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: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: Complement is fixed on KEL RBCs in-vivo and in-vitro in response to polyclonal anti-KEL (KELIg). (A) General in-vivo experimental design: recipients were treated with or without KELIg and transfused with DiO labeled KEL RBCs. (B) C3, C4, and Factor B were measured on recovered DiO positive KEL RBCs 1-hour post-transfusion; black filled histogram shows condition without KELIg; grey shaded histogram shows condition with KELIg. (C) In-vitro experiments were completed with KELIg incubated with KEL RBCs in the presence (darkest histogram) or absence (lightest histogram) of serum; an additional condition included saline incubated with KEL RBCs in the presence of serum (medium grey histogram). (D) Additional in-vitro conditions included KELIg incubated with KEL RBCs in the presence of serum (darkest histogram), in the presence of serum with EGTA-Mg (lightest histogram) or serum with EDTA (medium grey histogram). These data are representative of 3 independent experiments; p < 0.05 for C3 detection on RBCs in the presence or absence of KELIg.

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: In Vivo, In Vitro, Labeling, Incubation, Saline

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: Recipient C3 and C1q contribute to KELIg immunoprophylaxis failure in the setting of poly (I:C). (A) General in-vivo experimental design: recipients were infused with KELIg and transfused the following day in the absence or presence of poly (I:C) treatment, with anti-KEL responses evaluated longitudinally. (B) Anti-KEL IgG responses in wild type compared with C3 -/- recipients transfused in the absence of poly (I:C). (C) Anti-KEL IgG responses in wild type compared with C3 -/- recipients transfused in the presence of poly (I:C). (D) Anti-KEL IgG responses in wild type compared with C1q -/- recipients transfused in the presence of poly (I:C). (E) Baseline impact of recipient treatment with poly (I:C) on alloimmune responses to KEL RBCs in the absence of KELIg. These data are representative of 2-3 independent experiments, with 3-5 mice/group/experiment; error bars indicate standard deviation between individual mice. *p < 0.05 (C, D) d14,21,28, and all comparisons for (E) .

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: In Vivo, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: Recipient complement, KEL RBC clearance, and KEL antigen expression. KEL RBCs were labeled with DiO and mixed with wild type RBCs labeled with DiI; this mixture was transfused into recipients that had been infused with KELIg and treated with poly (I:C). (A) shows wild type compared with C3 -/- recipients; (B) shows wild type compared with C1q -/- recipients. Recovered DiO labeled RBCs were then evaluated for KEL glycoprotein expression by flow cytometry, after incubation with KELIg and fluorescently conjugated anti-mouse IgG; (C) shows wild type recipients compared with C3 -/- recipients; (D) shows wild type recipients compared with C1q -/- recipients. These data are representative of 2-3 independent experiments with 3 mice/group/experiment; error bars indicate standard deviation between mice. *p < 0.05 for all comparisons in (C) and p = ns, not significant for all comparisons in (D) .

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: Expressing, Labeling, Flow Cytometry, Incubation, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: Poly (I:C) and KELIg immunoprophylaxis result in increased transfused KEL RBC consumption by splenic inflammatory monocytes in wild type compared with C3 -/- mice. DiO labeled KEL RBCs were transfused to wild type or C3 -/- mice treated with KELIg in the presence or absence of poly (I:C) and splenic cell subsets were evaluated at 1 and 16 hours post-transfusion (A, B) . (C) Representative histograms for DiO RBC fluorescence patterns of inflammatory monocytes at 16 hours post-transfusion, after first excluding TER119 positive RBCs on the exterior of the splenic cells; black open histogram is KELIg in wild type, dotted open histogram is KELIg and PIC in wild type; white shaded histogram is KELIg in C3 -/- and dotted shaded histogram is KELIg and PIC in C3 -/- . DiO mean fluorescence intensity (MFI) of the splenic cell subsets was evaluated at 1 hour (D) and 16 hours (E) post-transfusion. These data are representative of 2-3 independent experiments with 3 mice/group/experiment; error bars indicate standard deviation between mice.

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: Labeling, Fluorescence, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: Complement-fixed KEL RBCs bind in-vitro to wild type donor derived B-cells but not to CR1/2 -/- B-cells. DiO labeled KEL RBCs were incubated with KELIg in the presence of sera, followed by incubation with peripheral blood derived WBCs from donor mice. (A) In wells using WBCs from wild type donor mice, CD19+B220+ B-cells cells were separated by DiO positivity and then by C3 positivity. The CD19+B220+ cells were next separated by their CD23 and CD21/35 expression, with the cells highest for CD23 and CD21/35 gated in the larger gate and those less strongly positive for CD23 and CD21/35 gated in the smaller gate; the DiO positive and negative populations were then evaluated for their C3 positivity. Shaded histograms are the DiO positive population, open histograms are the DiO negative population. (B) In wells using WBCs from CR1/2 -/- mice, CD19+B220+ B-cells cells were separated by DiO positivity and then by C3 positivity. Shaded histograms are the DiO positive population, open histograms are the DiO negative population. These data are representative of more than 3 independent experiments, with 2 involving CD23 and CD21/35 staining.

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: In Vitro, Derivative Assay, Labeling, Incubation, Expressing, Staining

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: CR1/2 are required for KELIg immunoprophylaxis failure in the setting of poly (I:C). (A) KEL RBCs were transfused into wild type recipients or recipients lacking CR1/2, in the presence or absence of poly (I:C). (B) RBCs were labeled with DiO and mixed with wild type RBCs labeled with DiI; this mixture was transfused into wild type or CR1/2 -/- recipients that had been infused with KELIg and treated with poly (I:C). Recovered DiO positive KEL RBCs were evaluated for (C) KEL glycoprotein expression, and (D) bound complement C3. (E) Wild type or CR1/2 -/- recipients were infused with KELIg and transfused the following day in the absence or presence of poly (I:C), with anti-KEL responses evaluated longitudinally. *p < 0.05 for d14, 21 and 28 of (A) between CR1/2 -/- mice treated with or without poly (I:C); p < 0.05 for 10 min and 1-hour timepoints in (C) ; p < 0.05 for d14, 21, and 28 in (E) . These data are representative of 2-3 independent experiments with 3 mice/group/experiment; error bars indicate standard deviation between mice. ns, not significant.

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: Labeling, Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: Early structural changes of the heart after experimental polytrauma and hemorrhagic shock

doi: 10.1371/journal.pone.0187327

Figure Lengend Snippet: Levels of (A) haemoglobin (Hb) in blood and (B) macrophage chemotactic protein 1 (MCP-1), (C) keratinocyte chemoattractant (KC) and (D) complement component C3a in plasma of animals 4 h after infliction of polytrauma and hemorrhagic shock (PTHS; n = 7 for Hb, n = 8 for MCP-1, KC and C3a) and native control animals (CTRL; n = 8 for Hb, n = 4 for MCP-1, n = 5 for C3a and KC). Results (B, C, D) are presented as amount of protein per total protein in plasma to unmask diluting effects from volume resuscitation. For statistical comparison of experimental means, unpaired t-tests (A, C, D) and Mann-Whitney rank sum test (B) were performed. *: p<0.05.

Article Snippet: Recombinant mouse complement component C3a (R&D Systems, Germany) served for calculation of a standard curve.

Techniques: Clinical Proteomics, Control, Comparison, MANN-WHITNEY

Journal: Cell reports

Article Title: Autocrine Effects of Tumor-Derived Complement

doi: 10.1016/j.celrep.2014.02.014

Figure Lengend Snippet: (A) We measured total tumor weight in an orthotopic murine model of ovarian cancer induced by ID8-VEGF murine ovarian cancer cells in C3 −/− and WT control mice, both in C57BL/6 background. n.s, not significant. (B) Immunostaining of tumors induced by ID8-VEGF in WT and C3 −/− mice, using anti-C3 antibody compared to negative control stain (secondary antibody alone). Scale bar length is 100 μm. (C) Quantitative real-time PCR for C3 mRNA on RNA isolated from murine and human ovarian cancer cell lines. Expression of C3 mRNA in cancer cell lines was compared to that in MOECs and normal human ovarian surface epithelial cell lines (HIO 180) (n = 3; **p ≤ 0.01, t test). (D) C3 gene knockdown in SKOV3ip1 human ovarian cancer cells using C3 siRNA, reduced proliferation, migration, and invasion of these cells in vitro. Results of three independent experiments (each of them in triplicate) are summarized as bar graphs (**p ≤ 0.01, t test). (E) C3 gene knockdown in SKOV3ip1-induced tumors by intraperitoneal injection of hC3 siRNA into tumor-bearing NU/NU mice reduced total weight (*p = 0.017) and number of tumor nodules (*p = 0.05). (F) Representative immunostaining for C3, Ki67, and CD31 in tumors resected from hC3 siRNA-injected and scrambled siRNA-injected mice. Scale bar, 100 μm. (G) The proliferation index in resected tumors was quantified as the percentage of Ki67 positivity shown in dot plots (39% in C3 siRNA versus 74% in scrambled siRNA, n = 5 mice in each group; *p = 0.05, t test). The number of blood vessels in resected tumors was quantified by counting the number of CD31+ lumen structures in five high-power fields (HPFs) per section and in five sections per tumor nodule and in five mice per group. Average number of CD31+ lumens per HPF is shown as dot plots (22/HPF in C3 siRNA versus 42/HPF in scrambled siRNA; *p = 0.05, t test). (H) We investigated the effect of complement on proliferation of endothelial cells by measuring the proliferation rate of RF24 endothelial cells after transfection with C3 siRNA. C3 knockdown did not reduce the proliferation rate in RF24 endothelial cells (n = 3; p = 0.07, t test).

Article Snippet: Rabbit anti-mouse C3 antibody (Abgent), mouse anti-hC3 antibody (Acris Antibodies), rabbit anti-hC5 antibody (Abcam), rabbit polyclonal anti-Ki67 antibody (Thermo Scientific/Lab Vision), rat anti-mouse CD31 antibody (BD Biosciences, BD PharMingen), mouse anti-C5b-9 antibody (Dako), mouse anti-C9 antibody (Hycult Biotech), anti-CD8 and anti-CD11b antibodies (AbD Serotec), rabbit-anti-h AKT, p-AKT, P85, and p-P85 antibodies (Cell Signaling Technology), C5aR antagonist (W-54011) ( ):N-((4-dimethylami-nophenyl)methyl)-7-methoxy-1,2,3,4-tetrahydronaphthalen-1-carboxamide, HCl, and C3aR antagonist (SB290157) ( ):N2-((2,2-dipheny-lethoxy)acetyl)-L-arginine (EMD Chemicals), 3,3′-diaminobenzidine (DAB; Open Biosystems), and Gill’s #3 hematoxylin (Sigma-Aldrich) were purchased from the perspective commercial sources.

Techniques: Control, Immunostaining, Negative Control, Staining, Real-time Polymerase Chain Reaction, Isolation, Expressing, Knockdown, Migration, In Vitro, Injection, Transfection

Journal: Journal of Extracellular Vesicles

Article Title: Yersinia enterocolitica O:3 Outer Membrane Vesicles as a Platform for Complement Activation

doi: 10.1002/jev2.70270

Figure Lengend Snippet: (A) The impact of OMVs on germicidal activity of serum against YeO3‐c bacteria. The influence of OMVs‐associated with: LPS polysaccharide chain length (a) and pYV‐coded factors (b). Data from one of at least two experiments with the use of separately prepared culture supernatants with similar results are presented. 1 sterile cell culture supernatants (secreted by bacteria grown to OD 600 = 0.6) were used as a source of OMVs; 2 expression at 37°C only; 3 NHS inactivated 30 min at 56°C. (B) Y. enterocolitica O:3 OMV‐induced complement activation in the presence of calcium and magnesium chelators. ELISA plates were coated with 10 8 of OMVs secreted by YeS‐c bacteria grown at 4°C, 22°C and 37°C and with OMVs of YeRa‐c, YeRd1‐c and YeRe‐c variants propagated at 37°C. After incubation with/without EDTA, EGTA or EGTA/Mg 2+ (EGTA supplemented with Mg 2+ ions), products of C3 activation were detected with specific antibodies. Data from one of two experiments with similar results are presented. (C) Comparison of complement activation and MBL binding by Yersinia enterocolitica O:3 bacterial cells, LPS and OMVs. ELISA plates were coated with 50 ng/well of bacteria, LPS or OMVs. The deposition of C3 (a, possible AP, CP and LP involvement), C4 (b, possible CP and LP involvement) or C4 LP‐dependent (d) activation products, TCC formation (c, possible AP, CP and LP involvement) and MBL‐binding (e) was analyzed after preincubation with normal human serum (filled columns) or with EDTA‐treated NHS (stripped columns) or without serum (open columns, negative control). Data from one of two experiments with similar results are presented. Dots represent individual OD values for each well. (D) Recognition of Y. enterocolitica O:3 OMVs by human mannose‐binding lectin. YeS‐c_37°C bacteria (1), OMVs (2) and LPS (3) were separated in SDS‐PAGE. After transfer to nitrocellulose and incubation with NHS, the bound MBL was detected with specific mAb (a). The experiment was performed at least 5 times. To control the loading of bacteria, LPS or OMVs the presence of OPS in separated samples was confirmed with anti‐6‐deoxy‐L‐altropyranose mAbs (b).

Article Snippet: Depletion of functional C3 from serum was verified in Western blot [acc. to Younger et al. ( )], using goat antibodies against mouse C3 (MP Biomedicals, USA), HRP‐conjugated anti‐goat Ig (Dako) for detection of intact C3 α‐chain and ECL detection system.

Techniques: Activity Assay, Bacteria, Sterility, Cell Culture, Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Comparison, Binding Assay, Negative Control, SDS Page, Control