Journal: Molecular immunology

Article Title: Autoantibodies to CD59, CD55, CD46 or CD35 are not associated with atypical haemolytic uraemic syndrome (aHUS)

doi: 10.1016/j.molimm.2014.07.017

Figure Lengend Snippet: (a) Newcastle preparation, after dialysis (Lane 1, 20 µl and lane 2, 10 µl), or St. Louis prep (lane 5), were loaded in non-reducing SDS–PAGE buffer (irrelevant lanes are not shown). (b) 400 ng London (1), St. Louis (2) and Newcastle (3) rhCD46 prep., or BSA (4) were run on 15% SDS–PAGE and blotted. rhCD46 was detected using GB24 and shee panti-mouse – HRPO (both 1:1000) and a 1 min exposure is shown for each blot. (c) Again 400 ng of each preparation and BSA (1–4 as above) were loaded onto two identical15% gels and blotted. The Nitrocellulose was divided in two and then probed with GB24 or P1 sera (1/300) or P1 IgG (1/50) or BDC sera (1/300) as indicated. The four ‘strips’ were aligned and exposed for 5 min. (d) Coomassie stained gel of GB24 purified Newcastle produced rhCD46. “Page ruler markers” are indicated throughout.

Article Snippet: Testing and purification of E. coli produced recombinantCD46 (membrane cofactor protein, MCP) We originally received a batch of recombinant human CD46 (rhCD46) protein, generated in E. coli from Dr Claudia Kemper’s group (London, UK) and used this to screen our BDC and aHUS cohorts for the presence of autoantibodies to CD46.

Techniques: SDS Page, Staining, Purification, Produced

Journal: Molecular immunology

Article Title: Autoantibodies to CD59, CD55, CD46 or CD35 are not associated with atypical haemolytic uraemic syndrome (aHUS)

doi: 10.1016/j.molimm.2014.07.017

Figure Lengend Snippet: (a) A standard curve based on the mAb GB24 (1 µg/ml) binding to rhCD46 using ELISA is shown, allowing RU values to be assigned to test samples in the ELISA. (b) Indicates the relative unit (RU) values of 100 healthy blood donor controls (BDC) and 89 aHUS patient samples. Mean RU values are indicated by the solid line in each cohort and a dashed line representing the 97.5 percentile of the BDC group is also indicated. (c) The highest reacting BDC and aHUS samples, as indicated, were applied to nitrocellulose strips from a Western blotted rhCD46 prep gel. Each strip (delineated by vertical bars) was aligned prior to exposure for 20 min. Irrelevant intervening strips have been removed in this picture. GB24 was used as a positive control for protein loading and position. Molecular weight markers are shown and results are representative of two experiments.

Article Snippet: Testing and purification of E. coli produced recombinantCD46 (membrane cofactor protein, MCP) We originally received a batch of recombinant human CD46 (rhCD46) protein, generated in E. coli from Dr Claudia Kemper’s group (London, UK) and used this to screen our BDC and aHUS cohorts for the presence of autoantibodies to CD46.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Stripping Membranes, Positive Control, Molecular Weight

Journal: Xenotransplantation

Article Title: Synthetic Liver Function is Detectable in Transgenic Porcine Livers Perfused with Human Blood

doi: 10.1111/xen.12361

Figure Lengend Snippet: Experimental Genotypes and Drug Regimens

Article Snippet: Twenty transgenic pigs were obtained from Revivicor Inc (Blacksburg, VA) with the following genotypes: α1,3-galactosyl transferase knock out and human membrane cofactor (GalTKO.hCD46, “2-gene”, n=8), GalTKO.hCD46 and N-glycolylneuraminic acid knock out (GalTKO.hCD46.Neu5GCKO, “3-gene”, n=6), and GalTKO.hCD46 and human decay-accelerating factor (hCD55), endothelial protein C receptor (hEPCR), tissue factor pathway inhibitor (hTFPI), and integrin associated protein (hCD47) (GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47, “6-gene”, n=6).

Techniques:

Journal: Xenotransplantation

Article Title: Synthetic Liver Function is Detectable in Transgenic Porcine Livers Perfused with Human Blood

doi: 10.1111/xen.12361

Figure Lengend Snippet: Kaplan-Meier curves showing pig liver survival time for GalTKO.hCD46 (2-gene), GalTKO.hCD46.Neu5GcKO (3-gene), and GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47 (6-gene) livers.

Article Snippet: Twenty transgenic pigs were obtained from Revivicor Inc (Blacksburg, VA) with the following genotypes: α1,3-galactosyl transferase knock out and human membrane cofactor (GalTKO.hCD46, “2-gene”, n=8), GalTKO.hCD46 and N-glycolylneuraminic acid knock out (GalTKO.hCD46.Neu5GCKO, “3-gene”, n=6), and GalTKO.hCD46 and human decay-accelerating factor (hCD55), endothelial protein C receptor (hEPCR), tissue factor pathway inhibitor (hTFPI), and integrin associated protein (hCD47) (GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47, “6-gene”, n=6).

Techniques:

Journal: Xenotransplantation

Article Title: Synthetic Liver Function is Detectable in Transgenic Porcine Livers Perfused with Human Blood

doi: 10.1111/xen.12361

Figure Lengend Snippet: A: ALT levels rose slowly throughout perfusion and remained within normal limits in the GalTKO.hCD46.Neu5GcKO (3-gene) and GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47 (6-gene) groups. ALT levels rose more sharply in the GalTKO.hCD46 (2-gene) group. B: When controlling for one experiment in the GalTKO.hCD46 (2-gene) group that had disproportionately high ALT and AST levels for unknown reasons there was no difference in ALT levels between the three groups. C: AST levels remained low in the GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47 (6-gene) group. AST levels rose to above physiologically normal levels in the GalTKO.hCD46 (2-gene) and GalTKO.hCD46.Neu5GcKO (3-gene) groups with the highest levels seen in the GalTKO.hCD46 group (2-gene). D: When controlling for one experiment in the GalTKO.hCD46 (2-gene) group that had disproportionately high ALT and AST levels for unknown reasons the AST levels of the GalTKO.hCD46 (2-gene) and GalTKO.hCD46.Neu5GcKO (3-gene) groups were not significantly different.

Article Snippet: Twenty transgenic pigs were obtained from Revivicor Inc (Blacksburg, VA) with the following genotypes: α1,3-galactosyl transferase knock out and human membrane cofactor (GalTKO.hCD46, “2-gene”, n=8), GalTKO.hCD46 and N-glycolylneuraminic acid knock out (GalTKO.hCD46.Neu5GCKO, “3-gene”, n=6), and GalTKO.hCD46 and human decay-accelerating factor (hCD55), endothelial protein C receptor (hEPCR), tissue factor pathway inhibitor (hTFPI), and integrin associated protein (hCD47) (GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47, “6-gene”, n=6).

Techniques:

Journal: Xenotransplantation

Article Title: Synthetic Liver Function is Detectable in Transgenic Porcine Livers Perfused with Human Blood

doi: 10.1111/xen.12361

Figure Lengend Snippet: A, B: Representative blots of albumin production. First lane showing molecular weight marker (MW). Minimal pig albumin signal was detected in human plasma samples (Human), while pig plasma (Pig) was strongly positive. Albumin bands (red) increased in intensity over time in all experiments. An appropriate IgG loading control band (green) was observed in all experimental lanes. C: Band intensities were measured by densitometry and the background signal detected in human control plasma was subtracted from each time point in the same gel. Total albumin detected was then divided by the duration of perfusion to account for different lengths of perfusions and obtain intensity/hr units. The GalTKO.hCD46.Neu5GCKO (3-gene) livers had the most robust albumin production.

Article Snippet: Twenty transgenic pigs were obtained from Revivicor Inc (Blacksburg, VA) with the following genotypes: α1,3-galactosyl transferase knock out and human membrane cofactor (GalTKO.hCD46, “2-gene”, n=8), GalTKO.hCD46 and N-glycolylneuraminic acid knock out (GalTKO.hCD46.Neu5GCKO, “3-gene”, n=6), and GalTKO.hCD46 and human decay-accelerating factor (hCD55), endothelial protein C receptor (hEPCR), tissue factor pathway inhibitor (hTFPI), and integrin associated protein (hCD47) (GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47, “6-gene”, n=6).

Techniques: Molecular Weight, Marker

Journal: Science Advances

Article Title: Structural basis for HCMV Pentamer receptor recognition and antibody neutralization

doi: 10.1126/sciadv.abm2536

Figure Lengend Snippet: ( A ) HUVEC or ARPE-19 cells were infected for 48 hours with VR1814 virus that had been preincubated with different concentrations of an anti-NRP2 antibody (red open circles), soluble recombinant CD46 (blue full circle), NRP2 (red full circle), THBD (orange full circle), a mix of recombinant NRP2 with THBD proteins (violet full circle), or a mix of anti-NRP2 with recombinant THBD proteins (violet full circle). The percentage of infected cells plotted against antibody or protein concentration is shown. The data shown are the means of three independent experiments ± SD. ( B ) Histogram representing the percentage of infection of HAP-1 cells WT or NRP2-KO that have been transduced with either empty lentivirus vector, lentivirus vector encoding CD46, or lentivirus vector encoding THBD. The percentage of maximum infection (geometric means of four independent experiments ± SD) is shown. n.s., not significant. ( C ) Overlay of HCMV Pentamer in complex with NRP2 and THBD. The a1 domain is shown in surface representation. ( D ) Binding of HCMV Pentamer to NRP2-Fc WT and increasing concentrations of THBD starting at equimolar ratio or addition of 100-fold excess of transforming growth factor β receptor 3 (TGFβR3) as a control. ( E ) Overlay between the HCMV Pentamer–NRP2 dimer cryo-EM map and the dimeric structure of HCMV Pentamer–THBD. ( F ) HCMV Pentamer dimerization interface mediated by UL128. ( G ) Close-in view of the dimeric interaction interface mediated by UL128 [top view relative to view in (G)].

Article Snippet: For cell surface staining, adherent cells (ARPE-19, HUVEC, and HAP-1) were gently detached with a cell scraper, washed twice with PBS + 2% FBS, and incubated in PBS + 0.5% bovine serum albumin (BSA) + 2 mM EDTA with specific anti-NRP2 and anti-CD46 antibodies (AF2215 and AF2005, R&D Systems), anti-THBD antibody (clone 141C01, Abcam), or, as isotype control, normal sheep immunoglobulin G (IgG) affinity pure (R&D Systems) at 2 μg/ml for 30 min on ice.

Techniques: Infection, Virus, Recombinant, Protein Concentration, Transduction, Plasmid Preparation, Binding Assay, Control, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: CD46 facilitates entry and dissemination of human cytomegalovirus

doi: 10.1038/s41467-019-10587-1

Figure Lengend Snippet: Identification of inhibitory monoclonal antibodies and their cellular target. a Supernatants from cell-surface binding clones were subjected to a high-throughput infectivity assay (HTI) with TB40/E FLAG YFP infection of ARPE-19 cells using YFP fluorescence as readout for infection. The % infection was determined using virus incubated with media alone as 100%. b Clones demonstrating reduced infection were validated using the TB40/E FLAG YFP /ARPE-19 cells HTI with varying amounts (%) of supernatant. c Purified mAbs (10 μg ml −1 ) were analyzed using the TB40/E FLAG YFP /ARPE-19 HTI in technical triplicates. d Polypeptides recovered with mAb 2E7 and 12H8 (arrows) from ARPE-19 cells metabolically labeled with 35 S-methinionine (6 h) were resolved on a SDS-polyacrylamide gel and visualized on a radiographic film. Beads only was used as a control. e Polypeptides recovered with mAb 2E7 and 12H8 from ARPE-19 cells were subjected to immunoblot analysis using anti-CD46 antibodies. mAb W6/32 and total cell lysates (TCL) were included as controls. The polypeptides and molecular weight markers are indicated. s.d. is depicted in the experiment. * P < 0.05, ** P < 0.01 (Student’s two-tailed t test)

Article Snippet: Virus (MOI:0.5) incubated with respective mAbs (2 μg ml −1 , or otherwise noted) or soluble proteins Nrp2 (P) and CD46 (P) (Sino Biological (cat#: 10695-H08H and 12239-H08H) for 1 h 4 °C was used to infect ARPE-19, HTR-8/SVneo, or MRC5 cells (1 × 10 4 cell/well in a 96-well plate) for 2 h (37 °C) and then replaced with new media.

Techniques: Binding Assay, Clone Assay, High Throughput Screening Assay, Infection, Fluorescence, Incubation, Purification, Metabolic Labelling, Labeling, Western Blot, Molecular Weight, Two Tailed Test

Journal: Nature Communications

Article Title: CD46 facilitates entry and dissemination of human cytomegalovirus

doi: 10.1038/s41467-019-10587-1

Figure Lengend Snippet: CD46-dependent CMV entry in epithelial cells. a TB40/E wt was subjected to a mAb inhibition HTI in ARPE-19 cells with mAb 5C3 (anti-gH), mAb 2E7 (anti-CD46), mAb PY102 (non-binding control) or no-antibody control (20-0.01 μg ml −1 , in threefold dilutions). Virus infection from PY102-treated cells represents 100% infection. b Total cell lysates from uninfected, TB40/E wt and AD169 BADrUL131 infected ARPE-19 cells treated with mAbs 5C3 or 2E7 (10 and 2 μg ml −1 ), mAb PY102 (10 μg ml −1 ), and no mAb were subject to immunoblot analysis for IE1 and GAPDH. ( c ) CD46 protein model represents four consensus repeats (SCRs) where complement proteins C4b, C3b, and C4b bind, a serine/threonine/proline (STP)-rich region, an uncharacterized segment (U), a transmembrane domain (TM), and a cytoplasmic tail . Alternative splicing accounts for common isomers expressing either STP regions BC or just C and either a short cytoplasmic tail (CYT1) or long cytoplasmic tail (CYT2) . d Using flow cytometry, anti-CD46 mAbs TRA-2–10 and GB24 (2 μg ml −1 ) were analyzed on ARPE-19 cells in comparison to PY102. e ARPE-19 cells were incubated with labeled 2E7 647 (2 μg ml −1 ) and increasing concentrations (6.7–0.22 μg ml −1 , in 3-fold dilutions) of mAbs TRA-2-10 or GB24. The mean fluorescence intensity (MFI) of anti-mouse IgG Alexa647 was measured by flow cytometry with PY102 (−) and non-labeled 2E7 (+) as controls. f TB40/E wt and AD169 BADrUL131 infection of ARPE-19 cells treated with mAbs 5C3, 2E7, TRA-2-10, GB24, and PY102 (6.7–0.74 μg ml −1 , in threefold dilutions) and a no-antibody control were analyzed using an HTI. PY102-treated cells represents 100% infection. g ARPE-19 cells transfected with non-targeting siRNA (SCR) or siRNA targeting CD46 (CD46) were infected with TB40/E FLAG YFP and AD169 BADrUL131 and analyzed for infection (YFP fluorescent intensity) by flow cytometry. SCR-transfected cells represent 100% infection. h Cell lysates from ARPE-19 cells transfected with no siRNA, SCR, or CD46 targeting siRNA and infected with AD169 BADrUL131 or TB40/E wt (MOI:0.5) were subjected to anti-IE1 and GAPDH immunoblots. The polypeptides and molecular weight markers are indicated for the immunoblots. Infection experiments were performed in triplicate. s.d. is depicted in the experiment. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Student’s two-tailed t test)

Article Snippet: Virus (MOI:0.5) incubated with respective mAbs (2 μg ml −1 , or otherwise noted) or soluble proteins Nrp2 (P) and CD46 (P) (Sino Biological (cat#: 10695-H08H and 12239-H08H) for 1 h 4 °C was used to infect ARPE-19, HTR-8/SVneo, or MRC5 cells (1 × 10 4 cell/well in a 96-well plate) for 2 h (37 °C) and then replaced with new media.

Techniques: Inhibition, Binding Assay, Infection, Western Blot, Expressing, Flow Cytometry, Incubation, Labeling, Fluorescence, Transfection, Molecular Weight, Two Tailed Test

Journal: Nature Communications

Article Title: CD46 facilitates entry and dissemination of human cytomegalovirus

doi: 10.1038/s41467-019-10587-1

Figure Lengend Snippet: CD46 is an important factor for CMV infection and spread. a CD46 cell surface expression of wild-type ARPE-19 cells (Ewt), β 2 m Knock-out (KO) ARPE-19 cells (Eβ1), and three CD46-KO ARPE-19 cell clones (EC1, EC2, and EC3) was assessed by flow cytometry with mAbs against CD46 (2E7 and GB24), MHC class I (W6/32), and PY102 as a non-binding control. Following incubation with anti-mouse IgG Alexa647 , the normalized cell number was plotted based on Alexa647 fluorescence intensity. b The respective KO cells ( a ) were subjected to anti-CD46 and GAPDH immunoblots. c ARPE-19 wt and CD46- and β 2 m-KO cells infected with TB40/E wt or AD169 BADrUL131 were analyzed for infection by an HTI. Total number of infected cells was determined using anti-IE1 antibodies. d ARPE-19 wt and CD46- and β 2 m-KO cells infected with TB40/E wt, TB40/E MC UL99-eGFP , or AD169 BADrUL131 were subjected to a plaque assay, counted 14 dpi. e Supernatant from Ewt, Eβ1, and EC2 cells infected (E: epithelial cells) with AD169 BADrUL131 (MOI:0.1) from days 0–12 was added to MRC5 and analyzed for infection at 24 hpi using YFP fluorescent with a cytometer. The virus titre (infectious units (IU)/ml) was plotted for up to 12 dpi. f Cell lysates of wt HTR-8/SVneo (Twt) cells, β 2 m-KO HTR-8/SVneo (Tβ1) cells, and CD46-KO HTR-8/SVneo cell clones (TC1 and TC2, T: trophoblasts) were subjected to immunoblot using anti-CD46 and anti-GAPDH antibodies. g Cell lysates of AD169 BADrUL131 (MOI:2) infected Twt, Tβ1, TC1 and TC2 were subjected to anti-IE1 and GAPDH immunoblots. h ARPE-19 wt and Tβ1, TC1, and TC2 clones infected with AD169 BADrUL131 at varying MOIs were analyzed for infection by an HTI assay. The total number of infected cells was determined using a cytometer. i Representative cytometer images of ( h ) (overlay: Hoechst stain for nuclear stain (blue) and GFP expression (green) for virally infected cells) (MOI 2). The polypeptides and molecular weight markers are indicated for the immunoblots. Infection experiments (excluding westerns) were performed in triplicate. s.d. is depicted in the experiment. ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Student’s two-tailed t test)

Article Snippet: Virus (MOI:0.5) incubated with respective mAbs (2 μg ml −1 , or otherwise noted) or soluble proteins Nrp2 (P) and CD46 (P) (Sino Biological (cat#: 10695-H08H and 12239-H08H) for 1 h 4 °C was used to infect ARPE-19, HTR-8/SVneo, or MRC5 cells (1 × 10 4 cell/well in a 96-well plate) for 2 h (37 °C) and then replaced with new media.

Techniques: Infection, Expressing, Knock-Out, Clone Assay, Flow Cytometry, Binding Assay, Incubation, Fluorescence, Western Blot, Plaque Assay, Cytometry, Staining, Molecular Weight, Two Tailed Test

Journal: Nature Communications

Article Title: CD46 facilitates entry and dissemination of human cytomegalovirus

doi: 10.1038/s41467-019-10587-1

Figure Lengend Snippet: CD46 is involved in a post-binding entry step. a TB40/E wt infection of ARPE-19 cells untreated or treated at −0.5, 0.5, and 5hpi with mAbs targeting HCMV gH (5C3) and CD46 (2E7), heparin, a non-binding antibody control (PY102), or a no-antibody control (10 μg ml −1 ) was analyzed with a HTI. PY102-treated cells was used as 100% infection. b TB40/E wt infection of ARPE-19 cells and ( c ) AD169 BADrUL131 infection of HTR-8/SVneo cells incubated with mAbs 5C3, 2E7, and anti-Nrp2 (αNrp2), or soluble CD46 and Nrp2 proteins (P) (20–0.24 μg ml −1 , in threefold dilutions) were subjected to the HTI assay. The % infection was determined using incubation with PY102 as 100% infection. Experiments were performed in triplicate. s.d. is depicted in the experiment. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Student’s two-tailed t test)

Article Snippet: Virus (MOI:0.5) incubated with respective mAbs (2 μg ml −1 , or otherwise noted) or soluble proteins Nrp2 (P) and CD46 (P) (Sino Biological (cat#: 10695-H08H and 12239-H08H) for 1 h 4 °C was used to infect ARPE-19, HTR-8/SVneo, or MRC5 cells (1 × 10 4 cell/well in a 96-well plate) for 2 h (37 °C) and then replaced with new media.

Techniques: Binding Assay, Infection, Incubation, Two Tailed Test