Journal: Journal of biomedical science

Article Title: Neutrophil-derived reactive agents induce a transient SpeB negative phenotype in Streptococcus pyogenes.

doi: 10.1186/s12929-023-00947-x

Figure Lengend Snippet: Fig. 1 Reversible loss of SpeB is associated with tissue pathology and inflammation. A Distribution of SpeB+ and SpeB− GAS clones directly isolated from NSTI patient tissue biopsies (n = 23). B Percentage of SpeB+ and SpeB− GAS clones after the passage in THY media (p1, passage 1; p2, passage 2). Representative analysis of 2006 GAS patient isolate is shown. C–G Correlation analysis of bacterial load (left panel; n = 81 biopsies) or percentage of SpeB− clones (right panel; n = 23 biopsies) with the presence of HMGB1 (C), IL-8 (D), infiltrating neutrophils (E), and resistin (F) in patient biopsies. Correlation was determined using Spearman test. Semiquantitative acquired computerized image analyses (ACIA) of immuno-histochemical staining were performed as described in the methods section. The cell area was defined by the hematoxylin counterstaining, and the results are presented as percent positively stained area × mean intensity of positive staining

Article Snippet: The following antibodies were used for immunohistochemistry: anti-human HMGB1 (clone EPR3507; Abcam), anti-human IL-8 (clone NAP-1; Invitrogen), anti-human resistin (clone 184,305; R&D systems), and anti-human neutrophilelastase (clone NP57; DAKO).

Techniques: Clone Assay, Isolation, Staining

Journal: Cardiovascular research

Article Title: Resistin is secreted from macrophages in atheromas and promotes atherosclerosis.

doi: 10.1016/j.cardiores.2005.09.015

Figure Lengend Snippet: Fig. 1. Immunohistochemical staining of aneurysms with antibodies to resistin and CD68. The various types of human vessels were stained with antibodies against resistin or CD68, the latter of which is a marker of monocytes/macrophages. Mayer’s hematoxylin was used for counterstaining. The left upper region of each picture indicates the endothelial lining. Resistin-positive areas are seen in atherosclerotic lesions as dark staining beneath the endothelial lining (C and D). Resistin-positive areas are distributed along inflammatory cells and stained positively for CD68 (G, H). No immunoreactive area was observed for either of the two antibodies in normal vessels (A, B, E, F). Staining without primary Abs was performed as a negative control (I, J, K, L).

Article Snippet: The sectionswere then incubated with primary antibodies, namely, monoclonal antihuman resistin (R&D Systems, Minneapolis, clone 184335), and anti-human CD68 (DakoCytomation, Glostrup, Denmark, clone KP1), the latter of which is a marker for monocytes/macrophages [22].

Techniques: Immunohistochemical staining, Staining, Marker, Negative Control

Journal: Cardiovascular research

Article Title: Resistin is secreted from macrophages in atheromas and promotes atherosclerosis.

doi: 10.1016/j.cardiores.2005.09.015

Figure Lengend Snippet: Fig. 3. Resistin mRNA expression measured by real-time PCR. (A) Resistin mRNA expression in aortic aneurysms. Total RNA was purified from aneurysm centers (n =4) and veins (n =3). Real-time quantitative PCR revealed the overexpression of resistin in aneurysm centers. The y axis represents resistin expression normalized against GAPDH expression. The data illustrated on the graph represent meansTSEM of 2deltadeltaCt of the samples. (B) Resistin mRNA expression in vascular cells. Isolated human VSMCs and commercial HUVECs were incubated with 10 ng/mL of TNF-a for 12 h. Human monocytes were isolated and incubated for 36 h, and total RNA was purified and resistin mRNA levels were measured by real-time RT-PCR, normalized with respect to the GAPDH mRNA levels. The data illustrated on the graph represent meansTSEM of 2deltadeltaCt of three different experiments.

Article Snippet: The sectionswere then incubated with primary antibodies, namely, monoclonal antihuman resistin (R&D Systems, Minneapolis, clone 184335), and anti-human CD68 (DakoCytomation, Glostrup, Denmark, clone KP1), the latter of which is a marker for monocytes/macrophages [22].

Techniques: Expressing, Real-time Polymerase Chain Reaction, Purification, Over Expression, Isolation, Incubation, Quantitative RT-PCR

Journal: Cardiovascular research

Article Title: Resistin is secreted from macrophages in atheromas and promotes atherosclerosis.

doi: 10.1016/j.cardiores.2005.09.015

Figure Lengend Snippet: Fig. 2. Immunofluorescent double staining of aneurysms with antibodies to resistin and vascular cell markers. Green indicates vascular cell proteins, red resistin, and blue DAPI-stained cellular nuclei. The upper region in each picture indicates endothelial lining. Resistin protein was found to co-localize with CD68 (A, B). Smooth muscle (SM) actin was positive throughout the vessel thickness (D), whereas CD31 was not detected (G). The distribution of resistin (E, H) was not concordant with either D or G. A, B and C were magnified (J¨L) and this showed that resistin was present both intracellularly and extracellularly. The merged picture (M) shows yellow rings composed of red resistin-positive areas and green transmembrane CD68-positive areas (arrows).

Article Snippet: The sectionswere then incubated with primary antibodies, namely, monoclonal antihuman resistin (R&D Systems, Minneapolis, clone 184335), and anti-human CD68 (DakoCytomation, Glostrup, Denmark, clone KP1), the latter of which is a marker for monocytes/macrophages [22].

Techniques: Double Staining, Staining

Journal: Cardiovascular research

Article Title: Resistin is secreted from macrophages in atheromas and promotes atherosclerosis.

doi: 10.1016/j.cardiores.2005.09.015

Figure Lengend Snippet: Fig. 4. Induction of PAI-1 in HUVECs after resistin treatment. HUVECs were incubated with 10¨100 ng/mL of resistin for 4¨24 h or with 10 ng/mL of TNF- a for 12 h as a positive control. (A) PAI-1 protein levels in culture media were measured by ELISA and are presented as fold changes versus the negative control. (B) PAI-1 mRNA expression was analyzed by Northern blotting, and normalized against GAPDH mRNA. Results are presented as fold changes versus the negative control. The data illustrated on the graph represent the meansTSEM of three different experiments. *p <0.05 vs. without resistin treatment.

Article Snippet: The sectionswere then incubated with primary antibodies, namely, monoclonal antihuman resistin (R&D Systems, Minneapolis, clone 184335), and anti-human CD68 (DakoCytomation, Glostrup, Denmark, clone KP1), the latter of which is a marker for monocytes/macrophages [22].

Techniques: Incubation, Positive Control, Enzyme-linked Immunosorbent Assay, Negative Control, Expressing, Northern Blot

Journal: Cardiovascular research

Article Title: Resistin is secreted from macrophages in atheromas and promotes atherosclerosis.

doi: 10.1016/j.cardiores.2005.09.015

Figure Lengend Snippet: Fig. 6. Induction of VSMC migration by resistin. VSMCs in serum-free media were incubated with 10¨100 ng/mL resistin for 24 h or with 10% FBS for 24 h as a positive control. Cell migration was detected by scratched wound assays and results are presented as fold changes versus the negative control. The data illustrated on the graph represent meansTSEM of three different experiments. *p <0.05 vs. without resistin treatment.

Article Snippet: The sectionswere then incubated with primary antibodies, namely, monoclonal antihuman resistin (R&D Systems, Minneapolis, clone 184335), and anti-human CD68 (DakoCytomation, Glostrup, Denmark, clone KP1), the latter of which is a marker for monocytes/macrophages [22].

Techniques: Migration, Incubation, Positive Control, Negative Control

Journal: Cardiovascular research

Article Title: Resistin is secreted from macrophages in atheromas and promotes atherosclerosis.

doi: 10.1016/j.cardiores.2005.09.015

Figure Lengend Snippet: Fig. 5. Induction of ET-1 in HUVECs after resistin treatment. HUVECs were incubated with 10¨100 ng/mL of resistin for 4¨24 h or with 10 ng/mL of TNF-a for 12 h as a positive control. ET-1 mRNA levels were analyzed by Northern blotting and normalized against GAPDH mRNA, and are presented as fold changes versus the negative control. The data illustrated on the graph represent the meansTSEM of three different experiments. *p <0.05 vs. without resistin treatment.

Article Snippet: The sectionswere then incubated with primary antibodies, namely, monoclonal antihuman resistin (R&D Systems, Minneapolis, clone 184335), and anti-human CD68 (DakoCytomation, Glostrup, Denmark, clone KP1), the latter of which is a marker for monocytes/macrophages [22].

Techniques: Incubation, Positive Control, Northern Blot, Negative Control

Journal: Journal of cellular and molecular medicine

Article Title: Resistin competes with lipopolysaccharide for binding to toll-like receptor 4.

doi: 10.1111/j.1582-4934.2009.00899.x

Figure Lengend Snippet: Fig. 1 Resistin binding to human leucocytes and epithelial cells. (A) Human leucocytes obtained from peripheral blood were incubated for 30 min with human recombinant resistin (500 ng/ml), washed and cell bound resistin was visualized by anti-resistin antibodies using flow cytometry. Pronounced binding of resistin to lymphocyte and monocyte cell population was observed. (filled area). In contrast, neutrophils display significant expression of resistin on their cell surface prior to incubation with exogenous resistin. (B) THP1 monocytic cells bind exogenous resistin in a dose-dependent man- ner (B1). This resistin binding (filled area, B2 and B3) was abolished by pre-incubation of THP1 cells with TLR4 antibodies (5 g/ml) (white line, B2), but not with isotype-matched mouse IgG (white line, B3). (C) HEK293 epithelial cells transfected with TLR4 display significantly higher binding capac- ity for exogenous resistin (bold, C1) as compared to HEK-null cells transfected with empty vector (bold, C4). Binding of resistin to HEK-TLR4 cells is decreased by pre-treatment of cells with antibodies against TLR4 (C3) and CD14 (C2) prior to exposure to resistin. This is not observed in HEK-null cells treated with the same antibodies (C5, C6).

Article Snippet: Following centrifugation, cells were stained with biotinylated anti-human resistin antibodies (clone 184320, R&D Systems, Minneapolis, MN, USA), TLR4 antibodies (phycoerytrin conjugated, Serotec, Oxford, UK) or appropriate mouse IgG isotype controls (BD Diagnostics, Franklin Lakes, NJ, USA) and subjected to FACS analyses.

Techniques: Binding Assay, Incubation, Recombinant, Flow Cytometry, Expressing, Transfection, Plasmid Preparation

Journal: Journal of cellular and molecular medicine

Article Title: Resistin competes with lipopolysaccharide for binding to toll-like receptor 4.

doi: 10.1111/j.1582-4934.2009.00899.x

Figure Lengend Snippet: Fig. 2 Western blot analysis of the membrane fraction of THP1 cells (1 107) incubated with resistin (500 ng/ml). The equal protein amounts of membrane extracts were precipitated using mouse anti-resistin antibod- ies (7.5 g), anti-TLR4 antibodies (10 g) or non-specific mouse IgG (10 g), followed by incubation with protein A/protein G sepharose beads. Immunoprecipitates obtained by precipitation with either TLR4 ab or resistin ab were subjected to electrophoresis and immunoblotting using TLR4 ab (A). Immunoprecipitates obtained by precipitation with either TLR4 ab, resistin ab or non-specific control ab were subjected to electrophoresis and immunoblotting using resistin antibodies (B). Co- precipitation of TLR4 in the resistin bound membrane fraction is clearly visible. Sensitivity of the system for resistin was tested using various concentrations of human recombinant resistin (C).

Article Snippet: Following centrifugation, cells were stained with biotinylated anti-human resistin antibodies (clone 184320, R&D Systems, Minneapolis, MN, USA), TLR4 antibodies (phycoerytrin conjugated, Serotec, Oxford, UK) or appropriate mouse IgG isotype controls (BD Diagnostics, Franklin Lakes, NJ, USA) and subjected to FACS analyses.

Techniques: Western Blot, Membrane, Incubation, Electrophoresis, Control, Recombinant

Journal: Journal of cellular and molecular medicine

Article Title: Resistin competes with lipopolysaccharide for binding to toll-like receptor 4.

doi: 10.1111/j.1582-4934.2009.00899.x

Figure Lengend Snippet: Fig. 3 Resistin-induced production of cytokines in human PBMC is dependent on TLR4 expression. Secretion of (A) IL-6 and (B) IL-8 in human PBMC (1 106/ml, n 9) following resistin stimu- lation was significantly decreased in the PBMC cultures pre-treated with TLR4 antibodies (*P 0.001). This decrease of cytokine expression was not observed in the PBMC cultures pre- treated with TLR2 antibodies or isotype matched mouse IgG. (C) HEK293 cells transfected with TLR4 display signifi- cantly higher secretion of IL-8 following stimulation with resistin (**P 0.01) as compared to HEK293 cells trans- fected with TLR2 or those transfected with an empty vector, HEK-null cells. Ligands specific for TLR2 (PamCys, 2 ng/ml) and TLR4 (LPS, 100 ng/ml) were used as positive controls.

Article Snippet: Following centrifugation, cells were stained with biotinylated anti-human resistin antibodies (clone 184320, R&D Systems, Minneapolis, MN, USA), TLR4 antibodies (phycoerytrin conjugated, Serotec, Oxford, UK) or appropriate mouse IgG isotype controls (BD Diagnostics, Franklin Lakes, NJ, USA) and subjected to FACS analyses.

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: Journal of cellular and molecular medicine

Article Title: Resistin competes with lipopolysaccharide for binding to toll-like receptor 4.

doi: 10.1111/j.1582-4934.2009.00899.x

Figure Lengend Snippet: Fig. 5 TLR4 is required for the induction of IL-8 expression in HEK293 cells in response to stimulation with resistin. (A) Down-regulation of TLR4 and MyD88 synthesis was achieved by transfecting HEK-TLR4 cells with siRNA sequences specific for these genes. This resulted with a decrease of IL-8 production in response to resistin as well as LPS stimulation in the siRNA transfected cell cultures (*P 0.05). (B) Down- regulation of MyD88 synthesis in HEK-TLR4 cells transfected with MyD88-specific siRNA and not non-targeting siRNA sequence is shown following 48 and 72 hrs incubation time. (C) Resistin-induced secretion of IL-8 in HEK-TLR4 cells is significantly reduced following incubation with anti-CD14 and anti-resistin antibodies. In contrast, transfection of HEK293 cells with MD2/CD14 in the absence of TLR4 did not lead to resistin-induced activation of HEK293 cells.

Article Snippet: Following centrifugation, cells were stained with biotinylated anti-human resistin antibodies (clone 184320, R&D Systems, Minneapolis, MN, USA), TLR4 antibodies (phycoerytrin conjugated, Serotec, Oxford, UK) or appropriate mouse IgG isotype controls (BD Diagnostics, Franklin Lakes, NJ, USA) and subjected to FACS analyses.

Techniques: Expressing, Transfection, Sequencing, Incubation, Activation Assay

Journal: Journal of cellular and molecular medicine

Article Title: Resistin competes with lipopolysaccharide for binding to toll-like receptor 4.

doi: 10.1111/j.1582-4934.2009.00899.x

Figure Lengend Snippet: Fig. 4 Resistin competes with LPS for binding to TLR4. (A) Treatment of human PBMC (n 3) with resistin (250 and 1000 ng/ml) significantly diminished secretion of IL-6 in response to LPS. (B) Analogous reduc- tion of LPS-induced production of IL-8 was observed in HEK-TLR4 cells cultured in the presence of resistin.

Article Snippet: Following centrifugation, cells were stained with biotinylated anti-human resistin antibodies (clone 184320, R&D Systems, Minneapolis, MN, USA), TLR4 antibodies (phycoerytrin conjugated, Serotec, Oxford, UK) or appropriate mouse IgG isotype controls (BD Diagnostics, Franklin Lakes, NJ, USA) and subjected to FACS analyses.

Techniques: Binding Assay, Cell Culture

Journal: Journal of cellular and molecular medicine

Article Title: Resistin competes with lipopolysaccharide for binding to toll-like receptor 4.

doi: 10.1111/j.1582-4934.2009.00899.x

Figure Lengend Snippet: Fig. 6 Intracellular regulation of resistin-mediated effects. Human PBMC (1 10 6/ml) were treated with specific inhibitors of signalling pathways including NF-B (Parthenolide, 25 M), p38 MAP-kinase (SB203580, 50 M), p44/p42 MAP-kinase (PD98059, 50 M) and PI3-kinase (LY294002, 50 M) prior to stimualtion with resistin (500 ng/ml). Cell cultures were evaluated for the expression of IL-6, TNF-, and IL-1 on mRNA level (A–C) and on protein level (D–F). We observed that inhibitors of NF-B and MAP-kinases significantly diminished resistin-induced expression of cytokines, while the inhibitor of PI3-kinase increased resistin stimulation. Differences in cytokine expression as compared to cultures stimulated with resistin are indicated by asterisk (*P 0.05, and **P 0.01). Abbreviations: R, resistin; Par, parthrnolide; Ly, LY294002; SB, SB203580; PD, PD98059.

Article Snippet: Following centrifugation, cells were stained with biotinylated anti-human resistin antibodies (clone 184320, R&D Systems, Minneapolis, MN, USA), TLR4 antibodies (phycoerytrin conjugated, Serotec, Oxford, UK) or appropriate mouse IgG isotype controls (BD Diagnostics, Franklin Lakes, NJ, USA) and subjected to FACS analyses.

Techniques: Expressing

Journal: Journal of cellular and molecular medicine

Article Title: Resistin competes with lipopolysaccharide for binding to toll-like receptor 4.

doi: 10.1111/j.1582-4934.2009.00899.x

Figure Lengend Snippet: Fig. 7 Structural requirements of resistin-TLR4 interaction. HEK293 cells transfected with TLR4, TLR2 or empty vector were stimulated with equimolar concentrations of resistin and resistin peptides, assigned as aa23–42, aa43–64, aa51–108, aa64–88 (50 nM) (A). Localization of the peptides in the whole resistin sequence is schematically shown above the graphs. Differences of IL-8 production in comparison to HEK- null cells are indicated by asterisk (*P 0.05 and **P 0.01). Inhibition of IL-8 production by antibodies against resistin and CD14 are evaluated in HEK-TLR4 cells (B). Differences of IL-8 production in HEK-TLR4 cells in the presence of antibodies are indicated by asterisk (*P 0.05 and **P 0.01).

Article Snippet: Following centrifugation, cells were stained with biotinylated anti-human resistin antibodies (clone 184320, R&D Systems, Minneapolis, MN, USA), TLR4 antibodies (phycoerytrin conjugated, Serotec, Oxford, UK) or appropriate mouse IgG isotype controls (BD Diagnostics, Franklin Lakes, NJ, USA) and subjected to FACS analyses.

Techniques: Transfection, Plasmid Preparation, Sequencing, Comparison, Inhibition

Journal: Journal of cellular and molecular medicine

Article Title: Resistin competes with lipopolysaccharide for binding to toll-like receptor 4.

doi: 10.1111/j.1582-4934.2009.00899.x

Figure Lengend Snippet: Fig. 8 Overall structure of TLR4- resistin complex. Side view of the complex (A) on the cell surface TLR4 is associated with MD-2 and adopts the horseshoe-like shape forming an interface for LPS bind- ing. CD14 delivers LPS molecules to the TLR4-MD-2 complex. Dimerization of TLR4-MD-2 com- plex is followed by recruitment of adaptor proteins MyD88 and IRAK initiating intracellular signalling. We show that interaction between resistin and TLR4 reduces LPS effects, potentially by interfering with its binding to TLR4-MD-2 complex. Top view (B) shows potential binding sites of resistin. R1, resistin occupies LPS binding site in the convex of TLR4 associ- ated to MD-2; R2, resistin binds to the upper central part of the TLR4 convex facilitating dimerization and recruitment of adaptor proteins; R3, resistin stretches through the cen- tral part of TLR4 forming several interactions by identical C-terminal loops of its hexamer.

Article Snippet: Following centrifugation, cells were stained with biotinylated anti-human resistin antibodies (clone 184320, R&D Systems, Minneapolis, MN, USA), TLR4 antibodies (phycoerytrin conjugated, Serotec, Oxford, UK) or appropriate mouse IgG isotype controls (BD Diagnostics, Franklin Lakes, NJ, USA) and subjected to FACS analyses.

Techniques: Binding Assay