Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A second stimulus required for enhanced antifungal activity of human neutrophils in blood is provided by anaphylatoxin C5a.

doi: 10.4049/jimmunol.1401845

Figure Lengend Snippet: FIGURE 2. C5a contributes to the early PMN activation during whole-blood infection with C. albicans. Human whole blood was treated with an anti- C5a Ab (IFX-1) prior to inoculation of C. albicans for 10 and 60 min to investigate the impact of C5a on activation of blood PMN. Samples were compared with either nontreated or IgG4-containig C. albicans–infected blood. (A) The percentages of ROI formation in samples inoculated with C. albicans were calculated relative to basal levels in the mock-infected sample (set to 100%). The bars show mean 6 SD of at least seven independent experiments with blood from different donors; **p , 0.01. (B) Differential regulation of surface exposure of degranulation marker CD66b is shown in histograms. Gray filled histograms indicate basal expression on PMN in the mock-infected sample. Black open histograms indicate surface levels following C. albicans infection and differentiate between PMN without fungal contact (dashed line) and PMN that interact with C. albicans (dotted line). C. albicans samples were either nontreated (a) or preincubated with IgG4 (b) or anti-C5a (c). (C) Plasma samples of whole-blood infection experiments were analyzed for secretion of antimicrobial peptides from primary (MPO, elastase-2) and secondary neutrophilic granules (NGAL, lactoferrin) and the release of IL-8. In comparison with non- (white bars) and IgG4-treated infected samples (striped bars), presence of anti-C5a Ab (black bars) significantly prevented the increase in plasma levels of lactoferrin, NGAL and IL-8 in response to C. albicans (t = 60 min). Gray bars indicate basal plasma levels within mock-infected blood. Bars show means 6 SD of nine independent experiments with whole blood from different donors. **p , 0.01. ns, nonsignificant.

Article Snippet: In selected experiments, recombinant human C5a (Hycult Biotech) was added at different concentrations (as indicated) and PMN activation was analyzed by flow cytometry following 30 min of incubation.

Techniques: Activation Assay, Infection, Marker, Expressing, Clinical Proteomics, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A second stimulus required for enhanced antifungal activity of human neutrophils in blood is provided by anaphylatoxin C5a.

doi: 10.4049/jimmunol.1401845

Figure Lengend Snippet: FIGURE 3. Both C5a and C. albicans phagocytosis trigger decrease in C5aR surface expression on PMN. Comparison of C5aR expression on primary and blood PMN after 10 min [(a), top and bottom, respectively] and 60 min [(b), top and bottom, respectively] confrontation with C. albicans. Gray filled histograms show basal expression in mock-infected samples. Black open histograms indicate surface levels following C. albicans infection and differentiate between PMN without fungal contact (dashed line) and PMN that interact with C. albicans (dotted line). Data from one of at least three independent experiments with virtually identical results are shown.

Article Snippet: In selected experiments, recombinant human C5a (Hycult Biotech) was added at different concentrations (as indicated) and PMN activation was analyzed by flow cytometry following 30 min of incubation.

Techniques: Expressing, Comparison, Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A second stimulus required for enhanced antifungal activity of human neutrophils in blood is provided by anaphylatoxin C5a.

doi: 10.4049/jimmunol.1401845

Figure Lengend Snippet: FIGURE 4. Effects of C5a on C. albicans–induced PMN activation in whole blood are mediated by C5aR. Presence of an anti-C5aR Ab during whole- blood infection with C. albicans showed that C5a acts via C5aR to modulate early PMN activation. (A) ROI formation in C. albicans samples (non-, IgG2a-, or anti–C5aR-treated) were normalized against basal levels in mock-infected blood (set to 100%). The bars show mean 6 SD of five independent experiments with blood from different donors. *p , 0.05. (B) Surface expression of CD66b is shown in histograms. Gray filled histograms indicate basal expression on PMN within mock-infected sample. Black open histograms indicate surface levels following C. albicans infection and differentiate between PMN without fungal contact (dashed line) and PMN that interact with C. albicans (dotted line). C. albicans–infected samples were either nontreated (a) or preincubated with IgG2a (b) or anti-C5aR (c). Data from one of at least three independent experiments with virtually identical results are shown. ns, nonsignificant.

Article Snippet: In selected experiments, recombinant human C5a (Hycult Biotech) was added at different concentrations (as indicated) and PMN activation was analyzed by flow cytometry following 30 min of incubation.

Techniques: Activation Assay, Infection, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A second stimulus required for enhanced antifungal activity of human neutrophils in blood is provided by anaphylatoxin C5a.

doi: 10.4049/jimmunol.1401845

Figure Lengend Snippet: FIGURE 5. Blocking of C5a or C5aR decreases initial phagocytosis of C. albicans by PMN. Presence of the anti-C5a (A) or the anti-C5aR Ab (B) signifi- cantly reduced phagocytosis of C. albicans by blood PMN after 10 min of infection but not after 60 min. Non- and isotype control–treated C. albicans samples served as references. C. albicans association with blood PMN was determined by flow cytometry. Each dot represents the percentage of fungal cells phagocytosed by PMN relative to total C. albicans in blood (set to 100%) of independent experiments with blood from different donors. The mean 6 SD is given. *p , 0.05, ***p , 0.001. ns, nonsignificant.

Article Snippet: In selected experiments, recombinant human C5a (Hycult Biotech) was added at different concentrations (as indicated) and PMN activation was analyzed by flow cytometry following 30 min of incubation.

Techniques: Blocking Assay, Infection, Control, Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A second stimulus required for enhanced antifungal activity of human neutrophils in blood is provided by anaphylatoxin C5a.

doi: 10.4049/jimmunol.1401845

Figure Lengend Snippet: FIGURE 6. C5a influences CD11b ex- pression on PMN in infected blood. CD11b expression on PMN in whole blood was analyzed 10 and 60 min after inoculation of C. albicans and compared with basal levels in mock-infected blood (gray filled histo- grams). Black open histograms indicate surface levels following C. albicans infec- tion and differentiate between PMN without fungal contact (dashed line) and PMN that interact with C. albicans (dotted line). C. albicans–infected samples were either nontreated [(a), top and bottom, respec- tively] or preincubated with IgG4 [(b), top and bottom, respectively] or anti-C5a [(c), top and bottom, respectively]. Data from one of at least three independent experiments with virtually identical results are shown.

Article Snippet: In selected experiments, recombinant human C5a (Hycult Biotech) was added at different concentrations (as indicated) and PMN activation was analyzed by flow cytometry following 30 min of incubation.

Techniques: Infection, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A second stimulus required for enhanced antifungal activity of human neutrophils in blood is provided by anaphylatoxin C5a.

doi: 10.4049/jimmunol.1401845

Figure Lengend Snippet: FIGURE 9. C5a induces PMN to phagocytose C. albicans in a mor- photype-independent manner. (A) C. albicans wild-type (;) and the nonfilamentous mutant cph1D/efg1D (d) resulted in equal C5a plasma levels during whole-blood infection. Dots show means 6 SD of five in- dependent experiments with whole blood from different donors. (B and C) Samples of human whole blood were infected with either FITC-labeled C. albicans wild-type and cph1D/efg1D (B) or killed GFP-expressing C. albicans wild-type yeast cells and filaments (C). Association with blood PMN after 60 min of confrontation was determined in the presence of IFX- 1 and compared with either IgG4-containig or nontreated C. albicans– infected blood (set to 100%) using flow cytometry. The bars show mean 6 SD of three independent experiments with blood from different donors. *p , 0.05.

Article Snippet: In selected experiments, recombinant human C5a (Hycult Biotech) was added at different concentrations (as indicated) and PMN activation was analyzed by flow cytometry following 30 min of incubation.

Techniques: Mutagenesis, Clinical Proteomics, Infection, Labeling, Expressing, Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Urine proteins reveal distinct coagulation and complement cascades underlying acute versus chronic lupus nephritis

doi: 10.1172/JCI186143

Figure Lengend Snippet: ( A ) Shown are representative fields from 3 independent experiments. Complement proteins C3a and C5a increased the expression of ECM proteins in THP1 macrophages, BMDMs, and HK2 proximal tubule cells after 72 hours of treatment in serum-free medium. Scale bars: 50 μm. ( B – D ) The scatter plots show the mean staining intensity per THP-1 macrophage ( B ), BMDM ( C ), and HK2 proximal tubule cell ( D ), normalized to expression levels in their respective vehicle-treated groups. Each data point corresponds to quantified fluorescence intensity in a single field of view (FOV) from the microscope, and the larger dots represent the average of FOVs in biological replicates, each of which is color coded. RT-qPCR analysis of ECM protein coding genes were measured in BMDMs ( E ) and in HK2 proximal tubule cells ( F ). Gene expression was normalized to the expression of 18S ribosomal RNA in the same sample and then normalized to the expression level of vehicle-treated group ( n = 4). * P < 0.05, ** P < 0.01, and *** P < 0.001 by 2-tailed Student’s t test.

Article Snippet: After 3 days of differentiation, the medium was replaced with serum-free medium for 24 hours, after which the cells were treated for 72 hours with either vehicle or 10 ng/mL C3a (R&D Systems 3677-C3-025) or 10 ng/mL of C5a (R&D Systems 2037-C5-025/CF).

Techniques: Expressing, Staining, Fluorescence, Microscopy, Quantitative RT-PCR, Gene Expression

Journal: The Journal of Clinical Investigation

Article Title: Urine proteins reveal distinct coagulation and complement cascades underlying acute versus chronic lupus nephritis

doi: 10.1172/JCI186143

Figure Lengend Snippet: Circulating immune complexes and Abs planted directly within glomerular and tubulo-interstitial regions of the kidneys may fix complement, resulting in complement activation. The alternative pathway may further amplify complement activation within the kidneys. The products of C3 and C5 convertases, including the anaphylatoxins C3a and C5a, engage cognate receptors on a wide spectrum of immune cells, leading to immune cell activation, release of cytokines and chemokines, and acute inflammation, leading to high AI, as depicted on the left. Long-standing, unresolved complement activation and eventual formation of MAC may additionally engage and activate more immune and renal-resident cells, leading to tissue damage and repair, ECM deposition, and renal fibrosis, leading to high CI, as depicted on the right.

Article Snippet: After 3 days of differentiation, the medium was replaced with serum-free medium for 24 hours, after which the cells were treated for 72 hours with either vehicle or 10 ng/mL C3a (R&D Systems 3677-C3-025) or 10 ng/mL of C5a (R&D Systems 2037-C5-025/CF).

Techniques: Activation Assay

Journal: Cells

Article Title: Extracellular Vesicles Derived from Endothelial Progenitor Cells Protect Human Glomerular Endothelial Cells and Podocytes from Complement- and Cytokine-Mediated Injury

doi: 10.3390/cells10071675

Figure Lengend Snippet: Time- and dose-dependent cytotoxicity and apoptosis assays on Glomerular Endothelial Cells (GECs) and Podocytes. GECs. Time-dependent XTT citotoxity assay on GECs ( A ) and podocytes ( B ) incubated with a medium containing CK (20 ng/mL TNF-α, 2.5 ng/mL IL-6, and 50 ng/mL human recombinant C5a protein) or vehicle alone at different time-points (12, 18, 24, 36, 48 h) Significant differences in viability were observed on both GECs and podocytes at 18, 24, 36, and 48 h (* p < 0.05, CK vs. vehicle). At 12 h, only the GECs (A) had different viability between treated (CK) and control cells (vehicle, * p < 0.05, CK vs. vehicle). Cytotoxicity tests by XTT ( C ) and apoptosis by TUNEL assay ( D ), evaluating the protective dose of EPC-derived EVs on GECs (black columns) and podocytes (white columns) after 24 h. Incubation with CK alone was effective in inducing cytotoxic (C) and apoptotic damage (D) in both cell types (* p < 0.05, CK vs. vehicle). Adding 1 or 10 μg/mL EPC-derived EVs to cells incubated with CK did not significantly reduce cell damage (* p < 0.05, CK + 1 μg/mL EV and CK + 10 μg/mL EV vs. vehicle), except with GEC incubated with 10 μg/mL EV in TUNEL assay (§ CK + 10 μg/mL EV vs. CK). In contrast, the adding of 25 or 50 μg/mL EPC-derived EVs to cells incubated with CK significantly reduced cell damage at the level of GECs and podocytes (§ p < 0.05, CK + 25 μg/mL EV and CK + 50 μg/mL EV vs. CK). XTT assays are reported as average optical density (O.D.) intensity ± 1SD. For TUNEL assays, data are expressed as the average number of green fluorescent apoptotic cells ± 1 SD. We performed three experiments for each test and the statistical analysis by ANOVA with Newmann–Keuls multiple comparison test.

Article Snippet: In selected experiments, we incubated cells in an appropriate medium containing 20 ng/mL tumor necrosis factor (TNF)-α (Sigma Aldrich), 2.5 ng/mL IL-6, and 50 ng/mL human recombinant C5a protein (R&D Systems, Minneapolis, MN, USA) in the presence or absence of different concentrations of EPC-derived EVs assessed by Nanosight analysis.

Techniques: Incubation, Recombinant, Control, TUNEL Assay, Derivative Assay, Comparison

Journal: Cells

Article Title: Extracellular Vesicles Derived from Endothelial Progenitor Cells Protect Human Glomerular Endothelial Cells and Podocytes from Complement- and Cytokine-Mediated Injury

doi: 10.3390/cells10071675

Figure Lengend Snippet: EPC-derived EVs protected GECs from complement- and cytokine-induced damage ( A , B ). Graphs showing GEC cytotoxicity by XTT assay ( A ) and apoptosis by TUNEL assay ( B ). For XTT assays, data are expressed as average OD intensity ± 1 SD, whereas we expressed TUNEL assays data as the average number of green fluorescent apoptotic cells ± 1 SD. We performed three experiments with similar results for all the assays and the statistical analysis by ANOVA with Newmann–Keuls multiple comparison test and Student’s t -test. ( C , D ) FACS analysis ( C ) and representative micrographs ( D ) of ROS expression of GEC (green staining) by confocal microscopy studies (magnification ×400, scale bar 50 μm). Nuclei were counterstained in blue by 2.5 μg/mL Hoechst. We performed three experiments with similar results for all the assays, and we performed the statistical analysis by ANOVA with Newmann–Keuls multiple comparison test and the Kolmogorov–Smirnov test. Incubation with cytokines 20 ng/mL TNF -α, 2.5 ng/mL IL-6, plus 50 ng/mL human recombinant C5a protein CKs significantly increased GEC vitality ( A ), inhibited resistance to apoptosis ( B ), and increased oxidative stress (C) in comparison to treatment with vehicle alone (vehicle, * p < 0.05 CK vs. vehicle). EV stimulation significantly inhibited these effects (§ p < 0.05 CK + EV vs. CK), but not EV were pre-treated with 1 U/mL RNase (# p < 0.05 CK + EV RNase vs. CK + EV).

Article Snippet: In selected experiments, we incubated cells in an appropriate medium containing 20 ng/mL tumor necrosis factor (TNF)-α (Sigma Aldrich), 2.5 ng/mL IL-6, and 50 ng/mL human recombinant C5a protein (R&D Systems, Minneapolis, MN, USA) in the presence or absence of different concentrations of EPC-derived EVs assessed by Nanosight analysis.

Techniques: Derivative Assay, XTT Assay, TUNEL Assay, Comparison, Expressing, Staining, Confocal Microscopy, Incubation, Recombinant

Journal: Cell Death & Disease

Article Title: Paclitaxel binds and activates C5aR1: A new potential therapeutic target for the prevention of chemotherapy-induced peripheral neuropathy and hypersensitivity reactions

doi: 10.1038/s41419-022-04964-w

Figure Lengend Snippet: Selected potential paclitaxel targets and their predicted affinity.

Article Snippet: The reagents that have been used are: recombinant human C5a anaphylotoxin chemotactic receptor 2 (C5AR2) (CUSABIO TECHNOLOGY, Houston, TX, US), recombinant protein of human complement component 5a receptor 1 (C5AR1) (Origene, Rockville, MD, US), anti-6X His tag antibody (Abcam, Cambridge, UK), Anti-Myc/c-Myc Antibody (9E10) (Santa Cruz Biotechnology, Dallas, Texas, US) and CD88 (clone S5/1) antibody (344302, Biolegend, San Diego, California, US), complement C5a des-Arg, Human (Merck, Burlington, Massachusetts, USA).

Techniques:

Journal: Cell Death & Disease

Article Title: Paclitaxel binds and activates C5aR1: A new potential therapeutic target for the prevention of chemotherapy-induced peripheral neuropathy and hypersensitivity reactions

doi: 10.1038/s41419-022-04964-w

Figure Lengend Snippet: ( A ) Binding of paclitaxel on immobilized C5aR1, C5aR2, C5a, and C5a des-Arg. ( B ) Dose-dependent binding of paclitaxel on immobilized C5aR1. ( C , D ) Co-injection of paclitaxel and C5aR1 on immobilized C5a and relative IC50. ( E , F ) Co-injection of paclitaxel and C5aR1 on immobilized C5a des-Arg and relative IC50. G , H Co-injection of paclitaxel and C5aR1 on immobilized monoclonal antibody anti-C5aR1 and relative IC50.

Article Snippet: The reagents that have been used are: recombinant human C5a anaphylotoxin chemotactic receptor 2 (C5AR2) (CUSABIO TECHNOLOGY, Houston, TX, US), recombinant protein of human complement component 5a receptor 1 (C5AR1) (Origene, Rockville, MD, US), anti-6X His tag antibody (Abcam, Cambridge, UK), Anti-Myc/c-Myc Antibody (9E10) (Santa Cruz Biotechnology, Dallas, Texas, US) and CD88 (clone S5/1) antibody (344302, Biolegend, San Diego, California, US), complement C5a des-Arg, Human (Merck, Burlington, Massachusetts, USA).

Techniques: Binding Assay, Injection

Journal: Cell Death & Disease

Article Title: Paclitaxel binds and activates C5aR1: A new potential therapeutic target for the prevention of chemotherapy-induced peripheral neuropathy and hypersensitivity reactions

doi: 10.1038/s41419-022-04964-w

Figure Lengend Snippet: Representative trace of electrophysiological recordings in DRG cells maintained under basal conditions ( A ), and in cells challenged with paclitaxel (10 nM) (PAC) ( B ), paclitaxel+DF3966A (1 µM) ( C ), C5a (100 nM) ( D ) or C5a + DF3966A ( E ). Quantitative evaluation of action potential firing rate in DRG exposed to paclitaxel alone or in combination with DF3966A ( F ), and C5a alone or in combination with DF3966A ( G ). DRG-derived neurons were cultured for 7 days. Cells were treated for 1 min 30 s (short) or 5 min (long). ** P < 0.005 and * P < 0.05 vs respective basal condition; + P < 0.05 vs paclitaxel or C5a. Data are mean ± SEM of n = 3 different experiments.

Article Snippet: The reagents that have been used are: recombinant human C5a anaphylotoxin chemotactic receptor 2 (C5AR2) (CUSABIO TECHNOLOGY, Houston, TX, US), recombinant protein of human complement component 5a receptor 1 (C5AR1) (Origene, Rockville, MD, US), anti-6X His tag antibody (Abcam, Cambridge, UK), Anti-Myc/c-Myc Antibody (9E10) (Santa Cruz Biotechnology, Dallas, Texas, US) and CD88 (clone S5/1) antibody (344302, Biolegend, San Diego, California, US), complement C5a des-Arg, Human (Merck, Burlington, Massachusetts, USA).

Techniques: Derivative Assay, Cell Culture

Journal: Cell Death & Disease

Article Title: Paclitaxel binds and activates C5aR1: A new potential therapeutic target for the prevention of chemotherapy-induced peripheral neuropathy and hypersensitivity reactions

doi: 10.1038/s41419-022-04964-w

Figure Lengend Snippet: Proteome profiles of cytokines released from RAW 264.7 ( A , B ) and human ( C , D ) macrophages challenged with C5a ( A , C ) or paclitaxel ( B , D ) alone or in combination with a C5AR1 antibody (anti C5AR), Avacopan or DF3966A. RAW 264.7 and human macrophages cells were treated with C5a (10 nM), or paclitaxel (PAC) (10 nM), or DF3966A (1 μM) or Avacopan (1 μM) and C5aR1 antibody (1 μM) for 24 h. Data are mean ± SEM of n = 3 different experiments.

Article Snippet: The reagents that have been used are: recombinant human C5a anaphylotoxin chemotactic receptor 2 (C5AR2) (CUSABIO TECHNOLOGY, Houston, TX, US), recombinant protein of human complement component 5a receptor 1 (C5AR1) (Origene, Rockville, MD, US), anti-6X His tag antibody (Abcam, Cambridge, UK), Anti-Myc/c-Myc Antibody (9E10) (Santa Cruz Biotechnology, Dallas, Texas, US) and CD88 (clone S5/1) antibody (344302, Biolegend, San Diego, California, US), complement C5a des-Arg, Human (Merck, Burlington, Massachusetts, USA).

Techniques:

Journal: bioRxiv

Article Title: Asparagine couples group A Streptococcal metabolism to virulence

doi: 10.1101/2024.07.08.602371

Figure Lengend Snippet: (a,b) Degradation of recombinant C5a by cultures supernatant containing the C5a-peptidase enzyme (ScpA). GAS was grown in CDM without or with Asn or/and LL-37. Supernatants of the indicated strains were incubated with recombinant human C5a, resolved on Tris-tricine gels, and visualized by Coomassie blue staining. M represents a marker, and an empty arrow is the cleaved C5a. The data are representative of 2 independent experiments. (c,d) Quantitation of IL-8 degradation by ScpC present in the supernatants of the indicated strains by ELISA. n=2 . The data shown represent the means ± S.D. (e) Asn does not affect in-vitro CovR phosphorylation . Purified CovR was incubated in the absence (−) and presence (+) of acetyl phosphate (Ac-P) as a phosphate donor and the indicated concentrations of Asn (µg ml -1 ). The protein samples were resolved on Phos-tag SDS-PAGE gel and visualized.

Article Snippet: The cell-free supernatants were incubated with 1 mg/ml of purified recombinant human complement component C5a (R&D Systems, USA) at 37°C for 2 h. The samples were heated at 100°C for 5 min with 4x Tricine loading buffer (0.4 M Tris HCl pH 6.8, 80% glycerol, 4% SDS, and 0.08% Coomassie blue) to stop the reaction.

Techniques: Recombinant, Incubation, Staining, Marker, Quantitation Assay, Enzyme-linked Immunosorbent Assay, In Vitro, Phospho-proteomics, Purification, SDS Page