Journal: European journal of immunology

Article Title: ? 2 integrins inhibit TLR responses by regulating NF-?B pathway and p38 MAPK activation

doi: 10.1002/eji.201242550

Figure Lengend Snippet: (A) Bone marrow-derived macrophages were rested overnight and stimulated with TLR agonists for 24 hours. The concentration of IL-12 p40, TNF and IL-6 in the supernatants was measured by ELISA. The results are displayed as relative cytokine levels, with the values found for WT macrophages at the highest concentration set to 1. Data are mean +/- SEM of 5 independent experiments. (B) Bone marrow-derived macrophages were stimulated with 1 ng/ml LPS for the indicated times and concentrations of cytokines in the supernatants were measured by ELISA. Relative values were derived by setting the WT time 0 value to 1. Data are mean +/- SEM of 3 independent experiments. (C and D) Macrophages were stimulated with 1 ng/ml LPS for indicated times and Brefeldin A was added for the last 2 hours of stimulation. TNF and IL-12 p40 production was measured by intracellular cytokine staining and flow cytometry. (C) Representative data of IL-12 p40 and TNF staining at 8 hours of LPS stimulation. Gating shows cytokine positive cells. (D) The kinetics of IL-12 p40- and TNF-positive macrophages after LPS stimulation as assessed by the gating strategy in (C) The data in (D) are shown as mean +/- SEM of 3 independent experiments. (E) Relative IFNP gene expression in macrophages stimulated with 1 ng/ml LPS was analyzed by qPCR, with results normalized to GAPDH mRNA levels and the WT 8 hour time point set to 1. The data shown are mean +/- SEM of 4 independent experiments. *p< 0.05, **p<0.01 and ***p<0.001.

Article Snippet: Total RNA from LPS or TNF (Peprotech) stimulated macrophages was isolated using the RNeasy Plus kit (QIAGEN) and reverse transcribed with Superscript III reverse transcriptase (Invitrogen).

Techniques: Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Gene Expression

Journal: European journal of immunology

Article Title: ? 2 integrins inhibit TLR responses by regulating NF-?B pathway and p38 MAPK activation

doi: 10.1002/eji.201242550

Figure Lengend Snippet: (A-B) D5 thioglycollate-elicited cells were enriched for F4/80+ macrophages, rested overnight before treatment with 1 ng/ml LPS with Brefeldin A for the last 2 hours of stimulation, and cytokine levels were determined by flow cytometry. (A) Representative histograms of IL-12 p40, IL-6 and TNF staining of F4/80+ cells at 4 hours after LPS treatment. (B) The kinetics of cytokine-producing inflammatory macrophages based on the gating strategy found in (A). The data in (B) are displayed as mean +/- SEM of 4 independent experiments. (C) WT (n=11) and Itgb2-/- (n=11) mice were injected i.p. with 1 mg/kg LPS. Blood was collected at the indicated time points and serum cytokine levels were measured by ELISA. These data are displayed as mean +/- SEM. * p<0.05, ** P<0.01, and ***p<0.001.

Article Snippet: Total RNA from LPS or TNF (Peprotech) stimulated macrophages was isolated using the RNeasy Plus kit (QIAGEN) and reverse transcribed with Superscript III reverse transcriptase (Invitrogen).

Techniques: Flow Cytometry, Staining, Injection, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: ? 2 integrins inhibit TLR responses by regulating NF-?B pathway and p38 MAPK activation

doi: 10.1002/eji.201242550

Figure Lengend Snippet: (A) Bone marrow-derived macrophages were stimulated with either LPS or CpG DNA for 24 hours and IL-10 secretion was measured by ELISA. Data are shown as mean +/- SEM of 3 independent experiments. (B) WT (n=3) and Itgb2-/- (n=3) mice were injected i.p with 1 mg/kg LPS. Blood was collected at indicated time points and IL-10 concentration was determined by ELISA. Data are shown as mean +/- SEM from one representative experiment. (C) Macrophages were treated with 10 ng/mL IL-10 for 30 minutes prior to stimulation with 1 ng/ml LPS. Cytokine production was assessed by ELISA. Data are shown as mean + SD of triplicate wells from one experiment, and are representative of 3 independent experiments. (D) The percent decrease in cytokine production was determined by normalizing to IL-10 production generated by TLR stimulation without exogenous IL-10 treatment. Results are displayed as mean +/- SEM of 3 independent experiments. (E) Relative inhibitor expression was determined by qPCR after stimulation of macrophages with 1 ng/ml LPS for the indicated time points. Expression levels for each inhibitor were normalized to GAPDH with the WT 4 hour time point set to 1, and the results are displayed as mean +/- SEM of 3 or 4 independent experiments. (F) and (G) Western blot for A20 (F) and phospho-p38 and phospho-ERK (G) expression in 1 ng/mL LPS-stimulated macrophages, with β actin as a loading control. The data are representative of 2 (F) and 3 (G) independent experiments. *p< 0.05.

Article Snippet: Total RNA from LPS or TNF (Peprotech) stimulated macrophages was isolated using the RNeasy Plus kit (QIAGEN) and reverse transcribed with Superscript III reverse transcriptase (Invitrogen).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Injection, Concentration Assay, Generated, Expressing, Western Blot, Control

Journal: European journal of immunology

Article Title: ? 2 integrins inhibit TLR responses by regulating NF-?B pathway and p38 MAPK activation

doi: 10.1002/eji.201242550

Figure Lengend Snippet: (A) Bone marrow-derived macrophages from WT, Itgal-/- (CD11a-deficient) and Itgam-/- (CD11b-deficient), and Itgb2-/- mice were treated with 1 ng/ml LPS, 100 nM CpG DNA or 100 μg/ml zymosan particles for 24 hours. IL-12 p40 concentrations in the supernatants were determined by ELISA and values are set relative to WT results for each stimulation condition. Results are shown as mean +/- SEM of 4 independent experiments. (B) F4/80+ thioglycollate-elicited peritoneal macrophages were treated with 1 ng/ml LPS and IL-12 p40 production was determined by flow cytometry as in Fig. 2. Data is shown as mean +/- SEM of cytokine-positive cells from 3 independent experiments. (C) Cytokine production from bone marrow-derived macrophages was measured by ELISA as in (A). Results are displayed as mean +/- SEM of 3 independent experiments. (D) The kinetics of cytokine-producing thioglycollate-elicited peritoneal macrophages was determined by flow cytometry as in (B). Data in (D) are shown as mean +/- SEM of 3 independent experiments.

Article Snippet: Total RNA from LPS or TNF (Peprotech) stimulated macrophages was isolated using the RNeasy Plus kit (QIAGEN) and reverse transcribed with Superscript III reverse transcriptase (Invitrogen).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: European journal of immunology

Article Title: ? 2 integrins inhibit TLR responses by regulating NF-?B pathway and p38 MAPK activation

doi: 10.1002/eji.201242550

Figure Lengend Snippet: (A) and (C) Bone marrow-derived macrophages were stimulated with 1 ng/mL LPS for the indicated times and cytoplasmic lysates were assayed for IκBα, with β actin used as a loading control. (B) and (D) Densitometry analysis for IκBα levels following TLR4 stimulation. Data are shown as mean +/- SEM of relative ratios of intensity (IκBα/β actin) for 3 independent experiments, with WT time 0 set to 1. (E) and (F) Relative IκBα mRNA expression following LPS stimulation, with data normalized to GAPDH expression and WT time 30 minutes (E) and WT at time 4 hours (F) set to 1. Results are shown as mean +/- SEM of 2 (E) and 4 (F) independent experiments.

Article Snippet: Total RNA from LPS or TNF (Peprotech) stimulated macrophages was isolated using the RNeasy Plus kit (QIAGEN) and reverse transcribed with Superscript III reverse transcriptase (Invitrogen).

Techniques: Derivative Assay, Control, Expressing

Journal: European journal of immunology

Article Title: ? 2 integrins inhibit TLR responses by regulating NF-?B pathway and p38 MAPK activation

doi: 10.1002/eji.201242550

Figure Lengend Snippet: (A) and (B) Macrophages were stimulated with 1.0 ng/ml LPS. Expression levels of the indicated genes were determined by qPCR, with results normalized to GAPDH expression, with WT at time 4 hours set to 1. The data are shown as mean +/- SEM of 3 (A) or 4 (B) independent experiments. (C) ChIP analysis of LPS-stimulated macrophages for p65/RelA recruitment to the Il12b locus, which encodes for IL-12 p40. Results were normalized to input chromatin levels and set relative to WT at time 0. Data are mean +/- SD of 2 independent experiments. *p<0.05.

Article Snippet: Total RNA from LPS or TNF (Peprotech) stimulated macrophages was isolated using the RNeasy Plus kit (QIAGEN) and reverse transcribed with Superscript III reverse transcriptase (Invitrogen).

Techniques: Expressing

Journal: Frontiers in Molecular Biosciences

Article Title: Glycosaminoglycans and fucoidan have a protective effect on experimental glomerulonephritis

doi: 10.3389/fmolb.2023.1223972

Figure Lengend Snippet: Albuminuria and plasma cytokine levels in mice with LPS-induced glomerulonephritis are in part normalized by treatment with unstimulated HS glx , LPS HS glx , fucoidan or sulodexide in mice. (A) . Urinary albumin/creatinine ratio and (B) . Renal function, as measured by BUN of mice injected with LPS (48 h) and treated with unstimulated HS glx , LPS HS glx , fucoidan or sulodexide. (C) . MCP-1, (D) . IL-6, and (E) . IL-10 plasma protein levels of mice injected with LPS (48 h) and treated with unstimulated HS glx , LPS HS glx , fucoidan or sulodexide. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001. n ≥ 8. Unstimulated HS glx , HS extracted from unstimulated endothelial glycocalyx; LPS HS glx , HS extracted from LPS stimulated endothelial glycocalyx; ACR, urinary albumin/creatinine ratio; BUN, blood urea nitrogen plasma level; MCP-1, monocyte chemoattractant protein-1.

Article Snippet: The mice were treated with 11 μg of HS extracted from unstimulated endothelial glycocalyx (Unstimulated HS glx ), HS extracted from LPS stimulated endothelial glycocalyx (LPS HS glx ), fucoidan extracted from Laminaria japonica (kind gift of Dr. Bob Long, MicroVascular Health Solutions LLC, Alpine, UT) or sulodexide (kind gift of Dr. Enrique Poradosu, Keryx Biopharmaceuticals, Boston, United States) in PBS via i. v. Injection.

Techniques: Clinical Proteomics, Injection

Journal: Frontiers in Molecular Biosciences

Article Title: Glycosaminoglycans and fucoidan have a protective effect on experimental glomerulonephritis

doi: 10.3389/fmolb.2023.1223972

Figure Lengend Snippet: Cortical mRNA expression levels of IL-6, ICAM-1 and Nephrin in mice with LPS-induced glomerulonephritis are affected by treatment with unstimulated HS glx , LPS HS glx , fucoidan or sulodexide. (A) . MCP-1, (B) . IL-6, (C) . IL-10, (D) . ICAM-1, (E) . VCAM-1, (F) . Nephrin, and (G) . HPSE1 cortical mRNA expression of mice injected with LPS (48 h) and treated with unstimulated HS glx , LPS HS glx , fucoidan or sulodexide. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001. n ≥ 4. Unstimulated HS glx , HS extracted from unstimulated endothelial glycocalyx; LPS HS glx , HS extracted from LPS stimulated endothelial glycocalyx; MCP-1, monocyte chemoattractant protein-1; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule-1; HPSE1, heparanase.

Article Snippet: The mice were treated with 11 μg of HS extracted from unstimulated endothelial glycocalyx (Unstimulated HS glx ), HS extracted from LPS stimulated endothelial glycocalyx (LPS HS glx ), fucoidan extracted from Laminaria japonica (kind gift of Dr. Bob Long, MicroVascular Health Solutions LLC, Alpine, UT) or sulodexide (kind gift of Dr. Enrique Poradosu, Keryx Biopharmaceuticals, Boston, United States) in PBS via i. v. Injection.

Techniques: Expressing, Injection

Journal: Frontiers in Molecular Biosciences

Article Title: Glycosaminoglycans and fucoidan have a protective effect on experimental glomerulonephritis

doi: 10.3389/fmolb.2023.1223972

Figure Lengend Snippet: Endothelial ICAM-1 expression is increased in LPS-induced glomerulonephritis and is not affected by treatment with unstimulated HS glx , LPS HS glx , fucoidan or sulodexide. (A) . Expression of ICAM-1 in control kidneys and kidneys of mice with LPS-induced glomerulonephritis combined with agrin staining to visualize the glomerular basement membrane (B) . Semiquantitative analysis of the glomerular expression of ICAM-1 in control mice and mice with LPS induced glomerulonephritis, untreated and treated with unstimulated HS glx , LPS HS glx , fucoidan or sulodexide. (C) . Expression of VCAM-1 and the mesangial cell marker PDGFR shows that VCAM-1 is upregulated in mesangial cells in LPS-induced glomerulonephritis. IF staining is presented on subsequent kidney sections, in which the same glomerulus was imaged because the antibodies for VCAM-1 and PDGFR could not be combined on a single kidney section. Data are expressed as mean ± SEM. **** p < 0.0001. n ≥ 4. Unstimulated HS glx , HS extracted from unstimulated endothelial glycocalyx; LPS HS glx , HS extracted from LPS stimulated endothelial glycocalyx; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule-1; PDGFR, platelet-derived growth factor receptor; AU, arbitrary units.

Article Snippet: The mice were treated with 11 μg of HS extracted from unstimulated endothelial glycocalyx (Unstimulated HS glx ), HS extracted from LPS stimulated endothelial glycocalyx (LPS HS glx ), fucoidan extracted from Laminaria japonica (kind gift of Dr. Bob Long, MicroVascular Health Solutions LLC, Alpine, UT) or sulodexide (kind gift of Dr. Enrique Poradosu, Keryx Biopharmaceuticals, Boston, United States) in PBS via i. v. Injection.

Techniques: Expressing, Control, Staining, Membrane, Marker, Derivative Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Glycosaminoglycans and fucoidan have a protective effect on experimental glomerulonephritis

doi: 10.3389/fmolb.2023.1223972

Figure Lengend Snippet: Treatment with unstimulated HS glx , fucoidan or sulodexide resulted in a decreased renal influx of immune cells in LPS-induced glomerulonephritis. Quantitative analysis of the influx of immune cells, specifically granulocytes and monocytes using flow cytometry and immunofluorescence. (A) . CD45 + leukocyte cell percentage of all gated cells using flow cytometry analysis. (B) . Neutrophil as percentage of all gated cells using flow cytometry analysis. (C) . Glomerular granulocyte influx as stained for with the marker Gr-1 in immunofluorescence. (D) . Quantitative analysis of the glomerular influx of granulocytes in mice injected with LPS and treated with unstimulated HS glx , LPS HS glx , fucoidan or sulodexide. (E) . Monocyte Ly6C high cell percentage of all gated cells using flow cytometry analysis. (F) . Glomerular macrophage influx as stained for with the marker CD68 in immunofluorescence. (G) . Quantitative analysis of the glomerular influx of macrophages (CD68) in mice with LPS-induced glomerulonephritis, untreated and treated with unstimulated HS glx , LPS HS glx , fucoidan or sulodexide. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01. n ≥ 4. Unstimulated HS glx , HS extracted from unstimulated endothelial glycocalyx; LPS HS glx , HS extracted from LPS stimulated endothelial glycocalyx; Gr-1, granulocyte-differentiation antigen; Ly6C, lymphocyte antigen 6C.

Article Snippet: The mice were treated with 11 μg of HS extracted from unstimulated endothelial glycocalyx (Unstimulated HS glx ), HS extracted from LPS stimulated endothelial glycocalyx (LPS HS glx ), fucoidan extracted from Laminaria japonica (kind gift of Dr. Bob Long, MicroVascular Health Solutions LLC, Alpine, UT) or sulodexide (kind gift of Dr. Enrique Poradosu, Keryx Biopharmaceuticals, Boston, United States) in PBS via i. v. Injection.

Techniques: Flow Cytometry, Immunofluorescence, Staining, Marker, Injection

Journal: Frontiers in Molecular Biosciences

Article Title: Glycosaminoglycans and fucoidan have a protective effect on experimental glomerulonephritis

doi: 10.3389/fmolb.2023.1223972

Figure Lengend Snippet: HPSE activity is dose-dependently inhibited by unstimulated HS glx , LPS HS glx , fucoidan or sulodexide. Dose response inhibition of recombinant human HPSE with (A) unstimulated HS glx (B) . LPS HS glx , (C) . fucoidan and (D) . sulodexide. Data are expressed as mean ± SEM. Unstimulated HS glx , HS extracted from unstimulated endothelial glycocalyx; LPS HS glx , HS extracted from LPS stimulated endothelial glycocalyx; HPSE, heparanase-1.

Article Snippet: The mice were treated with 11 μg of HS extracted from unstimulated endothelial glycocalyx (Unstimulated HS glx ), HS extracted from LPS stimulated endothelial glycocalyx (LPS HS glx ), fucoidan extracted from Laminaria japonica (kind gift of Dr. Bob Long, MicroVascular Health Solutions LLC, Alpine, UT) or sulodexide (kind gift of Dr. Enrique Poradosu, Keryx Biopharmaceuticals, Boston, United States) in PBS via i. v. Injection.

Techniques: Activity Assay, Inhibition, Recombinant

Journal: Frontiers in Molecular Biosciences

Article Title: Glycosaminoglycans and fucoidan have a protective effect on experimental glomerulonephritis

doi: 10.3389/fmolb.2023.1223972

Figure Lengend Snippet: LPS-induced ICAM-1 mRNA expression in cultured mouse glomerular endothelial cells is normalized by pre-treatment with fucoidan. qPCR analysis of (A) . ICAM-1, (B) . IL-6, (C) . MCP-1 mRNA expression and protein secretion levels of (D) . MCP-1 measured in culture supernatant by ELISA of mouse glomerular endothelial cells pre-treated with 10 μg/mL unstimulated HS glx , LPS HS glx , fucoidan or sulodexide for 1 h and subsequently stimulated with 100 ng/mL LPS for 24 h. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. n ≥ 3. Unstimulated HS glx , HS extracted from unstimulated endothelial glycocalyx; LPS HS glx , HS extracted from LPS stimulated endothelial glycocalyx; ICAM-1, intercellular adhesion molecule 1; MCP-1, monocyte chemoattractant protein-1.

Article Snippet: The mice were treated with 11 μg of HS extracted from unstimulated endothelial glycocalyx (Unstimulated HS glx ), HS extracted from LPS stimulated endothelial glycocalyx (LPS HS glx ), fucoidan extracted from Laminaria japonica (kind gift of Dr. Bob Long, MicroVascular Health Solutions LLC, Alpine, UT) or sulodexide (kind gift of Dr. Enrique Poradosu, Keryx Biopharmaceuticals, Boston, United States) in PBS via i. v. Injection.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Glycosaminoglycans and fucoidan have a protective effect on experimental glomerulonephritis

doi: 10.3389/fmolb.2023.1223972

Figure Lengend Snippet: LPS-induced IL-6, IL-10 and TNF protein secretion is not affected by pre-treatment with unstimulated HS glx , LPS HS glx , fucoidan or sulodexide in human peripheral blood mononuclear cells. Protein secretion levels of (A) . IL-6, (B) . IL-10 and (C) . TNF measured in culture supernatant of human peripheral blood mononuclear cells pre-treated with 10 μg/mL unstimulated HS glx , LPS HS glx , fucoidan or sulodexide for 1 h and subsequently stimulated with 10 ng/mL LPS for 24 h. Data are expressed as mean ± SEM. * p < 0.05, **** p < 0.0001. n ≥ 3. Unstimulated HS glx , HS extracted from unstimulated endothelial glycocalyx; LPS HS glx , HS extracted from LPS stimulated endothelial glycocalyx.

Article Snippet: The mice were treated with 11 μg of HS extracted from unstimulated endothelial glycocalyx (Unstimulated HS glx ), HS extracted from LPS stimulated endothelial glycocalyx (LPS HS glx ), fucoidan extracted from Laminaria japonica (kind gift of Dr. Bob Long, MicroVascular Health Solutions LLC, Alpine, UT) or sulodexide (kind gift of Dr. Enrique Poradosu, Keryx Biopharmaceuticals, Boston, United States) in PBS via i. v. Injection.

Techniques: