Journal: Journal of Clinical Investigation

Article Title: Complement activation on endothelium initiates antibody-mediated acute lung injury

doi: 10.1172/jci138136

Figure Lengend Snippet: Figure 7. High endothelial HLA class I expression in human lungs and complement activation after transfusion in clinical TRALI. Samples of human lungs were dissociated for measurement of cell surface HLA class I (HLA-ABC) expression using flow cytometry. (A) Gating strategy, (B) representative histograms of fully stained cells relative to FMO controls, and (C) quantification of HLA class I expression on different cells expressed as MFI (D–G) (n = 4). EC, endothelial cell. Plasma content of the classical/lectin pathway complement component C4 and its activation and degradation product C4d were mea- sured using pre- and posttransfusion samples from patients diagnosed with TRALI or control patients who did not develop TRALI after transfusion. (D) Concentrations of C4d and (E) fold change in C4d after transfusion, and (F) C4d/C4 ratios and (G) fold change in C4d/C4 ratios after transfusion. Normal- ized data (E and G) provided for enhanced display of individual changes after transfusion. Mean (D and F, bars) or median (E and G, lines) and individual values (D and F). In D and F, a 2-way repeated-measures mixed-model approach was used with pretransfusion values set as covariates, followed by Holm’s multiplicity correction for comparisons of effect of TRALI development after transfusion and effect of transfusion within the control group and the TRALI group. Control group, n = 19; TRALI group, n = 9. P values < 0.05 are shown.

Article Snippet: ELISAs for mouse C3 (ab157711, Abcam), human C4 (ab108824, Abcam), and human C4d (A009, Quidel) were used according to manufacturers’ instructions.

Techniques: Expressing, Activation Assay, Flow Cytometry, Staining, Clinical Proteomics, Control

Journal: Journal of Clinical Investigation

Article Title: Complement activation on endothelium initiates antibody-mediated acute lung injury

doi: 10.1172/jci138136

Figure Lengend Snippet: Figure 8. Anti-HLA antibodies in lung transplant recipients activate complement on pulmonary endothelium and increase risk of CLAD or death. Serial sections of a transbronchial biopsy from a lung transplant recipient diagnosed with antibody-mediated rejection (AMR) showing (A) diffuse, strong linear microvascular endothelial C4d positivity (immunohistochemistry: C4d = brown, hematoxylin counterstain = blue), and (B) organizing acute lung injury with increased neutrophils (H&E staining). (C) Chest computed tomography (CT) scan from lung transplant recipient diagnosed with AMR showing bilateral consolidations. (D and E) Kaplan-Meier curves showing chronic lung allograft dysfunction–free (CLAD-free) survival over time in lung transplant recipients comparing effects of (D) number of episodes of anti-HLA donor-specific antibodies (DSAs) (adjusted curves from Cox proportional hazards model using serologic detection of DSAs as a time-dependent variable), or (E) different reactivities of de novo anti-HLA DSAs (actual CLAD-free survival within groups). (D and E) P values are from log-rank tests from whole Cox models, n = 215 with 15 patients with anti-HLA class I DSAs only, 22 with anti-HLA class II DSAs only, and 7 with detected DSAs against both HLA classes.

Article Snippet: ELISAs for mouse C3 (ab157711, Abcam), human C4 (ab108824, Abcam), and human C4d (A009, Quidel) were used according to manufacturers’ instructions.

Techniques: Immunohistochemistry, Staining, Computed Tomography

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Antibodies for flow cytometric analysis on CiGEnCs.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques:

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Baseline characteristics of aABMR, aTCMR, and NR patients.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: Transplantation Assay

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Detailed immunological, histopathological and clinical information of individual patients included.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: Tandem Mass Spectroscopy

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Complement Levels in Plasma of aABMR, aTCMR, and NR patients. Plasma levels were measured for C3 (A) , C3d (B) , and C5b-9 (D) . Ratios were calculated from respective C3 and C3d levels (C) , indicating systemic complement consumption. Circles represent individual patients with open circles indicating C4d-positive patients and filled circles symbolizing C4d-negative patients. P values depict results from Mann-Whitney U statistical analysis. Statistical significance is defined as P < 0.05. aABMR, active antibody-mediated Rejection; aTCMR, acute T-cell mediated rejection; NR, non-rejection; ns, not significant.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Semiquantitative scores for C3d and C5b-9 deposition in renal biopsy specimens of aABMR, aTCMR, and NR patients. Semiquantitative scores range from 0 to 4, with higher scores indicating increased positivity in biopsy. Groups are compared regarding glomerular C3d deposition (A) , peritubular capillary C3d (B) , glomerular C5b-9 (C) , and peritubular capillary C5b-9 (D) deposition. Data represent staining results of individual patients. Error bars represent median with interquartile range. Unfilled circles symbolize biopsies of C4d-positive patients, filled circles C4d-negative patients. P-values are derived from Mann-Whitney U tests with statistical significance defined as *P < 0.05, **P < 0.001; aABMR, active antibody-mediated rejection; aTCMR, acute T-cell mediated rejection; NR, non-rejection; ns, not significant.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: Staining, Derivative Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Renal biopsy staining for complement factors in a KTR diagnosed with C4d-positive, aABMR. Selected section of biopsy slide stained for C3d in glomeruli (A) and in peritubular capillaries (B) . Selected section of biopsy slide stained for C5b-9 in glomeruli (C) and in peritubular capillaries (D) . White arrows indicate glomeruli and black arrows indicate peritubular capillaries. Double-compound arrows indicate C5b-9 negative peritubular capillaries (D) . Semi-quantitative scores (0-4) are indicated in the lower right corner of each picture. KTR, kidney transplant recipients; aABMR, active antibody-mediated rejection; s, semi-quantitative score.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: CD59 staining in vivo . Renal biopsy staining for complement regulator CD59 in glomeruli (A, C) and peritubular capillaries (B, D) . Staining was performed on human kidney before transplantation as control (A, B) , on biopsy specimen from a C4d-positive (C4d+) aABMR (C, D) . Black arrows point to CD59-positive peritubular capillaries, double-compound arrows to CD59-negative peritubular capillaries. aABMR, active antibody-mediated rejection.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: Staining, In Vivo, Transplantation Assay

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Complement system activation on conditionally immortalized glomerular endothelial cells in vitro in flow cytometric analysis. Complement factors C3 (activated), C4d, and C5b-9 on conditionally immortalized glomerular endothelial cells in vitro in flow cytometric analysis are depicted in (A) with the four different incubation conditions plotted on the y-axis. Deposition of complement regulator, CD59, was measured in flow-cytometry under five different incubation conditions (B) . cABO, ABO-compatible; HLA, Human Leucocyte Antigen; Abs, antibodies; IgG, Immunoglobulin G; FITC, Fluorescein isothiocyanate; iABO, ABO-incompatible.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: Activation Assay, In Vitro, Incubation, Flow Cytometry

Journal: Annals of Translational Medicine

Article Title: Characteristics of pre-sensitization-related acute antibody-mediated rejection in a rat model of orthotopic liver transplantation

doi: 10.21037/atm-22-4311

Figure Lengend Snippet: Levels of C4d were increased in sensitized followed transplant rats. (A) Lymphocytes of LEW rats were isolated for C4d detect using anti-C4d antibody and secondary antibody with FITC. (B) Flow data are presented on the statistical graph (n=3). Significant differences between each group were marked by ***, P<0.001, ****, P<0.0001. LEW, Lewis; FITC, fluorescein isothiocyante.

Article Snippet: At 14 days after liver transplantation, splenic cells of LEW rats (recipients) were isolated and detected for C4d expression using rabbit anti-rat C4d (Cat. No. HP8034-100UG; Hycult Biotech, Beutelsbach, Germany) and goat anti-rabbit IgG Antibody (FITC conjugate) (Cat. No. AP132F; Millipore, Darmstadt, Germany).

Techniques: Isolation

Journal: Annals of Translational Medicine

Article Title: Characteristics of pre-sensitization-related acute antibody-mediated rejection in a rat model of orthotopic liver transplantation

doi: 10.21037/atm-22-4311

Figure Lengend Snippet: Accelerate liver rejection and C4d deposition occurred in sensitized followed transplant rats. (A) Inflammation of portal areas, bile ducts, vascular endothelium, and capillaries is shown by HE staining in each group. (B) C4d deposition is shown in IHC. LEW, Lewis; HE, hematoxylin and eosin; IHC, immunohistochemistry.

Article Snippet: At 14 days after liver transplantation, splenic cells of LEW rats (recipients) were isolated and detected for C4d expression using rabbit anti-rat C4d (Cat. No. HP8034-100UG; Hycult Biotech, Beutelsbach, Germany) and goat anti-rabbit IgG Antibody (FITC conjugate) (Cat. No. AP132F; Millipore, Darmstadt, Germany).

Techniques: Staining, Immunohistochemistry

Journal: Annals of Translational Medicine

Article Title: Characteristics of pre-sensitization-related acute antibody-mediated rejection in a rat model of orthotopic liver transplantation

doi: 10.21037/atm-22-4311

Figure Lengend Snippet: AMR was occurred in sensitized followed transplant rats

Article Snippet: At 14 days after liver transplantation, splenic cells of LEW rats (recipients) were isolated and detected for C4d expression using rabbit anti-rat C4d (Cat. No. HP8034-100UG; Hycult Biotech, Beutelsbach, Germany) and goat anti-rabbit IgG Antibody (FITC conjugate) (Cat. No. AP132F; Millipore, Darmstadt, Germany).

Techniques: