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: 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