Journal: Hepatology Communications

Article Title: Hyperoxidized Albumin Modulates Platelets and Promotes Inflammation Through CD36 Receptor in Severe Alcoholic Hepatitis

doi: 10.1002/hep4.1440

Figure Lengend Snippet: Validation of platelet functionality in SAH. (A) Platelet activation/aggregation in SAH histogram shows the percent expression of PAC‐1, P‐selectin (CD62p), and intracellular Ca +2 (secretion) in SAH and HC. (B) Increased expression (relative change) of various mRNA in SAH platelets linked to platelet and complement activation. Protein expression of Gp2b/3a/β‐actin ratio in platelets of patients with SAH. Results expressed as mean ± SEM (* P < 0.05, ** P < 0.01 for SAH versus HC). (C) Patients with SAH showed higher plasma levels of PF‐4 (65.85 ± 0.9457 versus 81.13 ± 6.595 ng/mL, P < 0.02), P‐selectin (6.360 ± 0.1639 versus 10.118 ± 2.1492 ng/mL, P < 0.0001), and soluble cluster of differentiation 40 ligand (1,726 ± 134.4 versus 2,528 ± 1.525.4 pg/mL, P < 0.05). Circulating levels of PF‐4, P‐selectin, and sCD40L correlated with the MELD score in SAH (r > 0.5, P < 0.05). (D) Representative western blot of SNARE complex proteins in platelets isolated from SAH versus HC (n = 5). Results are expressed as mean ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001 as indicated).

Article Snippet: Plasma‐citrated blood samples were centrifuged for 15 minutes at 3,000 g , and levels of PF‐4 (E‐EL‐H2216; Elabscience, Houston, TX), P‐selectin (E‐EL‐H0917; Elabscience), and sCD40L (E‐EL‐H0035; Elabscience) were measured.

Techniques: Biomarker Discovery, Activation Assay, Expressing, Clinical Proteomics, Western Blot, Isolation

Journal: Particle and Fibre Toxicology

Article Title: Intranasal exposure to amorphous nanosilica particles could activate intrinsic coagulation cascade and platelets in mice

doi: 10.1186/1743-8977-10-41

Figure Lengend Snippet: Examination of intrinsic cascade pathways. (a) In vitro changes in blood coagulation factor XII activity in human plasma by silica particles of various sizes. The initial rate of reaction of coagulation factor XII was obtained from human plasma, which was obtained from a control sample of human plasma, which did not contain any silica particles, and was measured by fluorescence intensity. Results are expressed as mean ± S.D. ( n = 5). ** and * represent significant differences from the control (** p < 0.01, * p < 0.05). RFU: relative fluorescent unit. (b) The level of soluble CD40 ligand (sCD40L) and (c) the level of von Willebrand factor (vWF) in mouse plasma. BALB/c mice were intranasally exposed to nSP30, nSP70, mSP300, nSP100 or mSP1000 at a concentration of 500 μg/mouse for 7 days. Twenty-four hours after the final administration, the levels of sCD40L and vWF in the mouse plasma were determined using ELISA. Results are expressed as mean ± S.E. ( n = 4–5). ** and * represent significant differences from the control (** p < 0.01, * p < 0.05).

Article Snippet: TF, sCD40L, and vWF levels in plasma were determined using enzyme-linked immunosorbent assay (ELISA) kits (TF: Mouse Tissue Factor ELISA kit (Cusabio, Newark, DE, USA); sCD40L: Mouse sCD40L ELISA (eBioscience, San Diego, CA, USA); vWF: Mouse von Willebrand Factor ELISA kit (Cusabio, Newark, DE, USA)).

Techniques: In Vitro, Coagulation, Activity Assay, Clinical Proteomics, Control, Fluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay

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 impairs TNF-α-induced cell-surface expression of DC maturation markers. Dendritic cells were derived from human monocytes cultured for 5 days with GM-CSF (1000 IU/mL) and IL-4 (25 ng/mL). Maturation was induced after an additional 48-h culture with GM-CSF and 20 ng/mL TNF-α, in the absence (IL-4 − ) or presence (IL-4 + ) of 25 ng/mL IL-4. a Cells were stained with specific mAbs directed against CD83, CD86, HLA-DR or CD25 ( bold lines ) or their respective isotype controls ( thin lines ) and analyzed by flow cytometry. The figure in each histogram indicates the percentage of positive cells. The results are representative of 10 independent experiments. b Mean fluorescence intensity (MFI) for CD83, CD86, HLA-DR and CD25 expressed as the ratio of fluorescence intensity of specific labeling to background staining. The results are the mean ± SEM of MFI of ten separate experiments. *P < 0.05, compared to IL-4 − -DC. c Cells were double-stained with FITC-anti-CD83 and APC-anti-CD25 antibodies and analyzed by flow cytometry. d The results are expressed as the mean ± SEM % of IL-4 − and IL-4 + -DC expressing the indicated markers in five separate experiments. *P < 0.05, compared to IL-4 − -DC. e IL-4 − and IL-4 + -DC were stimulated during an additional 24-h culture with 250 ng/mL recombinant soluble CD40L and underwent ELISA for IL-12p70 and IL-10 release. The results are the mean ± SEM of six to eight independent experiments. *P < 0.05, compared to IL-4 − -DC

Article Snippet: 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: Expressing, Derivative Assay, Cell Culture, Staining, Flow Cytometry, Fluorescence, Labeling, Recombinant, Enzyme-linked Immunosorbent Assay

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 < 0.05, compared to DC activated in the absence of IL-4

Article Snippet: 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, Fluorescence, Labeling, Cell Culture

Journal:

Article Title: Critical role of the CD40-CD40-ligand pathway in regulating mucosal inflammation-driven angiogenesis in inflammatory bowel disease

doi: 10.1136/gut.2006.111989

Figure Lengend Snippet: Figure 1 VEGF production by CD40L‐stimulated HIFs. Panel A: Human intestinal fibroblasts (HIFs) were activated with soluble CD40 ligand (sCD40L) or were co‐cultured either with CD40+ D1.1 cells or with CD40− Jurkat cells for 24 hours, before measuring vascular endothelial growth factor (VEGF) release in culture supernatants. In selected experiments, blocking antibodies (Ab) to either CD40 (M2) or CD40L (M90) were used to interfere with the CD40–CD40L interaction. Results are representative of eight independent experiments performed in duplicate and are expressed as mean and standard deviation. *p<0.01 compared with VEGF release by HIFs not activated with sCD40L; **p<0.001 compared with VEGF release by HIFs cultured in the absence of CD40+ D1.1 cells; §p<0.001 compared with VEGF release in the absence of blocking antibodies to either CD40 or CD40L. Panel B: HIFs were co‐cultured either with unstimulated lamina propria T (LPT) cells or with CD40L+‐activated LPT cells. In selected experiments, blocking antibodies to either CD40 (M2) or CD40L (M90) were used to interfere with the CD40–CD40L interaction. **p<0.001 compared with VEGF release by HIFs cultured in the absence of CD40+ LPT; §p<0.001 compared with VEGF release in the absence of blocking antibodies to either CD40 or CD40L.

Article Snippet: 12 , 27 Trimeric sCD40L and antibodies against CD40 (M2 clone) were provided by Immunex.

Techniques: Cell Culture, Blocking Assay, Standard Deviation

Journal:

Article Title: Critical role of the CD40-CD40-ligand pathway in regulating mucosal inflammation-driven angiogenesis in inflammatory bowel disease

doi: 10.1136/gut.2006.111989

Figure Lengend Snippet: Figure 2 IL‐8 production by CD40L‐stimulated HIFs. Panel A: Human intestinal fibroblasts (HIFs) were activated with soluble CD40 ligand (sCD40L) or were co‐cultured either with CD40+ D1.1 cells or with CD40− Jurkat cells for 24 hours, before measuring interleukin‐8 (IL‐8) release in culture supernatants. In selected experiments, blocking antibodies (Ab) to either CD40 (M2) or CD40L (M90) were used to interfere with the CD40–CD40L interaction. Results are representative of eight independent experiments performed in duplicate and are expressed as mean and standard deviation. *p<0.001 compared with IL‐8 release by HIFs not activated with sCD40L; **p<0.001 compared with IL‐8 release by HIFs cultured in the absence of CD40+ D1.1 cells; §p<0.001 compared with IL‐8 release in the absence of blocking antibodies to either CD40 or CD40L. Panel B: HIFs were co‐cultured either with unstimulated lamina propria T (LPT) cells or with CD40L+ activated LPT cells. In selected experiments, blocking antibodies to either CD40 (M2) or CD40L (M90) were used to interfere with the CD40–CD40L interaction. **p<0.001 compared with IL‐8 release by HIFs cultured in the absence of CD40+ LPT cells; §p<0.001 compared with IL‐8 release in the absence of blocking antibodies to either CD40 or CD40L

Article Snippet: 12 , 27 Trimeric sCD40L and antibodies against CD40 (M2 clone) were provided by Immunex.

Techniques: Cell Culture, Blocking Assay, Standard Deviation

Journal:

Article Title: Critical role of the CD40-CD40-ligand pathway in regulating mucosal inflammation-driven angiogenesis in inflammatory bowel disease

doi: 10.1136/gut.2006.111989

Figure Lengend Snippet: Figure 3 HGF production by CD40L‐stimulated HIFs. Panel A: Human intestinal fibroblasts (HIFs) were activated with soluble CD40 ligand (sCD40L) or were co‐cultured either with CD40+ D1.1 cells or with CD40− Jurkat cells for 24 hours, before measuring hepatocyte growth factor (HGF) release in culture supernatants. In selected experiments, blocking antibodies to either CD40 (M2) or CD40L (M90) were used to interfere with the CD40–CD40L interaction. Results are representative of eight independent experiments performed in duplicate and are expressed as mean and standard deviation. *p<0.001 compared with HGF release by HIFs not activated with sCD40L; **p<0.001 compared with HGF release by HIFs cultured in the absence of CD40+ D1.1 cells; §p<0.001 compared with HGF release in the absence of blocking antibodies to either CD40 or CD40L. Panel B: HIFs were co‐cultured either with unstimulated lamina propria T (LPT) cells or with CD40L+ activated LPT cells. In selected experiments, blocking antibodies to either CD40 (M2) or CD40L (M90) were used to interfere with the CD40–CD40L interaction. **p<0.001 compared with HGF release by HIFs cultured in the absence of CD40+ LPT cells; §p<0.001 compared with HGF release in the absence of blocking antibodies to either CD40 or CD40L.

Article Snippet: 12 , 27 Trimeric sCD40L and antibodies against CD40 (M2 clone) were provided by Immunex.

Techniques: Cell Culture, Blocking Assay, Standard Deviation

Journal:

Article Title: Critical role of the CD40-CD40-ligand pathway in regulating mucosal inflammation-driven angiogenesis in inflammatory bowel disease

doi: 10.1136/gut.2006.111989

Figure Lengend Snippet: Figure 4 Effects of CD40 engagement on HIMEC chemotaxis. Migration of human intestinal microvascular endothelial cells (HIMECs) was measured after activation with either soluble CD40 ligand (sCD40L) or exogenously added vascular endothelial growth factor (VEGF). Additionally, cell migration was assessed after culturing with supernatants from sCD40L‐activated human intestinal fibroblasts (HIFs), either in the absence or in the presence of blocking antibodies to VEGF, interleukin‐8 (IL‐8) or hepatocyte growth factor (HGF). *p<0.001 compared with baseline HIMEC migration. Results are representative of six independent experiments performed in duplicate and are expressed as mean and standard deviation. §p<0.001 compared with cultures performed in the absence of blocking antibodies to VEGF, IL‐8 or HGF.

Article Snippet: 12 , 27 Trimeric sCD40L and antibodies against CD40 (M2 clone) were provided by Immunex.

Techniques: Chemotaxis Assay, Migration, Activation Assay, Blocking Assay, Standard Deviation

Journal:

Article Title: Critical role of the CD40-CD40-ligand pathway in regulating mucosal inflammation-driven angiogenesis in inflammatory bowel disease

doi: 10.1136/gut.2006.111989

Figure Lengend Snippet: Figure 5 Supernatants of CD40L‐activated HIF promote in vitro tube formation. Tube formation assays were performed with Matrigel™, as detailed in Materials and Methods. Inverted‐phase contrast microscopy was used to assess the formation of endothelia cell (EC)‐like tube structures. Either vascular endothelial growth factor (VEGF) or soluble CD40 ligand (sCD40L) were used as positive controls for EC formation. Results are representative of three independent experiments. Five high‐power fields per culture condition were examined. Magnification ×40. HIFs, human intestinal fibroblasts; HIMECs, human intestinal microvascular endothelial cells.

Article Snippet: 12 , 27 Trimeric sCD40L and antibodies against CD40 (M2 clone) were provided by Immunex.

Techniques: In Vitro, Microscopy