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 Journal of Clinical Investigation

Article Title: C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps

doi: 10.1172/JCI163105

Figure Lengend Snippet: An ELISA assay was performed to measure the concentrations of ( A ) C5a, ( B ) factor Bb, and ( C ) C3a in the BAL fluid from patients with influenza ( n = 16) and patients with COVID- 19 ( n = 16). ( B ) Paired concentrations of ( D ) C5a and ( E ) factor Bb in the plasma and BAL fluid from patients with COVID-19 were determined by ELISA. ( F ) A different cohort from a previously published data set was reanalyzed and the t-SNE analysis of total cells (65,166) from BAL fluid of patients with non-COVID-19 pneumonia ( n = 13) and COVID-19 ( n = 22) is shown. ( G ) Dot plots display the highlighted distribution of C5AR1 for each indicated cell population. ( H ) Violin plots showing the expression levels of C5aR1 in each type of cell from patients with COVID-19 or with non-COVID-19 pneumonia. ( I ) The dot plot depicts the scaled and centered expression of an average cell in each cluster and therefore contains negative and positive values. The average expression reflects the mean expression of C5AR1 in each cluster compared with all other cells. ( J ) Number of cells per cell population [neutrophils (Neu), monocytes/macrophages (Mo/Mac), and dendritic cells (cDC)] that express C5AR1 in the groups of patients with COVID-19 and non-COVID-19 pneumonia. ( K ) Average expression of C5AR1 per cell for each cell population [neutrophils (Neu), monocytes/macrophages (Mo/Mac), and dendritic cells (cDC)] in the groups of patients with COVID-19 and non-COVID-19 pneumonia. Data are shown as the mean ± SEM. P values were determined by 2-tailed unpaired ( A – D , J , and K ) or paired ( D and E ) Student’s t tests followed by Wilcoxon matched-pairs signed rank tests.

Article Snippet: C5a, factor Bb, and C3a ELISA assays were performed using, respectively, the human complement component C5a duoset ELISA kit from R&D Systems (DY2037), the MicroVue Bb plus fragment EIA kit from Quidel (A027), and the Complement C3a human ELISA kit from Thermo Fisher Scientific (BMS2089).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Expressing

Journal: The Journal of Clinical Investigation

Article Title: C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps

doi: 10.1172/JCI163105

Figure Lengend Snippet: ( A ) Tg mice were infected with SARS-CoV-2 (2 × 104 PFU, intranasally). ELISA assay to measure levels of ( B ) C5a in the lung homogenate of infected animals ( n = 14) or mock control ( n = 11). ( C ) factor Bb and ( D ) C3a levels in the lung homogenate of infected animals ( n = 8) or mock control ( n = 5). ( E ) Representative confocal images of the presence of C5aR1 expression in the lung tissue of Tg fl/fl mice ( C5ar1 -eGFP mice) infected with SARS-CoV-2 (5 dpi). Tissue slices were costained for nuclei (DAPI, blue), Iba-1 (macrophages, red) and NE (neutrophils, red) markers. Scale bar: 50 μm. ( F ) Percentage of cells expressing C5aR1 in the lung tissue of Tg fl/fl mice infected with SARS-CoV-2 ( n = 4 mice/4 randomized field). ( G ) Representative H&E staining from the lung of SARS-CoV-2-infected Tg fl/fl ( n = 6) or Tg cKO mice ( n = 6). A mock-infected group was used as control ( n = 6). Scale bars: 200 μm (4 ×), 100 μm(10 ×). ( H ) TUNEL staining (red) for detection of apoptotic cells in situ from lung tissue of SARS-CoV-2–infected Tg fl/fl ( n = 5) or Tg cKO mice ( n = 6). Mock-infected Tg mice were used as a control ( n = 5/group). ( I ) Quantification of the lung septal area fraction. ( J ) Percentage of TUNEL positive cells in lung tissue. Scale bar: 50 μm. ( K ) ELISA assays were performed to detect CCL2, CCL3, CCL4, CXCL1, and IL-6 levels in the lung tissue of Tg fl/f ( n = 8) or Infected Tg cKO mice ( n = 7). Mock-infected Tg mice were used as a control ( n = 5). Data are shown as the mean ± SEM. P values were determined by ( B – D ) Student’s 2-tailed t test and ( F , I , J , and K ) 1-way ANOVA followed by Bonferroni’s posthoc test.

Article Snippet: C5a, factor Bb, and C3a ELISA assays were performed using, respectively, the human complement component C5a duoset ELISA kit from R&D Systems (DY2037), the MicroVue Bb plus fragment EIA kit from Quidel (A027), and the Complement C3a human ELISA kit from Thermo Fisher Scientific (BMS2089).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Control, Expressing, Staining, TUNEL Assay, In Situ

Journal: The Journal of Clinical Investigation

Article Title: C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps

doi: 10.1172/JCI163105

Figure Lengend Snippet: ( A ) Tg mice were infected with SARS-CoV-2 (2 x 104 PFU, intranasally) and treated with DF2593A (3 mg/kg, p.o) 1 hour before SARS-CoV-2 infection and once a day up to the day of sample collection (5 dpi). ( B ) Body weight, clinical score, and oxygen saturation were measured daily after infection ( n = 11/group, pooled from 2 independent experiments). ( C ) Representative H&E staining from the harvested lung of the COVID-19 mouse model treated ( n = 4) or not ( n = 6) with DF2593A. A mock-infected group was used as control ( n = 5). Scale bars: 200 μm (4 ×); 100 μm (10 ×). ( D ) TUNEL staining (green) for detection of apoptotic cells in situ from lung tissue of mice ( n = 5/group). ( E ) Quantification of the lung septal area fraction. ( F ) Percentage of TUNEL-positive cells in lung tissue. Scale bar: 50 μm. ( G ) ELISA assays were performed to detect CCL2, CCL3, CCL4, CXCL1, and IL-6 levels in lung homogenate ( n = 6/group). A mock-infected group was used as the control group. Data are shown as the mean ± SEM. P values were determined by 1-way ANOVA followed by Bonferroni’s posthoc test ( E – G ).

Article Snippet: C5a, factor Bb, and C3a ELISA assays were performed using, respectively, the human complement component C5a duoset ELISA kit from R&D Systems (DY2037), the MicroVue Bb plus fragment EIA kit from Quidel (A027), and the Complement C3a human ELISA kit from Thermo Fisher Scientific (BMS2089).

Techniques: Infection, Staining, Control, TUNEL Assay, In Situ, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps

doi: 10.1172/JCI163105

Figure Lengend Snippet: ( A ) C57/BL6 mice were treated twice with vehicle, DNAse (Pulmozyme, 10 mg/kg, s.c.), or C5aR1 antagonist (DF2593A, 3 mg/kg, orally), 24 hours and 1 hour before the intratracheal instillation of rmC5a (400 ng). ( B ) Lung slices from the control group or mice challenged with rmC5a and treated with vehicle, DNAse, or C5aR1 antagonist (DF2593A) were stained with H&E for evaluation of histological changes. ( C ) Quantification of the lung septal area fraction ( n = 5/group). ( D ) Lung slices from the control group or from mice challenged with rmC5a and treated with vehicle, DNAse, or C5aR1 antagonist (DF2593A) were costained for nuclei (DAPI, blue), H3Cit (green), and MPO (red) markers. ( E ) NET quantification by the MPO-DNA PicoGreen assay in the supernatant of the lung homogenate ( n = 5–6/group). ( F ) ELISA assays were performed to detect CCL2, CCL3, CCL4, CXCL1, and IL-6 levels in lung homogenate ( n = 5–6/group). Data are shown as the mean ± SEM. P values were determined by 1-way ANOVA followed by Bonferroni’s posthoc test ( C , E , and F ).

Article Snippet: C5a, factor Bb, and C3a ELISA assays were performed using, respectively, the human complement component C5a duoset ELISA kit from R&D Systems (DY2037), the MicroVue Bb plus fragment EIA kit from Quidel (A027), and the Complement C3a human ELISA kit from Thermo Fisher Scientific (BMS2089).

Techniques: Control, Staining, Picogreen Assay, Enzyme-linked Immunosorbent Assay

Journal: Lupus

Article Title: Increased C1q, C4 and C3 deposition on platelets in patients with systemic lupus erythematosus--a possible link to venous thrombosis?

doi: 10.1177/0961203312457210

Figure Lengend Snippet: Figure 2 Representative flow cytometry plots of a systemic lupus erythematosus (SLE) patient with increased complement deposition on platelets. A) Platelets were gated through forward- and side-scatter properties and initially confirmed to be platelets by the expression of CD42a. Platelet deposition of B) C1q, C) C4d neo, D) C3d and E) C3a was measured by flow cytometry. The black lines represent an isotype antibody and the red lines the complement deposition on the platelet.

Article Snippet: The platelets, 4 ml, were incubated with anti-C1q- fluorescein isocyanate (FITC) (Dako, Glostrup, Denmark) or antibodies against C3d, C4d, C3a and C4d neo (Quidel, Lupus at GEORGIAN COURT UNIV on April 11, 2015lup.sagepub.comDownloaded from San Diego, CA, USA) in HEPES-buffer at a total volume of 50 ml for 40 minutes at room temperature.

Techniques: Flow Cytometry, Expressing

Journal: Lupus

Article Title: Increased C1q, C4 and C3 deposition on platelets in patients with systemic lupus erythematosus--a possible link to venous thrombosis?

doi: 10.1177/0961203312457210

Figure Lengend Snippet: Figure 4 Representative flow cytometry plots of in vitro complement deposition on fixed heterologous platelets. A) Platelets were gated based on forward- and side-scatter properties and analyzed for B) C1q, C) C3a, D) C3d, E) C4d and F) C4d neo-deposition by flow cytometry. The black line represents an isotype antibody, the blue line serum from a systemic lupus erythematosus (SLE) patient, and the red line the same SLE serum treated with 10 mM ethylenediaminetetraacetic acid (EDTA) to inhibit complement activation.

Article Snippet: The platelets, 4 ml, were incubated with anti-C1q- fluorescein isocyanate (FITC) (Dako, Glostrup, Denmark) or antibodies against C3d, C4d, C3a and C4d neo (Quidel, Lupus at GEORGIAN COURT UNIV on April 11, 2015lup.sagepub.comDownloaded from San Diego, CA, USA) in HEPES-buffer at a total volume of 50 ml for 40 minutes at room temperature.

Techniques: Flow Cytometry, In Vitro, Activation Assay

Journal: Lupus

Article Title: Increased C1q, C4 and C3 deposition on platelets in patients with systemic lupus erythematosus--a possible link to venous thrombosis?

doi: 10.1177/0961203312457210

Figure Lengend Snippet: Figure 5 In vitro complement deposition on platelets. Heterologous paraformaldehyde-fixed platelets were incubated with serum and analyzed for the ability to support complement deposition on the platelets. A) Systemic lupus erythematosus (SLE) patients were divided into patients being positive or negative for anti-cardiolipin antibodies (aCL), lupus anticoagulant (LA) or antipho- spholipid antibody syndrome (APS) and the ability of sera to support complement activation on platelets measured by flow cytometry. Serum from normal healthy individuals (NHS) served as controls. B) C4d deposition on platelets with serum from SLE patients with and without a deep venous thrombosis (DVT). The values are expressed as the mean fluorescent intensity (MFI) ratio for the target molecule as compared to an isotype antibody.

Article Snippet: The platelets, 4 ml, were incubated with anti-C1q- fluorescein isocyanate (FITC) (Dako, Glostrup, Denmark) or antibodies against C3d, C4d, C3a and C4d neo (Quidel, Lupus at GEORGIAN COURT UNIV on April 11, 2015lup.sagepub.comDownloaded from San Diego, CA, USA) in HEPES-buffer at a total volume of 50 ml for 40 minutes at room temperature.

Techniques: In Vitro, Incubation, Activation Assay, Flow Cytometry