Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) Model of SPD-gp100-CD40L. Amino acids 25 to 596 (sequence KVPRNQD to EAGLGQV) of human gp100 were inserted between amino acids 105 and 106 of murine SPD within the SPD-CD40L fusion construct. (b) Cartoon of expected SPD-gp100-CD40L 4-trimer structure showing trimers of CD40L (gray circles) and trimers of gp100 (black circles) extending from the SPD collagen-like domain. (c) Western blot analysis. 293T cells were transfected with DNA plasmid encoding gp100 or SPD-gp100-CD40L fusion protein. After 48-hour culture, supernatant was collected and run on an SDS-PAGE gel in the presence of reducing agent. Western blot was performed using a polyclonal antibody to gp100.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: Sequencing, Construct, Western Blot, Transfection, Plasmid Preparation, SDS Page

Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) Representative SEC elution profile of SPD-gp100-CD40L as monitored by the scattering intensity, expressed in terms of the excess Rayleigh ratio (R(θ)), at three scattering angles of 42° (tall gray line), 90° (black line) and 138° (short gray line) as a function of elution volume (V). Note that SPD-gp100-CD40L apparently elutes as a major species (megamer) and as a minor species (polymer, evident as a shoulder of the major elution peak). (b) Representative partial Zimm plot obtained from analytical SLS measurements at a specific protein concentration for the SPD-gp100-CD40L complex. The line through the data points—collected at three scattering angles of 42°, 90° and 138°—represents a linear fit. (c) Representative autocorrelation function plot obtained from analytical DLS measurements at a specific protein concentration for the SPD-gp100-CD40L complex. The line through the data points—collected at a scattering angle of 90°—represents non-linear least squares fit.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: Polymer, Protein Concentration

Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) In vitro activity of SPD-CD40L and SPD-gp100-CD40L was determined using a cell-based CD40 NF-κB enzymatic reporter system. An equivalent amount of 293T supernatant from pcDNA3.1, pSPD-CD40L or pSPD-gp100-CD40L transfected cells was incubated with 293-CD40-SEAP NF-κB reporter cells at various dilutions. (b) In vitro activity of supernatant from SPD-gp100-CD40L transfected 293T cells was evaluated on mouse bone marrow derived mouse DC and compared to supernatant from 293T cells transfected with empty vector pcDNA3.1. Error bars represent mean + SEM (n=3). * p<0.05, ** p<0.01 by Student’s t test compared to pcDNA3.1 supernatant.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: In Vitro, Activity Assay, Transfection, Incubation, Derivative Assay, Plasmid Preparation

Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) Immunization schedule for B16-F10 tumor challenge and DNA/GVAX therapeutic vaccination, as indicated by arrows. B16-F10 cells (50,000) were injected i.d. into the left flank of mice on day 0. Mice were then immunized by i.m. injection of PBS or pSPD-gp100-CD40L on day 3, 10, and 17. GVAX, B16-F10 tumor cells expressing GM-CSF, were irradiated at 5,000 rad and 1 × 106 cells injected subcutaneously on day 3, 6, and 9. (b) Tumor growth analysis. Each point represents the mean tumor volume per group (n=5). (c) Survival analysis was based on the date of death or when tumor size reached >1500 cm2.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: Injection, Expressing, Irradiation

Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) Immunization schedule for B16-F10 tumor challenge and DNA or GVAX vaccination, as indicated by arrows. B16-F10 cells (50,000) were injected i.d. into the left flank of the mice on day 0. Mice were immunized i.m. with PBS, pSPD-gp100-CD40L + pIL-12, pSPD-gp100-CD40L + pGM-CSF, or pSPD-gp100-CD40L + pIL-12 + pGM-CSF on day 3, 10, and 17. For GVAX therapy, B16-F10 tumor cells expressing GM-CSF (GVAX), were irradiated at 5,000 rad and 1 × 106 cells were injected subcutaneously on day 3, 6, and 9. (b) Tumor growth analysis. Each point represents the mean tumor volume of animals in each group (n=5). (c) Survival analysis of mice. (d) Tumor growth kinetics of individual mice from each treatment arm.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: Injection, Expressing, Irradiation

Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) Immunization schedule for B16-F10 tumor challenge and DNA vaccination, as indicated by arrows. B16-F10 cells (50,000) were injected into the left flank of the mice on day 0. Mice were then immunized i.m. with PBS, pgp100, pgp100 + pIL-12, pgp100 + pGM-CSF, pgp100 + pIL-12 + pGM-CSF, pgp100-IRES-SPD-CD40L + pIL-12 + pGM-CSF, or pSPD-gp100-CD40L + pIL-12 + pGM-CSF on day 3, 10, and 17. (b) Tumor growth analysis. Each point represents the mean tumor volume of animals in each group (n=5). (c) Survival analysis of mice.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: Injection

Journal: PLoS ONE

Article Title: 17β-Estradiol Enhances the Response of Plasmacytoid Dendritic Cell to CpG

doi: 10.1371/journal.pone.0008412

Figure Lengend Snippet: The PDCs were gated to fall within established viable cell forward and side scatter parameters and to eliminate the cell aggregates. The surface expression of costimulatory molecules CD80, CD86 and CD40 on purified pDCs under the treatment with E2 and/or CpG in vitro were further analyzed in a FL1 density plot. Data are reported as positive PDC cells and MFI. Histograms shown are representative of three experiments with homogenous results. Comparisons between the different stimuli are indicated by *, ** and ***: p<.05, .01 and .005, respectively; n = 3.

Article Snippet: FITC anti-mouse CD40, CD80, CD86, B220, IFN-α and IgG2b, and PE anti-mouse CD19 were bought from eBioscience Inc (San Diego, CA, USA).

Techniques: Expressing, Purification, In Vitro

Journal:

Article Title: A defect in bone marrow derived dendritic cell maturation in the nonobesediabetic mouse

doi: 10.1046/j.1365-2249.2001.01473.x

Figure Lengend Snippet: Surface phenotype of bone marrow derived DC. The DC consisting of 90–95% CD11c+ cells were analysed by flow cytometry for the expression of I-A, β2-microglobulin, CD80, CD86 and CD40 (▪) or by staining with secondary antibody alone (□). Two I-A specific mAbs, Ox-6 (shown here) or 10·2.16 were used with identical result. The results are representative of three different experiments carried out three weeks apart.

Article Snippet: The antibodies used in the study were the MHC class II specific rat antimouse I-A k clone MRC Ox-6 (Serotec, Oxford, UK) and anti-I-A g7 clone 10·2.16 (kindly provided by EP Reich, Anergen Inc), rat antimouse CD80 (B7·1) clone IG10 (Pharmingen, San Diego, USA), rat antimouse CD86 (B7·2) clone GL1 (Cambridge Bioscience, UK); rat antimouse CD40 clone 3/23 (Serotec, UK), hamster antimouse CD11c clone N-418 (produced by ICRF, London, UK); FITC-conjugated rabbit antirat Ig (Dako, Ely, UK); FITC-conjugated rabbit anti‐mouse Ig (Dako, Denmark); and mouse anti‐mouse β2-microglobulin clone S19·8 (kindly provided by M Millrain, CSC, UK).

Techniques: Derivative Assay, Flow Cytometry, Expressing, Staining