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: 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

Journal: Clinical Medicine Insights. Arthritis and Musculoskeletal Disorders

Article Title: Macroscopical, Histological, and In Vitro Characterization of Nonosteoarthritic Versus Osteoarthritic Hip Joint Cartilage

doi: 10.4137/CMAMD.S29844

Figure Lengend Snippet: Expression of complement receptors (C5aR and C3aR) and CRPs (CD46, CD55, and CD59) in cartilage in situ. FNF and OA cartilage sections immunolabeled for ( A 1–2 ) C5aR, ( B 1–2 ) C3aR, ( C 1–2 ) CD46, ( D 1–2 ) CD55, and ( E 1–2 ) CD59. Notes: Cell nuclei were counterstained using DAPI (blue). Scale bar: 200 µm. Negative controls (B 3 ) performed by using only the secondary antibody revealed no unspecific staining.

Article Snippet: The primary antibodies (rabbit anti-human C3aR [1:30, Assay Biotechnology], mouse anti-human C5aR [1:100, GeneTex, Biozol], mouse anti-human CD46, CD59 [1:20 and 1:40, both AbD Serotec], and goat anti-human CD55 [1:40, R&D Systems]) were diluted in blocking buffer containing 0.1% Triton X-100 (Sigma-Aldrich) and incubated overnight at 4 °C in a humidified chamber.

Techniques: Expressing, In Situ, Immunolabeling, Staining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Preclinical safety, pharmacokinetics, pharmacodynamics, and biodistribution studies with Ad35K++ protein: a novel rituximab cotherapeutic

doi: 10.1038/mtm.2016.13

Figure Lengend Snippet: Characterization of Ad35K++. ( a ) Polyacrylamide sodium dodecyl sulfate (SDS) gel analysis of Ad35K++. Five micrograms of protein samples were loaded in Laemmli buffer containing β-mercaptoethanol with or without boiling for 5 minutes. The right graphic shows a schematic of Ad35K++ in the various states: Native Ad35K++ contains three monomers comprising the C-terminal, affinity-enhanced fiber knob, which interacts with CD46 and a shaft motif required for trimerization. During production, the monomer spontaneously trimerizes. When boiled under denaturing and reducing conditions, the trimer dissociates into monomers. ( b ) Ad35K++ activity in the rituximab complement-dependent cytotoxicity assay using Raji cells. Raji cells were incubated with Ad35K++ (25 µg/ml) for 8 hours followed by rituximab (15 µg/ml). After 30 minutes, normal human serum (NHS) was added and viable cells were counted 3 hours later based on trypan blue exclusion. Controls included incubation with phosphate-buffered saline (PBS)/NHS or rituximab/NHS. The viability of PBS/NHS-treated cells was taken as 100%. N = 3 independent experiments. Rituximab/NHS versus Ad35K++/rituximab/NHS: P < 0.001.

Article Snippet: The mouse anti-human CD46 monoclonal antibody (clone 344519) and the goat anti-human CD46 polyclonal antibody were from R&D Systems (Minneapolis, MN).

Techniques: SDS-Gel, Activity Assay, CDC Assay, Incubation, Saline

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Preclinical safety, pharmacokinetics, pharmacodynamics, and biodistribution studies with Ad35K++ protein: a novel rituximab cotherapeutic

doi: 10.1038/mtm.2016.13

Figure Lengend Snippet: Pharmacokinetics (PK) and biodistribution studies in CD46tg mice. ( a ) Ad35K++ serum clearance. Ad35K++ serum concentrations were measured by enzyme-linked immunosorbent assay. Left panel: Summary of data from six animals. Right panel: Semi-log plots of average serum concentrations versus time profile (used for calculating the PK values). ( b ) Ad35K++ concentration in tissues at 6 hours after intravenous injection into hCD46 transgenic mice. Shown are Ad35K++ concentrations normalized to total protein as measured using the Bradford assay. N = 3.

Article Snippet: The mouse anti-human CD46 monoclonal antibody (clone 344519) and the goat anti-human CD46 polyclonal antibody were from R&D Systems (Minneapolis, MN).

Techniques: Drug discovery, Enzyme-linked Immunosorbent Assay, Concentration Assay, Injection, Transgenic Assay, Bradford Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Preclinical safety, pharmacokinetics, pharmacodynamics, and biodistribution studies with Ad35K++ protein: a novel rituximab cotherapeutic

doi: 10.1038/mtm.2016.13

Figure Lengend Snippet: In vivo activity of Ad35K++ in the Raji tumor xenograft mouse model. Immunodeficient CB17-SCID/beige mice received human lymphoma Raji cells via tail vein injection. ( a ) Three cycles of treatment were given at days 8, 11, and 14 after Raji cell injection (see arrows). Treatment groups were: (i) phosphate-buffered saline (PBS), (ii) Ad35K++ (2 mg/kg), (iii) rituximab (2 mg/kg), (iv) Ad35K++ followed 10 hours later by rituximab (“Ad35K++ → rituximab”), and (v) Ad35K++ given together with rituximab (“Ad35K++ plus rituximab”). Kaplan-Meier survival analysis with days of onset of hind leg paralysis as endpoints. N = 5. ( b ) CD46 shedding after Ad35K++ injection. Mice with established Raji tumors were intravenously injected with Ad35K++ at 2 mg/kg on day 17 after Raji cell injection (see arrow). The control group received PBS. Serum samples before and after Ad35K++/PBS injection were collected and CD46 was measured by enzyme-linked immunosorbent assay using antibodies that recognize the extracellular domain of CD46. N = 5.

Article Snippet: The mouse anti-human CD46 monoclonal antibody (clone 344519) and the goat anti-human CD46 polyclonal antibody were from R&D Systems (Minneapolis, MN).

Techniques: In Vivo, Activity Assay, Injection, Saline, Control, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Preclinical safety, pharmacokinetics, pharmacodynamics, and biodistribution studies with Ad35K++ protein: a novel rituximab cotherapeutic

doi: 10.1038/mtm.2016.13

Figure Lengend Snippet: Soluble CD46 serum levels in M. fascicularis . CD46 was measured by enzyme-linked immunosorbent assay with human CD46 extracellular domain (ECD)-specific antibodies that cross-react with macaque CD46. ( a ) Results from animals that were in the dose finding study which went for 7 days. ( b,c ) Results from animals that were in the 14-day efficacy study ( b ) and 6-hour biodistribution study ( c ). Ad35K++ doses are indicated in each graph.

Article Snippet: The mouse anti-human CD46 monoclonal antibody (clone 344519) and the goat anti-human CD46 polyclonal antibody were from R&D Systems (Minneapolis, MN).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Preclinical safety, pharmacokinetics, pharmacodynamics, and biodistribution studies with Ad35K++ protein: a novel rituximab cotherapeutic

doi: 10.1038/mtm.2016.13

Figure Lengend Snippet: Flow cytometry on M. fascicularis PBMC. ( a ) CD46 levels were assessed based on the mean fluorescence intensity (MFI) after staining with a goat polyclonal anti-CD46 antibody followed by an anti-goat Alexa Fluor 488 antibody. The relative percentage of CD46 MFI compared to pretreatment controls is shown. ( b ) Percentage relative to pretreated controls of CD20-, CD4-, CD8-, and PD-1-positive PBMC subsets. Differences in the baseline levels of subsets between individual animals are common in NHPs.

Article Snippet: The mouse anti-human CD46 monoclonal antibody (clone 344519) and the goat anti-human CD46 polyclonal antibody were from R&D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Fluorescence, Staining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Preclinical safety, pharmacokinetics, pharmacodynamics, and biodistribution studies with Ad35K++ protein: a novel rituximab cotherapeutic

doi: 10.1038/mtm.2016.13

Figure Lengend Snippet: Effect of Ad35K++ on rituximab killing of CD20 cells in M. fascicularis . Ad35K++ (4 mg/kg) was injected 10 minutes after preinjection samples (pre) were collected. Rituximab (0.01 mg/kg) was injected 10 minutes after day 2 blood samples were collected. The upper graph shows CD46 levels on CD20 cells (red lines) and the percentage of CD20+ cells compared to baseline (blue line). The lower graphs show a similar analysis of CD4+ cells.

Article Snippet: The mouse anti-human CD46 monoclonal antibody (clone 344519) and the goat anti-human CD46 polyclonal antibody were from R&D Systems (Minneapolis, MN).

Techniques: Injection

Journal: BMC Biology

Article Title: Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library

doi: 10.1186/s12915-020-00905-1

Figure Lengend Snippet: Selected Cas9-expressing single cell clones show stronger editing efficiency compared to a Cas9 bulk population. a Workflow for the selection of Cas9 single-cell clones (SCCs). SCCs were sorted from the HAP1 Cas9 bulk population and further characterized. Cas9 editing was assessed by cell surface marker knockout followed by FACS staining and cell viability upon knockout of a core essential gene. Two highly editing single-cell clones (SCC11 and SCC12) were selected for further experiments. b HAP1 Cas9 bulk, Cas9 SCC11, and Cas9 SCC12 cells were transfected with the HDCRISPRv1 vector encoding an sgRNA targeting either the safe harbor locus AAVS1 as a control or the core essential gene RNA Polymerase 2 subunit E ( POLR2E ). Editing efficiency based on cell viability of sgPOLR2E-transfected cells in comparison to sgAAVS1 control cells was addressed by crystal violet staining. The number of surviving cells was strongly reduced in cells transfected with an sgRNA directed against POLR2E ( n = 3 for each cell line and sgRNA). c Editing efficiency was furthermore assessed upon transduction of HAP1 Cas9 bulk, Cas9 SCC11, and Cas9 SCC12 cells with the HDCRISPRv1 vector expressing sgRNAs targeting the surface marker CD46 , followed by FACS staining of residual CD46 protein to address knockout efficiency. Antibody staining of the non-edited cell lines was used as a control. Lines represent the mean of independent measurements ( n = 3 for each cell line and condition)

Article Snippet: Five days after transduction, cells were harvested and the respective surface markers stained with an APC anti-human CD46 antibody (Biozol Diagnostica, Cat. No. 352405, RRID AB_2564356) or an APC anti-human CD81 antibody (Biozol Diagnostica, Cat. No BLD-349510, RRID AB_2564021).

Techniques: Expressing, Clone Assay, Selection, Marker, Knock-Out, Staining, Transfection, Plasmid Preparation, Transduction

Journal:

Article Title: Distinct Proinflammatory Host Responses to Neisseria gonorrhoeae Infection in Immortalized Human Cervical and Vaginal Epithelial Cells

doi: 10.1128/IAI.69.9.5840-5848.2001

Figure Lengend Snippet: Immunocytochemical analysis of adhesion molecule expression by cervicovaginal epithelial cells in vitro and in vivo. Positive cells appear red. (A and B) Constitutive expression of CD46 in endocervical tissue (A) and endocervical cell culture (B). (C to G) Expression of CD66 in vaginal tissue (C), ectocervical tissue (D), endocervical tissue (E), uninfected endocervical cell culture (F), and endocervical cell culture after 8 h of infection with the N. gonorrhoeae F62 piliated variant (G). (H to K) Expression of ICAM-1 in endocervical cell cultures with no infection (H), following 8 h of TNF-α stimulation (I), following 8 h of infection with N. gonorrhoeae piliated F62 (J), or following 8 h of infection with N. gonorrhoeae nonpiliated F62 (K). L. lumenal epithelial surface: B. basal epithelial layers. Magnification, ×125 (A, C to E, and H to K) and ×250 (B, F, and G).

Article Snippet: Anti-human CD46 antibody (mouse monoclonal MAS657) was purchased from Accurate Chemical and Scientific Corporation, Westbury, N.Y. Fluorescein isothiocyanate (FITC)-conjugated pan-CD66 antibody (mouse monoclonal F7112) recognizing an epitope common to four members of the CD66 family (CD66a, -b, -c, and -e) was purchased from Dako, Carpinteria, Calif.

Techniques: Expressing, In Vitro, In Vivo, Cell Culture, Infection, Variant Assay