Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 2. In vitro function of islets pre-cultured with exogenous complement component C3a. Insulin release at 2 mmol/L and 20 mmol/L glucose of 1020 replicates of three mouse islets per Eppendorf tube: (A) pre-cultured alone, with 10 nmol/L C3a alone or with 100 nmol/L C3a alone, for 48 h followed by subsequent GSIS assays in the absence of exogenous C3a; (B) pre-cultured alone, with 5 nmol/L ANXA1 alone or with 10 nmol/L C3a alone for 48 h followed by subsequent GSIS assays in the absence of exogenous C3a or ANXA1; (C) pre-cul- tured alone, with 5 nmol/L ANXA1 alone or with a dual combination of 5 nmol/L ANXA1 and 10 nmol/L C3a for 48 h followed by subse- quent GSIS assays in the absence of exogenous C3a and/or ANXA1, P < 0.05 versus islets pre-cultured alone at the same glucose concentration. (D) Protection from cytokine-induced apoptosis following a 48 h pre-culture with 5 nmol/L ANXA1-alone, 10 nmol/L C3a alone or a dual combination of 5 nmol/L ANXA1 and 10 nmol/L C3a, and the subsequent presence of specified MSC biotherapeutics during the final 20 h cytokine incubation. Eight to 12 replicates of five islets per well in each culture group assayed, *P < 0.05 versus islets pre-cul- tured alone in the presence of cytokines for the final 20 h of the 3-day culture period. The P values (AD) were calculated using two-way ANOVA Bonferroni’s post hoc test.

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: In Vitro, Cell Culture, Concentration Assay, Incubation

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 3. Pre-culturing islets with a cocktail of MSC secretory factors ensures sustained improvements to islet insulin secretory function and protection from cytokine-induced apoptosis. (A, B) Insulin release at 2 and 20 mmol/L glucose of 30 replicates of three mouse islets per Eppendorf tube, pre-cultured alone, with 5 nmol/L ANXA1 alone, with 5 nmol/L ANXA1 and 10 nmol/L SDF-1, with 5 nmol/L ANXA1 and 10 nmol/L C3a, or with a cocktail of 5 nmol/L ANXA1, 10 nmol/L SDF-1 and 10 nmol/L C3a, for 48 h, before removal of the MSC- derived biotherapeutics for 1 day (A) or 3 days (B), *P < 0.05 and **P < 0.01 versus islets cultured alone at the same glucose concentration. (C, D) Protection of islets from cytokine-induced apoptosis after pre-culture with MSC-derived biotherapeutics alone, in dual combination or a cocktail of all three factors (as of legend) for 48 h, before removal of the MSC-derived biotherapeutics for 1 day (C) or 3 days (D), 8 to 12 replicates of five islets per well were assayed, *P < 0.05 and **P < 0.01 versus islets cultured alone with cytokines, +P < 0.05 vs. islets cul- tured alone without cytokines. The P values (AD) were calculated using two-way ANOVA with Bonferroni post hoc test.

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: Cell Culture, Derivative Assay, Concentration Assay

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 4. In vivo function of islets pre-cultured alone, with ANXA1 alone or with a cocktail of MSC secretory factors. (A) Average blood glucose concentrations of STZ diabetic mice trans- planted with 150 islets pre-cultured for 48 h alone, with ANXA1 alone or with a cocktail of ANXA1/SDF-1/C3a, *P < 0.05 versus mice transplanted with islets pre-cultured alone (repeated-meas- urements ANOVA with Bonferroni post hoc test, n = 79). (B) Area under the curve (AUC) of STZ diabetic mice transplanted with 150 islets pre-cultured for 48 h alone, with ANXA1 alone or with a cocktail of ANXA1/SDF-1/C3a, *P < 0.05 versus mice transplanted with islets pre-cultured alone (one-way ANOVA with Dunn’s post hoc test, n = 79).

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: In Vivo, Cell Culture

Journal: The FASEB Journal

Article Title: C‐reactive protein inhibits C3a/C3aR‐dependent podocyte autophagy in favor of diabetic kidney disease

doi: 10.1096/fj.202200198r

Figure Lengend Snippet: FIGURE 1 The expression of complement effectors C3a, C5a and C5b-9 in peripheral blood and renal biopsies. (A–C) Serum levels of complement C3a, C5a and C5b-9 were examined in healthy controls (n = 20), DM patients (n = 20) and DN patients (n = 20) by ELISA. (D) Representative IHC staining images of complement C3a, C5a and C5b-9 from healthy controls and DN patients. (E) Quantitative analysis of C3a, C5a and C5b-9 staining in renal tissues by Image-Pro Plus 6.0 (n = 10–15). (F) Serum levels of CRP in healthy control (n = 20), DM patients (n = 20) and DN patients (n = 20) detected by ELISA. 400× original magnification; scale bar, 100 µm. Data are presented as the means ± SEM, and p < .05 was considered to be statistically significant

Article Snippet: Usually, the cells were seeded at 1 × 10 5/well in a 12- well plate, and then treated with low glucose (5 mM), high glucose (33 mM), recombinant mouse C3a (100 nM, HY- P7863, MCE, NJ, USA) and recombinant mouse C5a (100 nM, HY- P7695, MCE) for 24 h for qPCR and 72 h for Western blotting.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Control

Journal: The FASEB Journal

Article Title: C‐reactive protein inhibits C3a/C3aR‐dependent podocyte autophagy in favor of diabetic kidney disease

doi: 10.1096/fj.202200198r

Figure Lengend Snippet: FIGURE 2 Differences between human CRP and rat CRP in terms of structure and complement activation in vitro. (A) Human CRP and rat CRP migrated in 1/20 SDS-PAGE as pentamers and in SDS-PAGE as monomers. (B) The pentameric structure of human and rat CRP was shown by transmission electron microscopy (TEM). (C) Autologous complement activation of human CRP in human serum and rat CRP in rat serum (n = 6). (D) Interspecific complement activation of human CRP in rat serum and rat CRP in human serum (n = 6). Data are presented as the means ± SEM, and p < .05 was considered to be statistically significant

Article Snippet: Usually, the cells were seeded at 1 × 10 5/well in a 12- well plate, and then treated with low glucose (5 mM), high glucose (33 mM), recombinant mouse C3a (100 nM, HY- P7863, MCE, NJ, USA) and recombinant mouse C5a (100 nM, HY- P7695, MCE) for 24 h for qPCR and 72 h for Western blotting.

Techniques: Activation Assay, In Vitro, SDS Page, Transmission Assay, Electron Microscopy

Journal: The FASEB Journal

Article Title: C‐reactive protein inhibits C3a/C3aR‐dependent podocyte autophagy in favor of diabetic kidney disease

doi: 10.1096/fj.202200198r

Figure Lengend Snippet: FIGURE 3 The expression of complement C3a, C5a and C5b-9 in STZ-diabetic DKD rats. (A) Twenty-four-hour microalbuminuria was detected at 14-week with a commercial ELISA kit (n ≥ 8). (B) Blood urea nitrogen, serum CRE and urine CRE were monitored with an automated biochemistry analyzer (n ≥ 8). (C and D) Representative images and quantitative analysis of complement C3a, C5a and C5b-9 in rat kidney tissues by IHC staining (n = 4, more than 20 glomeruli). (E) Serum levels of complement C3a, C5a and C5b-9 were examined in rats with SZT-induced DKD by ELISA (n = 8). 400× original magnification; scale bar, 50 µm. Data are presented as the means ± SEM, and p < .05 was considered to be statistically significant

Article Snippet: Usually, the cells were seeded at 1 × 10 5/well in a 12- well plate, and then treated with low glucose (5 mM), high glucose (33 mM), recombinant mouse C3a (100 nM, HY- P7863, MCE, NJ, USA) and recombinant mouse C5a (100 nM, HY- P7695, MCE) for 24 h for qPCR and 72 h for Western blotting.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: The FASEB Journal

Article Title: C‐reactive protein inhibits C3a/C3aR‐dependent podocyte autophagy in favor of diabetic kidney disease

doi: 10.1096/fj.202200198r

Figure Lengend Snippet: FIGURE 6 Anaphylatoxin C3a induced podocyte autophagy under high-glucose conditions. (A) Quantitative RT–PCR analysis of LC3B and p62 expression under low-glucose and high-glucose conditions with C3a and C5a treatment (n = 3). (B and C) Western blot and quantitative analysis of LC3B and p62 expression under high-glucose conditions with C3a treatment (n = 4). (D) Representative images of autophagy flux changes with C3a and C5a treatment after Ad-mCherry-GFP-LC3B infection (n = 3). 600× original magnification; scale bar, 10 µm. Data are presented as the means ± SEM, p < .05 was considered to be statistically significant

Article Snippet: Usually, the cells were seeded at 1 × 10 5/well in a 12- well plate, and then treated with low glucose (5 mM), high glucose (33 mM), recombinant mouse C3a (100 nM, HY- P7863, MCE, NJ, USA) and recombinant mouse C5a (100 nM, HY- P7695, MCE) for 24 h for qPCR and 72 h for Western blotting.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Infection

Journal: The FASEB Journal

Article Title: C‐reactive protein inhibits C3a/C3aR‐dependent podocyte autophagy in favor of diabetic kidney disease

doi: 10.1096/fj.202200198r

Figure Lengend Snippet: FIGURE 7 C3a-induced podocyte autophagy was regulated in stable CRP-overexpression and CRP-knockout cell lines. (A and B) Quantitative RT-PCR analysis of LC3B and p62 expression in the CRP-overexpression experiment and CRP-knockout experiment (n = 3). (C and D) Western blot and quantitative analysis of LC3B and p62 in the CRP-overexpression experiment and CRP-knockout experiment (n = 4). (E and F) Autophagic flux was detected in a stable CRP-overexpression cell line and a stable CRP-knockout cell line under high-glucose conditions with C3a treatment (n = 3). 600× original magnification; scale bar, 10 µm. Data are presented as the means ± SEM, and p < .05 was considered to be statistically significant

Article Snippet: Usually, the cells were seeded at 1 × 10 5/well in a 12- well plate, and then treated with low glucose (5 mM), high glucose (33 mM), recombinant mouse C3a (100 nM, HY- P7863, MCE, NJ, USA) and recombinant mouse C5a (100 nM, HY- P7695, MCE) for 24 h for qPCR and 72 h for Western blotting.

Techniques: Over Expression, Knock-Out, Quantitative RT-PCR, Expressing, Western Blot

Journal: The FASEB Journal

Article Title: C‐reactive protein inhibits C3a/C3aR‐dependent podocyte autophagy in favor of diabetic kidney disease

doi: 10.1096/fj.202200198r

Figure Lengend Snippet: FIGURE 8 CRP suppressed C3aR expression under high-glucose conditions. (A) western blot and quantitative analysis of C3aR expression in CRP-overexpression and CRP-knockout experiments (n = 4). (B) IF staining of LC3B in cultured CRP-overexpression and CRP-knockout cell lines with C3a treatment (n = 3). (C) A C3aR agonist active peptide was used to reverse changes in LC3B and p62 expression in CRP-FL cells (n = 4). (D) A C3aR antagonist SB290157 was used to reverse changes in LC3B and p62 expression in CRP- KO2 cells (n = 4). 200× original magnification; scale bar, 10 µm. Data are presented as the means ± SEM, and p < .05 was considered to be statistically significant

Article Snippet: Usually, the cells were seeded at 1 × 10 5/well in a 12- well plate, and then treated with low glucose (5 mM), high glucose (33 mM), recombinant mouse C3a (100 nM, HY- P7863, MCE, NJ, USA) and recombinant mouse C5a (100 nM, HY- P7695, MCE) for 24 h for qPCR and 72 h for Western blotting.

Techniques: Expressing, Western Blot, Over Expression, Knock-Out, Staining, Cell Culture

Journal: Mucosal immunology

Article Title: Activation of C3a receptor is required in cigarette smoke-mediated emphysema

doi: 10.1038/mi.2014.118

Figure Lengend Snippet: Purified human C3 (100μg/ml) was cleaved with different concentrations of human ( a ) NE (10μg/ml, 1μg/ml, 0.1μg/ml) and ( b ) MMP12 (10μg/ml, 1μg/ml, 0.1μg/ml) for 4 hours at 37°C. Cleavage products were separated using 10% non-reducing Tricine gels, and detected by Western blot using anti-C3a antibody; purified C3a, NE and MMP12 were loaded as controls. ( c ) Bone marrow-derived dendritic cells (BMDCs; 5×10 4 ) and ( d ) myeloid-derived dendritic cells (MDDCs; 5×10 4 ) were suspended in media (RPMI-1640) and were placed on 48-well chemotaxis chambers for 1hr in the presence of intact or MMP12, NE cleaved C3 protein; control conditions included NE and MMP12. Transmigrating cells were detected in stained membranes visualized under microscope (20x) and reported as the average number of cells/field (n=4-6). **P<0.01, as determined by one-way ANOVA with Bonferroni's multiple comparison. Results are represented as mean±s.e.m, from 3 independent experiments.

Article Snippet: Antibodies used for western blotting and immunohistochemistry of C3 and monoclonal anti-human complement component C3a antibody were purchased from R&D Systems (Minneapolis, MN); mouse anti-human complement C3b-alpha monoclonal antibody for detection of C3b and C3c was purchased from Chemicon, Millipore (Billerica, MA); mouse anti-human C3d were purchased from AbD Serotec (Raleigh, NC).

Techniques: Purification, Western Blot, Derivative Assay, Chemotaxis Assay, Control, Staining, Microscopy, Comparison

Journal: Mucosal immunology

Article Title: Activation of C3a receptor is required in cigarette smoke-mediated emphysema

doi: 10.1038/mi.2014.118

Figure Lengend Snippet: WT and C3 deficient mice were exposed to cigarette smoke or air for 6 months. ( a ) Expression of C3ar1 mRNA in BAL cells isolated from WT or C3 −/− mice exposed to air or cigarette smoke was measured by quantitative reverse transcription PCR (qPCR). ***P<0.001, as determined by the one-way ANOVA with Bonferroni's multiple comparison. Representative ( b ) and cumulative data ( c ) measuring C3aR mean fluorescent intensity (MFI) in single lung cells gated on B220 − CD11c + population using flow cytometry. *P<0.05, as determined by the one-way ANOVA with Bonferroni's multiple comparison. ( d ) Representative photomicrograph of WT and C3 −/− mouse lung tissue exposed to six months of smoke or air immunostained for expression of C3aR (green) or nuclei (blue; DAPI). Scale bar: 20μm. Green arrows indicate C3aR + cells. ( e ) to ( h ) Mouse bone marrow-derived dendritic cells (BMDCs; 2×10 5 ) were treated with C3aR Agonist (CAS 944997-60-8; 20ng/ml) or vehicle (2% DMSO) for 48 hours. Expression level of C3aR1, Il6, Mmp9 and Mmp12 mRNA were measured using qPCR (n=4 in each group; **P<0.01, ***P<0.001, as determined by student t test. ( i ) Human CD1a + lung mDCs (2×10 5 ) were treated with purified human C3a (40ng/ml) for 24 hours or vehicle (media). Expression level of C3AR1 mRNA was measured by quantitative reverse transcription PCR (qPCR). n=3; **P<0.01, as determined by student t test. All gene expressions were normalized to 18S ribosomal RNA expression and analyzed by ΔΔCt . Results are represented as mean±s.e.m, from 3 independent experiments with 4-5 mice in each group ( a-d ).

Article Snippet: Antibodies used for western blotting and immunohistochemistry of C3 and monoclonal anti-human complement component C3a antibody were purchased from R&D Systems (Minneapolis, MN); mouse anti-human complement C3b-alpha monoclonal antibody for detection of C3b and C3c was purchased from Chemicon, Millipore (Billerica, MA); mouse anti-human C3d were purchased from AbD Serotec (Raleigh, NC).

Techniques: Expressing, Isolation, Reverse Transcription, Comparison, Flow Cytometry, Derivative Assay, Purification, RNA Expression

Journal: Journal of Alzheimer's Disease

Article Title: Impact of Preanalytical Procedures on Complement Biomarkers in Cerebrospinal Fluid and Plasma from Controls and Alzheimer’s Disease Patients

doi: 10.3233/JAD-240287

Figure Lengend Snippet: Sensitivity, precision and specificity results for the complement assays

Article Snippet: The C3 assay measures full-length C3 using anti-human C3a polyclonal antibody (R& D Systems, catalog AF3677) as a capture antibody and polyclonal anti-human C3 antibody for detection (MP Bio, catalog 0855033).

Techniques:

Journal: Journal of Alzheimer's Disease

Article Title: Impact of Preanalytical Procedures on Complement Biomarkers in Cerebrospinal Fluid and Plasma from Controls and Alzheimer’s Disease Patients

doi: 10.3233/JAD-240287

Figure Lengend Snippet: (a) Schematic of the study design to assess complement stability in plasma or CSF from HC and AD subjects. CSF and blood were collected from 9 donors, the biofluids were centrifuged and aliquoted at the clinical site without (No Addition) or with supplementation of the complement-stabilizing agents EDTA, EDTA + FUT and PIC. The CSF and plasma aliquots underwent 1-4 FT cycles. C4a, C3a, Bb, C4, C3, and FB concentrations were assessed by immunoassay. Created with BioRender.com. (b) AD diagnostic biomarkers Aβ 42 and Aβ 40 were measured by immunoassay in non-supplemented CSF samples from each donor. Aβ 42 :Aβ 40 ratio (AU) in HC (amyloid negative, N = 4) and AD (amyloid positive, N = 5) subjects; horizontal and vertical lines represent the mean and SD respectively. The dashed line represents the diagnostic cutoff value of 0.055.

Article Snippet: The C3 assay measures full-length C3 using anti-human C3a polyclonal antibody (R& D Systems, catalog AF3677) as a capture antibody and polyclonal anti-human C3 antibody for detection (MP Bio, catalog 0855033).

Techniques: Diagnostic Assay

Journal: Journal of Alzheimer's Disease

Article Title: Impact of Preanalytical Procedures on Complement Biomarkers in Cerebrospinal Fluid and Plasma from Controls and Alzheimer’s Disease Patients

doi: 10.3233/JAD-240287

Figure Lengend Snippet: (a–b) Impact of the different additives on complement levels in plasma (a) and CSF (b). Shown are the % recovery from the unsupplemented condition (No Addition) in the supplemented conditions (EDTA, EDTA + FUT, and PIC) for C4a, C3a, Bb, C4, C3, and FB at FT1. The dashed line represents the ‘No Addition’ reference (100%). Box plots shown comprise data from N = 9 individual donors. Boxes represent the median and interquartile range (IQR); The lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles). The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR from the hinge. The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge. The diamond shape represents the mean. Individual data points are plotted over the box plots.

Article Snippet: The C3 assay measures full-length C3 using anti-human C3a polyclonal antibody (R& D Systems, catalog AF3677) as a capture antibody and polyclonal anti-human C3 antibody for detection (MP Bio, catalog 0855033).

Techniques: Whisker Assay

Journal: Journal of Alzheimer's Disease

Article Title: Impact of Preanalytical Procedures on Complement Biomarkers in Cerebrospinal Fluid and Plasma from Controls and Alzheimer’s Disease Patients

doi: 10.3233/JAD-240287

Figure Lengend Snippet: Impact of FT cycles on complement levels in plasma. Shown are the % recovery from FT1 in the FT2, FT3, and FT4 unsupplemented (No Addition) or supplemented aliquots (EDTA, EDTA + FUT, PIC) for C4a, C3a, Bb, C4, C3, and FB. The dashed line represents the ‘FT1’ reference (100%). Box plots shown comprise data from N = 9 individual donors. Boxes represent the median and interquartile range (IQR); The lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles). The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR from the hinge. The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge. The diamond shape represents the mean. Individual data points are plotted over the box plots.

Article Snippet: The C3 assay measures full-length C3 using anti-human C3a polyclonal antibody (R& D Systems, catalog AF3677) as a capture antibody and polyclonal anti-human C3 antibody for detection (MP Bio, catalog 0855033).

Techniques: Whisker Assay

Journal: Journal of Alzheimer's Disease

Article Title: Impact of Preanalytical Procedures on Complement Biomarkers in Cerebrospinal Fluid and Plasma from Controls and Alzheimer’s Disease Patients

doi: 10.3233/JAD-240287

Figure Lengend Snippet: Impact of FT cycles on complement levels in CSF. Shown are the % recovery from FT1 in the FT2, FT3 and FT4 unsupplemented (No Addition) or supplemented aliquots (EDTA, EDTA + FUT, PIC) for C4a, C3a, Bb, C4, C3, and FB. The dashed line represents the ‘FT1’ reference (100%). Box plots shown comprise data from N = 9 individual donors. Boxes represent the median and interquartile range (IQR); The lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles). The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR from the hinge. The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge. The diamond shape represents the mean. Individual data points are plotted over the box plots.

Article Snippet: The C3 assay measures full-length C3 using anti-human C3a polyclonal antibody (R& D Systems, catalog AF3677) as a capture antibody and polyclonal anti-human C3 antibody for detection (MP Bio, catalog 0855033).

Techniques: Whisker Assay

Journal: Journal of Alzheimer's Disease

Article Title: Impact of Preanalytical Procedures on Complement Biomarkers in Cerebrospinal Fluid and Plasma from Controls and Alzheimer’s Disease Patients

doi: 10.3233/JAD-240287

Figure Lengend Snippet: (a–b) Rank order preservation of complement levels between the first (FT1) and second (FT2) freeze-thaw cycle in plasma (a) and CSF (b). Lollipop plot showing the Spearman Correlation Coefficient between the concentrations at FT1 and the concentrations at FT2 in unsupplemented (No Addition) or supplemented aliquots (EDTA, EDTA + FUT, PIC) for C4a, C3a, Bb, C4, C3, and FB.

Article Snippet: The C3 assay measures full-length C3 using anti-human C3a polyclonal antibody (R& D Systems, catalog AF3677) as a capture antibody and polyclonal anti-human C3 antibody for detection (MP Bio, catalog 0855033).

Techniques: Preserving