purified human c3a  (Alpha Diagnostics)

Journal: Science Immunology

Article Title: SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation

doi: 10.1126/sciimmunol.abg0833

Figure Lengend Snippet: ( A-D ) Confocal images ( A and C ) and quantification ( B and D ) from n =2 independent experiments showing expression of C3a and SARS-CoV-2 N-protein in SARS-CoV-2-treated or mock-infected Calu-3 cells ( A and B ) or induced pleuripotent stem cell-derived alveolar epithelial type 2 cells (iAEC2s) ( C and D ). Scale bars in A and C indicates 100m. Cell numbers are indicated below each violin and median values denoted by dots in B and D . ( E-F ) correlation between SARS-CoV-2 N-protein intensity and C3a intensity on a per cell basis in Calu-3 cells ( E ) and iAEC2s ( F ). Indicated are Pearson correlation coefficients and associated p-values. Infected and uninfected cells in ( B-D ) have been distinguished by red and blue fills, respectively. ****p<0.0001 by ANOVA.

Article Snippet: A C3a standard curve was constructed using purified human C3a (ComTech, A118) in concentrations ranging from 10nM to 0.00977nM.

Techniques: Expressing, Infection, Derivative Assay

Journal: Science Immunology

Article Title: SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation

doi: 10.1126/sciimmunol.abg0833

Figure Lengend Snippet: ( A ) chemoproteomic profiling of CFBi identified complement factor as the only target. Shown is the dose-dependent reduction of bead-binding of complement factor B from protein extracts of cells. Shown are mean and s.d. from 3 independent experiments. ( B) C3a ELISA in plasma treated with zymosan (an alternative complement pathway activator) in the presence of increasing concentrations of EDTA (a chelator of divalent cations, which stops convertase activity), a CFB blocking antibody or isotype control, the chemical CFBi or its carrier, DMSO. Bars show mean + sem; dots represent individual experiments. ( C ) confocal images (left) and quantifications (right) showing generation of C3a in mock- or SARS-CoV-2-infected iAEC2s treated with CFBi, ruxolitinib or a combination of ruxolitinib and remdesivir. Scale bar indicates 100m. Data are from n =2 independent experiments; 18191 + 660 (mean + sd) cells per condition. Bars indicate mean + sd ( A ) or sem ( B-C ). *p<0.05, ***p<0.001, ***p<0.0001 by ANOVA.

Article Snippet: A C3a standard curve was constructed using purified human C3a (ComTech, A118) in concentrations ranging from 10nM to 0.00977nM.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Activity Assay, Blocking Assay, Control, Infection

Journal: Science Immunology

Article Title: SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation

doi: 10.1126/sciimmunol.abg0833

Figure Lengend Snippet: Schematic model of SARS-CoV-2-induction of complement in respiratory epithelial cells . SARS-CoV-2 infects respiratory epithelial cells and induces an interferon response. IFNs signal via the IFN receptor to activate STAT1 via JAK1/2. STAT1 co-operates with RELA to induce transcription of IL6 and complement genes including C3 , CFB , C1R and C1S . CFB acts as an alternative pathway C3 convertase to cleave C3 intracellularly to C3a and C3b. C3a engages C3aR and C3b engages CD46 on leukocyte subsets in the lungs to drive inflammation. These events can be pharmacologically targeted with antivirals (e.g., remdesivir), JAK-STAT inhibitors (e.g., ruxolitinib) and/or cell permeable complement inhibitors, including CFBi.

Article Snippet: A C3a standard curve was constructed using purified human C3a (ComTech, A118) in concentrations ranging from 10nM to 0.00977nM.

Techniques:

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: (A) IFN-γ secretion (left) and intracellular C3a generation (right) by healthy donor CD4+ T cells activated as shown in the presence and absence of ICAM-1 with and without a cell-permeable cathepsin L inhibitor (CTSLi) for 72h (n=4 independent experiments). (B) Representative flow cytometry plots from n=4 independent experiments showing C3 mRNA and the mTOR down-stream target S6 kinase phosphorylated at ser235/ser236 (p-S6) in healthy donor CD4+ T cells activated as shown in the presence or absence of ICAM-1 for 36h. (C) Extracellular acidification rate (ECAR, a measure of glycolysis) and oxygen consumption rate (OCR, a measure of oxidative phosphorylation) in healthy donor naive CD4+ T cells activated as shown in the presence or absence of ICAM-1 with and without a CTSL inhibitor for 36h. Shown are representative ECAR (above) and OCR (middle) graphs together with cumulative data of the maximal respiration and glycolysis (below) from n=3 independent experiments. NA, non-activated; FCCP, p-trifluoromethoxyphenyl hydrazone; OM, oligomycin; ROT, rotenone. Bars show mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. See also Figure S4.

Article Snippet: Purified human C3a , CompTech , Cat. #A118.

Techniques: Flow Cytometry, Phospho-proteomics

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: (A) Steady-state expression of C3 and IFNG mRNA in paired CD4+ T cells drawn from blood and synovial fluid of pediatric patients with juvenile idiopathic oligo-arthritis (n=4; Table S2). Bars show mean + SEM. (B) IFN-γ secretion by paired CD4+ T cells drawn from blood and synovial fluid of two patients with rheumatoid arthritis (RA) activated in vitro with antibodies against CD3 with, or without, ICAM-1. (C-D) C3 mRNA expression by synovial T cells of patients with either oligo-arthritis (OA), non-inflamed RA or inflamed RA derived from an independent dataset (Zhang et al., 2019) (C) and receiver operating characteristic curves showing performance of C3 and IFNG mRNA expression in synovial T cells as biomarkers to distinguish inflamed from un-inflamed RA (D). (E) LFA-1 expression (top), C3 mRNA (middle) and IFN-γ secretion (bottom) by peripheral blood CD4+ T cells from patients with LFA-1 mutations (LAD-1 disease) (see Table S3) and age- and sex-matched controls activated in vitro as shown with, and without ICAM-1 for 72h. Data are from three patients, two of whom donated twice, and five controls. Bars represent mean of duplicate measurements per subject. (F) regression lines showing correlation between IFN-γ and LFA-1 expression (left) and between C3 mRNA and LFA-1 expression (right) from primary data in (E). 95% confidence intervals of the regression line are shown and individual donors are marked. (G) IFN-γ secretion by peripheral blood CD4+ T cells from three patients with LAD-1 disease after transduction with control adenovirus or adenovirus encoding C3a, electroporation with mRNA encoding GFP or C3a or electroporation with BSA or C3H2O protein, as indicated. Cells were activated with anti-CD3+CD46+ICAM-1 after transduction or electroporation. Each patient has been labelled, two of whom donated three times, and the over-expression method shown by color-coding. *p<0.05. See also Figure S6 and Tables S2 and S3.

Article Snippet: Purified human C3a , CompTech , Cat. #A118.

Techniques: Expressing, In Vitro, Derivative Assay, Transduction, Control, Electroporation, Over Expression

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Purified human C3a , CompTech , Cat. #A118.

Techniques: Virus, Generated, Control, Isolation, Recombinant, Purification, Staining, Diagnostic Assay, Cell Isolation, Enzyme-linked Immunosorbent Assay, Labeling, Gene Expression, Reverse Transcription, Synthesized, Software, Transfection, Imaging, Flow Cytometry, Microscopy, Western Blot

Journal: Molecular Medicine Reports

Article Title: Sustained activation of C3aR in a human podocyte line impairs the morphological maturation of the cells

doi: 10.3892/mmr.2020.11626

Figure Lengend Snippet: Primers used for reverse transcription-PCR.

Article Snippet: In the C3a stimulation tests, purified human C3a (Merck KGaA) was directly added to the cell culture medium once every two days at 37°C for a total of 6 days at a final concentration of 100 nM.

Techniques: Sequencing

Journal: Molecular Medicine Reports

Article Title: Sustained activation of C3aR in a human podocyte line impairs the morphological maturation of the cells

doi: 10.3892/mmr.2020.11626

Figure Lengend Snippet: Results of RT-qPCR, western blotting and immunofluorescence demonstrated that C3aR is expressed by HPC cells. Results for (A) RT-qPCR, (B) western blotting and (C) immunofluorescence staining for C3aR. Scale bar, 50 µM. Homologous serum was used instead of anti-C3aR antibody as the negative control in (C). RT-qPCR, reverse transcription-quantitative PCR; C3aR, human C3a anaphylatoxin receptor; HPC, human podocyte cell line; Md, DNA molecular weight; Neg, negative control; Mp protein molecular weight.

Article Snippet: In the C3a stimulation tests, purified human C3a (Merck KGaA) was directly added to the cell culture medium once every two days at 37°C for a total of 6 days at a final concentration of 100 nM.

Techniques: Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Negative Control, Real-time Polymerase Chain Reaction, Molecular Weight

Journal: Molecular Medicine Reports

Article Title: Sustained activation of C3aR in a human podocyte line impairs the morphological maturation of the cells

doi: 10.3892/mmr.2020.11626

Figure Lengend Snippet: C3a was proved to be overexpressed in HPC-C3a cells compared with HPC-NC and untransfected HPC cells. The overexpression of C3a in HPC-C3a cells was confirmed at the (A) mRNA level by reverse transcription-PCR and the (B) protein level by measuring C3a in the cultured medium using an ELISA kit. ***P<0.001 vs. the HPC group; ### P<0.001 vs. the HPC-NC group. C3a, human C3A anaphylatoxin; HPC, human podocyte cell line; NC, negative control.

Article Snippet: In the C3a stimulation tests, purified human C3a (Merck KGaA) was directly added to the cell culture medium once every two days at 37°C for a total of 6 days at a final concentration of 100 nM.

Techniques: Over Expression, Cell Culture, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Molecular Medicine Reports

Article Title: Sustained activation of C3aR in a human podocyte line impairs the morphological maturation of the cells

doi: 10.3892/mmr.2020.11626

Figure Lengend Snippet: Influence of C3A anaphylatoxin overexpression on the morphology and adhesion ability of HPC cells during maturation. (A) Representative images taken under a phase contrast microscope demonstrating the morphology of HPC, NC, C3a cells and C3a cells treated with 1 µM of SB290157 cultured during the maturation condition for 0, 2, 4, 7 and 14 days. Scale bar, 100 µM. (B) Representative images taken with a fluorescence microscope revealing the morphology of NC, C3a and SB cells cultured during the maturation condition for 0, 2, 4, 7 and 14 days. As normal HPC cells do not exhibit fluorescence, the untransfected HPC cells were not included in the cell morphology observational experiments under fluorescence microscopy. Scale bar, 50 µM. (C) Results of adhesion analysis. The adhesion ability of HPC, NC, C3a and SB cells was analyzed following culturing in the maturation condition for 6 days. **P<0.01 vs. the HPC group; ## P<0.01 vs. the NC group; && P<0.01 vs. the C3a group. C3a, HPC cells overexpressing human C3A anaphylatoxin; HPC, human podocyte cell; NC, HPC cells transfected with negative control vector; SB, C3a cells treated with SB290157.

Article Snippet: In the C3a stimulation tests, purified human C3a (Merck KGaA) was directly added to the cell culture medium once every two days at 37°C for a total of 6 days at a final concentration of 100 nM.

Techniques: Over Expression, Microscopy, Cell Culture, Fluorescence, Transfection, Negative Control, Plasmid Preparation

Journal: Molecular Medicine Reports

Article Title: Sustained activation of C3aR in a human podocyte line impairs the morphological maturation of the cells

doi: 10.3892/mmr.2020.11626

Figure Lengend Snippet: Overexpression of C3a markedly alters the immunostaining pattern of VCL. All cells (HPC, NC, C3a and SB) were grown on glass coverslips. Following culturing in the maturation condition for 6 days, the cells were fixed and stained for VCL. Homologous serum was used instead of anti-VCL antibody as the negative control (negative immuno-staining group). Scale bar, 50 µM. C3a, human C3A anaphylatoxin; VCL, vinculin; HPC, human podocyte cell line; NC, negative control; SB, SB290157.

Article Snippet: In the C3a stimulation tests, purified human C3a (Merck KGaA) was directly added to the cell culture medium once every two days at 37°C for a total of 6 days at a final concentration of 100 nM.

Techniques: Over Expression, Immunostaining, Staining, Negative Control

Journal: Molecular Medicine Reports

Article Title: Sustained activation of C3aR in a human podocyte line impairs the morphological maturation of the cells

doi: 10.3892/mmr.2020.11626

Figure Lengend Snippet: Expression levels of cell adhesion-associated genes.

Article Snippet: In the C3a stimulation tests, purified human C3a (Merck KGaA) was directly added to the cell culture medium once every two days at 37°C for a total of 6 days at a final concentration of 100 nM.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Sustained activation of C3aR in a human podocyte line impairs the morphological maturation of the cells

doi: 10.3892/mmr.2020.11626

Figure Lengend Snippet: Overexpression of C3a significantly decreased the levels of VCL, PTK2 and ITGB1 in HPC cells. All cells (HPC, NC, C3a and SB) were cultured in the maturation condition for 6 days. Total proteins were extracted and (A-C) western blotting and (D-F) statistical analysis for VCL, PTK2 and ITGB1 was performed. *P<0.05, **P<0.01 vs. the HPC group; # P<0.05, ## P<0.01 vs. HPC-NC group; & P<0.05, && P<0.01 vs. the C3a group. C3a, human C3A anaphylatoxin; PTK2, protein tyrosine kinase 2; ITGB1, integrin β1; HPC, human podocyte cell line; NC, negative control; SB, SB290157; VCL, vinculin.

Article Snippet: In the C3a stimulation tests, purified human C3a (Merck KGaA) was directly added to the cell culture medium once every two days at 37°C for a total of 6 days at a final concentration of 100 nM.

Techniques: Over Expression, Cell Culture, Western Blot, Negative Control

Journal: Molecular Medicine Reports

Article Title: Sustained activation of C3aR in a human podocyte line impairs the morphological maturation of the cells

doi: 10.3892/mmr.2020.11626

Figure Lengend Snippet: Addition of C3a to the medium only partially mimics the effect of overexpression of C3a in HPC cells. HPC cells were divided into three groups: HPC, HPC+SB+C3a and HPC+C3a. Morphological changes were observed under a phase contrast microscope. (A) Reverse transcription-PCR analysis of the expression of focal adhesion-associated genes. *P<0.05, **P<0.01 vs HPC group; # P<0.05, ## P<0.01 vs HPC+C3a group. (B) Immunofluorescence staining for VCL. Scale bar, 50 µM. (C) Representative images taken under phase contrast microscopy demonstrated cell morphology. Scale bar, 100 µM. (D) Results of adhesion analysis. COL1A1, collagen I α1; FN1, fibronectin 1; LAMA1, laminin α1; LAMB1, laminin β1; LAMC1, laminin γ1; ITGA1, integrin α1; ITGA2, integrin α2; ITGB1, integrin β1; PTK2, protein tyrosine kinase 2; NRP1, neuropilin 1; TLN2, talin 2; FERMT2, fermitin family member 2; VCL, vinculin; C3a, human C3a anaphylatoxin; HPC, human podocyte cell; SB, SB290157.

Article Snippet: In the C3a stimulation tests, purified human C3a (Merck KGaA) was directly added to the cell culture medium once every two days at 37°C for a total of 6 days at a final concentration of 100 nM.

Techniques: Over Expression, Microscopy, Expressing, Immunofluorescence, Staining