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: Journal of Biological Chemistry

Article Title: A potent complement factor C3–specific nanobody inhibiting multiple functions in the alternative pathway of human and murine complement

doi: 10.1074/jbc.ra117.001179

Figure Lengend Snippet: FIGURE 1. The alternative pathway and negative stain EM analysis. (A) The AP can be initiated by tick-over generated C3(H2O) or from C3b generated within the classical or lectin activation pathways. Both C3b or C3(H2O) can form the proconvertase with FB. C3b can be degraded to iC3b by FI assisted by a cofactor. The (P) signifies that the AP convertases may bind and be stabilized by properdin. (B) Negative stain EM 2D classes of the complex between hC3Nb1 and C3b. The TE domain adopts multiple alternative positions as compared to the C3b MG1-8 domain (Scale bar, 100 Å). (C) 3D reconstruction of negative stain EM particles of the C3b-hC3Nb1 complex with the crystal structure of C3b (PDB entry 5FO7) fitted to the envelope. Excess density corresponding to the Nb is marked by *.

Article Snippet: The differences were the use of a final concentration of 5 % (v/v) serum from C57Bl6 male mice and the use of anti-mouse C3 antibody (Cederlane, # CL7503NA) for detection of deposited C3 fragments.

Techniques: Staining, Generated, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: A potent complement factor C3–specific nanobody inhibiting multiple functions in the alternative pathway of human and murine complement

doi: 10.1074/jbc.ra117.001179

Figure Lengend Snippet: FIGURE 3. The hC3Nb1 is a potent inhibitor of the alternative pathway. (A) Assay in human serum on a zymosan-coated AP activating surface. The horizontal axis gives the concentration of nanobodies present in the serum. The vertical axis shows the level of C3 fragment deposition with 100 % deposition defined as the signal from serum without added nanobody. The dotted line indicates the final molar concentration of C3 in the assay based on a concentration of 5.3 μM in undiluted plasma (61). (B) Multiple sequence alignment of the hC3Nb1 epitope from human C3 and four animals of relevance for animal models for which residues marked gray differ from the human sequence. Colored triangles indicate residues involved in interactions with hC3Nb1. (C) Assay in mouse serum on a zymosan-coated alternative pathway activating surface depicted as in panel A. The dotted line represents the final molar concentration of C3 in the assay assuming 4.2 μM C3 in 100 % serum from C57BL6 male mice (62).

Article Snippet: The differences were the use of a final concentration of 5 % (v/v) serum from C57Bl6 male mice and the use of anti-mouse C3 antibody (Cederlane, # CL7503NA) for detection of deposited C3 fragments.

Techniques: Concentration Assay, Clinical Proteomics, Sequencing

Journal: Journal of Biological Chemistry

Article Title: A potent complement factor C3–specific nanobody inhibiting multiple functions in the alternative pathway of human and murine complement

doi: 10.1074/jbc.ra117.001179

Figure Lengend Snippet: FIGURE 5. SPR analysis of the interaction of hC3Nb1 with C3b and C3. Representative SPR concentration series for C3b (A) or C3 (B) are displayed. The measured (gray) and fitted (red) SPR curves are shown for 50, 20, 10, 5, 2, 1, 0.25 and 0.1 nM for C3b or 20, 10, 5, 2.5, 1.25, 0.61, 0.32 and 0.15 nM for C3. The ka, kd and KD values are given as their average fitted from three independent concentration series ± the standard deviation. (C) Competition SPR assay with 50 nM C3b preincubated with 500 nM of different hC3Nb1 mutants. (D) Competition SPR assay with 50 nM C3b preincubated with 500 nM FB or FH. In panel C the curve for hC3Nb1(W102A) is hidden below the C3b curve.

Article Snippet: The differences were the use of a final concentration of 5 % (v/v) serum from C57Bl6 male mice and the use of anti-mouse C3 antibody (Cederlane, # CL7503NA) for detection of deposited C3 fragments.

Techniques: Concentration Assay, Standard Deviation, SPR Assay

Journal: Journal of Biological Chemistry

Article Title: A potent complement factor C3–specific nanobody inhibiting multiple functions in the alternative pathway of human and murine complement

doi: 10.1074/jbc.ra117.001179

Figure Lengend Snippet: FIGURE 6. The hC3Nb1 nanobody also inhibits C3 cleavage at the substrate level. (A) C3 cleavage assay with the CVFBb convertase revealing that also for this convertase not containing C3b, hC3Nb1 is an inhibitor of C3 cleavage. (B) C5 cleavage assay with the CVFBb convertase demonstrating that hC3Nb1 has no effect on C5 cleavage. The bands around at and below the 200 kDa marker represents C5b not completely reduced/denatured. (C) Assay in 1 % human serum on a mannan-coated LP activating surface. The horizontal axis gives the concentration of nanobodies added to the serum. The vertical axis shows the level of C3 fragment deposition with 100 % deposition defined as the signal from serum without added nanobody. The dotted line indicates the final molar concentration of C3 in the assay based on a concentration of 5.3 μM in undiluted plasma.

Article Snippet: The differences were the use of a final concentration of 5 % (v/v) serum from C57Bl6 male mice and the use of anti-mouse C3 antibody (Cederlane, # CL7503NA) for detection of deposited C3 fragments.

Techniques: Cleavage Assay, Marker, Concentration Assay, Clinical Proteomics

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: The lectin-like domain of thrombomodulin interferes with complement activation and protects against arthritis.

doi: 10.1111/j.1538-7836.2006.02033.x

Figure Lengend Snippet: Fig. 4. Complement C3 deposition in joints. Healthy 5–8-week old female TMwt/wt (A) and TMLeD/LeD (B) mice were killed and knee joints were processed for immunohistochemical staining to detect complement factor C3 deposition. These sections are representative of three mice. No C3 was detectable in the joints of TMwt/wt mice (A). C3 was readily observed on the articular surface (small arrows) and in the bone marrow space of TMLeD/LeD mice (large arrows) (B). Higher power views of the articular surface are shown in the right upper corner of each panel.

Article Snippet: Specific immunostaining of histologic sections was achieved by overnight incubation with the following primary antibodies: CD45 (rat, 1:100; BecktonDickinson, Erembodegem, Belgium, no. 553076);Mac3 (rat, 1:300; BecktonDickinson, no. 553322); MPO (rabbit, 1:100, Dako A/S, Glostrup, Denmark, no. A0398); HMGB1 (rabbit; 1:250, Santa Cruz Biotechnology, Boechout, Belgium, no. sc12523); and complement factor C3 (rat, 1:200; Cedarlane,Uden, theNetherlands, no. CL7503AP), which recognizes intact C3, C3b, iC3b and C3d, but not C3a.

Techniques: Immunohistochemical staining, Staining

Journal: eBioMedicine

Article Title: Reprogramming astrocytic NDRG2/NF-κB/C3 signaling restores the diabetes-associated cognitive dysfunction

doi: 10.1016/j.ebiom.2023.104653

Figure Lengend Snippet: Excluded variables of multivariate logistic regression analyses.

Article Snippet: The Human C3 (Complement Component 3) ELISA Kit (E-EL-H6054) and Human INS (Insulin) ELISA Kit (E-EL-H2665c) were purchased from Elabscience, Wuhan, China.

Techniques:

Journal: eBioMedicine

Article Title: Reprogramming astrocytic NDRG2/NF-κB/C3 signaling restores the diabetes-associated cognitive dysfunction

doi: 10.1016/j.ebiom.2023.104653

Figure Lengend Snippet: NDRG2 reshapes the pathological structure of astrocytes by inhibiting NF-κB/C3 signaling. (a) GSEA of proteome indicated complement and coagulation cascade signaling were upregulated in diabetic mice (∗∗∗ p = 0.0002 between vehicle and STZ) and downregulated after exercise (∗∗ p = 0.0035 between STZ and STZ + Run). The cumulative enrichment scores were normalized (NES) ( n = 3/group). (b) Heatmaps showing the fold changes of significantly altered proteins in complement and coagulation cascades ( n = 3/group). (c – d) Representative immunofluorescent staining of intact C3 and its cleaved products in the hippocampus. Red: C3, green: GFAP, blue: DAPI. Scale bar, 50 μm. (∗∗∗ p = 0.0006 between vehicle and STZ, ∗∗∗ p = 0.0006 between STZ and STZ + Run) (e – f) Representative immunoblots of complement C3 in the hippocampus were increased in the STZ group compared to vehicle mice (∗∗∗∗ p < 0.0001 between vehicle and STZ), which was downregulated after exercise ( n = 6/group) (∗∗∗ p = 0.0008 between STZ and STZ + Run). (g – k) Representative immunoblots of astrocytic NDRG2 ( g, i ), complement C3 ( g, h ), NF-κB, and p–NF–κB ( j, k ) after NDRG2 loss of function ( n = 6/group) (∗ p = 0.0120 (h) , ∗∗∗∗ p < 0.0001 (i) , ∗∗ p = 0.0012 (k) between STZ + Run + acNDRG2 KO and STZ + Run + control). (l – n) Representative immunoblots of astrocytic NDRG2 ( l ), complement C3 ( m ), NF-κB, and p–NF–κB ( n ) in the hippocampus after NDRG2 gain of function ( n = 6/group) (∗∗ p = 0.0064 ( l ), ∗∗∗ p = 0.0001 ( m ), ∗∗ p = 0.0029 ( n ) between vehicle + AAV-Ctrl and STZ + AAV-Ctrl) (∗∗∗∗ p < 0.0001 ( l ), ∗ p = 0.0264 (m) , ∗∗ p = 0.0040 (n) between STZ + AAV-NDRG2 and STZ + AAV-Ctrl). Data are presented as mean ± SEM. GSEA analysis was performed in a . One-way ANOVA with Tukey's multiple comparisons test was performed in d, f, l–n . Two-tailed Student's t-test was performed in h, i, k .

Article Snippet: The Human C3 (Complement Component 3) ELISA Kit (E-EL-H6054) and Human INS (Insulin) ELISA Kit (E-EL-H2665c) were purchased from Elabscience, Wuhan, China.

Techniques: Coagulation, Staining, Western Blot, Control, Two Tailed Test

Journal: eBioMedicine

Article Title: Reprogramming astrocytic NDRG2/NF-κB/C3 signaling restores the diabetes-associated cognitive dysfunction

doi: 10.1016/j.ebiom.2023.104653

Figure Lengend Snippet: C3aR blockade rescues dendritic spine loss in diabetic mice, and C3 may be a biomarker to predict the progression of DACD in humans. (a) Schematic representing chronological order of STZ injection, C3aR antagonist, and behavioral testing. We used C3aR antagonists to clarify whether C3aR blockade could mimic the protective effect of NDRG2 overexpression on DACD. (b) Y-maze alternation triplet (%) was improved in the STZ + C3aRA group compared with STZ + PBS mice ( n = 7/group, n = 8 vehicle + PBS) (∗∗∗ p = 0.0006 between vehicle + PBS and STZ + PBS) (∗∗∗∗ p < 0.0001 between STZ + C3aRA and STZ + PBS). (c – d) Y-maze total distance ( c ) and total arm entries ( d ) ( n = 7/group, n = 8 vehicle + PBS). (e) Escape latency of MWM test was shorten at the STZ + C3aRA group compared with STZ + PBS mice ( n = 7/group, n = 8 vehicle + PBS) (∗ p = 0.0211 (day 1) , ∗ p = 0.0336 (day 2) , ∗∗ p = 0.0099 (day 3) , ∗∗ p = 0.0033 (day 4) between vehicle + PBS and STZ + PBS) ( ## p = 0.0021 (day 1) , # p = 0.0392 (day 3) between STZ + C3aRA and STZ + PBS). (f) Platform crossover of MWM test was upregulated at the STZ + C3aRA mice compared to STZ + PBS group ( n = 7/group, n = 8 vehicle + PBS) (∗∗ p = 0.0025 between vehicle + PBS and STZ + PBS) (∗∗ p = 0.0066 between STZ + C3aRA and STZ + PBS). (g) Representative images of 3D reconstruction of dendritic spines. Scale bar, 5 μm. (h – l) The densities of total spines ( h ), stubby spines ( i ), mushroom spines ( j ), long thin spines ( k ), and filopodia spines ( l ) ( n = 20/group) (∗∗∗∗ p < 0.0001 ( h ), ∗∗∗ p = 0.0008 ( i ), ∗ p = 0.0191 ( j ), ∗∗∗ p = 0.0006 ( k ) between vehicle + PBS and STZ + PBS) (∗∗∗∗ p < 0.0001 ( h ), ∗∗∗ p = 0.0005 ( i ), ∗ p = 0.0133 ( j ), ∗ p = 0.0153 ( l ) between STZ + C3aRA and STZ + PBS). (m) The DSST scores of diabetic patients ( n = 27) and non-diabetic peers ( n = 13) (∗∗∗∗ p < 0.0001 between Normal and Diabetes). (n) Fasting plasma glucose levels for diabetic patients ( n = 27) compared to non-diabetic patients ( n = 13) (∗∗∗∗ p < 0.0001 between Normal and Diabetes). (o) The serum levels of complement C3 in diabetic patients ( n = 27) and normal peers ( n = 13) (∗∗ p = 0.0072 between Normal and Diabetes). (p) The correlations between DSST score and increased C3 levels in diabetic patients ( n = 27) and normal peers ( n = 13). Correlations were found using linear regression, with r = −0.3315 and p = 0.0366. Values presented as mean ± SEM. Two-way ANOVA with Tukey's multiple comparisons test was performed in e . One-way ANOVA with Tukey's multiple comparisons test was performed in b, f–l . Two-tailed Student's t-test was performed in m–o .

Article Snippet: The Human C3 (Complement Component 3) ELISA Kit (E-EL-H6054) and Human INS (Insulin) ELISA Kit (E-EL-H2665c) were purchased from Elabscience, Wuhan, China.

Techniques: Biomarker Discovery, Injection, Over Expression, Clinical Proteomics, Two Tailed Test

Journal: eBioMedicine

Article Title: Reprogramming astrocytic NDRG2/NF-κB/C3 signaling restores the diabetes-associated cognitive dysfunction

doi: 10.1016/j.ebiom.2023.104653

Figure Lengend Snippet: Characteristics of the study population.

Article Snippet: The Human C3 (Complement Component 3) ELISA Kit (E-EL-H6054) and Human INS (Insulin) ELISA Kit (E-EL-H2665c) were purchased from Elabscience, Wuhan, China.

Techniques:

Journal: Frontiers in Immunology

Article Title: Comprehensive transcriptome profiling of urothelial cells following TNFα stimulation in an in vitro interstitial cystitis/bladder pain syndrome model

doi: 10.3389/fimmu.2022.960667

Figure Lengend Snippet: TNFα-treated RT4 urothelial cells upregulate the expression of genes involved in acute inflammatory response and complement and coagulation cascade. (A) Heatmap showing upregulation of transcripts (RNA seq), enriched in regulation of inflammatory and acute inflammatory response, in TNFα-treated urothelial cells (purple, n=3 independent experiments: A1, A2, A6) vs ctrl cells (green, n=3 independent experiments: B1, B2, B6). Rows are clustered using Euclidean distance and average linkage. Lower expression is indicated with blue, higher expression is indicated with red. (B) qPCR validation of selected genes of acute inflammatory response confirming their upregulation in TNFα-treated cells. Shown is mean ± SD fold change expression of target genes in TNFα-treated cells vs ctrl (set to 1; dotted red line), determined in 8 independent experiments. (C) Confirmatory analysis showing significantly higher protein levels of complement component C3 released in supernatants of TNFα-treated cells compared to controls (n=8 in each experimental condition). Shown are mean ± SD for control and TNFα-treated groups. *p<0.05; **p<0.01.

Article Snippet: The following ELISA kits were used in the present study: human IL8 ELISA MAXTM Deluxe Set, human IL1A MAXTM Deluxe Set, human IL32 MAXTM Deluxe Set, human CXCL10 ELISA MAXTM Deluxe Set (all Bio Legend, San Diego, CA, USA), human CXCL1 Duo Set Kit (R&D Systems, USA), human complement C3 ELISA Kit (Novus Biologicals, Englewood, CO, USA), and porcine IL8/CXCL8 Quantikine ELISA Kit (R&D Systems, USA).

Techniques: Expressing, Coagulation, RNA Sequencing, Biomarker Discovery, Control