Journal: Molecular Therapy

Article Title: A Novel C5a-neutralizing Mirror-image ( l -)Aptamer Prevents Organ Failure and Improves Survival in Experimental Sepsis

doi: 10.1038/mt.2013.178

Figure Lengend Snippet: NOX-D20 binds to C5 but does not inhibit complement-mediated hemolysis. SPR measurement of NOX-D20 binding to human ( a ) C5a, ( b ) C5a(desArg), and ( c ) C5. Kinetic rate constants k a and k d are shown as mean ± SEM. Data are representative for at least three individual measurements. ( d ) Human serum pretreated with NOX-D20 (black squares) or C5-binding aptamer C5C6 (black triangles) was incubated with opsonized sheep erythrocytes. Hemolysis was quantified by photometric measurement of hemoglobin in the supernatant at 405 nm. Normalized data representative for three independent experiments is shown. RU, response units.

Article Snippet: Recombinant human and mouse C5a was from R&D Systems (Wiesbaden, Germany), recombinant human C5a(desArg) from Hycult Biotech (Beutelsbach, Germany), and human C5 purified from serum from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: Binding Assay, Incubation

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: All inductions were performed for 3 days with 1 ×10 6 CD62L hi Foxp3 − CD25 − CD4 + T cells, rhIL-2 (5 ng/ml), and anti-CD3 + CD28 beads (per manufacturer’s instructions), experiments were repeated two times. ( a ) WT, C3ar1 −/− , C5ar1 −/− , or C3ar1 −/− C5ar1 −/− Foxp3 − cells were activated and assayed for percent Foxp3 + CD25 + by flow cytometry (*P<0.001, n=5). (b) Following iT reg induction (as in a ), flow sorted WT, C3ar1 −/− , C5ar1 −/− , or C3ar1 −/− C5ar1 −/− Foxp3 + CD25 + cells were incubated with 10 6 CellTracker Red™ labeled CD25 − Foxp3 − CD4 + WT cells in differing T eff /iT reg ratios + anti-CD3 (5 μg/ml) and 2.5×10 5 CD11c + DCs. Relative suppressive capacity was determined by percent Red-labeled dividers (n=5). (c) WT Foxp3 − CD4 + T cells were induced as in (a) in the absence or presence of anti-C3a (10 μg/ml), anti-C5a (10 μg/ml), or both or in the absence and presence of the antagonists C3aR-A (10 nM), C5aR-A (10 nM), or both. CD4 + T cells then were assayed for Foxp3 expression by flow cytometry (*P<0.001, n=6). (d) Following iT reg induction (as in a ), sorted WT and C3ar1 −/− C5ar1 −/− Foxp3 + CD25 + cells were washed and recultured for 24 hr in the absence of further stimulation. Culture supernatants were assayed for TGF-β, IL-6, and IL-10 by ELISA (*P<0.001, n=6). (e) C3ar1 −/− C5ar1 −/− iT regs were induced (as in a) in the absence and presence of anti-TGF-β mAb (5 μg/ml), TGF-βR1 inhibitor (10 nM), or Smad3 inhibitor (5 μM). Foxp3 + CD25 + T reg percentages were quantified by flow cytometry.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Flow Cytometry, Incubation, Labeling, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: iT reg induction and activation experiments were performed as in with 2.5×10 5 CD11c + DCs and anti-CD3 mAb (5 μg/ml) instead of anti-CD3+CD28 beads, experiments were repeated two times. (a) Sorted WT and C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with WT DCs ± TGF-β1 (5 ng/ml) and assayed for CD25 and Foxp3 expression by flow cytometry (*P<0.001, n=10). (b) (Left) WT Foxp3 − CD4 + T cells were incubated with C3 −/− C5 −/− DCs without TGF-β1 ± C3a/C5a (100 ng/ml) and Foxp3 + CD25 + cells quantified by flow cytometry. (Right) WT Foxp3 − CD4 + T cells were incubated in the presence of TGF-β1 (5 ng/ml) ± C3a/C5a (100 ng/ml) and percent Foxp3 + CD25 + cells quantified (*P<0.001, n=7). (c) Sorted WT or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with C3ar1 −/− C5ar1 −/− DCs in the absence or presence of C5a (100 ng/ml) after which percent Foxp3 + CD25 + cells was quantified. The % Foxp3 + cells with DKO or WT T cells did not significantly differ (P=0.54). (d) Sorted C3 −/− C5 −/− or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with Daf1 −/− DCs ± anti-C3a and anti-C5a mAbs after which percent Foxp3 + CD25 + cells was quantified (*P<0.001; n=6). (e) Sorted WT Foxp3 − or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated for 24 hr either in the absence of DCs or in the presence of WT DCs or C3ar1 −/− C5ar1 −/− DCs. Supernatants then were assayed for TGF-β1 and IL-6 by ELISA (*P<0.001, n=6). (f) C3ar1 −/− C5ar1 −/− Foxp3 − cells were incubated for 3 days with anti-CD3 (5 μg/ml), IL-2 (5 ng/ml), and WT DCs ± anti-TGF-β mAb (5 μg/ml), TGF-βR1 inhibitor (10 nM), or Smad3 inhibitor (5 μM). Following the 3 day induction, Foxp3 + CD25 + T reg percentages were determined by flow cytometry.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Activation Assay, Incubation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) WT CD4 + T cells were incubated for 30 min with anti-CD3+CD28 beads after which they were incubated for 10 min with Forskolin (30 μM) ± C3a (100 nM), C5a (100 nM), or both. Levels of cAMP activity were determined by cAMP-Glo assay (*P<0.001). (b) WT, C3ar1 −/− , C5ar1 −/− , and C3ar1 −/− C5ar1 −/− CD4 + T cells were stimulated with anti-CD3 mAb for 30 min after which PKA activity was quantified by PepTag assay. (c) iT regs generated from sorted WT Foxp3 − CD4 + T cells plus TGF-β1, iT regs derived from C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells, and conventional Foxp3 − CD4 + T cells were stimulated with anti-CD3+CD28 beads for 15 min and assayed for S 473 and T 308 p-AKT by Luminex assay (n=5). (d and e) WT or C3ar1 −/− C5ar1 −/− CD4 + T cells were incubated for 15 min ± anti-CD3+CD28 beads. Cells were extracted in phospho-lysis buffer and extracts assayed for (d) phospho-rbS6 and (e) phospho-Smad2 by immunoblotting. (f) iT regs induced from WT Foxp3 − CD4 + T cells plus TGF-β1 or from C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were assayed for phospho-STAT3 and phospho-STAT5 by flow cytometry (representative plots of n=6).

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Activity Assay, Glo Assay, Generated, Derivative Assay, Luminex, Lysis, Western Blot, Flow Cytometry

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) Sorted WT Foxp3 − cells were incubated for 1 hr with anti-CD3+CD28 beads (as in ) in the absence or presence of TGF-β1 (5 ng/ml) and then assayed for complement mRNA transcripts by qPCR (*P<0.001, n=5). (b) Sorted WT Foxp3 − CD4 + T cells were incubated for 1 hr with anti-CD3+CD28 beads plus TGF-β1 (5 ng/ml) alone, TGF-β1 (5 ng/ml) plus IL-6 (5 ng/ml), or IL-6 (5 ng/ml) alone, after which the cells were assayed for C3 mRNA expression by qPCR (*P<0.001, **P<0.02, n=5). (c and d) Sorted WT Foxp3 − CD4 + T cells were incubated for 48 hr with anti-CD3+CD28 beads plus TGF-β1 alone, TGF-β1+IL-6, or IL-6 alone as in (b) . Culture supernatants were assayed for (c) C3a and C5a generation by ELISAs, and (d) C3aR and C5aR surface expression by flow cytometry (P<0.01 for TGF-β1 alone vs TGF-β1+IL-6 or IL-6 alone, Mean Fluorescence Intensity (MFI) values; n=5). (e) Sorted WT Foxp3 − cells were activated for 48 hr with anti-CD3+CD28 beads ± C5a (100 ng/ml) or C3aR-A/C5aR-A (10 nM), after which culture supernatants were assayed for TGF-β1 or IL-6 by ELISA (P<0.05; n=6). (f) Sorted WT Foxp3 − CD4 + T cells were incubated with WT DCs or with C3 −/− C5 −/− DCs in the absence of TGF-β1 ( C3 −/− C5 −/− iT regs ). DCs (left side) were assayed for C5aR and C5L2 expression by gating on CD11c + cells. Responder T cells (right side) were assayed for C5aR and C5L2 expression by gating on Foxp3 − cells in the case of WT CD4 + T cells prepared with WT DCs and on Foxp3 + cells in the case of WT CD4 + prepared with C3 −/− C5 −/− DCs. (*P < 0.01; n=5). (g) Sorted WT or C5L2 −/− (encoded by Gpr77) Foxp3 − CD4 + T cells were incubated with WT DCs (Left) and sorted WT Foxp3 − CD4 + T cells were incubated with WT or C5L2 −/− DCs (right) both in the presence of anti-CD3 and TGF-β1 (5 ng/ml). Percent Foxp3 + CD25 + cells were assayed by flow cytometry. (*P<0.001, n=6). (h) WT Foxp3 − CD4 + T cells were incubated for 3 days with WT DCs plus TGF-β1 (5 ng/ml) after which Foxp3 + and Foxp3 − cells were sorted. Foxp3 − CD4 + T cells or Foxp3 + CD4 + iT regs were incubated for 5 min with anti-CD3+CD28 beads + biotin-labeled C5a, the cells chilled to 4°C, and plasma membrane fractions were isolated by ultracentrifugation. Following C5aR and C5L2 IP, bound proteins were eluted with alkaline solution, and equal amounts of protein (determined by A 280 ) were loaded onto SDS PAGE gels. Following electrophoresis, gels were blotted for biotinylated-C5a by adding streptavidin-HRP followed by ECL reagent.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Expressing, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Labeling, Membrane, Isolation, SDS Page, Electrophoresis

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) Flow sorted human CD45RA + CD25 − CD4 + T cells (1×10 6 ) were incubated for 3 days with soluble anti-CD3 mAb (3 μg/ml), rhIL-2 (5 ng/ml), and 2.5×10 5 autologous CD11c + DCs in the absence or presence of 1) TGF-β1 (5 ng/ml), 2) each of C3aR-A/C5aR-A (10 nM), or 3) each of anti-C3a/C5a mAbs (10 μg/ml). Percent Foxp3 + CD25 + CD4 + T cells then were determined by flow cytometry. (b) Flow sorted CD25 + cells from (a) were incubated for 3 days with in differing T eff /iT reg ratios with 1×10 6 CFSE labeled autologous naive CD25 − cells, anti-CD3 mAb (3 μg/ml), and 1×10 4 autologous CD11c + DCs. Percent dividers was determined by CFSE dilution. (c) CD45RA + CD25 − CD4 + T cells (1×10 6 ) were isolated from 5 healthy controls (NC) and 3 MS patients by flow sorting. The cells were incubated for 3 days with soluble anti-CD3 mAb (3 μg/ml), rhIL-2 (5 ng/ml), and 2.5×10 5 autologous DCs in the absence or presence of 1) rhTGF-β1 (5 ng/ml), 2) C3aR-A/C5aR-A (10 nM), or 3) anti-C3a/C5a mAbs (5 μg/ml). Cells were washed and sorted on CD25. After sorting, CD25 + (T reg ) were incubated for 3 days with anti-CD3 mAb, 2.5×10 5 autologous DCs, and 10 6 CD25 − (Effector) cells prelabeled with CFSE. Percent dividers was determined by CFSE dilution.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Flow Cytometry, Labeling, Isolation

Journal: Immunology

Article Title: Development and characterization of novel anti‐C5 monoclonal antibodies capable of inhibiting complement in multiple species

doi: 10.1111/imm.13083

Figure Lengend Snippet: Functional assays to determine whether monoclonal antibodies (mAb) 4G2, 7D4 and 10B6 inhibit complement in different species. (a–e) Classical pathway haemolysis (CH50). Sera tested were human (a), rat (b), rabbit (c), guinea pig (d) and mouse (e). Commercial mAb RO7112689 and Eculizumab were used as comparators. (f) Calculation of 50% inhibitory dose showed that human C5 inhibition by mAb 10B6 was equivalent to the two comparator mAb, RO7112689 and Eculizumab, and that 4G2 and 7D4 strongly inhibited rat C5. (g, h) Alternative pathway (AP) haemolysis (AH 50 ) assay using human (g) and rat (h) serum; all tested mAb inhibited AP in human serum, while 4G2 and 7D4 inhibited rat AP. (i) Inhibition of C5a generation by the novel mAb in a classical pathway assay with human serum; all three mAb efficiently inhibited C5a generation in a dose‐dependent manner. All experiments were repeated three times with the same results. The error bars are standard errors of triplicates. The dashed lines correspond to the comparator mAb.

Article Snippet: Blots were probed with anti‐C5a mAb (#HM2079; Hycult Biotech), detected using donkey anti‐mouse IgG‐HRP (Jackson ImmunoResearch, 715‐035‐150).

Techniques: Functional Assay, Inhibition

Journal: Immunology

Article Title: Development and characterization of novel anti‐C5 monoclonal antibodies capable of inhibiting complement in multiple species

doi: 10.1111/imm.13083

Figure Lengend Snippet: Impact of anti‐C5 monoclonal antibodies (mAb) on cleavage of C5 by neutrophil elastase. (a) Western blot of atypical cleavage of C5 by neutrophil elastase (NE). C5 was mixed with each mAb at 5× molar excess in HEPES‐buffered saline (HBS), NE was added at 420 n m , incubated and the reaction was stopped by addition of protease inhibitors. Samples (1 μg) were resolved on 4–20% SDS–PAGE gels under reducing (R) conditions and processed for WB to detect intact or cleaved C5 and C5a using an anti‐C5a mAb that also detects native C5 α chain (Hycult; mAb 2942). C5 α (115 000 MW), C5a (10·4 000 MW), CI; proteases cocktail inhibitors, Ecul.; Eculizumab, RO; RO7112689, 3D3; irrelevant non‐blocking anti‐C5 mAb. Note that numerous unidentified C5 cleavage products are present in all NE‐treated samples. (b) Densitometry analysis of the C5a band using imagej (expressed as % relative to the C5a generation in the absence of antibody; 100%) confirmed that mAb 4G2 and commercial mAb RO7112689 efficiently inhibited generation of C5a. (c) measurement of C5a generation by ELISA confirmed that mAb 4G2 and RO7112689 inhibited C5a generation while other mAb inhibited weakly. ELISA results are presented as percentage relative to C5a generation in the absence of any mAb (set as 100%; measured as 670 ng/ml in the ELISA). Results are representative of three independent experiments. The error bars are standard errors of triplicates.

Article Snippet: Blots were probed with anti‐C5a mAb (#HM2079; Hycult Biotech), detected using donkey anti‐mouse IgG‐HRP (Jackson ImmunoResearch, 715‐035‐150).

Techniques: Western Blot, Incubation, SDS Page, Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: Clinical and Experimental Immunology

Article Title: Complement component 5 does not interfere with physiological hemostasis but is essential for Escherichia coli‐ induced coagulation accompanied by Toll‐like receptor 4

doi: 10.1111/cei.13240

Figure Lengend Snippet: Effect of recombinant C5a on monocyte CD11b and tissue factor (TF) surface expression . Whole blood from healthy donors was incubated with phosphate‐buffered saline (PBS) or increasing concentrations of purified human C5a for 10 min at 37°C (CD11b analysis) or 120 min (TF analysis) followed by flow cytometry. The results from separate experiments using blood from healthy blood donors ( n = 4) are expressed as median fluorescence intensity (MFI) ± standard deviation (s.d.). The left y ‐axis indicates monocyte CD11b surface expression; samples added C5a (open circles) and samples added C5a and PMX53 (closed circle). The right y ‐axis indicates monocyte TF surface expression samples added C5a (open square) and samples added C5a and PMX53 (closed square).

Article Snippet: Increasing amounts of human recombinant C5a (Hycult Biotechnology, Uden, the Netherlands) were added to blood from healthy donors.

Techniques: Recombinant, Expressing, Incubation, Purification, Flow Cytometry, Fluorescence, Standard Deviation

Journal: Journal of Innate Immunity

Article Title: Functional Analysis of a Novel Complement C5a Receptor 1-Blocking Monoclonal Antibody

doi: 10.1159/000535084

Figure Lengend Snippet: Assay of C5aR1 inhibitors on iLite ® C5a Assay Ready Cells, displaying commercial mAb and small-molecule C5aR1 inhibitors ( a ) and in-house C5aR1 mAbs ( b ). Data were normalized to a stimulation control (added C5a) and analyzed by nonlinear curve fitting. Data points were collected in duplicates across multiple assay plates; each assay plate was repeated three times. Data are presented as mean ± SD.

Article Snippet: 40 µL of cell suspension was added to each well, followed by incubation at 37°C and 5% CO 2 for 30 min. C5a (R&D systems, cat. no.: 2037-C5) was added to the plate at a final concentration of 4 ng/mL (corresponding molarity: 481.93 p m ) in sample buffer.

Techniques: Control

Journal: Journal of Innate Immunity

Article Title: Functional Analysis of a Novel Complement C5a Receptor 1-Blocking Monoclonal Antibody

doi: 10.1159/000535084

Figure Lengend Snippet: Assay of C5aR1 inhibitors within a C5a-driven PMN calcium flux assay. A negative inhibition control (IgG1k isotype mAb) and a positive inhibition control (20 μ m avacopan) were included. After first recording the baseline, either C5a or buffer was added, followed by the addition of either ionomycin or buffer. Data are presented as fold increase based on the initially recorded baseline (mean FITC detector MFI). The experiment was repeated three times with the blood of a different anonymous healthy donor per repeat; data points were obtained in singlets. Data are presented as mean + SD (one-sided) for better visualization.

Article Snippet: 40 µL of cell suspension was added to each well, followed by incubation at 37°C and 5% CO 2 for 30 min. C5a (R&D systems, cat. no.: 2037-C5) was added to the plate at a final concentration of 4 ng/mL (corresponding molarity: 481.93 p m ) in sample buffer.

Techniques: Calcium Flux Assay, Inhibition, Control

Journal: Journal of Innate Immunity

Article Title: Functional Analysis of a Novel Complement C5a Receptor 1-Blocking Monoclonal Antibody

doi: 10.1159/000535084

Figure Lengend Snippet: Assay of C5aR1 inhibitors on C5a-stimulated PMNs ( a–c ), followed by titration of mAb 18-41-6-based F(ab’) 2 fragments versus equimolar concentrations of avacopan ( d , e ). a–c A negative control for C5aR1 inhibition (IgG1k isotype mAb) and a positive control (75 μ m avacopan) were included. Data points were normalized to a stimulation control sample (T10stim) with added C5a. a A baseline control (without incubation and without stimulation, Tb) and an incubation control (incubation and without stimulation, T10) are depicted. Titration curves of mAb 18-41-6-based F(ab’) 2 fragments and avacopan were analyzed by nonlinear curve fitting. The experiments were repeated three times with the blood of two different anonymous healthy donors ( a–c ) or one different anonymous healthy donor per repeat ( d , e ); data points were acquired in singlets. Data are presented as mean ± SD.

Article Snippet: 40 µL of cell suspension was added to each well, followed by incubation at 37°C and 5% CO 2 for 30 min. C5a (R&D systems, cat. no.: 2037-C5) was added to the plate at a final concentration of 4 ng/mL (corresponding molarity: 481.93 p m ) in sample buffer.

Techniques: Titration, Negative Control, Inhibition, Positive Control, Control, Incubation

Journal: BMC Oral Health

Article Title: Different concentrations of C5a affect human dental pulp mesenchymal stem cells differentiation

doi: 10.1186/s12903-021-01833-4

Figure Lengend Snippet: Morphological appearance of DPSCs cultured with different concentration of C5a for 2 weeks. A 50, B 100, C 200, D 300, E 400 ng/ml C5a groups and F Control group. DPSCs, dental pulp mesenchymal stem cells; C5a, complement component 5a

Article Snippet: Cells were cultured in a 37 °C, 5% CO 2 incubator and divided into six groups (n = 1 × 10 5 cells/group, using Corning® 25cm 2 Rectangular Canted Neck Cell Culture Flask with Vent Cap): (i) cells treated with basic cell culture medium containing 10 nmol/l dexamethasone, 5 mmol/l β-glycerophosphate and 50 mg/ml vitamin-C-phosphate as control; (ii) DPSCs treated with 50 ng/ml recombinant human complement component C5a protein (C5a; R&D Systems, Inc.); (iii) DPSCs treated with 100 ng/ml C5a; (iv) DPSCs treated with 200 ng/ml C5a; (v) DPSCs treated with 300 ng/ml C5a; and (vi) DPSCs treated with 400 ng/ml C5a.

Techniques: Cell Culture, Concentration Assay, Control

Journal: BMC Oral Health

Article Title: Different concentrations of C5a affect human dental pulp mesenchymal stem cells differentiation

doi: 10.1186/s12903-021-01833-4

Figure Lengend Snippet: Effect of different concentration of C5a stimulation on DSPP protein expression in DPSC. All 6 groups were cultured in dentinogenic medium. DSPP expression was determined by Western blot after 28 days of culture time. Results are expressed as relative expression to Actin. Data are presented as mean ± SEM of 3 independent experiments. *P < 0.05, *showed 100 ng/ml and 200 ng/ml groups expressed significantly lower DSP protein compared with other groups.. △ P < 0.05, △showed 400 ng/ml group expressed significantly higher DSP protein. DSPP, dentin sialophosphoprotein; DPSC, dental pulp mesenchymal stem cell

Article Snippet: Cells were cultured in a 37 °C, 5% CO 2 incubator and divided into six groups (n = 1 × 10 5 cells/group, using Corning® 25cm 2 Rectangular Canted Neck Cell Culture Flask with Vent Cap): (i) cells treated with basic cell culture medium containing 10 nmol/l dexamethasone, 5 mmol/l β-glycerophosphate and 50 mg/ml vitamin-C-phosphate as control; (ii) DPSCs treated with 50 ng/ml recombinant human complement component C5a protein (C5a; R&D Systems, Inc.); (iii) DPSCs treated with 100 ng/ml C5a; (iv) DPSCs treated with 200 ng/ml C5a; (v) DPSCs treated with 300 ng/ml C5a; and (vi) DPSCs treated with 400 ng/ml C5a.

Techniques: Concentration Assay, Expressing, Cell Culture, Western Blot

Journal: BMC Oral Health

Article Title: Different concentrations of C5a affect human dental pulp mesenchymal stem cells differentiation

doi: 10.1186/s12903-021-01833-4

Figure Lengend Snippet: Cell morphology and mineralized nodules of hDPSCs cultured with C5a and mineralized medium for 28 days. A 50 ng/ml C5a culture hDPSCs (arrows nodules). B 100 ng/ml C5a culture hDPSCs displayed smaller and less mineralized nodules (arrows nodules). C 200 ng/ml C5a culture hDPSCs, displayed smaller and less mineralized nodules (arrows nodules). D 300 ng/ml C5a culture hDPSCs (arrows nodules). E 400 ng/ml C5a culture hDPSCs displayed bigger and more mineralized nodules (arrows nodules). F Control group. C5a, complement component 5a, hDPSCs, human dental pulp mesenchymal stem cell (arrows nodules). G Quantification of mineralized nodule formation. Data are shown as mean OD/µg of total protein ± SD (n = 6). *Indicates significant differences compared to conrol group (P < 0.05)

Article Snippet: Cells were cultured in a 37 °C, 5% CO 2 incubator and divided into six groups (n = 1 × 10 5 cells/group, using Corning® 25cm 2 Rectangular Canted Neck Cell Culture Flask with Vent Cap): (i) cells treated with basic cell culture medium containing 10 nmol/l dexamethasone, 5 mmol/l β-glycerophosphate and 50 mg/ml vitamin-C-phosphate as control; (ii) DPSCs treated with 50 ng/ml recombinant human complement component C5a protein (C5a; R&D Systems, Inc.); (iii) DPSCs treated with 100 ng/ml C5a; (iv) DPSCs treated with 200 ng/ml C5a; (v) DPSCs treated with 300 ng/ml C5a; and (vi) DPSCs treated with 400 ng/ml C5a.

Techniques: Cell Culture, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: A Novel Cleavage Pattern of Complement C5 Induced by Chlamydia trachomatis Infection via the Chlamydial Protease CPAF

doi: 10.3389/fcimb.2021.732163

Figure Lengend Snippet: C. trachomatis infection yielded a novel undescribed C5 fragment independent of C3. (A) C . trachomatis -HeLa229 cell lysates (MOI=1) incubated with human purified C5 (400 nM) produced a previously undescribed ~38-kDa fragment encompassing C5a in Western blot analysis. (B) Schematic overview of proteolytical processing of C5 induced by traditional C5 convertases or C . trachomatis infection. (C) C . trachomatis infection generated a limited amount of C5a in the absence of C3 detected by ELISA. The C5 control group was taken as the baseline. Formation rates of C5a were calculated according to the concentration of human C5 protein before the reaction (400 nM). ns, not significant; *P < 0.05; **P < 0.01; ****P < 0.0001 (Dunnett’s multiple comparisons test, n =3).

Article Snippet: Quantitative analysis of C5a in the cleavage products were analyzed using a commercially available human C5a ELISA kit (CUSABIO BIOTECH, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Infection, Incubation, Purification, Produced, Western Blot, Generated, Enzyme-linked Immunosorbent Assay, Control, Concentration Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: A Novel Cleavage Pattern of Complement C5 Induced by Chlamydia trachomatis Infection via the Chlamydial Protease CPAF

doi: 10.3389/fcimb.2021.732163

Figure Lengend Snippet: Chlamydial infection after 24 hours post-infection but not EB cleaves C5 at K970 site. (A) The (C) trachomatis-HeLa229 cell lysates (MOI=1) collected at 0 h, 12 h, 24 h, 36h and 44 h post-infection were co-incubated with purified C5 (400 nM) for 120 min. Western blot analysis showed the fragments encompassing C5a. (B) The purified chlamydial EBs were co-incubated with purified C5 (400 nM) for 30 min. Western blot analysis showed the fragments encompassing C5a. (C) Penicillin treatment significantly reduced the consumption of the band of C5α chain, and also inhibited the expression of CPAF.

Article Snippet: Quantitative analysis of C5a in the cleavage products were analyzed using a commercially available human C5a ELISA kit (CUSABIO BIOTECH, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Infection, Incubation, Purification, Western Blot, Expressing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: A Novel Cleavage Pattern of Complement C5 Induced by Chlamydia trachomatis Infection via the Chlamydial Protease CPAF

doi: 10.3389/fcimb.2021.732163

Figure Lengend Snippet: CPAF induces a C5-derived novel fragment independent of C3. (A) Purified C5 was incubated with PBS, HeLa229 cells lysate, C . trachomatis -HeLa229 cells lysate, or C . trachomatis CPAF −/− -HeLa229 cells lysate, respectively. Products of the incubation were subjected to immunoblot analysis against C5a, MOMP, and CPAFc. (B) Lactacystin (500 μM) failed to inhibit C5 cleavage induced by 1:1 dilution of C. trachomatis -HeLa229 cell lysates, but slightly reduced consumption of the band corresponding to the C5α chain (115 kDa) in the group of 1:2 dilution of C. trachomatis -HeLa229 cell lysates.

Article Snippet: Quantitative analysis of C5a in the cleavage products were analyzed using a commercially available human C5a ELISA kit (CUSABIO BIOTECH, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Purification, Incubation, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: A Novel Cleavage Pattern of Complement C5 Induced by Chlamydia trachomatis Infection via the Chlamydial Protease CPAF

doi: 10.3389/fcimb.2021.732163

Figure Lengend Snippet: NM inhibits the C3-independent C5 activation triggered by chlamydial infection and has a reasonable inhibitory conformation with CPAF. (A) NM (400 μM) was pre-mixed with different dilutions of C . trachomatis -HeLa229 cell lysates. Purified C5 was then incubated with PBS or the C . trachomatis -HeLa229 cell lysates for 30 min, respectively. The resulting products were subjected to immunoblot analysis against C5a, MOMP, and CPAFc. (B) The 3D structure of NM-CPAF complex. (C) The surface of the active site of CPAF. (D) The detail binding mode of NM-CPAF complex. The backbone of CPAF was rendered in tube and colored in bright blue. NM was colored in yellow. The hydrogen bonds in NM-CPAF complex were shown in red dashed lines.

Article Snippet: Quantitative analysis of C5a in the cleavage products were analyzed using a commercially available human C5a ELISA kit (CUSABIO BIOTECH, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Activation Assay, Infection, Purification, Incubation, Western Blot, Binding Assay