Journal: PLoS ONE

Article Title: Nicotinamide Exacerbates Hypoxemia in Ventilator-Induced Lung Injury Independent of Neutrophil Infiltration

doi: 10.1371/journal.pone.0123460

Figure Lengend Snippet: (A) Mice were anesthetized and placed on mechanical ventilation (MV) with tidal volumes of 20 ml/kg and zero PEEP. After 1 hour of mechanical ventilation, mice received intraperitoneal injections of either 400 mg/kg nicotinamide (open squares, NAM+MV) or an equivalent volume of PBS (closed circles, PBS+MV), and mechanical ventilation was continued for a total of 6 hours. All mice were euthanized at the end of 6 hours mechanical ventilation or spontaneous breathing (control mice), which was 5 hours after NAM or PBS administration. Bronchoalveolar lavage (BAL) fluid was assayed for: (A) protein concentration; (B) neutrophils (PMNs); (D and E) chemokines KC and MIP2; and (G) cytokine IL-1 n = 4 /group (non-ventilated controls) and 7-8/group (mechanical ventilation). F) Oxygen saturation was measured each hour. n = 11-12/group. (C) Lung homogenate was assayed via ELISA for MPO content. n = 6-7/group. Data are representative of two (C-E, G) to four (A, B, F) separate experiments. Significance is indicated for comparisons between PBS and PBS+MV, and between PBS+MV and NAM+MV. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The cytokine concentrations in BAL were determined using Mouse IL-1β ELISA (eBioscience, San Diego, CA), and Mouse keratinocyte-derived chemokine (KC) and macrophage inflammatory protein 2 (MIP2) ELISA kits (R&D Systems, Minneapolis, MN).

Techniques: Control, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Targeting S1P1 receptor protects against murine immunological hepatic injury through myeloid-derived suppressor cells.

doi: 10.4049/jimmunol.1301193

Figure Lengend Snippet: FIGURE 2. Treatment with FTY720 increases the liver expression of chemokines that mediate CD11b+Gr1+ myeloid cell recruitment. (A–D) C57BL/6 mice were injected with PBS or FTY720 (1 mg/kg) daily starting at 6 h before the injection of Con A (15 mg/kg). (A) At 20 h after Con A injection, the percentage of peripheral blood CD11b+Gr1+ cells was determined by flow cytometry, and the absolute numbers of CD11b+Gr1+ cells were calculated accordingly (n = 5–8). (B) CD11b+Gr1+ cells in liver were determined by flow cytometry and are presented as a representative FACS plot (left panel) and the absolute numbers of CD11b+Gr1+ cells (n = 6–8) (right panel). (C) The concentration of CXCL1 and CXCL2 in serum (left panel) or the mRNA level of CXCL1 and CXCL2 in liver (right panel) CD11b+Gr1+ cells was determined by ELISA (n = 4) or quantitative PCR (n = 4). (D) CXCR2 expression in liver CD11b+Gr1+ cells was determined by intracellular staining, followed by flow cytometry analysis (n = 3). (E and F) CIH mice were administered 50 mg anti-CXCR2 mAb (242216) or IgG isotype control (54447; both from R&D Systems) via i.v. injection. (E) Mice were sacrificed at 20 h after Con A injection, and livers were analyzed using H&E staining. Scale bar, 50 mm. Arrowheads indicate the area of necrosis. (F) The percentage of necrotic area (left panel) was quantified using ImageJ software. For each experiment, three random sections/liver were taken from four livers/group. Serum ALT levels were assessed by ELISA (n = 4) (right panel). Data are representative of four (A, B) or two (C–F) independent experiments (mean 6 SD). *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: The concentrations of serum CXCL1 and CXCL2 and serum or culture supernatant IFN-g were quantified by sandwich ELISA (MKC00B, MM200, and MIF00; R&D Systems).

Techniques: Expressing, Injection, Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Staining, Control, Software

Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: Stanniocalcin-1 Attenuates Ischemic Cardiac Injury and Response of Differentiating Monocytes/Macrophages to Inflammatory Stimuli

doi: 10.1016/j.trsl.2016.06.011

Figure Lengend Snippet: (A) Schematic illustrating the strategy used to evaluate the effects of rSTC-1 pre-treatment on macrophage response to lipopolysaccharide (LPS). Human U937 monocytes were induced to differentiate into macrophages by treatment with 100 ng/ml of phorbol 12-myristate 13-acetate (PMA) in the presence or absence of 1 μg/ml of rSTC-1. After 48 hours, differentiated macrophages were harvested, plated, and incubated for another 3 hours. Then, macrophages were stimulated with 50 ng/ml of LPS. After 5 hours, media conditioned (CM) by unstimulated or LPS-stimulated macrophages were collected and used to evaluate changes in levels of secreted TNFα (B) and CXCL2 (C) by ELISA. (D) Schematic illustrating the strategy used to evaluate the effects of rSTC-1 on the response of differentiated macrophages to LPS. U937 monocytes were plated in macrophage medium supplemented with 100 ng/ml of PMA for 48 hours. Differentiated macrophages were harvested, re-plated, and incubated with or without 1 μg/ml of rSTC-1. After 3 hours, macrophages were stimulated with LPS for an additional 5 hours. The CM was collected, clarified by centrifugation and used to determine levels of TNFα (E) and CXCL2 (F) by ELISA. Values are presented as mean + SEM (n=3). Statistical significance was determined using ANOVA (B-C) or Student's t-test (E-F) (not significant, ns p≥ 0.05; ***p< 0.001).

Article Snippet: Levels of mouse myeloperoxidase (MPO), CD14, CXCL2, and IL-1β were measured using commercially available ELISA Kits (CD14 and IL-1β were form R&D Systems, CXCL2 from Abnova, and MPO from Hycult Biotech, Plymouth Meeting, PA).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Centrifugation

Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: Stanniocalcin-1 Attenuates Ischemic Cardiac Injury and Response of Differentiating Monocytes/Macrophages to Inflammatory Stimuli

doi: 10.1016/j.trsl.2016.06.011

Figure Lengend Snippet: Human U937 monocytes were incubated in macrophage medium supplemented with 100 ng/ml of phorbol 12-myristate 13-acetate (PMA) in the presence or absence of 1.0 μg/ml of rSTC-1. After 48 hours, morphology of differentiated macrophages was evaluated by light microscopy. Then, macrophages were harvested and processed for real-time RT-PCR or flow cytometry analysis. (A) Schematic showing the workflow. (B) Representative images of undifferentiated U937 monocytes (No Treatment), PMA-differentiated U937 macrophages (PMA), and U937 cells stimulated with PMA and rSTC-1 (PMA+ rSTC-1). Scale bar, 100 μm. (C) Cell surface expression of CD11b and CD14 was assessed by flow cytometry. Real-time RT-PCR for CD11b (D), CD14 (E), TLR4 (F), and TLR2 (G) in undifferentiated U937 monocytes and cells stimulated with PMA for 48 hours (with and without rSTC-1 treatment). (H) Representative images of U937 cells treated for 48 hours with PMA in the presence and absence of rSTC-1, MnTBAP, and paraquat. Scale bar, 50 μm. Real-time RT-PCR for CD11b (I) and CD14 (J) in U937 cells stimulated for 48 hours with the compounds described in panel H. Values are expressed as mean + SEM (n=3-5). Data was analyzed using ANOVA (not significant, ns p≥ 0.05, *p< 0.05, **p< 0.01, ***p< 0.001).

Article Snippet: Levels of mouse myeloperoxidase (MPO), CD14, CXCL2, and IL-1β were measured using commercially available ELISA Kits (CD14 and IL-1β were form R&D Systems, CXCL2 from Abnova, and MPO from Hycult Biotech, Plymouth Meeting, PA).

Techniques: Incubation, Light Microscopy, Quantitative RT-PCR, Flow Cytometry, Expressing

Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: Stanniocalcin-1 Attenuates Ischemic Cardiac Injury and Response of Differentiating Monocytes/Macrophages to Inflammatory Stimuli

doi: 10.1016/j.trsl.2016.06.011

Figure Lengend Snippet: Human U937 monocytes were stimulated with 5 μg/ml human high mobility group box1 (HMGB1) and 10 ng/ml each of recombinant human tumor necrosis factor alpha (TNFa), interleukin-1 beta (IL-1β), and IL-6 in the presence or absence of 1.0 μg/ml rSTC-1 treatment. After 48 hours, cells were lysed for real-time RT-PCR. (A) Schematic showing the workflow. The fold changes of mRNA levels for CD11b (B) and CD14 (C) were measured in unstimulated U937 monocytes, stimulated U937 monocytes, and stimulated cells treated simultaneously with rSTC-1. Values are expressed as mean + SEM (n=3). Data were analyzed with ANOVA (not significant, ns p≥ 0.05, *p< 0.05, **p< 0.01, ***p< 0.001).

Article Snippet: Levels of mouse myeloperoxidase (MPO), CD14, CXCL2, and IL-1β were measured using commercially available ELISA Kits (CD14 and IL-1β were form R&D Systems, CXCL2 from Abnova, and MPO from Hycult Biotech, Plymouth Meeting, PA).

Techniques: Recombinant, Quantitative RT-PCR

Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: Stanniocalcin-1 Attenuates Ischemic Cardiac Injury and Response of Differentiating Monocytes/Macrophages to Inflammatory Stimuli

doi: 10.1016/j.trsl.2016.06.011

Figure Lengend Snippet: NOD/SCID mice were subjected to ischemic cardiac injury by permanent ligation of the left descending coronary artery (LLDCA). At 1 hour and 24 hours after ligation, 2.0 mg/kg of rSTC-1 or equal volume of 0.9% sodium chloride (saline) was administered intravenously. Mice were euthanized up to 48 hours after LLDCA to collect heart tissue and assess inflammatory response. Blood was collected to measure serum levels of cardiac troponin (cTnI). (A) Diagram showing the workflow of the cardiac injury model. (B) Changes in expression of kidney UCP2 were determined by real-time RT-PCR. (C) Time-dependent change in serum level of cTnI was determined by ELISA. (D) Assessment of serum cTnI, 24 hours after permanent ligation and rSTC-1 treatment. Intravenous rSTC-1 suppressed levels of (E) CD14, inflammatory cytokines TNFα (F), CXCL2 (G), and IL-1β (H), and (I) myeloperoxidase (MPO) in heart tissue lysate prepared 48 hours after LLDCA. Mean value is illustrated by the black lines. Real-time RT-PCR of UCP2 in cardiac (J) and kidney (K) tissue, and (L) UCP3 in cardiac tissue. Statistical significance was determined using Student's t-test when 2 groups were compared, or ANOVA for analysis of more than 2 groups (not significant, ns p> 0.05, * p< 0.05, ** p< 0.01).

Article Snippet: Levels of mouse myeloperoxidase (MPO), CD14, CXCL2, and IL-1β were measured using commercially available ELISA Kits (CD14 and IL-1β were form R&D Systems, CXCL2 from Abnova, and MPO from Hycult Biotech, Plymouth Meeting, PA).

Techniques: Ligation, Saline, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: Stanniocalcin-1 Attenuates Ischemic Cardiac Injury and Response of Differentiating Monocytes/Macrophages to Inflammatory Stimuli

doi: 10.1016/j.trsl.2016.06.011

Figure Lengend Snippet: Tissue injury induces release of differentiation stimuli [such as pathogen-associated molecular pattern molecules (PAMPs), damage-associated molecular pattern molecules (DAMPs), cytokines, and chemokines] that promote monocyte differentiation and recruitment to the site of injury. Differentiation stimuli cause an increase in expression of surface molecules such as CD11b and CD14 in monocyte/macrophage and enhance secretion of inflammatory cytokines and chemokines (for example TNFα, IL-1β, and CXCL2) in these cells. Administration of rSTC-1 during the differentiation process reduces expression of CD14 by monocytes/macrophages, which subsequently decreases their response to inflammatory stimuli including DAMPS and PAMPS.

Article Snippet: Levels of mouse myeloperoxidase (MPO), CD14, CXCL2, and IL-1β were measured using commercially available ELISA Kits (CD14 and IL-1β were form R&D Systems, CXCL2 from Abnova, and MPO from Hycult Biotech, Plymouth Meeting, PA).

Techniques: Expressing

Journal: PLOS ONE

Article Title: ECRG4 mediates host response to cutaneous infection by regulating neutrophil recruitment and adhesion receptor expression

doi: 10.1371/journal.pone.0310810

Figure Lengend Snippet: Early neutrophil mobilization from the BM was determined using an intraperitoneal LPS injection model, with evaluation at 2 hours after challenge. Flow cytometry was used to quantify neutrophils in the BM (a) and blood (b) 2 hours after LPS challenge. The relative expression of key mediators of neutrophil retention in the BM, CXCR4 and CXCL12, as well as the receptors for the major chemokines/cytokines responsible for their mobilization, CXCR2 and G-CSF receptor ( csf3r ) were assessed in the BM by qPCR (c). (d) ELISA was used to evaluate plasma levels of key cytokines and chemokines 2 hours after LPS challenge, and to determine CXCL12 in the BM (e). N = 6 mice per group. 1-Way ANOVA, P = *<0.05 **<0.01, ***<0.001, ****<0.0001.

Article Snippet: ELISAs were done using the R&D Systems ELISA kits (TNFa # DY410, G-CSF # DY414, CXCL12 # MCX120, CXCL1 # DY453, CXCL2 # DY452, IL6 # M6000B, IL1b # MLB00C) according to manufacturers recommended procedures.

Techniques: Injection, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Clinical Proteomics