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 Clinical Investigation

Article Title: Endothelial C3a receptor mediates vascular inflammation and blood-brain barrier permeability during aging

doi: 10.1172/jci140966

Figure Lengend Snippet: Figure 1. C3a/C3aR signaling regulates age-associated endothelial VCAM1 expression. (A) ELISA measurement of C3 levels in WT mouse brain lysates at 2, 12, and 20 months (n = 8/group). (B) Immunofluorescence staining using anti-GFAP and anti-C3 antibodies demonstrated localization of C3 to astrocytes. (C) Quantification confirming increased C3 staining within GFAP + astrocytes in the hippocampus with age (n = 5/age). (D) Triple immunostaining of isolated vessels from WT and C3ar1 –/– brains using anti-GFAP, anti–VE-cadherin, and anti-C3aR antibodies showing positive C3aR staining along endothelial cell surface that was not present in C3ar1–/– vessels. (E) Triple immunostaining of brain tissue with anti-Glut1, anti-C3aR, and anti-CD31 or anti–PDGFR-β, anti-C3aR, and anti–Col IV, demonstrating expression of C3aR on brain endothelial cells but not pericytes. (F and G) Immunofluorescence staining and quantification using anti-CD31 and anti-VCAM1 antibodies of WT or C3ar1–/– mouse cortices at 2, 12, and 20 months demonstrated an increase in VCAM1 with age in WT mice, but VCAM1 was rescued in the absence of C3aR. All data represent the mean ± SEM. Significance was calculated using 1-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). Scale bars: 20 μm (B), 10 μm (D), 15 μm (E), and 50 μm (F).

Article Snippet: Confluent cells were treated with IL-1β (10 ng/mL, R&D Systems, 201-LB-005), C3a (500 nM, R&D Systems, 3677-C3-025), C5a (250 nM, R&D Systems, 2037-C5-025), ionomycin (10 μM, Cayman Chemical, 10004974), or in combination with one of the inhibitors SB290157 (5 μM, Calbiochem, 559410), W7 (50 μM, Tocris, 0369) or BAPTA-AM (1 μM, Tocris, 2787).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Triple Immunostaining, Isolation

Journal: Cellular & molecular medicine: open access

Article Title: Potential Mechanisms Underlying TGF-β-mediated Complement Activation in Lung Fibrosis

doi:

Figure Lengend Snippet: RNA interference suppresses local complement activation. BALF collected from Figure 1 were analyzed for C3a (A) and C5a (B) levels in the lung by ELISA. Values: Means ± SEM. (n=5-7 per group). One-way ANOVA, Newman-Keuls (A, B). Compared to bleomycin: ***: p<0.001; **: p<0.01; *: p<0.05. Results are representative of three independent experiments.

Article Snippet: These studies used recombinant human C3a and C5a (100 nM; Complement Technology, Inc., Tyler, TX), platelet-derived TGF-β1 (2 ng/ml; Roche Diagnostics, Germany).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Cellular & molecular medicine: open access

Article Title: Potential Mechanisms Underlying TGF-β-mediated Complement Activation in Lung Fibrosis

doi:

Figure Lengend Snippet: miRNA regulation in response to TGF-β, C3a and C5a for 24 h in normal primary human SAECs.

Article Snippet: These studies used recombinant human C3a and C5a (100 nM; Complement Technology, Inc., Tyler, TX), platelet-derived TGF-β1 (2 ng/ml; Roche Diagnostics, Germany).

Techniques:

Journal: Oncoimmunology

Article Title: Blocking C5aR signaling promotes the anti-tumor efficacy of PD-1/PD-L1 blockade

doi: 10.1080/2162402X.2017.1349587

Figure Lengend Snippet: Anti-PD-L1 treatment leads to activation of the complement system. MC38 tumor-bearing mice (∼100 mm3) were intraperitoneally injected with anti-PD-L1 antibodies. The tumors were removed at the indicated time points (n = 5–6 mice/group). (A) C3a and C5a levels in homogenates from tumors and spleens were quantified by ELISA. (B) C1q and C3b/iC3b/C3c staining of tumors and spleens were quantified by IHC. (C) C3b/iC3b/C3c levels of homogenates from tumors and spleens were quantified by ELISA. One-way ANOVA (A) and Multiple t-test (C) were used to evaluate statistical significance (*p < 0.05, **p < 0.01).

Article Snippet: Biotinylated Rat Anti-Mouse C3a (clone I87–419, BD, 2 μg/ml) was used for detection, and purified recombinant mouse C3a (BD) was used as a standard.

Techniques: Activation Assay, Injection, Enzyme-linked Immunosorbent Assay, Staining