Journal: Biomedicines

Article Title: Combination of Anti-CD40 and Anti-CD40L Antibodies as Co-Stimulation Blockade in Preclinical Cardiac Xenotransplantation.

doi: 10.3390/biomedicines12081927

Figure Lengend Snippet: Figure 4. Levels of non-Gal-α(1,3)-Gal xenoreactive IgM (a) and IgG (b) as measured by flow cytometry on porcine aortic endothelial cells (PAEC) from GGTA1-KO, hCD46/hTBM transgenic animals. The values from a previously investigated baboon that had rejected an intrathoracic heterotopically transplanted pig heart served as positive control (black) and showed a strong increase in both IgM and IgG, indicative of humoral rejection. Note: while animal #17020 revealed clinical and histological signs of humoral rejection, there was no increase in xenoreactive IgM and IgG.

Article Snippet: Immunohistochemistry was performed using the following primary antibodies: mouse anti-human CD46 monoclonal antibody (HM2103, Hycult Biotech, Uden, The Netherlands) and mouse anti-human thrombomodulin monoclonal antibody (sc-13164, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Flow Cytometry, Transgenic Assay, Positive Control

Journal: Biomedicines

Article Title: Combination of Anti-CD40 and Anti-CD40L Antibodies as Co-Stimulation Blockade in Preclinical Cardiac Xenotransplantation.

doi: 10.3390/biomedicines12081927

Figure Lengend Snippet: Figure 8. Immunofluorescence staining of post-mortem myocardial specimens. Antibody deposition (IgM) (a), complement deposition (C3b/c, C4c, C5b) (b,c), fibrin deposition (Fgn) (d), cardiomyocyte structure (WGA, a lectin staining N-acetyl-D-glucosamine and sialic acid on the cell membrane) (e) and macrophage infiltration (CD68) (f) were analyzed in all donor organs. Scale bar, 100 µm. n = 4 GGTA1-KO, hCD46/hTBM transgenic pigs; n = 1 wild-type pig (control).

Article Snippet: Immunohistochemistry was performed using the following primary antibodies: mouse anti-human CD46 monoclonal antibody (HM2103, Hycult Biotech, Uden, The Netherlands) and mouse anti-human thrombomodulin monoclonal antibody (sc-13164, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Immunofluorescence, Staining, Membrane, Transgenic Assay, Control

Journal: bioRxiv

Article Title: A novel Gorilla-derived oncolytic Adenovirus with natural selective replication in cancer cells

doi: 10.64898/2026.02.26.708271

Figure Lengend Snippet: (A) A549 cells were infected with GRAd32 or GRAd25 at MOI 50 in the presence (w) or absence (w/o) of an anti-CD46 blocking antibody. Images were acquired 3 days post-infection. Competition with CD46 resulted in a delayed onset of cytopathic effect (CPE) exclusively in GRAd25-infected cells. (B) Flow cytometry analysis of surface CAR and CD46 expression levels in lung cancer (A549, NCI-H727, NCI-H1975, NCI-H1299) and normal (MRC5, HUVEC) cell lines. (C) Infection of MRC5 and A549 cells with a replication-defective GRAd32 GFP reporter virus. GFP expression was evaluated at 48 hours post-infection. The presence of GFP signal confirms that GRAd32 is capable of entering MRC5 cells despite its inability to replicate in this cell line.

Article Snippet: For the evaluation of CD46 and CAR cell surface levels, 2-3 x 10 5 cells (MRC5, HUVEC, A549, NCI-H1299, NCI-H1975, NCI-H727) were collected and incubated with anti-human CD46 (1:50; mouse monoclonal; #12239-MM05, Sino Biological) or with anti-human CAR (1:50; rabbit monoclonal; #10799-R271, Sino Biological) for 30 min at 4°C.

Techniques: Infection, Blocking Assay, Flow Cytometry, Expressing, Virus

Journal: Xenotransplantation

Article Title: Glycocalyx dynamics and the inflammatory response of genetically modified porcine endothelial cells.

doi: 10.1111/xen.12820

Figure Lengend Snippet: FIGURE 1 Coverage of GlcNAc/Sia and HS on wild type and GTKO/hCD46/hTM arterial and venous porcine EC. Representative confocal images of EC from wild type and GTKO/hCD46/hTM pigs grown in Ibidi μ-Slides. Cells were kept 72 h under static or 10 dyn/cm2 shear stress conditions (flow). Cells were stained for (A, B, D, E) N-Acetylglucosamine/Sialic acid (GlcNAc/Sia) in green and (G, H, J, K) heparan sulfate (HS) in red. Nuclei are shown in blue (DAPI). All images were acquired with a confocal microscope (Zeiss LSM980). Scale bar: 50 μm. Coverage of GlcNAc/Sia (C, F) and HS (I, L) for each condition (four images/experiment) was calculated as percentage area positive for the respective staining normalized to the cell count. Dark grey bars represent data from wild type EC while light grey bars represent data from GTKO/hCD46/hTM EC. Data are from four or more independent experiments and at least two different porcine EC donors. One-Way ANOVA with multiple comparisons was used for statistical analysis.

Article Snippet: Additionally, transgene expression was evaluated on both arterial and venous isolated GTKO/hCD46/hTM EC by staining for hCD46 (Hycult Biotech HM2103, dilution 1:100) and hTM (Abcam ab6980, dilution 1:100).

Techniques: Shear, Staining, Microscopy, Cell Counting

Journal: Xenotransplantation

Article Title: Glycocalyx dynamics and the inflammatory response of genetically modified porcine endothelial cells.

doi: 10.1111/xen.12820

Figure Lengend Snippet: FIGURE 2 Cell surface expression of HS and E-selectin on wild type and GTKO/hCD46/hTM porcine arterial and venous EC after xenogeneic activation. Representative confocal images of Ibidi μ-slide channels containing (A, E) arterial and (C, G) venous EC from wild type and GTKO/hCD46/hTM pigs. Cells were continuously perfused for 72 h under 10 dyn/cm2 shear stress conditions and subsequently either left untreated (–) or activated with 10% human serum or 10% heat-inactivated human serum (HI human serum) during 2 h. (A, C) Heparan sulfate (HS) is shown in red, (E, G) E-selectin in green and nuclei are shown in blue (DAPI). Scale bar: 50 𝜇m. All images were acquired with a confocal microscope (Zeiss LSM980). (B, D, F, H) coverage of HS and E-selectin was calculated for each image (3–4 images/condition/experiment) as percentage of area positive for the respective staining normalized to the cell count. Data are from three or more independent experiments and at least two different porcine EC donors. One-way ANOVA with multiple comparisons was used for statistical analysis.

Article Snippet: Additionally, transgene expression was evaluated on both arterial and venous isolated GTKO/hCD46/hTM EC by staining for hCD46 (Hycult Biotech HM2103, dilution 1:100) and hTM (Abcam ab6980, dilution 1:100).

Techniques: Expressing, Activation Assay, Shear, Microscopy, Staining, Cell Counting

Journal: Xenotransplantation

Article Title: Glycocalyx dynamics and the inflammatory response of genetically modified porcine endothelial cells.

doi: 10.1111/xen.12820

Figure Lengend Snippet: FIGURE 3 Deposition of complement C3b/c on the surface of wild type and GTKO/hCD46/hTM porcine arterial and venous EC after xenogeneic activation. Representative confocal images of Ibidi μ-Slide channels containing (A) arterial and (B) venous EC from wild type and GTKO/hCD46/hTM pigs. Cells were continuously perfused for 72 h under 10 dyn/cm2 shear stress conditions and subsequently either left untreated (–) or activated with 10% human serum or 10% heat-inactivated human serum (HI human serum) during 2 h. C3b/c is shown in yellow, and nuclei are shown in blue (DAPI). Scale bar: 50 𝜇m. All images were acquired with a confocal microscope (Zeiss LSM980). (B, D) Coverage of C3b/c was calculated for each image (four images/condition/experiment) as percentage of area positive for C3b/c normalized to the cell count. Data are from four or more independent experiments and at least two different porcine EC donors. One-way ANOVA with multiple comparisons was used for statistical analysis.

Article Snippet: Additionally, transgene expression was evaluated on both arterial and venous isolated GTKO/hCD46/hTM EC by staining for hCD46 (Hycult Biotech HM2103, dilution 1:100) and hTM (Abcam ab6980, dilution 1:100).

Techniques: Activation Assay, Shear, Microscopy, Cell Counting

Journal: Xenotransplantation

Article Title: Glycocalyx dynamics and the inflammatory response of genetically modified porcine endothelial cells.

doi: 10.1111/xen.12820

Figure Lengend Snippet: FIGURE 4 Expression of HS, complement C3b/c deposition and E-selectin coverage on the cell surface of GTKO/hCD46/hTM porcine arterial and venous EC in an inflammatory xenogeneic environment. Representative confocal images of Ibidi μ-slide channels containing (A, D, G) arterial (C, F, I) venous GTKO/hCD46/hTM EC. Cells were continuously perfused for 72 h under 10 dyn/cm2 shear stress conditions and subsequently either perfused with only human serum or simultaneously perfused with rhTNF𝛼and human or porcine serum. To evaluate E-selectin expression, channels were perfused with rhTNF𝛼alone during 4 h (A, C) Heparan sulfate (HS) is shown in red, (D, F) complement C3b/c in yellow, (G, I) E-selectin in green and nuclei are shown in blue (DAPI). Scale bar: 50 𝜇m. All images were acquired with a confocal microscope (Zeiss LSM980). (B, E, H, J,) Coverage of HS, C3b/c and E-selectin was calculated for each image (3–4 images/condition/experiment) as percentage of area positive for the respective staining normalized to the cell count. Data are from three or more independent experiments and at least two different porcine EC donors. One-way ANOVA with multiple comparisons was used for statistical analysis.

Article Snippet: Additionally, transgene expression was evaluated on both arterial and venous isolated GTKO/hCD46/hTM EC by staining for hCD46 (Hycult Biotech HM2103, dilution 1:100) and hTM (Abcam ab6980, dilution 1:100).

Techniques: Expressing, Shear, Microscopy, Staining, Cell Counting

Journal: International journal of molecular sciences

Article Title: Peritoneal Expression of Membrane Complement Regulators Is Decreased in Peritoneal Dialysis Patients with Infected Peritonitis.

doi: 10.3390/ijms24119146

Figure Lengend Snippet: Figure 1. Correlations of expressions of membrane complement regulators (CRegs), CD46, CD55, and CD59, deposition of complement (C) activation products such as aC3 and C5b-9, and levels of sC5b-9 in peritoneal dialysis fluid (PDF) with tissue injury score and comparison of sC5-9 levels in PDF between Groups P and NP. Expressions of each of the CRegs CD46, CD55, and CD59 showed good inverse correlations with the severity score for peritoneal injuries ((A–C) respectively). In contrast, deposition of C activation products such as activated C3 (aC3) and C5b-9 correlated with the severity score of peritoneal tissue injures ((D,E) respectively). Adjusted PDF levels of sC5b-9 (sC5b-9/TP) also correlated with the severity score for peritoneal injures (F). Between Groups P and NP, the tissue injury score and PDF levels of sC5b-9/TP were significantly different ((G,H), respectively). Dots in the graph show outliers. Outliers were defined as values greater than the 75th percentile plus 1.5 times the interquartile range. #, p < 0.05.

Article Snippet: Monoclonal Ab (mAb) mouse antihuman CD46 (MEM 258) was purchased from BIO-RAD (Hercules, CA, USA).

Techniques: Membrane, Activation Assay, Comparison

Journal: International journal of molecular sciences

Article Title: Peritoneal Expression of Membrane Complement Regulators Is Decreased in Peritoneal Dialysis Patients with Infected Peritonitis.

doi: 10.3390/ijms24119146

Figure Lengend Snippet: Figure 4. Expressions of CRegs CD46, CD55, and CD59 in those with and without peritonitis and depositions of complement (C) activation products. Frames (A–E) and (F–J) represent peritoneal tissues in Group P and Group NP, respectively. Frames (A,F), frames (B,G), frames (C,H), frames (D,I), and frames (E,J) show expressions of CD46, CD55, and CD59 and depositions of activated C3 (aC3) and C5b- 9, respectively. Graphs (K–O) show results for expressions of CD46, CD55, and CD59 and deposition of aC3 and C5b-9, respectively. Original magnification of (A–C) and (F–H) is ×400, while that of (D,E,I,J) is ×100. Inserted frames in right upper corners of frames (F–H) are at double the original magnification. Scale bars of 50 µm and 200 µm are shown in the left upper corner of frames (A,D), respectively. * shows peritoneal cavity side. Dots in the graph show outliers. Outliers were defined as values greater than the 75th percentile plus 1.5 times the interquartile range. # p < 0.05.

Article Snippet: Monoclonal Ab (mAb) mouse antihuman CD46 (MEM 258) was purchased from BIO-RAD (Hercules, CA, USA).

Techniques: Activation Assay

Journal: International journal of molecular sciences

Article Title: Peritoneal Expression of Membrane Complement Regulators Is Decreased in Peritoneal Dialysis Patients with Infected Peritonitis.

doi: 10.3390/ijms24119146

Figure Lengend Snippet: Figure 5. Pathological changes with accumulation of inflammatory cells of peritonitis and expression of membrane complement regulators (CRegs) caused by Candida spp. or P. aeruginosa (Group P1) and Gram-positive cocci (Group P2). Graphs (A,B) show the tissue injury scores and the proportion of cytokeratin-positive mesothelial cells in the peritoneum, respectively. Graph (C–E) shows the numbers of esterase-positive neutrophils, CD3-positive pan-T cells, and CD68-positive macrophages, respectively. Graphs (F,G) show the scores of fibrin deposition and fibrosis in the peritoneum, respectively. Graphs (H–J) show CRegs CD46, CD55, and CD59, respectively. Group P1 shows cases with fungal infection or P. aeruginosa and Group P2 shows cases with Gram-positive cocci. Dots in the graph show outliers. Outliers were defined as values greater than the 75th percentile plus 1.5 times the interquartile range. #, p < 0.05.

Article Snippet: Monoclonal Ab (mAb) mouse antihuman CD46 (MEM 258) was purchased from BIO-RAD (Hercules, CA, USA).

Techniques: Expressing, Membrane, Infection

Journal: International Journal of Molecular Sciences

Article Title: Peritoneal Expression of Membrane Complement Regulators Is Decreased in Peritoneal Dialysis Patients with Infected Peritonitis

doi: 10.3390/ijms24119146

Figure Lengend Snippet: Correlations of expressions of membrane complement regulators (CRegs), CD46, CD55, and CD59, deposition of complement (C) activation products such as aC3 and C5b-9, and levels of sC5b-9 in peritoneal dialysis fluid (PDF) with tissue injury score and comparison of sC5-9 levels in PDF between Groups P and NP. Expressions of each of the CRegs CD46, CD55, and CD59 showed good inverse correlations with the severity score for peritoneal injuries (( A – C ) respectively). In contrast, deposition of C activation products such as activated C3 (aC3) and C5b-9 correlated with the severity score of peritoneal tissue injures (( D , E ) respectively). Adjusted PDF levels of sC5b-9 (sC5b-9/TP) also correlated with the severity score for peritoneal injures ( F ). Between Groups P and NP, the tissue injury score and PDF levels of sC5b-9/TP were significantly different (( G , H ), respectively). Dots in the graph show outliers. Outliers were defined as values greater than the 75th percentile plus 1.5 times the interquartile range. # , p < 0.05.

Article Snippet: Monoclonal Ab (mAb) mouse anti-human CD46 (MEM 258) was purchased from BIO-RAD (Hercules, CA, USA).

Techniques: Membrane, Activation Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Peritoneal Expression of Membrane Complement Regulators Is Decreased in Peritoneal Dialysis Patients with Infected Peritonitis

doi: 10.3390/ijms24119146

Figure Lengend Snippet: Expressions of CRegs CD46, CD55, and CD59 in those with and without peritonitis and depositions of complement (C) activation products. Frames ( A – E ) and ( F – J ) represent peritoneal tissues in Group P and Group NP, respectively. Frames ( A , F ), frames ( B , G ), frames ( C , H ), frames ( D , I ), and frames ( E , J ) show expressions of CD46, CD55, and CD59 and depositions of activated C3 (aC3) and C5b-9, respectively. Graphs ( K – O ) show results for expressions of CD46, CD55, and CD59 and deposition of aC3 and C5b-9, respectively. Original magnification of ( A – C ) and ( F – H ) is ×400, while that of ( D , E , I , J ) is ×100. Inserted frames in right upper corners of frames ( F – H ) are at double the original magnification. Scale bars of 50 μm and 200 μm are shown in the left upper corner of frames ( A , D ), respectively. * shows peritoneal cavity side. Dots in the graph show outliers. Outliers were defined as values greater than the 75th percentile plus 1.5 times the interquartile range. # p < 0.05.

Article Snippet: Monoclonal Ab (mAb) mouse anti-human CD46 (MEM 258) was purchased from BIO-RAD (Hercules, CA, USA).

Techniques: Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Peritoneal Expression of Membrane Complement Regulators Is Decreased in Peritoneal Dialysis Patients with Infected Peritonitis

doi: 10.3390/ijms24119146

Figure Lengend Snippet: Pathological changes with accumulation of inflammatory cells of peritonitis and expression of membrane complement regulators (CRegs) caused by Candida spp. or P. aeruginosa (Group P1) and Gram-positive cocci (Group P2). Graphs ( A , B ) show the tissue injury scores and the proportion of cytokeratin-positive mesothelial cells in the peritoneum, respectively. Graph ( C – E ) shows the numbers of esterase-positive neutrophils, CD3-positive pan-T cells, and CD68-positive macrophages, respectively. Graphs ( F , G ) show the scores of fibrin deposition and fibrosis in the peritoneum, respectively. Graphs ( H – J ) show CRegs CD46, CD55, and CD59, respectively. Group P1 shows cases with fungal infection or P. aeruginosa and Group P2 shows cases with Gram-positive cocci. Dots in the graph show outliers. Outliers were defined as values greater than the 75th percentile plus 1.5 times the interquartile range. # , p < 0.05.

Article Snippet: Monoclonal Ab (mAb) mouse anti-human CD46 (MEM 258) was purchased from BIO-RAD (Hercules, CA, USA).

Techniques: Expressing, Membrane, Infection