Journal: medRxiv

Article Title: Differential virulence potential of different clades of multidrug-resistant Klebsiella pneumoniae ST258

doi: 10.64898/2026.03.28.26349612

Figure Lengend Snippet: (A and B) Bacteria recovered after 30-minute exposure of 10 8 CFU/mL to 85% human serum. Limit of detection (LOD) is indicated with a dashed line. (A) 13 Clade 1 isolates and 36 Clade 2 isolates were tested, each with at least two biological replicates. (B) 6 biological replicates per strain were tested. (C and D) C3 binding to bacteria was measured using an indirect ELISA. (C) 13 Clade 1 isolates and 36 Clade 2 isolates were tested, each with at least two biological replicates. (D) 6 biological replicates were tested per strain, each with at least 5 technical replicates. (E) Correlation between C3 binding and serum survival of Clade 2 isolates was assessed using Spearman correlation. (F) Uronic acid was quantified from a selection of Clade 1 (n=6) and Clade 2 (n=10) isolates with 3 biological replicates per isolate. Significance was assessed with Welch’s t-test. (G) Correlation between uronic acid quantity and serum survival of 10 Clade 2 isolates was assessed using Spearman correlation. (H) OD-normalized bacteria were reacted with 5% human serum in PBS for 30 minutes. C5a from the supernatant of the reaction was quantified using a sandwich ELISA. At least two biological replicates per isolate were performed. For panels A-D and H, P ≤ 0.05: *, P ≤ 0.01: **, P ≤ 0.001: *** by Mann-Whitney test.

Article Snippet: C5a quantification was performed using the Human Complement Component C5a DuoSet ELISA from R&D systems.

Techniques: Bacteria, Binding Assay, Indirect ELISA, Selection, Sandwich ELISA, MANN-WHITNEY

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: 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: 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: