Journal: Journal of Translational Medicine

Article Title: Unexpected impairment of TNF-α-induced maturation of human dendritic cells in vitro by IL-4

doi: 10.1186/s12967-016-0848-2

Figure Lengend Snippet: IL-4 reduces CCR7 mRNA synthesis, intracellular and surface protein content in TNF-α-matured DC. a RT-PCR analysis of CCR7 and β-actin mRNA expression in 5 × 10 4 iDC, IL-4 − and IL-4 + -DC. b Quantification of RT-PCR analysis in ( a ). Data are expressed as the ratio of expression of CCR7 to β-actin. The results are representative of four independent experiments. c Interleukin-4 − and IL-4 + -DC were fixed, permeabilized and stained with rat PE-anti-CCR7 antibody (bold lines) or its isotype control (thin lines ) and analyzed by flow cytometry. The results are representative of six independent experiments. d Mean fluorescence intensity (MFI) of CCR7 intracellular content expressed as the ratio of fluorescence intensity of specific labeling to background staining. The results are the mean ± SEM of the MFI from six independent experiments. e Immature DC were cultured for 0, 8, 24 or 48 h with IL-4 (25 ng/mL) during maturation with GM-CSF and TNF-α. f Immature DC were cultured for 48 h, either in the absence of (0) or with increasing concentrations (0.5, 1, 5, 25 ng/mL) of IL-4 or IL-13, during maturation with GM-CSF and TNF-α. g Immature DC were activated with GM-CSF and TNF-α (20 ng/mL), LPS (50 ng/mL) or sCD40L (250 ng/mL) in the absence or presence of 25 ng IL-4. e – g Cells were stained with PE-anti-CCR7 antibody and analyzed by flow cytometry. The results are the mean ± SEM of the MFI from four to five separate experiments. *P

Article Snippet: Cytokine detection by enzyme-linked immunosorbent assay (ELISA) For the determination of IL-12p70 and IL-10 release, IL-4− and IL-4+ -DC were stimulated for 24 h with 250 ng/mL recombinant sCD40L (Alexis Biochemicals).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Flow Cytometry, Cytometry, Fluorescence, Labeling, Cell Culture

Journal: Redox Report : Communications in Free Radical Research

Article Title: Phospholipase C-γ2 via p38 and ERK1/2 MAP kinase mediates diperoxovanadate-asparagine induced human platelet aggregation and sCD40L release

doi: 10.1179/1351000213Y.0000000057

Figure Lengend Snippet: Schematic illustration of DPV-Asn-induced oxidative stress in platelets causing aggregation and sCD40L release. The model depicts that catalase-tolerant peroxide DPV-Asn mediates sequential induction of a signaling cascade in platelets, chiefly regulated by PLC-γ2, which apparently played a central role in upregulating dense granule secretion, calcium influx, p38 and ERK1/2 MAP kinase phosphorylation, and subsequently directed COX activation and enhanced TxA 2 generation to further amplify platelet aggregation and thrombus formation. DPV-Asn further augments GPIIbIIIa-dependent release of proinflammatory cytokine sCD40L, thereby exhibiting a thrombo-inflammatory phenotype. DPV-Asn, diperoxovanadate-asparagine; ROS, reactive oxygen species; NAC, N -acetyl cysteine; AA, arachidonic acid; COX, cycloxygenase; TxA 2 , thromboxane A 2 ; TP, thromboxane-prostanoid receptor.

Article Snippet: Results Platelet aggregation induced by DPV-Asn was chiefly regulated by dense granule secretion, thromboxane A2 (TxA2 ) generation, intra-platelet [Ca2+ ] influx, GPIIbIIIa activation and sCD40L release, which were significantly reduced in presence of U73122 (PLC inhibitor), aspirin (COX), SB203580 (p38 inhibitor), and PD98059 (ERK inhibitor).

Techniques: Planar Chromatography, Activation Assay

Journal: Redox Report : Communications in Free Radical Research

Article Title: Phospholipase C-γ2 via p38 and ERK1/2 MAP kinase mediates diperoxovanadate-asparagine induced human platelet aggregation and sCD40L release

doi: 10.1179/1351000213Y.0000000057

Figure Lengend Snippet: DPV-Asn-induced release of sCD40L is regulated by PLC-γ2 and thromboxane A 2 . (A) DPV-Asn, H 2 O 2 (8 mM) or collagen (6 µg/ml)-induced release of soluble CD40L (sCD40L) from human washed platelets. The soluble CD40L (sCD40L) released from activated platelets in the presence of DPV-Asn, H 2 O 2 or collagen was measured by enzyme-linked immunosorbent assay. (B) Effect of various pharmacologic interventions on DPV-Asn-induced sCD40L release from platelets (NAC (5 mM), aspirin (100 µM), U73122 (1 µM), SB203580 (10 µM), PD98059 (10 µM), eptifibatide (10 µM), SQ29548 (100 nM)). Data have been presented as mean ± SEM of amount of sCD40L released (pg/ml) of four independent experiments (* P

Article Snippet: Results Platelet aggregation induced by DPV-Asn was chiefly regulated by dense granule secretion, thromboxane A2 (TxA2 ) generation, intra-platelet [Ca2+ ] influx, GPIIbIIIa activation and sCD40L release, which were significantly reduced in presence of U73122 (PLC inhibitor), aspirin (COX), SB203580 (p38 inhibitor), and PD98059 (ERK inhibitor).

Techniques: Planar Chromatography, Enzyme-linked Immunosorbent Assay