Figure Lengend Snippet: RF induces low-level local inflammation. Dorsal skin of C57BL/6 mice were exposed to RF or intradermally injected with 20 µl Alum (1:1 volume ratio in PBS), AddaVax (50%, vol/vol in PBS), or MPL (25 µg). Adjuvant-treated and non-treated skins were dissected at indicated times. a , b Heat map of relative cytokine ( a ) and chemokine ( b ) gene expression. Total RNA was extracted followed by reverse transcription and real-time PCR analysis of cytokine and chemokine gene expression using GAPDH as an internal control. The baseline gene expression level was set at 1. c – g Different innate immune cell levels in RF and adjuvant-treated skin. Skin was digested in collagenase D and dispase to prepare single-cell suspensions. Cells were then stained with fluorescence-conjugated antibodies followed by flow cytometry analysis of levels of different cell types: neutrophils ( c ), monocytes ( d ), macrophages ( e ), eosinophils ( f ), and mDCs ( g ) (Supplementary Fig. 2). n = 4. Student’s t -test was used to compare differences between groups at 48 and 96 h. *, p

Article Snippet: Skin was digested in 0.2% collagenase D (Roche Diagnostics, Mannheim, Germany) and 0.6U/ml dispase (Gibco, Invitrogen, Carlsbad, CA) in PBS at 37˚C for 3 h with intermittent vortexing.

Techniques: Mouse Assay, Injection, Expressing, Real-time Polymerase Chain Reaction, Staining, Fluorescence, Flow Cytometry, Cytometry

Journal: Nature Communications

Article Title: Augmentation of vaccine-induced humoral and cellular immunity by a physical radiofrequency adjuvant

doi: 10.1038/s41467-018-06151-y

Figure Lengend Snippet: RF increases antigen uptake of DCs in skin. Lateral back skin of C57BL/6 mice was exposed to RF or sham treatment followed by ID injection of 2 µg AF647-OVA into RF or sham-treated skin. ID injection of PBS served as control. Skin was harvested 24 h later and digested in collagenase D and dispase to prepare single-cell suspensions. Skin cells were then stained with fluorescence-conjugated antibodies followed by flow cytometry analysis. a Live cells were gated and plotted based on CD11c and MHC II expression. CD11c + MHC II + cells were gated and plotted based on Langerin expression. b Langerin + cells (gate 1) were plotted based on CD11b and CD103 expression into Langerin + CD11b − CD103 + (I) and Langerin + CD11b + CD103 − subsets (II), whereas Langerin − cells (gate 2) were plotted based on CD11b expression into Langerin − CD11b + (III) and Langerin − CD11b − subsets (IV). c Percentage of AF647 + cells was analyzed for each DC subset. n = 4 for PBS control and 6 for sham and RF groups. Student’s t -test was used to compare differences between RF and sham groups. * p

Article Snippet: Skin was digested in 0.2% collagenase D (Roche Diagnostics, Mannheim, Germany) and 0.6U/ml dispase (Gibco, Invitrogen, Carlsbad, CA) in PBS at 37˚C for 3 h with intermittent vortexing.

Techniques: Mouse Assay, Injection, Staining, Fluorescence, Flow Cytometry, Cytometry, Expressing

Journal: PLoS ONE

Article Title: In Vitro Study of Novel Collagenase (XIAFLEX(R)) on Dupuytren's Disease Fibroblasts Displays Unique Drug Related Properties

doi: 10.1371/journal.pone.0031430

Figure Lengend Snippet: Measurement of comparative effect of Xiaflex and Collagenase A on mRNA steady-state levels of primary DD fibroblasts isolated from different anatomical sites. A. Collagen I; B. Collagen III. #p

Article Snippet: Xiaflex® versus Collagenase A The relative and dose-dependent effects of Xiaflex® (Auxilium Pharmaceuticals, USA) and a commercially available reagent grade collagenase, Collagenase A (Roche Diagnostics, UK), on DD and CT primary cultured fibroblasts from each anatomical site were assessed.

Techniques: Isolation

Journal: PLoS ONE

Article Title: In Vitro Study of Novel Collagenase (XIAFLEX(R)) on Dupuytren's Disease Fibroblasts Displays Unique Drug Related Properties

doi: 10.1371/journal.pone.0031430

Figure Lengend Snippet: Comparison of the effect of Xiaflex® and Collagenase A on cell cycle regulation. Cell cycle gene (PCNA, Cyclin D1 and Cyclin D2) were assessed at mRNA and protein levels using qRT-PCR and In-Cell Western blotting respectively. A. mRNA steady-state levels of cell cycle genes (PCNA, Cyclin D1 and Cyclin D2) after treatment with Xiaflex® and Collagenase A at various concentrations as indicated in the graphs. All the cell cycle genes were dose-dependently down regulated by Xiaflex® and Collagenase A compared to the untreated control group. B. Relative protein expression of cell cycle proteins (PCNA and Cyclin D) after treatment with Xiaflex® and Collagenase A. *p

Article Snippet: Xiaflex® versus Collagenase A The relative and dose-dependent effects of Xiaflex® (Auxilium Pharmaceuticals, USA) and a commercially available reagent grade collagenase, Collagenase A (Roche Diagnostics, UK), on DD and CT primary cultured fibroblasts from each anatomical site were assessed.

Techniques: Quantitative RT-PCR, In-Cell ELISA, Expressing

Journal: PLoS ONE

Article Title: In Vitro Study of Novel Collagenase (XIAFLEX(R)) on Dupuytren's Disease Fibroblasts Displays Unique Drug Related Properties

doi: 10.1371/journal.pone.0031430

Figure Lengend Snippet: Real-Time Cell Analaysis (RTCA) monitoring of Xiaflex® and Collagenase A effects on DD-primary fibroblasts obtained from different anatomical sites. This diagram demonstrates average cell indeces (CI) of untreated and treated cell groups taken from six independent RTCA experiments, which have been plotted.

Article Snippet: Xiaflex® versus Collagenase A The relative and dose-dependent effects of Xiaflex® (Auxilium Pharmaceuticals, USA) and a commercially available reagent grade collagenase, Collagenase A (Roche Diagnostics, UK), on DD and CT primary cultured fibroblasts from each anatomical site were assessed.

Techniques:

Journal: PLoS ONE

Article Title: In Vitro Study of Novel Collagenase (XIAFLEX(R)) on Dupuytren's Disease Fibroblasts Displays Unique Drug Related Properties

doi: 10.1371/journal.pone.0031430

Figure Lengend Snippet: Comparison of the effect of Xiaflex® and Collagenase A on Type I and Type III collagen protein synthesis by DD fibroblasts. DD fibroblasts from passages 1–4 were cultured in a 96 well plate (1.5×10 4 cells/well) and treated with both drugs at various concentrations as indicated in the figure. Cell lysates from treated and untreated cells were subjected to capture sandwich ELISA (for the detection of collagen I) and Indirect ELISA (for the detection of collagen III) as described previously. A. Collagen I and B. Collagen III. *p

Article Snippet: Xiaflex® versus Collagenase A The relative and dose-dependent effects of Xiaflex® (Auxilium Pharmaceuticals, USA) and a commercially available reagent grade collagenase, Collagenase A (Roche Diagnostics, UK), on DD and CT primary cultured fibroblasts from each anatomical site were assessed.

Techniques: Cell Culture, Sandwich ELISA, Indirect ELISA

Journal: PLoS ONE

Article Title: In Vitro Study of Novel Collagenase (XIAFLEX(R)) on Dupuytren's Disease Fibroblasts Displays Unique Drug Related Properties

doi: 10.1371/journal.pone.0031430

Figure Lengend Snippet: Quantitative In-Cell Western blotting assay for the analysis of protein expression upon treatment with Xiaflex® and Collagenase A by DD fibroblasts. Primary fibroblasts were seeded in 96 well plate (1.5×10 4 cells/well) and allowed to grow for ∼7–8 hours. The cells were then treated with various concentrations of Xiaflex® and Collagenase A as indicated. At 24 hours drug treatment, the cells were fixed in 4% formaldehyde/PBS. In-Cell Western blotting was performed. Representative output infrared images of treated and untreated fibroblasts, stained for protein expression (visible in green/red) from different anatomical sites are shown. Bar graphs represent the quantification of average protein expression in different treatments from three independent experiments after normalization to loading control beta-actin. A. Collagen I; B. Collagen III; C. Fibronectin; D. α-SMA; E. Desmin; F. Tenascin; G. Collagen IV; H. CTGF; I. MMP-9. *p

Article Snippet: Xiaflex® versus Collagenase A The relative and dose-dependent effects of Xiaflex® (Auxilium Pharmaceuticals, USA) and a commercially available reagent grade collagenase, Collagenase A (Roche Diagnostics, UK), on DD and CT primary cultured fibroblasts from each anatomical site were assessed.

Techniques: In-Cell ELISA, Expressing, Staining

Journal: PLoS ONE

Article Title: In Vitro Study of Novel Collagenase (XIAFLEX(R)) on Dupuytren's Disease Fibroblasts Displays Unique Drug Related Properties

doi: 10.1371/journal.pone.0031430

Figure Lengend Snippet: Detection of Early Apoptosis and Necrosis using Annexin V and PI. Fibroblasts from different anatomical sites (Nodule, Cord, Fat and Skin) were treated with various concentration of Xiaflex® and Collagenase A as indicated in the graphs. 24 hours post-treatment, cells were harvested and labeled with Annexin V-FITC and PI. A. FITC-conjugated annexin V staining for untreated cells, upper left plot (labeled-untreated), compared with the viable control cells, upper right plot (unlabeled cells). Dual-staining of treated cells (lower panel): the quadrant analysis shows viable cells negative for annexin V and PI in the lower left, R3. Apoptotic cells stained with annexin V but excluding PI are shown in the lower right, R4. Secondary necrotic cells (i.e. necrosis after apoptosis) positive for both PI and annexin V are shown in upper right, R2. Necrotic or mechanically damaged cells positive for PI only are shown in upper left, R1. Representative data are shown from three independent experiments in triplicates. B. Annexin V and PI positive cells after 24 hrs treatment with Xiaflex® and Collagenase A at various concentrations as indicated in bar graph. Positive cells were counted from three independent experiments and plotted on the graph as an average means ± SEM. *p

Article Snippet: Xiaflex® versus Collagenase A The relative and dose-dependent effects of Xiaflex® (Auxilium Pharmaceuticals, USA) and a commercially available reagent grade collagenase, Collagenase A (Roche Diagnostics, UK), on DD and CT primary cultured fibroblasts from each anatomical site were assessed.

Techniques: Concentration Assay, Labeling, Staining

Journal: PLoS ONE

Article Title: In Vitro Study of Novel Collagenase (XIAFLEX(R)) on Dupuytren's Disease Fibroblasts Displays Unique Drug Related Properties

doi: 10.1371/journal.pone.0031430

Figure Lengend Snippet: Real-Time Monitoring of Xiaflex® and Collagenase A effect on DD primary fibroblasts from different anatomical sites using MESA. Primary fibroblasts of DD (Nodule, Cord, Fat and Skin) were seeded onto the E-plate and cells were allowed to grow prior to the introduction of Xiaflex® and Collagenase A at various concentrations. After drug addition, cells were allowed to grow for 24 hours in the presence of drugs. After 24 hrs, the drugs were removed and the cells were fed with fresh supWillE media for 24 hrs to assess the reversibility of the inhibitory effect of the drugs. Cell Indexes were recorded every 15 minutes. Each trace at each concentration was an average of three replicates. A. Effect of Xiaflex® and Collagenase A on DD-Nodule. B. Effect of Xiaflex® and Collagenase A on DD-Cord.

Article Snippet: Xiaflex® versus Collagenase A The relative and dose-dependent effects of Xiaflex® (Auxilium Pharmaceuticals, USA) and a commercially available reagent grade collagenase, Collagenase A (Roche Diagnostics, UK), on DD and CT primary cultured fibroblasts from each anatomical site were assessed.

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: In Vitro Study of Novel Collagenase (XIAFLEX(R)) on Dupuytren's Disease Fibroblasts Displays Unique Drug Related Properties

doi: 10.1371/journal.pone.0031430

Figure Lengend Snippet: Effect of Xiaflex® and Collagenase A on cell membrane integrity (cytotoxicity detection) and cell viability/metabolic activity measured by LDH and WST-1 assays. A. LDH (lactose dehydrogenase) leakage assay for cell membrane integrity assessed the cytotoxic effect of the drugs. B. WST-1 (water soluble-tetrazolium salt-1) assayed for cell viability/metabolic activity and cell death. *p

Article Snippet: Xiaflex® versus Collagenase A The relative and dose-dependent effects of Xiaflex® (Auxilium Pharmaceuticals, USA) and a commercially available reagent grade collagenase, Collagenase A (Roche Diagnostics, UK), on DD and CT primary cultured fibroblasts from each anatomical site were assessed.

Techniques: Activity Assay