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 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: eLife

Article Title: REV-ERBα mediates complement expression and diurnal regulation of microglial synaptic phagocytosis

doi: 10.7554/eLife.58765

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used (with dilution): Gfap (Rabbit polyclonal, 1:2500, Dako/Agilent Cat# Z0334), C3 (Rat monoclonal, 1:500, Novus Biologicals Cat# NB200-540), Iba1 (Goat polyclonal, 1:500, Abcam Cat# ab5076), Synaptophysin (Mouse monoclonal, 1:100, Abcam Cat# ab8049), CD68 (Rat monoclonal, 1:250, BioRad, Cat# MCA1957), Homer1 (Rabbit polyclonal, 1:500, Synaptic Systems Cat# 160 003), Synaptoporin (Rabbit polyclonal, 1:1000, Synaptic Systems Cat# 102 002), NeuN (Mouse monoclonal, 1:1000, EMD/Millipore Cat# 14-5698-82).

Techniques: Sequencing, Software

Journal: Anesthesia & Analgesia

Article Title: Analgesic Effect of Exercise on Neuropathic Pain via Regulating the Complement Component 3 of Reactive Astrocytes

doi: 10.1213/ane.0000000000006884

Figure Lengend Snippet: Figure 5. Effect of C3 on exercise-induced analgesic effect. A and B, The dose-dependent effect of rC3 (i.t.) on mechanical (A) and cold (B) pain behaviors. One-way ANOVA was used to test the statistical difference and the post hoc Bonferroni test indicates that 50 ng rC3 induced both mechanical (** P = .0062) and cold pain behaviors (** P = .0028). C–E, Analgesic effect of exercise on rC3-induced mechanical and cold allodynia. Schematic of experimental approach (C) and bar graph (D to E) showing significantly higher PWT (* P = .0168) and decreased cold pain behavior (** P = .0025) in the rC3 combined exercise group than rC3 alone group. F–H, The effect of subeffective dose of rC3 on pain threshold of SNI mice after 2 weeks training. Schematic of experimental approach (F) and the changes of PWT (* P = .0348) (G) and cold sensitivity (* P = .0191) (H) were significant after 10 ng rC3 administration; n=7–8 per group. D–H, 2-way ANOVA followed by Bonferroni post hoc test was used to analyze the statistical difference. Data are represented as mean ± SEM. ANOVA indicates analysis of variance; PWT, paw withdrawal threshold; rC3, recombinant C3; SEM, standard error of the mean; SNI, spared nerve injury.

Article Snippet: Intrathecal Injection Intrathecal injections were performed under isoflurane anesthesia as described above.14 10 μL recombinant mouse C3 (rC3, R&D Systems Inc, 8085) or phosphate-buffered saline (PBS) solution was injected into intervertebral space by using a 10-μL microinjection syringe as previously described.

Techniques: Recombinant