Journal: Infection and Immunity

Article Title: Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

doi: 10.1128/iai.00322-07

Figure Lengend Snippet: FIG. 1. Flt3L-Ag85B is expressed by DNA vaccines in a functional form. (A) Schematic representation of pCDNA3 vectors expressing Flt3L, M. tuberculosis Ag85B (p85B), and the Flt3L-Ag85B fusion protein (pFlt-85). (B) Secretion of Flt3L by DNA-transfected HEK 293 cells. Flt3L in the culture medium of cells 3 days after transfection was detected by ELISA. (C) Generation of DCs from bone marrow progenitors using culture medium from DNA-transfected HEK 293 cells. The generation of CD11c MHC-II cells on day 6 after addition of HEK 293 supernatants to bone marrow cells was determined by flow cytometry. (D) Total number of CD11c MHC-II cells in culture.

Article Snippet: Lysates and supernatants were analyzed for expression of recombinant Flt3L by immunoblotting using the anti-myc monoclonal antibody (MAb) 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA) or by a murine Flt3L-specific enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN).

Techniques: Vaccines, Functional Assay, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Cytometry

Journal: Infection and Immunity

Article Title: Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

doi: 10.1128/iai.00322-07

Figure Lengend Snippet: FIG. 2. Immunogenicity and protective efficacy of DNA encoding murine Flt3L and M. tuberculosis Ag85B. (A) Splenocytes from immunized mice were cultured with 3 g/ml Ag85B, and the level of IFN- released was determined by ELISA. (B) For determination of protective efficacy, 4 weeks following the final vaccination mice were infected by the aerosol route with 100 CFU of M. tuberculosis H37Rv. Four weeks after challenge, the bacterial load, expressed as the log10 CFU (mean standard error of the mean), was analyzed in the lung. The significance of differences between groups was determined by ANOVA. The error bars indicate standard errors of the means, and the data are representative of two separate experiments.

Article Snippet: Lysates and supernatants were analyzed for expression of recombinant Flt3L by immunoblotting using the anti-myc monoclonal antibody (MAb) 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA) or by a murine Flt3L-specific enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN).

Techniques: Immunopeptidomics, Cell Culture, Enzyme-linked Immunosorbent Assay, Infection, Aerosol

Journal: Infection and Immunity

Article Title: Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

doi: 10.1128/iai.00322-07

Figure Lengend Snippet: FIG. 3. Construction and in vivo immunogenicity of BCG:Flt3L. BCG Pasteur was transformed with the control pMV261 plasmid (BCG:Ct) or plasmid pJEX73 (BCG:Flt3L), and the presence of Flt3L in cell lysates was determined by immunoblotting with the anti-c-myc MAb 9E10 (A). The presence of Flt3L in the culture supernatants of rBCG strains was determined by murine Flt3L-specific ELISA (B). To assess the immunogenicity, mice were not vaccinated (striped bar) or were vaccinated with 5 105 CFU of BCG:Ct (open bars) or BCG:Flt3L (solid bars), and at the indicated time points splenocytes (C) or DLN cells (D) were stimulated with 1 g/ml BCG lysate. The number of IFN--secreting cells was determined by an enzyme-linked immunospot assay. The significance of differences between BCG:Flt3L-vaccinated animals and BCG:Ct- vaccinated animals (asterisk, P 0.05) was determined by ANOVA. The data are representative of two separate experiments.

Article Snippet: Lysates and supernatants were analyzed for expression of recombinant Flt3L by immunoblotting using the anti-myc monoclonal antibody (MAb) 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA) or by a murine Flt3L-specific enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN).

Techniques: In Vivo, Immunopeptidomics, Transformation Assay, Control, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Infection and Immunity

Article Title: Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

doi: 10.1128/iai.00322-07

Figure Lengend Snippet: FIG. 4. Influence of BCG:Flt3L on DC numbers in vaccinated mice. Mice were vaccinated subcutaneously with 5 105 CFU of rBCG, and at the indicated time points the numbers of CD11c MHC-II cells in the spleen (A, C, and E) and DLNs (B, D, and F) were determined by flow cytometry. The cells were further defined as cells that were B220 (C and D) or B220 (E and F). Striped bars, unvaccinated mice; open bars, BCG:Ct-vaccinated animals; solid bars, BCG:Flt3L-vaccinated animals. The significance of differences between BCG:Flt3L-vaccinated animals and BCG:Ct-vaccinated animals (asterisk, P 0.05) was determined by ANOVA. The data are representative of two separate experiments.

Article Snippet: Lysates and supernatants were analyzed for expression of recombinant Flt3L by immunoblotting using the anti-myc monoclonal antibody (MAb) 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA) or by a murine Flt3L-specific enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN).

Techniques: Cytometry

Journal: Infection and Immunity

Article Title: Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

doi: 10.1128/iai.00322-07

Figure Lengend Snippet: FIG. 5. Protective efficacy of BCG secreting Flt3L. Mice were immunized subcutaneously with 5 105 CFU of BCG:Flt3L or BCG:Ct, and 12 weeks after immunization they were challenged by the aerosol route with M. tuberculosis H37Rv. Four weeks postchallenge the bacterial loads, expressed as log10 CFU (means standard errors of the means), were analyzed in the (A) lungs and (B) spleens of mice. The statistical significance between naı¨ve and rBCG-vaccinated groups (asterisk, P 0.01) was analyzed by ANOVA. The data are representative of three individual experiments. Unv, unvaccinated mice. (C and D) For assessment of rBCG growth in vivo, mice were infected i.v. with 1 106 CFU of BCG:Flt3L (F) or BCG:Ct (E). At 1, 14, 28, and 56 days postinfection the bacterial loads were assessed in the lungs (C) and spleens (D). The statistical significance between groups (asterisk, P 0.05) was analyzed by ANOVA.

Article Snippet: Lysates and supernatants were analyzed for expression of recombinant Flt3L by immunoblotting using the anti-myc monoclonal antibody (MAb) 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA) or by a murine Flt3L-specific enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN).

Techniques: Aerosol, In Vivo, Infection

Journal: Infection and Immunity

Article Title: Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

doi: 10.1128/iai.00322-07

Figure Lengend Snippet: FIG. 6. Safety of BCG:Flt3L in immunodeficient mice. (A) RAG- 1/ mice were infected i.v. with 1 106 CFU of BCG:Flt3L (F) or BCG:Ct (E), and survival was monitored over time. The significance of differences in survival was determined by the Mantel-Cox log rank test (asterisk, P 0.001). (B and C) RAG-1/ mice were vaccinated subcutaneously with 5 105 CFU of rBCG strains, and the numbers of CD11c MHC-II cells in the DLNs (B) and spleens (C) of the vaccinated mice were determined by flow cytometry. Striped bars, unvaccinated mice; open bars, BCG:Ct-vaccinated mice; solid bars, BCG:Flt3L-vaccinated mice. There were no statistically significant dif- ferences between groups as determined by ANOVA. The data are representative of one of two individual experiments.

Article Snippet: Lysates and supernatants were analyzed for expression of recombinant Flt3L by immunoblotting using the anti-myc monoclonal antibody (MAb) 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA) or by a murine Flt3L-specific enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN).

Techniques: Infection, Cytometry

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Regression of human mammary adenocarcinoma by systemic administration of a recombinant gene encoding the hFlex-TRAIL fusion protein.

doi: 10.1006/mthe.2001.0280

Figure Lengend Snippet: FIG. 2. Western blot analysis of the fusion protein expressed in the BL6 cell line following transfection with the various recombinant DNA constructs. (A) Detection of hFlex, F81T, F81TZ, FET, and FETZ fusion proteins in the cell lysates by anti-Flt3L (left) and anti-TRAIL (right). (B) Presence of the FET and FETZ fusion proteins in cell culture medium. The F81T and F81TZ fusion proteins were not detectable in cell culture medium. (C) Analysis of FET and FETZ proteins by native 6% PAGE. Native FETZ was assembled in stable trimers compared to the monomeric FET.

Article Snippet: The membrane was then incubated with 5 mg/ml rabbit polyclonal anti-TRAIL antibody or goat polyclonal anti-Flt3L antibody (Santa Cruz Biotech, Santa Cruz, CA) for 1.5 h at room temperature.

Techniques: Western Blot, Transfection, Recombinant, Construct, Cell Culture

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Regression of human mammary adenocarcinoma by systemic administration of a recombinant gene encoding the hFlex-TRAIL fusion protein.

doi: 10.1006/mthe.2001.0280

Figure Lengend Snippet: FIG. 3. Determination of the concentration of the fusion proteins in Flt3L ELISA. (A) Cell medium was collected 24 h after transfection of BL6 cells with the recombinant genes and (B) serum was collected from mice 24 h after injection with 10 mg of recombinant genes. Each bar represents the mean and standard deviation of three independent samples.

Article Snippet: The membrane was then incubated with 5 mg/ml rabbit polyclonal anti-TRAIL antibody or goat polyclonal anti-Flt3L antibody (Santa Cruz Biotech, Santa Cruz, CA) for 1.5 h at room temperature.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Recombinant, Injection, Standard Deviation

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Regression of human mammary adenocarcinoma by systemic administration of a recombinant gene encoding the hFlex-TRAIL fusion protein.

doi: 10.1006/mthe.2001.0280

Figure Lengend Snippet: FIG. 4. Time course of expression of the fusion protein following a single injection of different amounts of pFETZ plasmid DNA in SCID mice. The level of FETZ in the serum was determined using Flt3L ELISA. Each time point represents the mean and standard deviation of three mice.

Article Snippet: The membrane was then incubated with 5 mg/ml rabbit polyclonal anti-TRAIL antibody or goat polyclonal anti-Flt3L antibody (Santa Cruz Biotech, Santa Cruz, CA) for 1.5 h at room temperature.

Techniques: Expressing, Injection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Regression of human mammary adenocarcinoma by systemic administration of a recombinant gene encoding the hFlex-TRAIL fusion protein.

doi: 10.1006/mthe.2001.0280

Figure Lengend Snippet: FIG. 5. Ability of FETZ to induce cell killing of human mammary adenocar- cinoma MDA-231 cells. Cells were incubated with serum, containing fusion proteins (1 mg/ml), obtained from mice 12 h following injection with the various recombinant TRAIL DNA constructs. The concentration of the fusion protein was determined by Flt3L ELISA. Commercial human recombinant TRAIL (hTrail) protein was diluted in normal mouse serum.

Article Snippet: The membrane was then incubated with 5 mg/ml rabbit polyclonal anti-TRAIL antibody or goat polyclonal anti-Flt3L antibody (Santa Cruz Biotech, Santa Cruz, CA) for 1.5 h at room temperature.

Techniques: Incubation, Injection, Recombinant, Construct, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Characterisation of Dendritic Cells Arising from Progenitors Endogenous to Murine Spleen

doi: 10.1371/journal.pone.0088311

Figure Lengend Snippet: ( A ) D0, D2, D4, D5, D6, D7 and adult spleen cells were cultured with Flt3L or GM-CSF/IL-4. Cell production was assessed using antibody staining and multicolor flow cytometry. Gates were set on bivariate plots using isotype control antibodies, and numbers in gates reflect % positive cells. Production of CD11b + CD11c − and CD11b + CD11c + cells was calculated in terms of proportion of each subset amongst all CD11c + and/or CD11b + cells. Representation of CD11b + CD11c + MHC-II − (L-DC) and CD11b + CD11c + MHC-II + (cDC-like) cells was calculated in terms of proportion amongst CD11b + CD11c + cells. ( A ) Proportion of cells induced by Flt3L. ( B ) Proportion of cells induced by GM-CSF/IL-4.

Article Snippet: Flt3L (Genzyme: Cambridge, MA, USA) was added to cultures at a final concentration of 200 ng/ml.

Techniques: Cell Culture, Staining, Flow Cytometry, Control

Journal: PLoS ONE

Article Title: Characterisation of Dendritic Cells Arising from Progenitors Endogenous to Murine Spleen

doi: 10.1371/journal.pone.0088311

Figure Lengend Snippet: Spleen cell cultures were supplemented cultures with Flt3L and GM-CSF/IL-4 were analysed for production of CD11c + CD11b + DC. These subsets were further distinguished by expression of MHC-II, F4/80, Ly6C, CD8, Ly6G and B220.

Article Snippet: Flt3L (Genzyme: Cambridge, MA, USA) was added to cultures at a final concentration of 200 ng/ml.

Techniques: Expressing