Journal: Viruses

Article Title: Systemic Inflammation and Complement Activation Parameters Predict Clinical Outcome of Severe SARS-CoV-2 Infections

doi: 10.3390/v13122376

Figure Lengend Snippet: Characteristics as well as systemic and experimental laboratory blood parameters of patients and healthy controls. Values represent baseline parameters acquired at first sampling time point. Data are presented as mean ± SD or n (percentage). Differences between both groups were analyzed by unpaired t -test, Mann–Whitney U test, or Fisher’s exact test, as appropriate.

Article Snippet: Monoclonal anti-human C5a antibody (Hycult Biotech, Uden, The Netherlands) was coated on 96-well, medium-binding microplates (Greiner Bio-One, St. Gallen, Switzerland).

Techniques: Sampling, Mann-Whitney U-Test

Journal: Viruses

Article Title: Systemic Inflammation and Complement Activation Parameters Predict Clinical Outcome of Severe SARS-CoV-2 Infections

doi: 10.3390/v13122376

Figure Lengend Snippet: Kinetics of systemic activation of different parameters in blood. Levels of ( a ) complement protein C5a, ( b ) neutrophilic extracellular traps (NETs), ( c ) terminal complement complex (TCC), and ( d ) CRP were measured during the course of mechanical ventilation of COVID-19 patients that survived ( n = 5) or died ( n = 7). Data are shown as mean ± SD. Dotted lines indicate the mean value in the control group. p -Values were calculated by linear mixed model of clustered log-transformed data.

Article Snippet: Monoclonal anti-human C5a antibody (Hycult Biotech, Uden, The Netherlands) was coated on 96-well, medium-binding microplates (Greiner Bio-One, St. Gallen, Switzerland).

Techniques: Activation Assay, Transformation Assay

Journal: Viruses

Article Title: Systemic Inflammation and Complement Activation Parameters Predict Clinical Outcome of Severe SARS-CoV-2 Infections

doi: 10.3390/v13122376

Figure Lengend Snippet: Correlation matrix of activation in systemic parameters. Complement activation products, C5a and TCC, and NETs release correlated with common inflammation and coagulation markers in mechanically ventilated patients with SARS-CoV-2 infection. Correlation coefficient was assessed using the Spearman rank correlation test. Values indicate Spearman r. CRP, C-reactive protein; PCT, procalcitonin; IL-6, interleukin 6; WBC, white blood cell count; INR, international normalized rate; TCC, terminal complement complex; NETs, neutrophilic extracellular traps. * p < 0.002 (significant after Bonferroni correction).

Article Snippet: Monoclonal anti-human C5a antibody (Hycult Biotech, Uden, The Netherlands) was coated on 96-well, medium-binding microplates (Greiner Bio-One, St. Gallen, Switzerland).

Techniques: Activation Assay, Coagulation, Infection, Cell Counting

Journal: Viruses

Article Title: Systemic Inflammation and Complement Activation Parameters Predict Clinical Outcome of Severe SARS-CoV-2 Infections

doi: 10.3390/v13122376

Figure Lengend Snippet: Complement, NETosis, and hemoglobin levels in tracheal fluid of patients and healthy controls. Values represent baseline levels at first sampling time point. Data are presented as mean ± SD or n (percentage). Differences between both groups were analyzed by unpaired t -test or Mann–Whitney U test, as appropriate.

Article Snippet: Monoclonal anti-human C5a antibody (Hycult Biotech, Uden, The Netherlands) was coated on 96-well, medium-binding microplates (Greiner Bio-One, St. Gallen, Switzerland).

Techniques: Sampling, Mann-Whitney U-Test

Journal: Frontiers in Immunology

Article Title: A Novel Role for C5a in B-1 Cell Homeostasis

doi: 10.3389/fimmu.2018.00258

Figure Lengend Snippet: Impact of the C5/C5a/C5aR/TLR2 axes on peritoneal CXCL13 production under steady-state and inflammatory conditions. (A) CXCL13 concentrations in the peritoneal lavage fluid of wild-type (wt), C5aR1-, C5aR2-, and C5-deficient mice (C57BL/6J background) under steady-state conditions. (B) CXCL13 concentrations in the peritoneal lavage fluid of wt, C5aR1- and C5aR2-deficient mice (BALB/c background) under steady-state conditions. (C) In vitro CXCL13 production by peritoneal macrophages from wt mice (C57BL/6J background) 24 h after stimulation with IL-10, Pam3CSK4, or Pam3CSK4 + IL-10. (D) CXCL13 production from wt, C5aR1 −/− , C5aR2 −/− , or C5 −/− peritoneal macrophages (all on C57BL/6J background) in response to Pam3CSK4 + IL-10. (E) CXCL13 concentrations in the peritoneal lavage fluid of wt mice (C57BL/6J background) 6 h after i.p. injection with 200 nM C5a or PBS. (F,G) In vitro CXCL13 (F) or IL-10 (G) production by total peritoneal cavity (PerC) cells from wt mice 24 h after stimulation with Pam3CSK4 compared to unstimulated (left panel) or Pam3CSK4-stimulated cells compared to simultaneous stimulation of Pam3CSK4 together with +anti-IL-10R Ab (right panel). (H) CXCL13 production from unstimulated (left panel) or Pam3CSK4 + IL-10-stimulated (right panel) adherent PerC cells from wt mice in the presence or absence of C5aRA (5 µM). (I) CXCL13 concentrations in the peritoneal lavage fluid of wt and TLR2-deficient mice (C57BL/6J background) under steady-state conditions (left). (J) Total B-1, B-1a, and B-1b cell numbers in the peritoneum of TLR2-deficient as compared to wt mice (both C57BL/6J background). Values shown are the mean ± SD (A,B,D,E,I,J) . Statistical differences between wt and knockout mice were determined using an unpaired t -test (A,B,I,J) or Mann–Whitney test (D) ; statistical differences between different stimulation conditions were determined using an unpaired (E) or paired t -test (C,F–H) . * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Briefly, the membrane was incubated with a biotinylated C5/C5a-specific antibody (Hycult Biotech, clone 557; 4 µg/ml) in TBS + Tween + milk powder for 2 h at room temperature.

Techniques: In Vitro, Injection, Knock-Out, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: A Novel Role for C5a in B-1 Cell Homeostasis

doi: 10.3389/fimmu.2018.00258

Figure Lengend Snippet: Impact of the C5/C5a/C5aR axes on IL-10 production from peritoneal B-1 cells under steady-state and inflammatory conditions. (A) IL-10 production from non-adherent and adherent PerC cells in response to Pam3CSK4. (B) Determination of IL-10 production in peritoneal B-1a, B-1b, and B-2 cells using GFP-IL-10 knockin mice (Vert-X) (grey histogram = wildtype control, blue line = unstimulated Vert-X mouse). The right panel shows the quantification of IL-10 production (shown as MFI of the GFP signal) from B-1a and B-1b cells. (C) Impact of IL-10 deletion in CD19 + B cells on the number of peritoneal B-1 (left), B-1a (middle), and B-1b (right) cells under steady-state conditions. (D) Impact of C5, C5aR1, or C5aR2 deficiency on IL-10 production from non-adherent PerC cells in response to Pam3CSK4 stimulation. (E) Impact of C5aR1/2 blockade (using the C5aRA A8 Δ71–73 ) on Pam3CSK4-induced IL-10 production. (F) GFP-C5aR1 expression (left panel) or C5aR1 surface expression (right panel) in peritoneal B-1 cells. Values shown in (A–C) are the mean ± SD. Statistical differences between non-adherent and adherent PerC cells with or without stimulation were determined using Kruskal–Wallis test with Dunn’s post hoc test (A) . Statistical differences between MFI expression in B-1a and B-1b cells were determined using unpaired t -test (B) ; statistical differences in B-1 cell numbers between control mice and B cell-specific IL-10 knockout mice were determined using Mann–Whitney test (C) ; statistical differences between different stimulation conditions were determined using a paired t -test (D,E) . * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Briefly, the membrane was incubated with a biotinylated C5/C5a-specific antibody (Hycult Biotech, clone 557; 4 µg/ml) in TBS + Tween + milk powder for 2 h at room temperature.

Techniques: Knock-In, Expressing, Knock-Out, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: A Novel Role for C5a in B-1 Cell Homeostasis

doi: 10.3389/fimmu.2018.00258

Figure Lengend Snippet: C5 production and non-canonical C5a generation in peritoneal macrophages following IL-10 and Pam3CSK4 stimulation. (A) Immunohistochemical analysis of C5 production from wild-type (wt) peritoneal macrophages (C57BL/6J background) in response to 24 h PBS, Pam3CSK4 or combined Pam3CSK4 + IL-10 stimulation (three pictures on the left). Quantification of C5 production based on the immunohistochemical analysis of C5 production (right panel). (B) Real-time RT-PCR for C5 using wt peritoneal macrophages (C57BL/6J background) 24 h after in vitro Pam3CSK4 + IL-10 stimulation compared to unstimulated controls. (C) Western Blot to determine C5 cleavage and C5a generation from wt peritoneal macrophages (C57BL/6J background) or their supernatant (SN) 24 h after in vitro Pam3CSK4 + IL-10 stimulation. Lane 1 = marker; lane 2 = Pam3CSK4 + IL-10-stimulated peritoneal macrophages in the presence of exogenous hC5; lane 3 = as in lane 2 but without cells; lane 4 = SN of Pam3CSK4 + IL-10-activated peritoneal macrophages incubated 4 h with exogenous hC5; lane 5 = as in lane 4 but without SN; lane 6 = hC5a purified from serum. The arrow depicts the monomeric C5a band. (D) Impact of MyD88-dependent cell signaling on Pam3CKS4 + IL-10-induced C5 production from peritoneal macrophages. The immunohistochemical analysis of C5 production from wt and MyD88 −/− peritoneal macrophages (both C57BL/6J background) is shown on the left. On the right, the quantification of the C5 production based on the evaluation of the immunohistochemical staining is shown. (E) CXCL13 production from unstimulated MyD88 −/− adherent PerC cells as well as after stimulation with IL-10, Pam3CSK4, or both compared to wt cells (both C57BL/6J background) and (F) from unstimulated (left panel) and Pam3CSK4 + IL-10-stimulated (right panel) wt adherent PerC cells in the presence or absence of the Stat-3 inhibitor Stattic (50 µM). Values shown in (A,B,D,E) are the mean ± SD. Statistical differences between unstimulated and stimulated samples or between wt and knockout cells were determined by Kruskal–Wallis test with Dunn’s post hoc test (A,E) , paired t -test (B,F) , or Mann–Whitney test (D) . * p < 0.05, ** p < 0.01.

Article Snippet: Briefly, the membrane was incubated with a biotinylated C5/C5a-specific antibody (Hycult Biotech, clone 557; 4 µg/ml) in TBS + Tween + milk powder for 2 h at room temperature.

Techniques: Immunohistochemical staining, Quantitative RT-PCR, In Vitro, Western Blot, Marker, Incubation, Purification, Staining, Knock-Out, MANN-WHITNEY

Journal:

Article Title: Functional basis for complement evasion by staphylococcal superantigen-like 7

doi: 10.1111/j.1462-5822.2010.01486.x

Figure Lengend Snippet: SSL7 inhibits generation of C5a during staphylococcal opsonisation. (A,B) Staphylococci were treated with 10% human serum for 30 minutes at 37°C in the presence of 0 – 450 nM SSL7 or SSL10. In one case (“SSL7 after”), 450 nM SSL7 was added to serum supernate after opsonisation. C5a generation was measured by using supernates as stimuli for calcium mobilization in U937-C5aR cells. Results depict in (A) representative calcium mobilization plots and in (B) mean values ± SEM of three independent experiments and are expressed relative to cells treated with supernate without staphylococcal proteins. In (A), “background” and “control” represent cells stimulated with untreated serum and serum after opsonisation without SSL7, respectively. (C) Cell-bound C5 convertases were constructed on staphylococci by initial deposition of C3b and subsequent addition of soluble fD, fB, and properdin. C5 was subsequently added in the presence of 0 – 450 nM SSL7, and C5a generation in the absence of IgA was analysed by using supernates as stimuli for calcium mobilization in U937-C5aR cells. Results represent mean values ± SEM of three independent experiments and are expressed relative to cells treated with supernate without SSL7.

Article Snippet: Bound C5 was detected using anti-C5/C5a (Hycult biotechnology, The Netherlands) and peroxidase (PO)-conjugated goat anti–mouse IgG.

Techniques: Construct

Journal:

Article Title: Functional basis for complement evasion by staphylococcal superantigen-like 7

doi: 10.1111/j.1462-5822.2010.01486.x

Figure Lengend Snippet: SSL7 effects are not dependent on IgA binding. (A-C) ELISA experiments showing binding of IgA1 (A), IgA2 (B), or C5 (C) to wells coated with 450 nM SSL7 and SSL7-IgA mutant. Results represent mean values ± SEM of two independent experiments. (D) Staphylococci were opsonized with 10% human serum in the presence of 450 nM SSL7 or SSL7-IgA mutant. Supernates were examined for generated C5a by Western blotting. Representative of three experiments.

Article Snippet: Bound C5 was detected using anti-C5/C5a (Hycult biotechnology, The Netherlands) and peroxidase (PO)-conjugated goat anti–mouse IgG.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Mutagenesis, Generated, Western Blot