Journal: Gene therapy

Article Title: Measles virus selectively blind to signaling lymphocyte activation molecule as a novel oncolytic virus for breast cancer treatment.

doi: 10.1038/gt.2012.44

Figure Lengend Snippet: Figure 2. PVRL4 and not CD46 is involved in the infection of breast cancer cell lines with rMV-SLAMblind. (a) Surface expression of CD46 and PVRL4 in breast cancer cell lines analyzed by flow cytometry. (Left) Cells incubated with anti-CD46 mouse MAb (gray histogram) or isotype control (white histogram) followed by incubation with Alexa-488-conjugated goat anti-mouse antibody. (Right) Cells incubated with anti- PVRL4 goat polyclonal antibody (gray histogram) or isotype control (white histogram) followed by incubation with Alexa-488-conjugated rabbit anti-goat antibody. (b) Cells were pretreated with anti-CD46 or anti-PVRL4 antibody and infected with rMV-EGFP, rMV-EGFP-SLAMblind or rMV-Edmonston at an MOI of 0.1. Cells were incubated in medium with or without antibody and were photographed at 2 (Vero) or 3 d.p.i. (the others). Cells infected with rMV-Edmonston were immunostained using anti-N rabbit polyclonal antibody. Magnification: 100. (c) CHO- K1 cells were transfected with pCAG-hCD46 or the empty vector (pCAGGS). After 2 days, they were infected with rMV, rMV-SLAMblind or rMV- Edmonston at an MOI of 1 and incubated in the presence of fusion inhibitory peptide. At 2 d.p.i., cells were fixed and immunostained using anti-N MAb and the number of infected cells was counted. Infectivity in cells transfected with pCAGGS was set as 100%. Error bars indicate s.e. *Po0.001 versus pCAGGS. (d) BHK cells were transfected with pCAG-hPVRL4 or pCAGGS. After two days, they were infected with rMV, rMV- SLAMblind or rMV-Edmonston at an MOI of 0.1 and incubated in the presence of fusion inhibitory peptide. At 2 d.p.i., infectivity was determined as described in (c). *Po0.001 versus pCAGGS.

Article Snippet: The following antibodies were used as primary antibodies: anti-human SLAM MAb (clone 7D4; Biolegend, San Diego, CA, USA), anti-human CD46 MAb, anti-PVRL4 goat polyclonal antibody (R&D Systems), mouse control IgG1 (R&D Systems) and goat control IgG (R&D Systems).

Techniques: Infection, Expressing, Cytometry, Incubation, Control, Transfection, Plasmid Preparation

Journal: Gene therapy

Article Title: Measles virus selectively blind to signaling lymphocyte activation molecule as a novel oncolytic virus for breast cancer treatment.

doi: 10.1038/gt.2012.44

Figure Lengend Snippet: Figure 3. Infection of CD46-positive normal human cells. (a) Surface expression of CD46, PVRL4 and SLAM in NHDFs analyzed by flow cytometry as described in Figures 1c and 2a. (b, c) NHDFs were infected with rMV-SLAMblind or rMV-Edmonston at an MOI of 1. (b) Cells fixed and immunostained with anti-N MAb at 3 d.p.i. Magnification: 100. (c) Cell viability measured at each time point by WST-1 assay.

Article Snippet: The following antibodies were used as primary antibodies: anti-human SLAM MAb (clone 7D4; Biolegend, San Diego, CA, USA), anti-human CD46 MAb, anti-PVRL4 goat polyclonal antibody (R&D Systems), mouse control IgG1 (R&D Systems) and goat control IgG (R&D Systems).

Techniques: Infection, Expressing, Cytometry, WST-1 Assay

Journal: Journal of Virology

Article Title: In Vitro and In Vivo Properties of Adenovirus Vectors with Increased Affinity to CD46

doi: 10.1128/jvi.01308-08

Figure Lengend Snippet: FIG. 1. Ad35 knob mutants with increased affinity to CD46. (A) Amino acid sequence of the Ad35 fiber knob. -sheet regions (A to J) are shown. Amino acid residues that, when mutated, can increase affinity to CD46 are marked by arrows. (B) Biacore response data for sCD46 binding to a biosensor surface containing Ad35 knob mutants. Experimental data represent the responses of duplicate injections of various concentrations of sCD46 (169 nM, 56 nM, 19 nM, 6 nM, and 2 nM). Note that the time scale for Asp207Gly plus Thr245Ala and Asp207Gly plus Thr245Ala plus Ile256Leu is 3,000 s and that one additional sCD46 concentration (1 nM) was used for these two knob mutants. wt, wild type.

Article Snippet: The blot was then incubated with anti-CD46 antibody (clone J4.48; Fitzgerald, Concord, MA) (1:50 dilution) in TBS and 3% milk for 1 h at RT and then washed three times for 10 min in TBS-T buffer.

Techniques: Sequencing, Binding Assay, Concentration Assay

Journal: Journal of Virology

Article Title: In Vitro and In Vivo Properties of Adenovirus Vectors with Increased Affinity to CD46

doi: 10.1128/jvi.01308-08

Figure Lengend Snippet: FIG. 2. Structural analysis of increased binding Ad35 fiber mu- tants. (A) Ribbon representation of the DE and HI loops of the Ad35 fiber with Asp or Gly residues shown at position 207. CD46 residues 8 to 37 and 104 to 117 are also present, with Ile13 shown. (B) Structural ribbon representation of the FG loop of the Ad35 fiber with Thr and Ala residues shown at position 245 and Thr246 also shown. CD46 residues 58 to 68 are present, with residues Glu63, Thr64, and Tyr67 shown. The orange dashed line shows an interaction previously de- scribed as important for Ad35-CD46 binding (36), while black lines indicate a region of polarity lost upon mutation of Thr245 to Ala245. (C) Structural ribbon representation of the G and H sheets of the Ad35 fiber (residues 249 to 277), with Ile and Leu residues shown at position 256 and Asn271 also shown. Representations of the Ad35 monomer and trimer are shown.

Article Snippet: The blot was then incubated with anti-CD46 antibody (clone J4.48; Fitzgerald, Concord, MA) (1:50 dilution) in TBS and 3% milk for 1 h at RT and then washed three times for 10 min in TBS-T buffer.

Techniques: Binding Assay, Mutagenesis

Journal: Journal of Virology

Article Title: In Vitro and In Vivo Properties of Adenovirus Vectors with Increased Affinity to CD46

doi: 10.1128/jvi.01308-08

Figure Lengend Snippet: FIG. 3. Ad vectors with increased affinity to CD46. (A) Analysis of vector genomes. (Top) Structure vector genomes. (Bottom) Restriction endonuclease analysis (X, XhoI; H, HindIII) of vectors genomes isolated from purified Add5/35, Ad5/35, and Ad5/35 particles. The distinguishing DNA fragments are, for Ad5/35, the 3.14- and 1.78-kb XhoI fragments and, for Ad5/35, the 2.53- and 2.14-kb HindIII fragments. M, 1-kb ladder marker. wt, wild type; CMV, cytomegalovirus; ITR, inverted terminal repeat. (B) Biacore analysis of Ad-sCD46 interactions. Purified Ad particles were immobilized, and binding to sCD46 was analyzed. Sensorgrams are shown for injections at seven different concentrations of sCD46 (50 to 0.781 g/ml in a twofold dilution series). The analysis was performed in triplicate. KD, equilibrium dissociation constant; ka, association rate constant; kd, dissociation rate constant.

Article Snippet: The blot was then incubated with anti-CD46 antibody (clone J4.48; Fitzgerald, Concord, MA) (1:50 dilution) in TBS and 3% milk for 1 h at RT and then washed three times for 10 min in TBS-T buffer.

Techniques: Plasmid Preparation, Isolation, Marker, Binding Assay

Journal: Journal of Virology

Article Title: In Vitro and In Vivo Properties of Adenovirus Vectors with Increased Affinity to CD46

doi: 10.1128/jvi.01308-08

Figure Lengend Snippet: FIG. 4. Transduction of CHO-CD46 cell clones. (A) CHO cells that did not express CD46 (N.D.) and CHO cell clones that expressed CD46 at different levels (1) were infected with Ad5/35 at the indicated MOIs, and the percentages of GFP-expressing cells (left) and GFP mean fluorescence (right) were measured at 24 h by flow cytometry. (B) CHO-CD46 cell clones were infected with Ad5/35, Ad5/35, and Ad5/35, and GFP expression was analyzed as described for panel A. Shown are the averages of three independent experiments. The standard deviations were less then 10% from the averages for all data points.

Article Snippet: The blot was then incubated with anti-CD46 antibody (clone J4.48; Fitzgerald, Concord, MA) (1:50 dilution) in TBS and 3% milk for 1 h at RT and then washed three times for 10 min in TBS-T buffer.

Techniques: Transduction, Clone Assay, Infection, Expressing, Cytometry

Journal: Journal of Virology

Article Title: In Vitro and In Vivo Properties of Adenovirus Vectors with Increased Affinity to CD46

doi: 10.1128/jvi.01308-08

Figure Lengend Snippet: FIG. 6. Analysis of intracellular trafficking in MO7e cells. (A) Attachment of 3H-labeled Ad particles to MO7e cells. MO7e cells were incubated with 3H-labeled Ads at different MOIs, and the numbers of VP bound per cell after 1 h of incubation on ice were determined. (B) Inhibition of Ad transduction by anti-CD46 antibodies. MO7e cells were incubated with 10 g/ml anti-human CD46 antibody (clone MEM-258; Serotec Ltd., Oxford, United Kingdom) or 10 g/ml anti-human isotype control antibody for 45 min on ice, washed once with PBS, and then infected with 40 PFU/cell of Ad5/35, Ad5/35, or Ad5/35. Twenty-four hours after infection, GFP expression was analyzed. Shown are the averages of three independent experiments. The standard deviations were less then 10% from the averages for all data points in panels A and B. (C) Immunoflu- orescence analysis of Ad particles (red) and the late endosomal marker cathepsin B (green) in MO7e cells 4 h after infection with Ad5/35 (left) or Ad5/35 (right) at an MOI of 4,000 VP per cell. (D) Electron microscopy analysis of intracellular distribution of Ad5/35 and Ad5/35 vector particles in MO7e cells 4 h after virus infection. Representative fields are shown. Low magnification, 6,000; high magnification, 20,000. (Right) Quantification of Ad particles inside cells (n 100 cells).

Article Snippet: The blot was then incubated with anti-CD46 antibody (clone J4.48; Fitzgerald, Concord, MA) (1:50 dilution) in TBS and 3% milk for 1 h at RT and then washed three times for 10 min in TBS-T buffer.

Techniques: Labeling, Incubation, Inhibition, Transduction, Control, Infection, Expressing, Marker, Electron Microscopy, Plasmid Preparation, Virus

Journal: Journal of Virology

Article Title: In Vitro and In Vivo Properties of Adenovirus Vectors with Increased Affinity to CD46

doi: 10.1128/jvi.01308-08

Figure Lengend Snippet: FIG. 7. Analysis of CD46 distribution in infected MO7e cells. (A) Colocalization of CD46 and virus. CD46 (green) was analyzed before infection and at 15 min and 4 h after infection with Ad5/35 and Ad5/35 at an MOI of 4,000 VP per cell. Cathepsin B-positive endosomal compartments are green. Confocal images of cells were taken in both green and red channels. Representative fields are shown. (B) Analysis of CD46 on the surfaces of MO7e cells and GFP expression at different time points after infection with Ad5/35, Ad5/35, or Ad5/35 at MOIs of 50 PFU/cell and 400 PFU/cell. Notably, staining for the viral hexon at 8 h postinfection did not reveal any signal at the cell surface, indicating that the decrease in CD46-PE fluorescence was not due to competition with Ad particles bound to surface CD46. Shown are the averages of three independent experiments. The standard deviations were less then 10% from the averages for all data points.

Article Snippet: The blot was then incubated with anti-CD46 antibody (clone J4.48; Fitzgerald, Concord, MA) (1:50 dilution) in TBS and 3% milk for 1 h at RT and then washed three times for 10 min in TBS-T buffer.

Techniques: Infection, Virus, Expressing, Staining

Journal: Journal of Virology

Article Title: In Vitro and In Vivo Properties of Adenovirus Vectors with Increased Affinity to CD46

doi: 10.1128/jvi.01308-08

Figure Lengend Snippet: FIG. 8. GFP expression in organs and liver metastases after intravenous injection of Ad5/35 and Ad5/35 into CD46-transgenic mice bearing CD46high liver metastases. Mice were intraportally injected with TC1-CD46 tumor cells. Three weeks later, when liver metastases were established, mice received tail vein injection of 3 109 PFU of Ad5/35 and Ad5/35 (n 4). Organ sections were analyzed for GFP expression 3 days later. (A) Representative sections. (B) Quantification of GFP-positive cells/mm2 in liver metastases (tumor), liver, and spleen (20 sections per mouse, four mice per virus). (C) Quantification of GFP-positive cells in cell suspensions of microdissected liver metastases. Tumor tissue was digested with collagenase and versene, and cell suspensions were analyzed by flow cytometry for GFP and CD46 expression. Representative samples are shown. Transduced TC1-CD46 cells appear as GFP CD46high cells (upper right quadrant). Cells in the upper and lower left quadrants represent cell debris that stained positive with propidium iodide (data not shown).

Article Snippet: The blot was then incubated with anti-CD46 antibody (clone J4.48; Fitzgerald, Concord, MA) (1:50 dilution) in TBS and 3% milk for 1 h at RT and then washed three times for 10 min in TBS-T buffer.

Techniques: Expressing, Injection, Transgenic Assay, Virus, Cytometry, Staining

Journal: Oncotarget

Article Title: Efficacy of CD46-targeting chimeric Ad5/35 adenoviral gene therapy for colorectal cancers.

doi: 10.18632/oncotarget.9427

Figure Lengend Snippet: Figure 1: Gene transduction efficacy of Ad5/35 is enhanced in CD46-expressing cells. A. Western blot analysis of CD46 expression in parental rodent BHK cells or BHK-CAR and BHK-CD46 cells which ectopically express the Ad receptors CAR and CD46, respectively. B. Flow cytometric analysis of CD46 expression in BHK, BHK-CAR and BHK-CD46 cells. C. Transduction analysis of BHK, BHK-CAR and BHK-CD46 cells using different doses of Ad5/35-GFP. Cells were analyzed for GFP reporter expression 24 hrs post transduction by flow cytometry. Numbers in parentheses indicate percentage of GFP positive cells. D. Cell killing assay in BHK, BHK- CAR and BHK-CD46 cells transduced with Ad5/35-tk and treated with GCV for 4 days. Cytotoxicity was analyzed by the MTT assay. Error bars represent SEM. Statistics: C-D, p<0.01 by 2-way ANOVA.

Article Snippet: Cells were harvested and incubated with rabbit polyclonal CD46 antibody (Origene, Rockville, MD, USA) for 30 min at 4°C.

Techniques: Transduction, Expressing, Western Blot, Flow Cytometry, MTT Assay

Journal: Oncotarget

Article Title: Efficacy of CD46-targeting chimeric Ad5/35 adenoviral gene therapy for colorectal cancers.

doi: 10.18632/oncotarget.9427

Figure Lengend Snippet: Figure 2: CD46 expression analysis and Ad5/35-mediated gene transduction in colorectal cancer cell lines. A. CD46 expression in colon cancer cell lines was analyzed by Western blot analysis. B. CD46-positive cells were detected by flow cytometry in response to different dose of Ad5/35-GFP. C-E. Colon cancer cells were applied to flow cytometry to demonstrate intensity of CD46 expression (C) or Ad5/35-mediated reporter transduction (D). Percentage of cells double-stained with CD46 and GFP was shown in E. Error bars represent SEM. Statistics: B, p<0.01 by 1-way ANOVA and post-hoc Tukey test; C-D, p=0.267 by 2-way ANOVA.

Article Snippet: Cells were harvested and incubated with rabbit polyclonal CD46 antibody (Origene, Rockville, MD, USA) for 30 min at 4°C.

Techniques: Expressing, Transduction, Western Blot, Flow Cytometry, Staining

Journal: Oncotarget

Article Title: Efficacy of CD46-targeting chimeric Ad5/35 adenoviral gene therapy for colorectal cancers.

doi: 10.18632/oncotarget.9427

Figure Lengend Snippet: Figure 4: CD46 promotes Ad5/35-tk-mediated cytotoxicity for tumor growth in vivo. A-B. Cells were transduced with Ad5/35-tk followed by GCV treatment. MTT in vitro proliferation assays were performed 5 days post infection. C-D. Cells were injected subcutaneously into nude mice (10-11 mice per group). Intra-tumor injections of Ad5/35-tk viruses (1.5 × 108 PFU) were made twice at the indicated time points followed by GCV injections (75 mg/kg) intraperitoneally on days 2–12. Tumor growth was measured by a caliper at the indicated time points. E. Cell proliferation of parental A549 and CD46-overexpressing A549 was measured by the MTT assay. Error bars represent SEM. Statistics: A-B, p<0.01 by 2-way ANOVA; D, p=0.005 by repeated-measures ANOVA; E, p=0.44 by 1-way ANOVA.

Article Snippet: Cells were harvested and incubated with rabbit polyclonal CD46 antibody (Origene, Rockville, MD, USA) for 30 min at 4°C.

Techniques: In Vivo, Transduction, In Vitro, Infection, Injection, MTT Assay

Journal: Oncotarget

Article Title: Efficacy of CD46-targeting chimeric Ad5/35 adenoviral gene therapy for colorectal cancers.

doi: 10.18632/oncotarget.9427

Figure Lengend Snippet: Figure 5: Immunohistochemical analysis of CD46 expression in patient colorectal cancers. Tumors were analyzed for the expression of CD46 by immunostaining. A, normal mucosa. B-C, well- differentiated tumors. D-F, poorly differentiated and highly invasive tumors. While CD46 was generally overexpressed in colorectal tumors compared to normal counterparts, some invasive cancers showed no expression of CD46. High-powered pictures are shown in inlets of selected areas.

Article Snippet: Cells were harvested and incubated with rabbit polyclonal CD46 antibody (Origene, Rockville, MD, USA) for 30 min at 4°C.

Techniques: Immunohistochemical staining, Expressing, Immunostaining

Journal: Oncotarget

Article Title: Efficacy of CD46-targeting chimeric Ad5/35 adenoviral gene therapy for colorectal cancers.

doi: 10.18632/oncotarget.9427

Figure Lengend Snippet: Figure 6: CD46 expression indicates better survival of CRC patients. Overall survivability was demonstrated by Kaplan-Meier curve and measured by the log-rank test (p=0.031).

Article Snippet: Cells were harvested and incubated with rabbit polyclonal CD46 antibody (Origene, Rockville, MD, USA) for 30 min at 4°C.

Techniques: Expressing

Journal: Heliyon

Article Title: Proteolytic shedding of CD46 from human hepatocytes indicates liver stress

doi: 10.1016/j.heliyon.2024.e40841

Figure Lengend Snippet: Establishing and validating an ELISA for soluble CD46 (sCD46) . (A) Signal:noise (S:N) values for different concentrations of CD46 capture and detection antibodies in Reagent Diluent 2 (n = 2). (B) Titration curve using recombinant human CD46 (rhCD46) as the analyte. Lower limit of quantification (LLOQ), upper limit of quantification (ULOQ) and limit of detection (LOD) are indicated (n = 3). (C) Accuracy (spike recovery) of human serum samples (n = 5). (D) Intra-assay precision, inter-assay precision and reproducibility of human serum sample at two different dilution factors. (E) Analysis of assay linearity (n = 4, Pearson correlation, indicating 90 % prediction bands). (F) Linear regression of the bias plotted against nominal titres (n = 4, Pearson correlation). (G) Robustness based on incubation timing (n = 4, one sample t -test to a mean of 1). (H) Stability of sCD46 (pg/ml) in representative cell culture supernatants over multiple freeze/thaw cycles depending on the storage temperature and the application of a protease inhibitor. (I) rhCD46 titration in ELISA and competitive FACS-based assay (n = 3). (J) Performance of commercial CD46 ELISA kits in detecting serum sCD46 compared to our in-house ELISA. Samples dilutions were based on manufacturers' recommendations.

Article Snippet: Next, 100 μl/well of samples, the corresponding 1:2 serial diluted standards of rhCD46 (2 ng/ml to 31.25 pg/ml, 10256-CD, R&D Systems) and a blank control are added and then incubated for 2 h. Then, 100 μl/well detection antibody (50 ng/ml, BAF2005, R&D Systems) is added followed by another incubation for 2 h. Next, 100 μl/well of Streptavidin-HRP (DY998, R&D Systems) is added followed by 100 μl/well of substrates (DY999, R&D Systems) for 20 and 30 min, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Titration, Recombinant, Intra Assay, Inter Assay, Incubation, Cell Culture, Protease Inhibitor