Journal: bioRxiv

Article Title: Engineering sialylation in pigs: A promising strategy for overcoming xenograft rejection and revolutionizing organ transplantation

doi: 10.1101/2025.04.30.651299

Figure Lengend Snippet: Schematic illustration of the in-vitro experimental procedure, measurements of human thrombin/anti-thrombin III complex (TAT), C3a, C4b, and C5b9 were taken from plasma in mixed human blood and porcine kidney and aorta tissues transfected with ST8Sia6. These were compared to controls porcine kidney and aorta endothelium transfected with GFP under in vitro conditions at three different time points (0, 3, and 6 hours). Result indicates that control GFP transfected Kidney and Aorta group (grey line) had increased complement activation. b-c, indicate C3a level in kidney and aorta condition respectively, similarly d-e, represent C4b level, f-g, represent C5b9 level from kidney and aorta experimental conditions. h-i, TAT level in plasma from kidney and aorta experimental conditions. In contrast, the ST8Sia6-transfected porcine kidney group (red line) showed a slight reduction in complement activation. Notably, the ST8Sia6- transfected aorta cells demonstrated significant reductions in complement activations. All graphs summarize the results from 4 donors (mean ± S.E) with 4 individual replicates. Significance differences in P value (P<0.05) were evaluated using ANOVA, and the P values are represented in the graphs.

Article Snippet: Plasma levels of thrombin/anti-thrombin III complex (TAT), C3a, C4d, and sC5b-9 were measured using commercially available ELISA kits: Human Thrombin-Antithrombin Complex ELISA Kit (Abcam AB1089071), Human C4b (Novus Biologicals™ NBP270046), Human C3a (Novus Biologicals™ NBP266755), and Human C5b-9 (Novus Biologicals™ NBP266708).

Techniques: In Vitro, Clinical Proteomics, Transfection, Control, Activation Assay

Journal: bioRxiv

Article Title: Evasion of serum antibodies and complement by Salmonella Typhi and Paratyphi A

doi: 10.1101/2025.01.20.633845

Figure Lengend Snippet: ( A) Restoration of S . Paratyphi A long and O9-antigen increases human C3a and (B) C5a production following incubation with human serum for 60 min at 37°C. The S. Typhi Vi capsule prevents C3a production but does not impact C5a release. Long O-antigen in S. Typhimurium is the primary determinant of C5a release. C3a and C5a release were measured by enzyme-linked immunosorbent assay (ELISA). For statistical comparison, individual replicates are normalized to the corresponding wild-type serovar and statistical analysis performed by one sample t test with a hypothetical value of 1.0, with P values in red indicating statistical significance as P < 0.05. Column bars represent the means, with error bars showing standard deviation. STm, S. Typhimurium; STy, S. Typhi; SPa, S. Paratyphi A.

Article Snippet: Human C3a and C5a production were measured using the Human Complement C3a ELISA Kit (Invitrogen, catalog #BMS2089) and the Human C5a ELISA Kit (Invitrogen, catalog #BMS2088), respectively, following the manufacturer’s instructions.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Comparison, Standard Deviation

Journal: bioRxiv

Article Title: Evasion of serum antibodies and complement by Salmonella Typhi and Paratyphi A

doi: 10.1101/2025.01.20.633845

Figure Lengend Snippet: ( A) O-antigen modifications by glycosyltransferases in S. Paratyphi A decrease complement activation as measured by C5a release and (B) IgM binding. (C) and (D) O-antigen modifications by glycosyltransferases in S. Paratyphi A do not impact complement deposition or (E) serum resistance. (F) O-antigen modifications by glycosyltransferases in S. Typhimurium do not impact complement activation, (H) and (I) C3b deposition, or (J) serum resistance, but (G) reduce IgM binding. Statistical analysis was performed by one-way ANOVA on 3-4 independent replicates, with P values in red indicating statistical significance with P <0.05. Column bars represent the means, with error bars displaying standard deviation. STm, S. Typhimurium; STy, S. Typhi; SPa, S. Paratyphi A.

Article Snippet: Human C3a and C5a production were measured using the Human Complement C3a ELISA Kit (Invitrogen, catalog #BMS2089) and the Human C5a ELISA Kit (Invitrogen, catalog #BMS2088), respectively, following the manufacturer’s instructions.

Techniques: Activation Assay, Binding Assay, Standard Deviation

Journal: International Journal of Clinical Practice

Article Title: Pleural Mesothelial Cells-Induced Monocytes to the Pleural Cavity through the Effect of C3 Lytic Products in Tuberculous Pleural Effusion

doi: 10.1155/2024/5544085

Figure Lengend Snippet: Figure 2: Te concentrations of complement pyrolysis products were found higher in pleural efusion than in plasma from TPE patients. (a) Complement pyrolysis products, including C3a, C3b, C3d, C5a, and opsonin receptors (CR1 and CR3) were detected in human tuberculosis pleural biopsy samples by immunohistochemistry. (Original magnifcation, ×200) (n 4). (b) Higher levels of complement pyrolysis products were found in pleural efusion than in plasma in TPE patients (n 20). Te concentrations of complement pyrolysis products in pleural fuid and plasma from TPE patients were measured by ELISA (n 20).

Article Snippet: Te concentrations of complement components and chemokines, including C1q (E-EL-H6053), factor B (E-ELH6056), factor (E-EL-H0817), MBL (E-EL-H1305), MAC (E-EL-H2376), CD46 (ab283877, Abcam), C3 (E-ELH6054), C5 (E-EL-H0810), C3a (E-EL-H0818), C5a (E-ELH0190), C3b (E-EL-H6054), C3d (E-EL-H5457), and CXCL12 (E-EL-H0052c), in both pleural fuids and plasma were measured by ELISA kits according to the manufacturer’s protocols (all kits were purchased from Elabscience and Abcam).

Techniques: Clinical Proteomics, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Clinical Practice

Article Title: Pleural Mesothelial Cells-Induced Monocytes to the Pleural Cavity through the Effect of C3 Lytic Products in Tuberculous Pleural Effusion

doi: 10.1155/2024/5544085

Figure Lengend Snippet: Figure 3: Monocyte migration was inhibited by antibodies that blocked CXCL12. (a) CXCL12 and CXCR4 staining by immunohisto- chemistry in human pleural biopsy (original magnifcation, ×200) (n 4). (b) Te concentration of CXCL12 in pleural fuid and plasma from TPE patients was measured by ELISA (n 20). (c) CXCL12 produced by PMCs was measured by PCR and ELISA after Mpt64 and anaphylatoxin activation. PMCs were incubated for 24 hours in control media or in media with Mpt64 (20 μg/ml) and with or without human C3a (100 nM) or C3aRA (100 nM) (n 4). (d) Coexpression of CXCL12-CXCR4 in PMCs and monocytes from TPE was detected by immunofuorescence (original magnifcation, ×400) (n 4). (e) Monocytes were seeded into the top chamber of a transwell system, and the supernatant from PMCs cultured with anti-CXCL12 antibody or PBS were placed in the bottom chamber. Te migratory index was calculated by dividing the number of monocytes that migrated in response to the supernatants from cultured PMCs by the number of monocytes that migrated in response to the control. ∗vs the MO-PBS group, ∗∗P < 0.01. #vs the MO-PMC group, ##P < 0.01 (n 4).

Article Snippet: Te concentrations of complement components and chemokines, including C1q (E-EL-H6053), factor B (E-ELH6056), factor (E-EL-H0817), MBL (E-EL-H1305), MAC (E-EL-H2376), CD46 (ab283877, Abcam), C3 (E-ELH6054), C5 (E-EL-H0810), C3a (E-EL-H0818), C5a (E-ELH0190), C3b (E-EL-H6054), C3d (E-EL-H5457), and CXCL12 (E-EL-H0052c), in both pleural fuids and plasma were measured by ELISA kits according to the manufacturer’s protocols (all kits were purchased from Elabscience and Abcam).

Techniques: Migration, Staining, Immunohistochemistry, Concentration Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Produced, Activation Assay, Incubation, Control, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps

doi: 10.1172/JCI163105

Figure Lengend Snippet: An ELISA assay was performed to measure the concentrations of ( A ) C5a, ( B ) factor Bb, and ( C ) C3a in the BAL fluid from patients with influenza ( n = 16) and patients with COVID- 19 ( n = 16). ( B ) Paired concentrations of ( D ) C5a and ( E ) factor Bb in the plasma and BAL fluid from patients with COVID-19 were determined by ELISA. ( F ) A different cohort from a previously published data set was reanalyzed and the t-SNE analysis of total cells (65,166) from BAL fluid of patients with non-COVID-19 pneumonia ( n = 13) and COVID-19 ( n = 22) is shown. ( G ) Dot plots display the highlighted distribution of C5AR1 for each indicated cell population. ( H ) Violin plots showing the expression levels of C5aR1 in each type of cell from patients with COVID-19 or with non-COVID-19 pneumonia. ( I ) The dot plot depicts the scaled and centered expression of an average cell in each cluster and therefore contains negative and positive values. The average expression reflects the mean expression of C5AR1 in each cluster compared with all other cells. ( J ) Number of cells per cell population [neutrophils (Neu), monocytes/macrophages (Mo/Mac), and dendritic cells (cDC)] that express C5AR1 in the groups of patients with COVID-19 and non-COVID-19 pneumonia. ( K ) Average expression of C5AR1 per cell for each cell population [neutrophils (Neu), monocytes/macrophages (Mo/Mac), and dendritic cells (cDC)] in the groups of patients with COVID-19 and non-COVID-19 pneumonia. Data are shown as the mean ± SEM. P values were determined by 2-tailed unpaired ( A – D , J , and K ) or paired ( D and E ) Student’s t tests followed by Wilcoxon matched-pairs signed rank tests.

Article Snippet: C5a, factor Bb, and C3a ELISA assays were performed using, respectively, the human complement component C5a duoset ELISA kit from R&D Systems (DY2037), the MicroVue Bb plus fragment EIA kit from Quidel (A027), and the Complement C3a human ELISA kit from Thermo Fisher Scientific (BMS2089).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Expressing

Journal: The Journal of Clinical Investigation

Article Title: C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps

doi: 10.1172/JCI163105

Figure Lengend Snippet: ( A ) Tg mice were infected with SARS-CoV-2 (2 × 104 PFU, intranasally). ELISA assay to measure levels of ( B ) C5a in the lung homogenate of infected animals ( n = 14) or mock control ( n = 11). ( C ) factor Bb and ( D ) C3a levels in the lung homogenate of infected animals ( n = 8) or mock control ( n = 5). ( E ) Representative confocal images of the presence of C5aR1 expression in the lung tissue of Tg fl/fl mice ( C5ar1 -eGFP mice) infected with SARS-CoV-2 (5 dpi). Tissue slices were costained for nuclei (DAPI, blue), Iba-1 (macrophages, red) and NE (neutrophils, red) markers. Scale bar: 50 μm. ( F ) Percentage of cells expressing C5aR1 in the lung tissue of Tg fl/fl mice infected with SARS-CoV-2 ( n = 4 mice/4 randomized field). ( G ) Representative H&E staining from the lung of SARS-CoV-2-infected Tg fl/fl ( n = 6) or Tg cKO mice ( n = 6). A mock-infected group was used as control ( n = 6). Scale bars: 200 μm (4 ×), 100 μm(10 ×). ( H ) TUNEL staining (red) for detection of apoptotic cells in situ from lung tissue of SARS-CoV-2–infected Tg fl/fl ( n = 5) or Tg cKO mice ( n = 6). Mock-infected Tg mice were used as a control ( n = 5/group). ( I ) Quantification of the lung septal area fraction. ( J ) Percentage of TUNEL positive cells in lung tissue. Scale bar: 50 μm. ( K ) ELISA assays were performed to detect CCL2, CCL3, CCL4, CXCL1, and IL-6 levels in the lung tissue of Tg fl/f ( n = 8) or Infected Tg cKO mice ( n = 7). Mock-infected Tg mice were used as a control ( n = 5). Data are shown as the mean ± SEM. P values were determined by ( B – D ) Student’s 2-tailed t test and ( F , I , J , and K ) 1-way ANOVA followed by Bonferroni’s posthoc test.

Article Snippet: C5a, factor Bb, and C3a ELISA assays were performed using, respectively, the human complement component C5a duoset ELISA kit from R&D Systems (DY2037), the MicroVue Bb plus fragment EIA kit from Quidel (A027), and the Complement C3a human ELISA kit from Thermo Fisher Scientific (BMS2089).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Control, Expressing, Staining, TUNEL Assay, In Situ

Journal: The Journal of Clinical Investigation

Article Title: C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps

doi: 10.1172/JCI163105

Figure Lengend Snippet: ( A ) Tg mice were infected with SARS-CoV-2 (2 x 104 PFU, intranasally) and treated with DF2593A (3 mg/kg, p.o) 1 hour before SARS-CoV-2 infection and once a day up to the day of sample collection (5 dpi). ( B ) Body weight, clinical score, and oxygen saturation were measured daily after infection ( n = 11/group, pooled from 2 independent experiments). ( C ) Representative H&E staining from the harvested lung of the COVID-19 mouse model treated ( n = 4) or not ( n = 6) with DF2593A. A mock-infected group was used as control ( n = 5). Scale bars: 200 μm (4 ×); 100 μm (10 ×). ( D ) TUNEL staining (green) for detection of apoptotic cells in situ from lung tissue of mice ( n = 5/group). ( E ) Quantification of the lung septal area fraction. ( F ) Percentage of TUNEL-positive cells in lung tissue. Scale bar: 50 μm. ( G ) ELISA assays were performed to detect CCL2, CCL3, CCL4, CXCL1, and IL-6 levels in lung homogenate ( n = 6/group). A mock-infected group was used as the control group. Data are shown as the mean ± SEM. P values were determined by 1-way ANOVA followed by Bonferroni’s posthoc test ( E – G ).

Article Snippet: C5a, factor Bb, and C3a ELISA assays were performed using, respectively, the human complement component C5a duoset ELISA kit from R&D Systems (DY2037), the MicroVue Bb plus fragment EIA kit from Quidel (A027), and the Complement C3a human ELISA kit from Thermo Fisher Scientific (BMS2089).

Techniques: Infection, Staining, Control, TUNEL Assay, In Situ, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps

doi: 10.1172/JCI163105

Figure Lengend Snippet: ( A ) C57/BL6 mice were treated twice with vehicle, DNAse (Pulmozyme, 10 mg/kg, s.c.), or C5aR1 antagonist (DF2593A, 3 mg/kg, orally), 24 hours and 1 hour before the intratracheal instillation of rmC5a (400 ng). ( B ) Lung slices from the control group or mice challenged with rmC5a and treated with vehicle, DNAse, or C5aR1 antagonist (DF2593A) were stained with H&E for evaluation of histological changes. ( C ) Quantification of the lung septal area fraction ( n = 5/group). ( D ) Lung slices from the control group or from mice challenged with rmC5a and treated with vehicle, DNAse, or C5aR1 antagonist (DF2593A) were costained for nuclei (DAPI, blue), H3Cit (green), and MPO (red) markers. ( E ) NET quantification by the MPO-DNA PicoGreen assay in the supernatant of the lung homogenate ( n = 5–6/group). ( F ) ELISA assays were performed to detect CCL2, CCL3, CCL4, CXCL1, and IL-6 levels in lung homogenate ( n = 5–6/group). Data are shown as the mean ± SEM. P values were determined by 1-way ANOVA followed by Bonferroni’s posthoc test ( C , E , and F ).

Article Snippet: C5a, factor Bb, and C3a ELISA assays were performed using, respectively, the human complement component C5a duoset ELISA kit from R&D Systems (DY2037), the MicroVue Bb plus fragment EIA kit from Quidel (A027), and the Complement C3a human ELISA kit from Thermo Fisher Scientific (BMS2089).

Techniques: Control, Staining, Picogreen Assay, Enzyme-linked Immunosorbent Assay