lipopolysaccharaide Search Results


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  • 99
    Thermo Fisher lps
    5-ASA and AZTP restore epithelial barrier. ( A ) Paracellular permeability assay using FITC-Dextran: Differentiated and polarized T-84 monolayer grown in Transwell was treated with TNF-α (10 ng/ml), <t>IFN-γ</t> (100U/ml) or <t>LPS</t> (10 ng/ml) with or without pretreatment (5 h) with 5-ASA (5 mM) or AZTP (10 μM) n = 5 each treatment. Experiments were performed in biological duplicates and repeated three times. p value (≤0.05), * con vs treatment; # vs TNF-α or IFN-γ. One-way ANOVA with multiple comparisons were done using Bonferroni’s post hoc analysis. ( B , C ) PCR array analysis of T-84 cells treated with 5-ASA (5 mM) and AZTP(10 μM), in the presence or absence of TNF-α (10 ng/ml) ( D ) Immunofluorescence analysis of T-84 monolayer for the representative proteins of adherens junctions; AJs (E-cadherin), tight junctions; TJs (occludin), Desmosomes (Desmoglein-2). TNF-α treatment disrupted all junctions and resulted in protein internalization as indicated by arrows. 5-ASA and AZTP restored membranous expression of these proteins. Image magnification 400x. Scale 50μm. E-cadherin and Desmoglein were visualized using Alexa Fluor 488 secondary antibody. Occludin was visualized using Alexa Fluor 568 ( E ) qRT-PCR analysis of intestinal alkaline phosphatase as a marker of cellular differentiation in T-84 cells. The experiment was performed twice.
    Lps, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2391 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore lps
    Effects of prenatal <t>LPS</t> (E15/16) and postnatal MP-III-022 (P21–28) treatments on distance traveled by female Wistar offspring at P60. Activity was recorded during 30 min of habituation and baseline activity (HAB trial), then 30 min after saline challenge (SAL trial) and finally 60 min after amphetamine (0.5 mg/kg, i.p.) administration (AMPH trial). Panel A: the main effects of between-subject factors (LPS and MP-III-022) in each trial, as analyzed by three-way RM ANOVA. Graphs B, C and D: pairwise multiple comparison (Student–Newman–Keuls method) of LPS and MP-III-022 effects on total distance traveled in each of three trials (HAB, SAL and AMPH, respectively). Graphs E and F: effects of MP-III-022 on distance per time bin analyzed by pairwise multiple comparison procedures (Student–Newman–Keuls method) within SAL or LPS cohorts, respectively. Data (mean value ± SEM) are shown as total distance per trial (B, C and D) or distance traveled in 10 min bins (E and F). *(p
    Lps, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 55449 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore lipopolysaccharide lps
    PS2 protein is increased in microglia activated by <t>IFNγ.</t> A) Western blot analysis demonstrating the PS2 C-terminus migrating at ∼20 kDa. Twenty-four hour stimulation with 10 u/ml IFNγ induces increased levels of PS2 expression in lysates prepared from primary microglia, while 100 ng/ml <t>LPS</t> does not induce PS2 expression. B) Quantitative densitometry on Western blots of lysates from 4 independent cultures shows significantly increased induction of PS2 by IFNγ. Data represent the mean ± SEM of 4 independent experiments (*p
    Lipopolysaccharide Lps, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore lipopolysaccharides lps
    Effusanin E affects activity of the COX-2 pathway. ( A ) NPC cells were pretreated with lipopolysaccharides <t>(LPS)</t> (2 µg/ml) for 8 hours then treated with effusanin E at 125 µM and 250 µM (EFE 125 µM or EFE 250 µM). At 24 hours after treatment, COX-2 protein expression was determined by western blotting. ( B ) NPC cells were transfected with a luciferase expression vector containing a COX-2 5-flanking fragment for 8 hours and treated with effusanin E (EFE) at the indicated doses. At 24 hours after treatment, COX-2 promoter activities were determined. ( C ) The effect of effusanin E was inhibited by an inhibitor of COX-2 in NPC cells. NPC cells were treated with <t>celecoxib</t> (CB) (20 µM or 50 µM) for 8 hours followed by effusanin E at 125 µM and 250 µM (EFE 125 µM or EFE 250 µM) for up to 24 hours or 48 hours. At the indicated time points, the cells were analyzed using the MTS assay. The data are presented as the mean ± S.D. of three separate experiments. * represent P
    Lipopolysaccharides Lps, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore e coli lps
    Serum levels of inflammation-related molecules in treated endotoxemic mice. (A) <t>Endotoxemia</t> was induced by the intraperitoneal injection of 10 mg/kg E. coli <t>LPS.</t> A control group was injected i.v. with PBS (n = 10, light gray bars), a second group
    E Coli Lps, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1622 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    InvivoGen lipopolysaccharide
    Costimulation and/or restimulation of TLR7 and TLR9 decreased the levels of proinflammatory cytokine production in mouse macrophages. (A) Mouse macrophages were stimulated with either an agonist for TLR7 (GDQ) or TLR9 (ODN 1826) or both (GDQ + ODN1826) overnight (1st), and the production of TNF-α in the supernatant was measured using ELISA. <t>LPS</t> stimulation was introduced as a positive control. □ Raw264.7 macrophage cell line, and ▪ BMDMs from Balb/c mice. (B) After the first stimulation, the cells were washed with PBS and incubated with culture media overnight. Subsequently, the cells were restimulated with TLR agonists for 1 h (2nd), and the production of TNF-α was measured using ELISA. The levels of IL-6 (C) and IL-10 (D) were measured in RAW264.7 cells under the same conditions. Significant differences were indicated as *P
    Lipopolysaccharide, supplied by InvivoGen, used in various techniques. Bioz Stars score: 92/100, based on 680 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Difco lipopolysaccharide
    Expression of various molecules on <t>DCs</t> pulsed with HTLV-1. Immature DCs were differentiated from normal healthy donor monocytes, pulsed with 20 CCID 50 of live HTLV-1 or equivalent dose of heat-inactivated virions, and matured by <t>LPS</t> (10 ng/ml). –––, control MAb; ———, indicated MAb. The number in each panel represents the mean fluorescence intensity.
    Lipopolysaccharide, supplied by Difco, used in various techniques. Bioz Stars score: 93/100, based on 226 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pro q emerald 300 lipopolysaccharide gel stain kit
    LPS profiles of Pseudomonas syringae pv. actinidiae ( Psa ) and P. syringae pv. actinidifoliorum ( Pfm ). (A) <t>Pro-Q™</t> Emerald 300 staining of lipopolysaccharide (LPS) preparations on a NuPAGE 4–12% gradient bis-tricine SDS gel from representatives of Pfm lineages (L3 ICMP 18807, lane 2; L1 ICMP 18803, lane 8), Psa biovars (BV1 ICMP 9617, lane 3; BV2 ICMP 19071, lane 4; BV3 ICMP 18884, lane 5; BV5 MAFF212063, lane 6; BV6 MAFF 212141, lane 7), with Escherichia coli standards (Std; lanes 1 and 9). A total of 0.5–15 μL LPS extract for each sample (normalized by trial runs) was applied per lane. (B) Western blot of LPS extracts from representatives of Psa biovars and Pfm lineages run on a NuPAGE 4– 12% gradient bis-tricine SDS gel, obtained by probing with polyclonal antibodies to Psa BV3 ICMP 18884 (Bi) , Psa BV1 ICMP 9617 (Bii) , or Pfm L1 ICMP 18803 (Biii) in a 5000:1 ratio.
    Pro Q Emerald 300 Lipopolysaccharide Gel Stain Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 252 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore salmonella enterica serotype typhimurium
    Cytokine production in LPS-stimulated peritoneal macrophages and phagocytosis function of control mice and mice with high-fat diet induced obesity, administered or not B. uniformis CECT 7771. SD: standard diet group (control) (n = 6); SD+B: standard diet group receiving a daily dose of 5.0×10 8 CFU B. uniformis CECT 7771 by gavage for 7 weeks (n = 6); HFD: high fat diet group (n = 6); HFD+B: high fat diet group receiving a daily dose of 5.0×10 8 CFU B. uniformis CECT 7771 by gavage for 7 weeks (n = 6). In the cytokine production study, peritoneal macrophages were stimulated with purified lipopolysaccharide (LPS) from S. <t>enterica</t> serotype <t>Typhimurium</t> ( Figure 6A ). Non-stimulated peritoneal macrophages were evaluated as controls of basal cytokine levels. In the phagocytosis study ( Figure 6B ), evidence of oxygen-radical production by macrophages was determined by the NBT test after in vitro interaction with a bacterial extract. Figure 6A: TNF- α and IL-10 cytokines produced by LPS-stimulated macrophages; Figure 6B: % NBT (+) cells. Data are expressed as mean and standard deviation of duplicate measurements determined in two independent experiments. Statistically significant differences of data are established at P
    Salmonella Enterica Serotype Typhimurium, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    InvivoGen ultrapure lipopolysaccharide
    CD14 bright /CD56+ monocyte subset is expanded in older healthy controls and produces more cytokines in response to <t>lipopolysaccharide.</t> (a) Representative dot plot of CD14 and CD56 expression on monocytes. Three relevant subpopulations are separated by the quadrants and marked by the arrows in the right panel. APC, allophycocyanin; FITC, fluorescein isothiocyanate; FL1-H, fluorescence intensity on channel that detects emissions from fluorescein isothiocyanate. (b) Scatterplot showing the correlation between age and the peripheral blood frequencies of CD14 bright /CD56+ monocytes in healthy controls. (c) Bar graphs depict the frequency of tumor necrosis factor-positive (TNF+) ( n = 5), interleukin 10-positive (IL-10+) ( n = 8) and IL-23+ ( n = 7) cells and the mean intracellular <t>IL-1β</t> content ( n = 7) in CD56+ and CD56– monocytes of healthy controls in response to lipopolysaccharide. (d) Spontaneous reactive oxygen intermediate (ROI) production of CD56– and CD56+ monocytes ( n = 4).
    Ultrapure Lipopolysaccharide, supplied by InvivoGen, used in various techniques. Bioz Stars score: 89/100, based on 189 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Enzo Biochem lipopolysaccharide lps
    OSM producing neutrophils did not express a classical N1 phenotype (A) OSM + neutrophils contained a distinct population of Arg1 + MPO hi cells, representative data. (B) 3.7±1.3% of cells in the CD45 gate were OSM + ; within the OSM + gate, 56.0±8.2% of the cells were CD16 + IL-5R− neutrophils. Within the neutrophil gate 72.56± 11.9% of the cells were Arg1 + MPO hi , and 22.1± 8.0% were Arg1 − MPO lo , n=5. (C) Blood neutrophils were either left untreated, or treated under N1 polarizing conditions <t>(LPS/IFNγ),</t> N2 polarizing conditions (IL-4/IL-13) or with GM-CSF. GM-CSF treated neutrophils secreted elevated levels of OSM into the cell culture supernatants, while N1 and N2 polarizing conditions did not induce OSM, (n=4–7, p
    Lipopolysaccharide Lps, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 181 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    5-ASA and AZTP restore epithelial barrier. ( A ) Paracellular permeability assay using FITC-Dextran: Differentiated and polarized T-84 monolayer grown in Transwell was treated with TNF-α (10 ng/ml), IFN-γ (100U/ml) or LPS (10 ng/ml) with or without pretreatment (5 h) with 5-ASA (5 mM) or AZTP (10 μM) n = 5 each treatment. Experiments were performed in biological duplicates and repeated three times. p value (≤0.05), * con vs treatment; # vs TNF-α or IFN-γ. One-way ANOVA with multiple comparisons were done using Bonferroni’s post hoc analysis. ( B , C ) PCR array analysis of T-84 cells treated with 5-ASA (5 mM) and AZTP(10 μM), in the presence or absence of TNF-α (10 ng/ml) ( D ) Immunofluorescence analysis of T-84 monolayer for the representative proteins of adherens junctions; AJs (E-cadherin), tight junctions; TJs (occludin), Desmosomes (Desmoglein-2). TNF-α treatment disrupted all junctions and resulted in protein internalization as indicated by arrows. 5-ASA and AZTP restored membranous expression of these proteins. Image magnification 400x. Scale 50μm. E-cadherin and Desmoglein were visualized using Alexa Fluor 488 secondary antibody. Occludin was visualized using Alexa Fluor 568 ( E ) qRT-PCR analysis of intestinal alkaline phosphatase as a marker of cellular differentiation in T-84 cells. The experiment was performed twice.

    Journal: Scientific Reports

    Article Title: Mesalamine and azathioprine modulate junctional complexes and restore epithelial barrier function in intestinal inflammation

    doi: 10.1038/s41598-019-39401-0

    Figure Lengend Snippet: 5-ASA and AZTP restore epithelial barrier. ( A ) Paracellular permeability assay using FITC-Dextran: Differentiated and polarized T-84 monolayer grown in Transwell was treated with TNF-α (10 ng/ml), IFN-γ (100U/ml) or LPS (10 ng/ml) with or without pretreatment (5 h) with 5-ASA (5 mM) or AZTP (10 μM) n = 5 each treatment. Experiments were performed in biological duplicates and repeated three times. p value (≤0.05), * con vs treatment; # vs TNF-α or IFN-γ. One-way ANOVA with multiple comparisons were done using Bonferroni’s post hoc analysis. ( B , C ) PCR array analysis of T-84 cells treated with 5-ASA (5 mM) and AZTP(10 μM), in the presence or absence of TNF-α (10 ng/ml) ( D ) Immunofluorescence analysis of T-84 monolayer for the representative proteins of adherens junctions; AJs (E-cadherin), tight junctions; TJs (occludin), Desmosomes (Desmoglein-2). TNF-α treatment disrupted all junctions and resulted in protein internalization as indicated by arrows. 5-ASA and AZTP restored membranous expression of these proteins. Image magnification 400x. Scale 50μm. E-cadherin and Desmoglein were visualized using Alexa Fluor 488 secondary antibody. Occludin was visualized using Alexa Fluor 568 ( E ) qRT-PCR analysis of intestinal alkaline phosphatase as a marker of cellular differentiation in T-84 cells. The experiment was performed twice.

    Article Snippet: Proinflammatory mediators used were human recombinant IFN-γ (100U/ml), LPS (10 ng/ml; eBioscience, Austria) and TNF-α (10 ng/ml; Miltenyi Biotec, Germany).

    Techniques: Permeability, Polymerase Chain Reaction, Immunofluorescence, Expressing, Quantitative RT-PCR, Marker, Cell Differentiation

    Effects of prenatal LPS (E15/16) and postnatal MP-III-022 (P21–28) treatments on distance traveled by female Wistar offspring at P60. Activity was recorded during 30 min of habituation and baseline activity (HAB trial), then 30 min after saline challenge (SAL trial) and finally 60 min after amphetamine (0.5 mg/kg, i.p.) administration (AMPH trial). Panel A: the main effects of between-subject factors (LPS and MP-III-022) in each trial, as analyzed by three-way RM ANOVA. Graphs B, C and D: pairwise multiple comparison (Student–Newman–Keuls method) of LPS and MP-III-022 effects on total distance traveled in each of three trials (HAB, SAL and AMPH, respectively). Graphs E and F: effects of MP-III-022 on distance per time bin analyzed by pairwise multiple comparison procedures (Student–Newman–Keuls method) within SAL or LPS cohorts, respectively. Data (mean value ± SEM) are shown as total distance per trial (B, C and D) or distance traveled in 10 min bins (E and F). *(p

    Journal: International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience

    Article Title: Positive modulation of α5 GABAA receptors in preadolescence prevents reduced locomotor response to amphetamine in adult female but not male rats prenatally exposed to lipopolysaccharide

    doi: 10.1016/j.ijdevneu.2017.06.001

    Figure Lengend Snippet: Effects of prenatal LPS (E15/16) and postnatal MP-III-022 (P21–28) treatments on distance traveled by female Wistar offspring at P60. Activity was recorded during 30 min of habituation and baseline activity (HAB trial), then 30 min after saline challenge (SAL trial) and finally 60 min after amphetamine (0.5 mg/kg, i.p.) administration (AMPH trial). Panel A: the main effects of between-subject factors (LPS and MP-III-022) in each trial, as analyzed by three-way RM ANOVA. Graphs B, C and D: pairwise multiple comparison (Student–Newman–Keuls method) of LPS and MP-III-022 effects on total distance traveled in each of three trials (HAB, SAL and AMPH, respectively). Graphs E and F: effects of MP-III-022 on distance per time bin analyzed by pairwise multiple comparison procedures (Student–Newman–Keuls method) within SAL or LPS cohorts, respectively. Data (mean value ± SEM) are shown as total distance per trial (B, C and D) or distance traveled in 10 min bins (E and F). *(p

    Article Snippet: On gestation (embryonic) days 15 and 16 (E15/16) pregnant dams were treated intraperitoneally either with LPS (from Escherichia coli, serotype 0111:B4, Sigma L2630) at a dose of 100 μg/kg per day, or with 0.9% saline (2 mL/kg per day).

    Techniques: Activity Assay

    Effect of O111:B4 LPS (0.2 ng.L −1 ) on whole blood (without thrombin), where dense matted deposits were spontaneously formed, not seen in control whole blood smears.

    Journal: bioRxiv

    Article Title: Acute induction of anomalous blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide (LPS)

    doi: 10.1101/053538

    Figure Lengend Snippet: Effect of O111:B4 LPS (0.2 ng.L −1 ) on whole blood (without thrombin), where dense matted deposits were spontaneously formed, not seen in control whole blood smears.

    Article Snippet: LPS types, purified fibrinogen and thrombin concentration used The LPS used was from E. coli O111:B4 (Sigma, L2630) and also E. coli O26:B6 (Sigma L2762).

    Techniques:

    The effect of 0.2 ng.L −1 O111:B4 LPS on the morphology of fibrin fibres in the platelet poor plasma (PPP) of healthy individuals (with added thrombin). A) Healthy fibres; B and C) PPP with added LPS. D) Fibre distribution of the control fibres and of controls with added LPS of 30 individuals. Note: in samples with added LPS there were areas of matted layers with no visible fibres to measure. Fibres were measured using ImageJ as described in Materials and Methods.

    Journal: bioRxiv

    Article Title: Acute induction of anomalous blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide (LPS)

    doi: 10.1101/053538

    Figure Lengend Snippet: The effect of 0.2 ng.L −1 O111:B4 LPS on the morphology of fibrin fibres in the platelet poor plasma (PPP) of healthy individuals (with added thrombin). A) Healthy fibres; B and C) PPP with added LPS. D) Fibre distribution of the control fibres and of controls with added LPS of 30 individuals. Note: in samples with added LPS there were areas of matted layers with no visible fibres to measure. Fibres were measured using ImageJ as described in Materials and Methods.

    Article Snippet: LPS types, purified fibrinogen and thrombin concentration used The LPS used was from E. coli O111:B4 (Sigma, L2630) and also E. coli O26:B6 (Sigma L2762).

    Techniques:

    ITC analysis of the LPS-fibrinogen interaction. A) Titration of 8.8 μM human plasma fibrinogen (black circles) or buffer (green open circles) into 100 μM of E. coli O111:B4 LPS. B) Titration of 50 μg/μl LPS (2.5 μM) or buffer into 3 μg/μl fibrinogen (8.8 μM) or buffer as indicated. Experiments were conducted at 37 °C in phosphate buffered saline.

    Journal: bioRxiv

    Article Title: Acute induction of anomalous blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide (LPS)

    doi: 10.1101/053538

    Figure Lengend Snippet: ITC analysis of the LPS-fibrinogen interaction. A) Titration of 8.8 μM human plasma fibrinogen (black circles) or buffer (green open circles) into 100 μM of E. coli O111:B4 LPS. B) Titration of 50 μg/μl LPS (2.5 μM) or buffer into 3 μg/μl fibrinogen (8.8 μM) or buffer as indicated. Experiments were conducted at 37 °C in phosphate buffered saline.

    Article Snippet: LPS types, purified fibrinogen and thrombin concentration used The LPS used was from E. coli O111:B4 (Sigma, L2630) and also E. coli O26:B6 (Sigma L2762).

    Techniques: Titration

    A) Purified fibrinogen with added thrombin but no LPS; B) purified fibrinogen with added O111:B4 LPS (30 seconds exposure) and 0.2 ng.L −1 LPS; C) as B but 10 minutes exposure. Scale bar: 1 μ m.

    Journal: bioRxiv

    Article Title: Acute induction of anomalous blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide (LPS)

    doi: 10.1101/053538

    Figure Lengend Snippet: A) Purified fibrinogen with added thrombin but no LPS; B) purified fibrinogen with added O111:B4 LPS (30 seconds exposure) and 0.2 ng.L −1 LPS; C) as B but 10 minutes exposure. Scale bar: 1 μ m.

    Article Snippet: LPS types, purified fibrinogen and thrombin concentration used The LPS used was from E. coli O111:B4 (Sigma, L2630) and also E. coli O26:B6 (Sigma L2762).

    Techniques: Purification

    In vitro infection of mouse peritoneal macrophages. C57BL/6 mouse peritoneal macrophages seeded on 96-well plates were pre-stimulated with 1 μg/ml E. coli lipopolysaccharide to induce the expression of pro-IL-1β for 3 h. The freshly cultured X4550(pYA3334-SspH2-EscI), X4550(pYA3334-SspH2) and X4550(pYA3334) were then added with the desired multiplicity of infection (MOI). The cell plate was centrifuged to enhance the contact of bacteria with the cells and the infected cells were then incubated for 30 min. The supernatants containing uninfected bacteria were replaced with RPMI 1640 complete medium (100 μl/well) containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml LPS prior to the start of the subsequent incubation. The uninfected cells were used as control. a Supernatant IL-1β and IL-18 levels at 4 h post-infection (hpi) with MOI 10, 50 and 100; b LDH release at 1, 3, 5 hpi with MOI 100 and 24 hpi with MOI 10, 50 and 100; c Intracellular caspase-1 activation at 1 h hpi with MOI 100; d Cell morphology at 24 hpi with MOI 100 (a, uninfection; b, infected with X4550(pYA3334); c, infected with X4550(pYA3334-SspH2); d, infected with X4550(pYA3334-SspH2-EscI), arrows show pyroptotic cell death); e The count of cells and intracellular bacteria at 24 hpi with MOI 100. The data shown are representative of three replicate experiments

    Journal: BMC Immunology

    Article Title: Recombinant Salmonella expressing SspH2-EscI fusion protein limits its colonization in mice

    doi: 10.1186/s12865-017-0203-2

    Figure Lengend Snippet: In vitro infection of mouse peritoneal macrophages. C57BL/6 mouse peritoneal macrophages seeded on 96-well plates were pre-stimulated with 1 μg/ml E. coli lipopolysaccharide to induce the expression of pro-IL-1β for 3 h. The freshly cultured X4550(pYA3334-SspH2-EscI), X4550(pYA3334-SspH2) and X4550(pYA3334) were then added with the desired multiplicity of infection (MOI). The cell plate was centrifuged to enhance the contact of bacteria with the cells and the infected cells were then incubated for 30 min. The supernatants containing uninfected bacteria were replaced with RPMI 1640 complete medium (100 μl/well) containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml LPS prior to the start of the subsequent incubation. The uninfected cells were used as control. a Supernatant IL-1β and IL-18 levels at 4 h post-infection (hpi) with MOI 10, 50 and 100; b LDH release at 1, 3, 5 hpi with MOI 100 and 24 hpi with MOI 10, 50 and 100; c Intracellular caspase-1 activation at 1 h hpi with MOI 100; d Cell morphology at 24 hpi with MOI 100 (a, uninfection; b, infected with X4550(pYA3334); c, infected with X4550(pYA3334-SspH2); d, infected with X4550(pYA3334-SspH2-EscI), arrows show pyroptotic cell death); e The count of cells and intracellular bacteria at 24 hpi with MOI 100. The data shown are representative of three replicate experiments

    Article Snippet: The adherent cells were pre-stimulated with 1 μg/ml E. coli lipopolysaccharide (LPS) (Sigma-Aldrich) to induce the expression of pro-IL-1β.

    Techniques: In Vitro, Infection, Expressing, Cell Culture, Incubation, Activation Assay

    RvD2 decreases TLR4 expression in time-dependent manner in THP-1 monocytic cells. A ) THP-1 cells were treated with 1–100 nM RvD2 followed by 20 ng/ml LPS. TLR4 mRNA was assessed 15 min, 1 h, and 4 h after LPS stimulation. B ) Effects of 1–100 nM RvD2 alone and with LPS treatment on TLR4 mRNA were also assessed 4 h after LPS exposure (same data shown as that for vehicle/vehicle, vehicle/LPS, and RvD2/LPS groups). C ) THP-1 cells were also treated with 100 nM RvD2 followed by 20 ng/ml LPS (either 0111:B4 or 055:B5) or hyaluronan, and TLR4 mRNA was assessed after 4 h. D ) TLR4 protein expression was determined by Western blot analysis 4 h after LPS exposure (representative image shown, densitometry for n = 4). Statistical significance was determined by 2-way ANOVA, n = 3–5 independent experiments. # P

    Journal: The FASEB Journal

    Article Title: Resolvin D2 decreases TLR4 expression to mediate resolution in human monocytes

    doi: 10.1096/fj.201600375R

    Figure Lengend Snippet: RvD2 decreases TLR4 expression in time-dependent manner in THP-1 monocytic cells. A ) THP-1 cells were treated with 1–100 nM RvD2 followed by 20 ng/ml LPS. TLR4 mRNA was assessed 15 min, 1 h, and 4 h after LPS stimulation. B ) Effects of 1–100 nM RvD2 alone and with LPS treatment on TLR4 mRNA were also assessed 4 h after LPS exposure (same data shown as that for vehicle/vehicle, vehicle/LPS, and RvD2/LPS groups). C ) THP-1 cells were also treated with 100 nM RvD2 followed by 20 ng/ml LPS (either 0111:B4 or 055:B5) or hyaluronan, and TLR4 mRNA was assessed after 4 h. D ) TLR4 protein expression was determined by Western blot analysis 4 h after LPS exposure (representative image shown, densitometry for n = 4). Statistical significance was determined by 2-way ANOVA, n = 3–5 independent experiments. # P

    Article Snippet: LPS 0111:B4 (L4391), anti-TLR4 antibody (PRS3141), LPS 055:B5 (6529), and hyaluronan (S0326) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Expressing, Western Blot

    Effect of O111:B4 LPS (0.2 ng l −1 ) on whole blood (without thrombin), where dense matted deposits were spontaneously formed, not seen in control whole blood smears.

    Journal: Journal of the Royal Society Interface

    Article Title: Acute induction of anomalous and amyloidogenic blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide

    doi: 10.1098/rsif.2016.0539

    Figure Lengend Snippet: Effect of O111:B4 LPS (0.2 ng l −1 ) on whole blood (without thrombin), where dense matted deposits were spontaneously formed, not seen in control whole blood smears.

    Article Snippet: Lipopolysaccharide types, purified fibrinogen and thrombin concentration used The LPS used was from E. coli O111:B4 (Sigma, L2630) and also E. coli O26:B6 (Sigma L2762).

    Techniques:

    ( a ) Purified fibrinogen with added thrombin but no LPS; ( b ) purified fibrinogen with added O111:B4 LPS (30 s exposure) and 0.2 ng l −1 LPS; ( c ) as panel ( b ) but 10 min exposure.

    Journal: Journal of the Royal Society Interface

    Article Title: Acute induction of anomalous and amyloidogenic blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide

    doi: 10.1098/rsif.2016.0539

    Figure Lengend Snippet: ( a ) Purified fibrinogen with added thrombin but no LPS; ( b ) purified fibrinogen with added O111:B4 LPS (30 s exposure) and 0.2 ng l −1 LPS; ( c ) as panel ( b ) but 10 min exposure.

    Article Snippet: Lipopolysaccharide types, purified fibrinogen and thrombin concentration used The LPS used was from E. coli O111:B4 (Sigma, L2630) and also E. coli O26:B6 (Sigma L2762).

    Techniques: Purification

    The effect of 0.2 ng l −1 O111:B4 LPS on the morphology of fibrin fibres in the platelet-poor plasma (PPP) of healthy individuals (with added thrombin). ( a ) Healthy fibres; ( b,c ) PPP with added LPS. ( d ) Fibre distribution of the control fibres and of controls with added LPS of 30 individuals. Note: in samples with added LPS, there were areas of matted layers with no visible fibres to measure. Fibres were measured using ImageJ as described in Material and methods.

    Journal: Journal of the Royal Society Interface

    Article Title: Acute induction of anomalous and amyloidogenic blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide

    doi: 10.1098/rsif.2016.0539

    Figure Lengend Snippet: The effect of 0.2 ng l −1 O111:B4 LPS on the morphology of fibrin fibres in the platelet-poor plasma (PPP) of healthy individuals (with added thrombin). ( a ) Healthy fibres; ( b,c ) PPP with added LPS. ( d ) Fibre distribution of the control fibres and of controls with added LPS of 30 individuals. Note: in samples with added LPS, there were areas of matted layers with no visible fibres to measure. Fibres were measured using ImageJ as described in Material and methods.

    Article Snippet: Lipopolysaccharide types, purified fibrinogen and thrombin concentration used The LPS used was from E. coli O111:B4 (Sigma, L2630) and also E. coli O26:B6 (Sigma L2762).

    Techniques:

    ITC analysis of the LPS–fibrinogen interaction. ( a ) Titration of 8.8 μM human plasma fibrinogen ( black circles ) or buffer ( green open circles ) into 100 µM of E. coli O111:B4 LPS. ( b ) Titration of 50 ng µl −1 LPS (2.5 µM) or buffer into 3 µg µl −1 fibrinogen (8.8 µM) or buffer as indicated. Experiments were conducted at 37°C in phosphate buffered saline. (Online version in colour.)

    Journal: Journal of the Royal Society Interface

    Article Title: Acute induction of anomalous and amyloidogenic blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide

    doi: 10.1098/rsif.2016.0539

    Figure Lengend Snippet: ITC analysis of the LPS–fibrinogen interaction. ( a ) Titration of 8.8 μM human plasma fibrinogen ( black circles ) or buffer ( green open circles ) into 100 µM of E. coli O111:B4 LPS. ( b ) Titration of 50 ng µl −1 LPS (2.5 µM) or buffer into 3 µg µl −1 fibrinogen (8.8 µM) or buffer as indicated. Experiments were conducted at 37°C in phosphate buffered saline. (Online version in colour.)

    Article Snippet: Lipopolysaccharide types, purified fibrinogen and thrombin concentration used The LPS used was from E. coli O111:B4 (Sigma, L2630) and also E. coli O26:B6 (Sigma L2762).

    Techniques: Titration

    PS2 protein is increased in microglia activated by IFNγ. A) Western blot analysis demonstrating the PS2 C-terminus migrating at ∼20 kDa. Twenty-four hour stimulation with 10 u/ml IFNγ induces increased levels of PS2 expression in lysates prepared from primary microglia, while 100 ng/ml LPS does not induce PS2 expression. B) Quantitative densitometry on Western blots of lysates from 4 independent cultures shows significantly increased induction of PS2 by IFNγ. Data represent the mean ± SEM of 4 independent experiments (*p

    Journal: PLoS ONE

    Article Title: Presenilin 2 Is the Predominant ?-Secretase in Microglia and Modulates Cytokine Release

    doi: 10.1371/journal.pone.0015743

    Figure Lengend Snippet: PS2 protein is increased in microglia activated by IFNγ. A) Western blot analysis demonstrating the PS2 C-terminus migrating at ∼20 kDa. Twenty-four hour stimulation with 10 u/ml IFNγ induces increased levels of PS2 expression in lysates prepared from primary microglia, while 100 ng/ml LPS does not induce PS2 expression. B) Quantitative densitometry on Western blots of lysates from 4 independent cultures shows significantly increased induction of PS2 by IFNγ. Data represent the mean ± SEM of 4 independent experiments (*p

    Article Snippet: Cytokine measurement Primary microglia plated at 5×104 cells per well in poly-D-lysine coated 96 well plates were cultured overnight followed by stimulation with vehicle, 10 u/ml IFNγ (R & D systems, Minneapolis MN), 100 ng/ml lipopolysaccharide (LPS) (catalog # L2654, strain 026:B6 Escherichia coli , Sigma-Aldrich, St. Louis, MO) or amyloid-β (1–42) (catalog #62-0-80, American Peptide, Sunnyvale, CA) for 24 hours.

    Techniques: Western Blot, Expressing

    Effusanin E affects activity of the COX-2 pathway. ( A ) NPC cells were pretreated with lipopolysaccharides (LPS) (2 µg/ml) for 8 hours then treated with effusanin E at 125 µM and 250 µM (EFE 125 µM or EFE 250 µM). At 24 hours after treatment, COX-2 protein expression was determined by western blotting. ( B ) NPC cells were transfected with a luciferase expression vector containing a COX-2 5-flanking fragment for 8 hours and treated with effusanin E (EFE) at the indicated doses. At 24 hours after treatment, COX-2 promoter activities were determined. ( C ) The effect of effusanin E was inhibited by an inhibitor of COX-2 in NPC cells. NPC cells were treated with celecoxib (CB) (20 µM or 50 µM) for 8 hours followed by effusanin E at 125 µM and 250 µM (EFE 125 µM or EFE 250 µM) for up to 24 hours or 48 hours. At the indicated time points, the cells were analyzed using the MTS assay. The data are presented as the mean ± S.D. of three separate experiments. * represent P

    Journal: PLoS ONE

    Article Title: Effusanin E Suppresses Nasopharyngeal Carcinoma Cell Growth by Inhibiting NF-κB and COX-2 Signaling

    doi: 10.1371/journal.pone.0109951

    Figure Lengend Snippet: Effusanin E affects activity of the COX-2 pathway. ( A ) NPC cells were pretreated with lipopolysaccharides (LPS) (2 µg/ml) for 8 hours then treated with effusanin E at 125 µM and 250 µM (EFE 125 µM or EFE 250 µM). At 24 hours after treatment, COX-2 protein expression was determined by western blotting. ( B ) NPC cells were transfected with a luciferase expression vector containing a COX-2 5-flanking fragment for 8 hours and treated with effusanin E (EFE) at the indicated doses. At 24 hours after treatment, COX-2 promoter activities were determined. ( C ) The effect of effusanin E was inhibited by an inhibitor of COX-2 in NPC cells. NPC cells were treated with celecoxib (CB) (20 µM or 50 µM) for 8 hours followed by effusanin E at 125 µM and 250 µM (EFE 125 µM or EFE 250 µM) for up to 24 hours or 48 hours. At the indicated time points, the cells were analyzed using the MTS assay. The data are presented as the mean ± S.D. of three separate experiments. * represent P

    Article Snippet: Reagents Ammonium pyrrolidinedithiocarbamate (PDTC), celecoxib (CB) and lipopolysaccharides (LPS) were purchased from Sigma (St. Louis, MO).

    Techniques: Activity Assay, Expressing, Western Blot, Transfection, Luciferase, Plasmid Preparation, MTS Assay

    Effusanin E affects the activity of the NF-κB pathway. ( A ) Effusanin E inhibited the expression of p50 and p65. NPC cells were treated with DMSO (basal) or LPS (2 µg/ml) for 8 hours followed by effusanin E treatment at125 µM or 250 µM (EFE 125 µM or EFE 250 µM) for up to 24 hours. At the indicated time points, the cells were detected by Western blot analysis. ( B ) The binding of p50 and p65 NF-κB to the biotin-labeled, COX-2 promoter probe was analyzed by streptavidin-agarose pulldown assays, and the levels of p50 and p65 protein expression were detected by Western blot analysis. ( C ) The effect of effusanin E was inhibited by an inhibitor of NF-κB in NPC cells. NPC cells were treated with PDTC (100 µM) for 8 hours followed by effusanin E at 125 µM and 250 µM (EFE 125 µM or EFE 250 µM) treatment for up to 24 hours or 48 hours. At the indicated time points, the cells were analyzed using the MTS assay. ( D ) The effect of effusanin E was blocked by an activator of NF-κB. NPC cells were treated with LPS (2 µg/ml) or ammonium pyrrolidinedithiocarbamate (PDTC) (100 µM) for 8 hours followed by effusanin E at125 µM or 250 µM (EFE 125 µM or EFE 250 µM) treatment for up to 24 hours or 48 hours. At the indicated time points, the cells were analyzed using the MTS assay. ( E ) Analysis of the reduced nuclear translocation of NF-κB p65 by immunofluorescence imaging (IFI). CNE1 and CNE2 cells were treated with effusanin E at 125 µM (EFE) or PDTC (100 µM) or PDTC (100 µM) for 8 hours followed by effusanin E at 125 µM (PDTC + EFE), and NF-κB nuclear translocation in CNE1 and CNE2 cells was determined by immunofluorescence imaging analysis. * represent P

    Journal: PLoS ONE

    Article Title: Effusanin E Suppresses Nasopharyngeal Carcinoma Cell Growth by Inhibiting NF-κB and COX-2 Signaling

    doi: 10.1371/journal.pone.0109951

    Figure Lengend Snippet: Effusanin E affects the activity of the NF-κB pathway. ( A ) Effusanin E inhibited the expression of p50 and p65. NPC cells were treated with DMSO (basal) or LPS (2 µg/ml) for 8 hours followed by effusanin E treatment at125 µM or 250 µM (EFE 125 µM or EFE 250 µM) for up to 24 hours. At the indicated time points, the cells were detected by Western blot analysis. ( B ) The binding of p50 and p65 NF-κB to the biotin-labeled, COX-2 promoter probe was analyzed by streptavidin-agarose pulldown assays, and the levels of p50 and p65 protein expression were detected by Western blot analysis. ( C ) The effect of effusanin E was inhibited by an inhibitor of NF-κB in NPC cells. NPC cells were treated with PDTC (100 µM) for 8 hours followed by effusanin E at 125 µM and 250 µM (EFE 125 µM or EFE 250 µM) treatment for up to 24 hours or 48 hours. At the indicated time points, the cells were analyzed using the MTS assay. ( D ) The effect of effusanin E was blocked by an activator of NF-κB. NPC cells were treated with LPS (2 µg/ml) or ammonium pyrrolidinedithiocarbamate (PDTC) (100 µM) for 8 hours followed by effusanin E at125 µM or 250 µM (EFE 125 µM or EFE 250 µM) treatment for up to 24 hours or 48 hours. At the indicated time points, the cells were analyzed using the MTS assay. ( E ) Analysis of the reduced nuclear translocation of NF-κB p65 by immunofluorescence imaging (IFI). CNE1 and CNE2 cells were treated with effusanin E at 125 µM (EFE) or PDTC (100 µM) or PDTC (100 µM) for 8 hours followed by effusanin E at 125 µM (PDTC + EFE), and NF-κB nuclear translocation in CNE1 and CNE2 cells was determined by immunofluorescence imaging analysis. * represent P

    Article Snippet: Reagents Ammonium pyrrolidinedithiocarbamate (PDTC), celecoxib (CB) and lipopolysaccharides (LPS) were purchased from Sigma (St. Louis, MO).

    Techniques: Activity Assay, Expressing, Western Blot, Binding Assay, Labeling, MTS Assay, Translocation Assay, Immunofluorescence, Imaging

    Serum levels of inflammation-related molecules in treated endotoxemic mice. (A) Endotoxemia was induced by the intraperitoneal injection of 10 mg/kg E. coli LPS. A control group was injected i.v. with PBS (n = 10, light gray bars), a second group

    Journal: Molecular Medicine

    Article Title: Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes

    doi: 10.2119/molmed.2014.00224

    Figure Lengend Snippet: Serum levels of inflammation-related molecules in treated endotoxemic mice. (A) Endotoxemia was induced by the intraperitoneal injection of 10 mg/kg E. coli LPS. A control group was injected i.v. with PBS (n = 10, light gray bars), a second group

    Article Snippet: Endotoxemia was induced by the intraperitoneal injection of 10 mg/kg E. coli LPS (B 055:B5, #L2880, Sigma-Aldrich).

    Techniques: Mouse Assay, Injection

    A single administration of Fe(II) exposure-modified IVIg overcomes the coagulation and complement abnormalities in LPS-induced sepsis. (A) Endotoxemia was induced to ICR mice by the intraperitoneal injection of 10 mg/kg E. coli LPS. A

    Journal: Molecular Medicine

    Article Title: Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes

    doi: 10.2119/molmed.2014.00224

    Figure Lengend Snippet: A single administration of Fe(II) exposure-modified IVIg overcomes the coagulation and complement abnormalities in LPS-induced sepsis. (A) Endotoxemia was induced to ICR mice by the intraperitoneal injection of 10 mg/kg E. coli LPS. A

    Article Snippet: Endotoxemia was induced by the intraperitoneal injection of 10 mg/kg E. coli LPS (B 055:B5, #L2880, Sigma-Aldrich).

    Techniques: Modification, Coagulation, Mouse Assay, Injection

    LPS induces SBD-1 expression mainly through P38 MAPK signaling but not through JNK or ERK signaling. a b OOECs were cultured with LPS (100 ng/mL), with or without P38 MAPK inhibitors SB203580 and SB202190 for 12 h. Total RNA was prepared and used for examination of SBD-1 mRNA expression by QRT-PCR or RT-PCR with specific primers for SBD-1 and GAPDH. c d OOECs were cultured with LPS (100 ng/mL), with or without JNK inhibitor SP600125 and ERK inhibitor PD98059 for 12 h. Total RNA was prepared and used for examination of SBD-1 mRNA expression by QRT-PCR or RT-PCR with specific primers for SBD-1 and GAPDH. All of the experiments were repeated at least three times. * p

    Journal: Lipids in Health and Disease

    Article Title: Lipopolysaccharide induces SBD-1 expression via the P38 MAPK signaling pathway in ovine oviduct epithelial cells

    doi: 10.1186/s12944-016-0294-4

    Figure Lengend Snippet: LPS induces SBD-1 expression mainly through P38 MAPK signaling but not through JNK or ERK signaling. a b OOECs were cultured with LPS (100 ng/mL), with or without P38 MAPK inhibitors SB203580 and SB202190 for 12 h. Total RNA was prepared and used for examination of SBD-1 mRNA expression by QRT-PCR or RT-PCR with specific primers for SBD-1 and GAPDH. c d OOECs were cultured with LPS (100 ng/mL), with or without JNK inhibitor SP600125 and ERK inhibitor PD98059 for 12 h. Total RNA was prepared and used for examination of SBD-1 mRNA expression by QRT-PCR or RT-PCR with specific primers for SBD-1 and GAPDH. All of the experiments were repeated at least three times. * p

    Article Snippet: Reagents LPS (Cat. No. L2880), the P38 MAPK inhibitors SB203580 (Cat. No. S8307) and SB202190 (Cat. No. S7067), the JNK inhibitor SP600125 (Cat. No. S5567), and the ERK1/2 inhibitor PD98059 (Cat. No. P215) were purchased from SIGMA-ALDRICH.

    Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

    LPS stimulates the expression of SBD-1 through TLR4. a Tlr4 mRNA expression of ovine oviduct stromal cells (OOSCs) and epithelial cells (OOECs) was assessed by qRT-PCR. b PCR was conducted to examine the expression of TLR4 in the ovine oviduct epithelial cells harvested at different times. c Western blotting was performed to examine the expression levels of P38 MAPK after treatment with LPS (100 ng/mL) or a TLR4 neutralizing antibody for 12 h. d The densitometric analysis of the bands on the western blotting showed that LPS could markedly activate P38 MAPK, while the separate addition of the TLR4 neutralizing antibody had no effect. However, treatment with the TLR4 neutralizing antibody could significantly decrease the levels of phosphorylated P38 induced by LPS. e QRT-PCR analysis was used to examine the mRNA levels of SBD-1 after treatment with the TLR4 neutralizing antibody for 12 h. Blocking TLR4 activity could inhibit the expression of SBD-1. All of the experiments were repeated at least three times. * p

    Journal: Lipids in Health and Disease

    Article Title: Lipopolysaccharide induces SBD-1 expression via the P38 MAPK signaling pathway in ovine oviduct epithelial cells

    doi: 10.1186/s12944-016-0294-4

    Figure Lengend Snippet: LPS stimulates the expression of SBD-1 through TLR4. a Tlr4 mRNA expression of ovine oviduct stromal cells (OOSCs) and epithelial cells (OOECs) was assessed by qRT-PCR. b PCR was conducted to examine the expression of TLR4 in the ovine oviduct epithelial cells harvested at different times. c Western blotting was performed to examine the expression levels of P38 MAPK after treatment with LPS (100 ng/mL) or a TLR4 neutralizing antibody for 12 h. d The densitometric analysis of the bands on the western blotting showed that LPS could markedly activate P38 MAPK, while the separate addition of the TLR4 neutralizing antibody had no effect. However, treatment with the TLR4 neutralizing antibody could significantly decrease the levels of phosphorylated P38 induced by LPS. e QRT-PCR analysis was used to examine the mRNA levels of SBD-1 after treatment with the TLR4 neutralizing antibody for 12 h. Blocking TLR4 activity could inhibit the expression of SBD-1. All of the experiments were repeated at least three times. * p

    Article Snippet: Reagents LPS (Cat. No. L2880), the P38 MAPK inhibitors SB203580 (Cat. No. S8307) and SB202190 (Cat. No. S7067), the JNK inhibitor SP600125 (Cat. No. S5567), and the ERK1/2 inhibitor PD98059 (Cat. No. P215) were purchased from SIGMA-ALDRICH.

    Techniques: Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Western Blot, Blocking Assay, Activity Assay

    LPS induces SBD-1 mRNA expression and activates the MAPK signaling pathway in ovine oviduct epithelial cells. a Immunofluorescence was performed to identify ovine oviduct epithelial cells. After 24 h in culture, cells were stained for an ovine oviduct epithelial cell marker CK-18 ( green ). Hoechst 33342 ( blue ) was used to counterstain the nuclear DNA. An isotype-matched IgG was used as the negative control. Scale bar: 100 μm. b Ovine oviduct epithelial cells (OOECs) were treated with the indicated concentrations of LPS for 24 h and compared with untreated controls. The QRT-PCR analysis showed that LPS induces SBD-1 expression in a concentration-dependent manner. c OOECs were treated with LPS (100 ng/mL) for various time intervals and compared with untreated controls. The QRT-PCR results indicated that LPS induces SBD-1 expression in a time-dependent manner. d OOECs were treated with LPS (100 ng/mL) and harvested at the indicated time points. Whole-cell lysates were prepared and used for western blot analysis with MAPK and P-MAPK antibodies. e A densitometric analysis of the optical density (OD) of different p-MAPKs relative to the OD of MAPKs. All of the experiments were repeated at least three times. * p

    Journal: Lipids in Health and Disease

    Article Title: Lipopolysaccharide induces SBD-1 expression via the P38 MAPK signaling pathway in ovine oviduct epithelial cells

    doi: 10.1186/s12944-016-0294-4

    Figure Lengend Snippet: LPS induces SBD-1 mRNA expression and activates the MAPK signaling pathway in ovine oviduct epithelial cells. a Immunofluorescence was performed to identify ovine oviduct epithelial cells. After 24 h in culture, cells were stained for an ovine oviduct epithelial cell marker CK-18 ( green ). Hoechst 33342 ( blue ) was used to counterstain the nuclear DNA. An isotype-matched IgG was used as the negative control. Scale bar: 100 μm. b Ovine oviduct epithelial cells (OOECs) were treated with the indicated concentrations of LPS for 24 h and compared with untreated controls. The QRT-PCR analysis showed that LPS induces SBD-1 expression in a concentration-dependent manner. c OOECs were treated with LPS (100 ng/mL) for various time intervals and compared with untreated controls. The QRT-PCR results indicated that LPS induces SBD-1 expression in a time-dependent manner. d OOECs were treated with LPS (100 ng/mL) and harvested at the indicated time points. Whole-cell lysates were prepared and used for western blot analysis with MAPK and P-MAPK antibodies. e A densitometric analysis of the optical density (OD) of different p-MAPKs relative to the OD of MAPKs. All of the experiments were repeated at least three times. * p

    Article Snippet: Reagents LPS (Cat. No. L2880), the P38 MAPK inhibitors SB203580 (Cat. No. S8307) and SB202190 (Cat. No. S7067), the JNK inhibitor SP600125 (Cat. No. S5567), and the ERK1/2 inhibitor PD98059 (Cat. No. P215) were purchased from SIGMA-ALDRICH.

    Techniques: Expressing, Immunofluorescence, Staining, Marker, Negative Control, Quantitative RT-PCR, Concentration Assay, Western Blot

    Schematic diagram of LPS regulating SBD-1 expression. LPS binds to TLR4 receptor, activates P38 MAPK signaling pathway, and subsequently stimulates SBD-1 expression in ovine oviduct epithelial cells

    Journal: Lipids in Health and Disease

    Article Title: Lipopolysaccharide induces SBD-1 expression via the P38 MAPK signaling pathway in ovine oviduct epithelial cells

    doi: 10.1186/s12944-016-0294-4

    Figure Lengend Snippet: Schematic diagram of LPS regulating SBD-1 expression. LPS binds to TLR4 receptor, activates P38 MAPK signaling pathway, and subsequently stimulates SBD-1 expression in ovine oviduct epithelial cells

    Article Snippet: Reagents LPS (Cat. No. L2880), the P38 MAPK inhibitors SB203580 (Cat. No. S8307) and SB202190 (Cat. No. S7067), the JNK inhibitor SP600125 (Cat. No. S5567), and the ERK1/2 inhibitor PD98059 (Cat. No. P215) were purchased from SIGMA-ALDRICH.

    Techniques: Expressing

    Nod1-dependent CCL2 induction in vivo. (A) WT and Nod1-deficient mice were administrated i.p. with 200 μg KF1B, iE-DAP, MDP, E. coli O55:B5 LPS, or PBS alone. The serum level of CCL2 was determined by ELISA. Because the relative levels of CCL2 induced by MDP was lower than those by KF1B, the CCL2 levels induced by MDP are also given (inset). The CCL2 levels are given with means ± SD from three mice. (B) The serum CXCL2 level from mice stimulated with 50 μg KF1B or PBS alone at indicated times after i.p. injection was determined by ELISA. Representative data from three independent experiments are shown. (C) The serum levels of CCL2 from WT and Nod1-deficient (KO) mice treated with 50 μg by a different administration method (i.n. [intranasal], i.p., and oral route) at 6 and 24 h were determined by ELISA. Representative data from four independent experiments are shown. (D) cytokine levels in tissues from mice stimulated with 200 μg KF1B or PBS alone (−) 6 h after i.p. injection was determined by ELISA. Representative data from three independent experiments are shown. The results are representative of at least three experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Nod1 acts as an intracellular receptor to stimulate chemokine production and neutrophil recruitment in vivo

    doi: 10.1084/jem.20051229

    Figure Lengend Snippet: Nod1-dependent CCL2 induction in vivo. (A) WT and Nod1-deficient mice were administrated i.p. with 200 μg KF1B, iE-DAP, MDP, E. coli O55:B5 LPS, or PBS alone. The serum level of CCL2 was determined by ELISA. Because the relative levels of CCL2 induced by MDP was lower than those by KF1B, the CCL2 levels induced by MDP are also given (inset). The CCL2 levels are given with means ± SD from three mice. (B) The serum CXCL2 level from mice stimulated with 50 μg KF1B or PBS alone at indicated times after i.p. injection was determined by ELISA. Representative data from three independent experiments are shown. (C) The serum levels of CCL2 from WT and Nod1-deficient (KO) mice treated with 50 μg by a different administration method (i.n. [intranasal], i.p., and oral route) at 6 and 24 h were determined by ELISA. Representative data from four independent experiments are shown. (D) cytokine levels in tissues from mice stimulated with 200 μg KF1B or PBS alone (−) 6 h after i.p. injection was determined by ELISA. Representative data from three independent experiments are shown. The results are representative of at least three experiments.

    Article Snippet: E. coli O55:B5 LPS was purchased from Sigma-Aldrich.

    Techniques: In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection

    Enhancement of LPS-induced IL-6, IL-1β, and CCL2 secretion by Nod1 stimulation in monocytic MonoMac6, macrophages, and DCs. MonoMac6 cells were treated with the indicated amounts per milliliter of compounds in the absence (−) and presence (+LPS) of 0.7 ng/ml E. coli O55:B5 LPS for 24 h. The levels of IL-6 (A) and IL-1β (B) in the medium were determined by ELISA. (C) BM-derived macrophages (mφ) were treated with 2 μg/ml KF-1B and/or E. coli O55:B5 LPS, and BM-derived DCs were treated with 2 μg/ml KF-1B and/or 10 ng/ml E. coli O55:B5 LPS for 24 h (D) BM-derived mφ from WT and Nod1-deficient (KO) mice were treated with 2 μg/ml KF-1B and/or 10 ng/ml E. coli O55:B5 LPS for 24 h. The levels of CCL2 (C) and IL-6 (D) in the medium were determined by ELISA. The results shown are given as means ± SD of triplicate cultures and are representative of three experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Nod1 acts as an intracellular receptor to stimulate chemokine production and neutrophil recruitment in vivo

    doi: 10.1084/jem.20051229

    Figure Lengend Snippet: Enhancement of LPS-induced IL-6, IL-1β, and CCL2 secretion by Nod1 stimulation in monocytic MonoMac6, macrophages, and DCs. MonoMac6 cells were treated with the indicated amounts per milliliter of compounds in the absence (−) and presence (+LPS) of 0.7 ng/ml E. coli O55:B5 LPS for 24 h. The levels of IL-6 (A) and IL-1β (B) in the medium were determined by ELISA. (C) BM-derived macrophages (mφ) were treated with 2 μg/ml KF-1B and/or E. coli O55:B5 LPS, and BM-derived DCs were treated with 2 μg/ml KF-1B and/or 10 ng/ml E. coli O55:B5 LPS for 24 h (D) BM-derived mφ from WT and Nod1-deficient (KO) mice were treated with 2 μg/ml KF-1B and/or 10 ng/ml E. coli O55:B5 LPS for 24 h. The levels of CCL2 (C) and IL-6 (D) in the medium were determined by ELISA. The results shown are given as means ± SD of triplicate cultures and are representative of three experiments.

    Article Snippet: E. coli O55:B5 LPS was purchased from Sigma-Aldrich.

    Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Mouse Assay

    Cytokine secretion from human intestinal epithelial LoVo cells. LoVo cells were treated with 2 μg/ml of each ligand, except 200 ng/ml for AcMTP and 10 ng/ml TNFα. The levels of IL-8 (A) and CXCL1 (E) were determined by ELISA. (B) LoVo cells were treated with 20 μg/ml KF1B, 5 μg/ml AcMTP, 1 μM CpG, 5 μg/ml polyIC, and 5 μg/ml E. coli O55:B5 LPS. The levels of IL-8 in medium at the indicated times were determined by ELISA. (C) SW620 cells were treated with 5 μg/ml of the indicated ligands. The secretion levels of IL-8 were determined by ELISA. (D) LoVo cells were treated transfected with NF-κB–dependent and –independent reporter by Lipofectamine 2000. 8 h after transfection, cells were treated with 2,000 ng/ml KF1B, iE-DAP, or control meso DAP or left untreated (−). (F) The mRNA levels of CD83, NFKBIA, IL-8, and CXCL1 from cells treated with 2 μg/ml KF1B (closed bars) for 60 min or left untreated (open bars) were determined by real-time PCR analysis. (G) CD83 in lysates from LoVo cells treated with 2,000 ng/ml KF1B for 24 h was detected by immunoblotting with anti-CD83 antibody. The results shown are given as means ± SD of triplicate cultures and were representative of five experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Nod1 acts as an intracellular receptor to stimulate chemokine production and neutrophil recruitment in vivo

    doi: 10.1084/jem.20051229

    Figure Lengend Snippet: Cytokine secretion from human intestinal epithelial LoVo cells. LoVo cells were treated with 2 μg/ml of each ligand, except 200 ng/ml for AcMTP and 10 ng/ml TNFα. The levels of IL-8 (A) and CXCL1 (E) were determined by ELISA. (B) LoVo cells were treated with 20 μg/ml KF1B, 5 μg/ml AcMTP, 1 μM CpG, 5 μg/ml polyIC, and 5 μg/ml E. coli O55:B5 LPS. The levels of IL-8 in medium at the indicated times were determined by ELISA. (C) SW620 cells were treated with 5 μg/ml of the indicated ligands. The secretion levels of IL-8 were determined by ELISA. (D) LoVo cells were treated transfected with NF-κB–dependent and –independent reporter by Lipofectamine 2000. 8 h after transfection, cells were treated with 2,000 ng/ml KF1B, iE-DAP, or control meso DAP or left untreated (−). (F) The mRNA levels of CD83, NFKBIA, IL-8, and CXCL1 from cells treated with 2 μg/ml KF1B (closed bars) for 60 min or left untreated (open bars) were determined by real-time PCR analysis. (G) CD83 in lysates from LoVo cells treated with 2,000 ng/ml KF1B for 24 h was detected by immunoblotting with anti-CD83 antibody. The results shown are given as means ± SD of triplicate cultures and were representative of five experiments.

    Article Snippet: E. coli O55:B5 LPS was purchased from Sigma-Aldrich.

    Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Real-time Polymerase Chain Reaction

    Inhibitory effect of L -Rhamnose monosaccharide and Rha-BSA on rHPL-LPS/bacteria interaction. A total of 0.5 µg of E. coli O55:B5 LPS (A), E. coli O26:B6 (B), S. typhimurium (C), P. aeruginosa (D), or 5×10 7 cells P. aeruginosa PAO1 (E) was coated on 96-well microplates and incubated at 37°C for 3 h or at 4°C overnight. The microplates with the immobilized bacteria were washed, and unbound regions were blocked with BSA. Various concentrations of glycans or glycan-protein conjugates were incubated with 1 µM rHPL and then added to microplates. Anti-His (1∶5000) was used to detect rHPL binding to bacterial cells. Blank refers to microplate wells containing only buffer. The values are the mean ± SD from triplicate experiments. * P

    Journal: PLoS ONE

    Article Title: A Recombinant Horseshoe Crab Plasma Lectin Recognizes Specific Pathogen-Associated Molecular Patterns of Bacteria through Rhamnose

    doi: 10.1371/journal.pone.0115296

    Figure Lengend Snippet: Inhibitory effect of L -Rhamnose monosaccharide and Rha-BSA on rHPL-LPS/bacteria interaction. A total of 0.5 µg of E. coli O55:B5 LPS (A), E. coli O26:B6 (B), S. typhimurium (C), P. aeruginosa (D), or 5×10 7 cells P. aeruginosa PAO1 (E) was coated on 96-well microplates and incubated at 37°C for 3 h or at 4°C overnight. The microplates with the immobilized bacteria were washed, and unbound regions were blocked with BSA. Various concentrations of glycans or glycan-protein conjugates were incubated with 1 µM rHPL and then added to microplates. Anti-His (1∶5000) was used to detect rHPL binding to bacterial cells. Blank refers to microplate wells containing only buffer. The values are the mean ± SD from triplicate experiments. * P

    Article Snippet: Lipopolysaccharides (LPSs) of E. coli O26:B6, E. coli O55:B5, P. aeruginosa sero 10, Salmonella enterica serovar typhimurium and L -Rhamnose (L -Rha) monosaccharide were purchased from Sigma.

    Techniques: Incubation, Binding Assay

    Costimulation and/or restimulation of TLR7 and TLR9 decreased the levels of proinflammatory cytokine production in mouse macrophages. (A) Mouse macrophages were stimulated with either an agonist for TLR7 (GDQ) or TLR9 (ODN 1826) or both (GDQ + ODN1826) overnight (1st), and the production of TNF-α in the supernatant was measured using ELISA. LPS stimulation was introduced as a positive control. □ Raw264.7 macrophage cell line, and ▪ BMDMs from Balb/c mice. (B) After the first stimulation, the cells were washed with PBS and incubated with culture media overnight. Subsequently, the cells were restimulated with TLR agonists for 1 h (2nd), and the production of TNF-α was measured using ELISA. The levels of IL-6 (C) and IL-10 (D) were measured in RAW264.7 cells under the same conditions. Significant differences were indicated as *P

    Journal: Molecules and Cells

    Article Title: The Early Induction of Suppressor of Cytokine Signaling 1 and the Downregulation of Toll-like Receptors 7 and 9 Induce Tolerance in Costimulated Macrophages

    doi: 10.14348/molcells.2015.2136

    Figure Lengend Snippet: Costimulation and/or restimulation of TLR7 and TLR9 decreased the levels of proinflammatory cytokine production in mouse macrophages. (A) Mouse macrophages were stimulated with either an agonist for TLR7 (GDQ) or TLR9 (ODN 1826) or both (GDQ + ODN1826) overnight (1st), and the production of TNF-α in the supernatant was measured using ELISA. LPS stimulation was introduced as a positive control. □ Raw264.7 macrophage cell line, and ▪ BMDMs from Balb/c mice. (B) After the first stimulation, the cells were washed with PBS and incubated with culture media overnight. Subsequently, the cells were restimulated with TLR agonists for 1 h (2nd), and the production of TNF-α was measured using ELISA. The levels of IL-6 (C) and IL-10 (D) were measured in RAW264.7 cells under the same conditions. Significant differences were indicated as *P

    Article Snippet: Reagents Gardiquimod (GDQ) (TLR7 agonist) and lipopolysaccharide (LPS) (TLR4 agonist) were purchased from InvivoGen (USA).

    Techniques: Enzyme-linked Immunosorbent Assay, Positive Control, Mouse Assay, Incubation

    Expression of various molecules on DCs pulsed with HTLV-1. Immature DCs were differentiated from normal healthy donor monocytes, pulsed with 20 CCID 50 of live HTLV-1 or equivalent dose of heat-inactivated virions, and matured by LPS (10 ng/ml). –––, control MAb; ———, indicated MAb. The number in each panel represents the mean fluorescence intensity.

    Journal: Journal of Virology

    Article Title: The Role of Human T-Lymphotropic Virus Type 1 (HTLV-1)-Infected Dendritic Cells in the Development of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis

    doi:

    Figure Lengend Snippet: Expression of various molecules on DCs pulsed with HTLV-1. Immature DCs were differentiated from normal healthy donor monocytes, pulsed with 20 CCID 50 of live HTLV-1 or equivalent dose of heat-inactivated virions, and matured by LPS (10 ng/ml). –––, control MAb; ———, indicated MAb. The number in each panel represents the mean fluorescence intensity.

    Article Snippet: Maturation of DCs was performed by addition of 10 ng of lipopolysaccharide (LPS) from Escherichia coli O111:B4 (Difco Laboratories, Detroit, Mich.) per ml for 24 h, and matured DCs were assessed for APC functions after treatment with 50 μg of mitomycin C per ml.

    Techniques: Expressing, Fluorescence

    Comparison of T-cell-stimulating activities of HTLV-1-infected DCs and inactivated virion-pulsed DCs. (a) Immature DCs were either infected with various doses of HTLV-1 or pulsed with an equivalent dose of heat-inactivated virions, subsequently matured by LPS, and cocultured with autologous unseparated, CD4 + and CD8 + T cells (10 5 /well) for 5 days at a DC:T-cell ratio of 1:20. (b) LPS-matured DCs were exposed to live or inactivated virus for 4 days and cocultured with various T cells (10 5 /well) for 5 days at a DC:T-cell ratio of 1:20. A representative result of three independent experiments is shown. Each experiment was performed in triplicate.

    Journal: Journal of Virology

    Article Title: The Role of Human T-Lymphotropic Virus Type 1 (HTLV-1)-Infected Dendritic Cells in the Development of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis

    doi:

    Figure Lengend Snippet: Comparison of T-cell-stimulating activities of HTLV-1-infected DCs and inactivated virion-pulsed DCs. (a) Immature DCs were either infected with various doses of HTLV-1 or pulsed with an equivalent dose of heat-inactivated virions, subsequently matured by LPS, and cocultured with autologous unseparated, CD4 + and CD8 + T cells (10 5 /well) for 5 days at a DC:T-cell ratio of 1:20. (b) LPS-matured DCs were exposed to live or inactivated virus for 4 days and cocultured with various T cells (10 5 /well) for 5 days at a DC:T-cell ratio of 1:20. A representative result of three independent experiments is shown. Each experiment was performed in triplicate.

    Article Snippet: Maturation of DCs was performed by addition of 10 ng of lipopolysaccharide (LPS) from Escherichia coli O111:B4 (Difco Laboratories, Detroit, Mich.) per ml for 24 h, and matured DCs were assessed for APC functions after treatment with 50 μg of mitomycin C per ml.

    Techniques: Infection

    LPS profiles of Pseudomonas syringae pv. actinidiae ( Psa ) and P. syringae pv. actinidifoliorum ( Pfm ). (A) Pro-Q™ Emerald 300 staining of lipopolysaccharide (LPS) preparations on a NuPAGE 4–12% gradient bis-tricine SDS gel from representatives of Pfm lineages (L3 ICMP 18807, lane 2; L1 ICMP 18803, lane 8), Psa biovars (BV1 ICMP 9617, lane 3; BV2 ICMP 19071, lane 4; BV3 ICMP 18884, lane 5; BV5 MAFF212063, lane 6; BV6 MAFF 212141, lane 7), with Escherichia coli standards (Std; lanes 1 and 9). A total of 0.5–15 μL LPS extract for each sample (normalized by trial runs) was applied per lane. (B) Western blot of LPS extracts from representatives of Psa biovars and Pfm lineages run on a NuPAGE 4– 12% gradient bis-tricine SDS gel, obtained by probing with polyclonal antibodies to Psa BV3 ICMP 18884 (Bi) , Psa BV1 ICMP 9617 (Bii) , or Pfm L1 ICMP 18803 (Biii) in a 5000:1 ratio.

    Journal: bioRxiv

    Article Title: Variation at the common polysaccharide antigen locus drives lipopolysaccharide diversity within the P. syringae species complex

    doi: 10.1101/2020.03.31.019141

    Figure Lengend Snippet: LPS profiles of Pseudomonas syringae pv. actinidiae ( Psa ) and P. syringae pv. actinidifoliorum ( Pfm ). (A) Pro-Q™ Emerald 300 staining of lipopolysaccharide (LPS) preparations on a NuPAGE 4–12% gradient bis-tricine SDS gel from representatives of Pfm lineages (L3 ICMP 18807, lane 2; L1 ICMP 18803, lane 8), Psa biovars (BV1 ICMP 9617, lane 3; BV2 ICMP 19071, lane 4; BV3 ICMP 18884, lane 5; BV5 MAFF212063, lane 6; BV6 MAFF 212141, lane 7), with Escherichia coli standards (Std; lanes 1 and 9). A total of 0.5–15 μL LPS extract for each sample (normalized by trial runs) was applied per lane. (B) Western blot of LPS extracts from representatives of Psa biovars and Pfm lineages run on a NuPAGE 4– 12% gradient bis-tricine SDS gel, obtained by probing with polyclonal antibodies to Psa BV3 ICMP 18884 (Bi) , Psa BV1 ICMP 9617 (Bii) , or Pfm L1 ICMP 18803 (Biii) in a 5000:1 ratio.

    Article Snippet: Gels were stained using the Pro- Q™ Emerald 300 Lipopolysaccharide Gel Stain Kit (Molecular Probes, OR, USA).

    Techniques: Staining, SDS-Gel, Western Blot

    Effects of CPA TET operon swap on LPS banding and immunological recognition in Pseudomonas syringae pv. actinidiae ( Psa ). (A) Genetic polymorphism of the TET operon for Psa BV1 (ICMP 9617) versus BV3 (ICMP 18884). Genetic sequences aligned in Geneious v10. Genome sequences are indicated by light grey bars with associated translated regions as black bars, operons are annotated in light blue, genes in light green with gene names indicated, coding sequences in red with predicted biochemical function indicated, the original TET operon from BV1 indicated in dark green with upstream (KO-5’) and downstream (KO-3’) regions for generating the LPS TET operon knock-out in grey, and the TET operon swap region by knock-in from BV3 indicated in dark blue. (B) LPS profiles of Psa BV3 (lane 1 and lane 8), BV1 (lane 2), BV1 TET operon knockout (Δ LPS ; lane 3), four BV1-to-BV3 TET operon complemented isolates (Δ LPS + LPS _BV3 1-2, 1-4, 2-1, and 2-5; lanes 4–7), and one BV1 revertant to knock-out isolates (ΔLPS-LPSrev 1-3; lane 9). Pro-Q™ Emerald 300 staining of LPS preparations on a 4–12% gradient Tris-glycine SDS gel. LPS extract (15 μL) for each sample was applied per lane. (C) Immuno-blot of LPS extracts from wild-type, knock-out (KO), KO-complemented, and KO-revertant strains run on a gradient SDS-PAGE gel as for (A), probed with polyclonal antibodies to Psa BV3 in a 5000:1 ratio.

    Journal: bioRxiv

    Article Title: Variation at the common polysaccharide antigen locus drives lipopolysaccharide diversity within the P. syringae species complex

    doi: 10.1101/2020.03.31.019141

    Figure Lengend Snippet: Effects of CPA TET operon swap on LPS banding and immunological recognition in Pseudomonas syringae pv. actinidiae ( Psa ). (A) Genetic polymorphism of the TET operon for Psa BV1 (ICMP 9617) versus BV3 (ICMP 18884). Genetic sequences aligned in Geneious v10. Genome sequences are indicated by light grey bars with associated translated regions as black bars, operons are annotated in light blue, genes in light green with gene names indicated, coding sequences in red with predicted biochemical function indicated, the original TET operon from BV1 indicated in dark green with upstream (KO-5’) and downstream (KO-3’) regions for generating the LPS TET operon knock-out in grey, and the TET operon swap region by knock-in from BV3 indicated in dark blue. (B) LPS profiles of Psa BV3 (lane 1 and lane 8), BV1 (lane 2), BV1 TET operon knockout (Δ LPS ; lane 3), four BV1-to-BV3 TET operon complemented isolates (Δ LPS + LPS _BV3 1-2, 1-4, 2-1, and 2-5; lanes 4–7), and one BV1 revertant to knock-out isolates (ΔLPS-LPSrev 1-3; lane 9). Pro-Q™ Emerald 300 staining of LPS preparations on a 4–12% gradient Tris-glycine SDS gel. LPS extract (15 μL) for each sample was applied per lane. (C) Immuno-blot of LPS extracts from wild-type, knock-out (KO), KO-complemented, and KO-revertant strains run on a gradient SDS-PAGE gel as for (A), probed with polyclonal antibodies to Psa BV3 in a 5000:1 ratio.

    Article Snippet: Gels were stained using the Pro- Q™ Emerald 300 Lipopolysaccharide Gel Stain Kit (Molecular Probes, OR, USA).

    Techniques: Knock-Out, Knock-In, Staining, SDS-Gel, SDS Page

    (A) Phylogenetic comparison of TET operons from various Pseudomonas syringae strains. Amino acid sequences from three proteins of the CPA locus (Wzt, WbdD and WbdA) were aligned in Geneious v10 and neighbour-joining phylogenetic trees built for their TET operons. Strains were grouped into numbered clades as indicated. Strains are coloured according to their core genome phylogenies as previously described ( Dillon et al. , 2019b ): phylogroup 1 blue; phylogroup 2 red; phylogroup 3 green; phylogroup 4 orange; phylogroup 5 purple; phylogroup 6 yellow; phylogroup 7 pink; phylogroup 9 grey; phylogroup 10 mauve; phylogroup 11 chocolate; phylogroup 13 claret. (B) LPS profiles of Pseudomonas syringae representing phylogenetic clades for the TET operon. Pro-Q™ Emerald 300 staining of lipopolysaccharide (LPS) preparations on a 4–12% gradient Tris-glycine SDS gel from representatives of P. syringae strains marked with asterisks from Supplementary Table 1 (top to bottom, lanes 1–11), with Escherichia coli control (Std, lane 12). Banding patterns were grouped into clades on their designations from Figure 7. A total of 2–15 μL LPS extract for each sample (normalized by trial runs) was applied per lane.

    Journal: bioRxiv

    Article Title: Variation at the common polysaccharide antigen locus drives lipopolysaccharide diversity within the P. syringae species complex

    doi: 10.1101/2020.03.31.019141

    Figure Lengend Snippet: (A) Phylogenetic comparison of TET operons from various Pseudomonas syringae strains. Amino acid sequences from three proteins of the CPA locus (Wzt, WbdD and WbdA) were aligned in Geneious v10 and neighbour-joining phylogenetic trees built for their TET operons. Strains were grouped into numbered clades as indicated. Strains are coloured according to their core genome phylogenies as previously described ( Dillon et al. , 2019b ): phylogroup 1 blue; phylogroup 2 red; phylogroup 3 green; phylogroup 4 orange; phylogroup 5 purple; phylogroup 6 yellow; phylogroup 7 pink; phylogroup 9 grey; phylogroup 10 mauve; phylogroup 11 chocolate; phylogroup 13 claret. (B) LPS profiles of Pseudomonas syringae representing phylogenetic clades for the TET operon. Pro-Q™ Emerald 300 staining of lipopolysaccharide (LPS) preparations on a 4–12% gradient Tris-glycine SDS gel from representatives of P. syringae strains marked with asterisks from Supplementary Table 1 (top to bottom, lanes 1–11), with Escherichia coli control (Std, lane 12). Banding patterns were grouped into clades on their designations from Figure 7. A total of 2–15 μL LPS extract for each sample (normalized by trial runs) was applied per lane.

    Article Snippet: Gels were stained using the Pro- Q™ Emerald 300 Lipopolysaccharide Gel Stain Kit (Molecular Probes, OR, USA).

    Techniques: Staining, SDS-Gel

    Cytokine production in LPS-stimulated peritoneal macrophages and phagocytosis function of control mice and mice with high-fat diet induced obesity, administered or not B. uniformis CECT 7771. SD: standard diet group (control) (n = 6); SD+B: standard diet group receiving a daily dose of 5.0×10 8 CFU B. uniformis CECT 7771 by gavage for 7 weeks (n = 6); HFD: high fat diet group (n = 6); HFD+B: high fat diet group receiving a daily dose of 5.0×10 8 CFU B. uniformis CECT 7771 by gavage for 7 weeks (n = 6). In the cytokine production study, peritoneal macrophages were stimulated with purified lipopolysaccharide (LPS) from S. enterica serotype Typhimurium ( Figure 6A ). Non-stimulated peritoneal macrophages were evaluated as controls of basal cytokine levels. In the phagocytosis study ( Figure 6B ), evidence of oxygen-radical production by macrophages was determined by the NBT test after in vitro interaction with a bacterial extract. Figure 6A: TNF- α and IL-10 cytokines produced by LPS-stimulated macrophages; Figure 6B: % NBT (+) cells. Data are expressed as mean and standard deviation of duplicate measurements determined in two independent experiments. Statistically significant differences of data are established at P

    Journal: PLoS ONE

    Article Title: Bacteroides uniformis CECT 7771 Ameliorates Metabolic and Immunological Dysfunction in Mice with High-Fat-Diet Induced Obesity

    doi: 10.1371/journal.pone.0041079

    Figure Lengend Snippet: Cytokine production in LPS-stimulated peritoneal macrophages and phagocytosis function of control mice and mice with high-fat diet induced obesity, administered or not B. uniformis CECT 7771. SD: standard diet group (control) (n = 6); SD+B: standard diet group receiving a daily dose of 5.0×10 8 CFU B. uniformis CECT 7771 by gavage for 7 weeks (n = 6); HFD: high fat diet group (n = 6); HFD+B: high fat diet group receiving a daily dose of 5.0×10 8 CFU B. uniformis CECT 7771 by gavage for 7 weeks (n = 6). In the cytokine production study, peritoneal macrophages were stimulated with purified lipopolysaccharide (LPS) from S. enterica serotype Typhimurium ( Figure 6A ). Non-stimulated peritoneal macrophages were evaluated as controls of basal cytokine levels. In the phagocytosis study ( Figure 6B ), evidence of oxygen-radical production by macrophages was determined by the NBT test after in vitro interaction with a bacterial extract. Figure 6A: TNF- α and IL-10 cytokines produced by LPS-stimulated macrophages; Figure 6B: % NBT (+) cells. Data are expressed as mean and standard deviation of duplicate measurements determined in two independent experiments. Statistically significant differences of data are established at P

    Article Snippet: Media were changed before stimulation and, then, cells were incubated in the presence of 100 µl of a cell suspension (1×107 cfu/ml) of each Bacteroides strain for 24 h. Purified LPS from Salmonella enterica serotype Typhimurium (Sigma Chemical Co, Madrid, Spain) was used at a concentration of 1 µg/ml as a positive control.

    Techniques: Mouse Assay, Purification, In Vitro, Produced, Standard Deviation

    Influence of LPS stimuli on cytokine production and activation of T-lymphocyte proliferation by dendritic cells (DCs) generated from control mice and mice with high-fat diet induced obesity, administered or not B. uniformis CECT 777. SD: standard diet group (control) (n = 6); SD+B: standard diet group receiving a daily dose of 5.0×10 8 CFU B. uniformis CECT 7771 by gavage for 7 weeks (n = 6); HFD: high fat diet group (n = 6); HFD+B: high fat diet group receiving a daily dose of 5.0×10 8 CFU B. uniformis CECT 7771 by gavage for 7 weeks (n = 6). In the cytokine production study, DCs were stimulated with purified lipopolysaccharide (LPS) from S. enterica serotype Typhimurium ( Figure 7A ). Non-stimulated DCs were evaluated as controls of basal cytokine levels. In the lymphocyte proliferation study ( Figure 7B ), matured DCs were used for priming a T-cell proliferative response at the following LT/CD ratios: 1∶1, 1∶2, 1∶4. Lymphocyte proliferation was measured with the cell proliferation ELISA BrdU-colorimetric assay. Figure 7A: TNF- α and IL-10 cytokines produced by LPS-stimulated CDs; Figure 7B: Lymphocyte proliferation. Data are expressed as means ± SD of duplicate measures determined in two independent experiments. Statistically significant differences of data are established at P

    Journal: PLoS ONE

    Article Title: Bacteroides uniformis CECT 7771 Ameliorates Metabolic and Immunological Dysfunction in Mice with High-Fat-Diet Induced Obesity

    doi: 10.1371/journal.pone.0041079

    Figure Lengend Snippet: Influence of LPS stimuli on cytokine production and activation of T-lymphocyte proliferation by dendritic cells (DCs) generated from control mice and mice with high-fat diet induced obesity, administered or not B. uniformis CECT 777. SD: standard diet group (control) (n = 6); SD+B: standard diet group receiving a daily dose of 5.0×10 8 CFU B. uniformis CECT 7771 by gavage for 7 weeks (n = 6); HFD: high fat diet group (n = 6); HFD+B: high fat diet group receiving a daily dose of 5.0×10 8 CFU B. uniformis CECT 7771 by gavage for 7 weeks (n = 6). In the cytokine production study, DCs were stimulated with purified lipopolysaccharide (LPS) from S. enterica serotype Typhimurium ( Figure 7A ). Non-stimulated DCs were evaluated as controls of basal cytokine levels. In the lymphocyte proliferation study ( Figure 7B ), matured DCs were used for priming a T-cell proliferative response at the following LT/CD ratios: 1∶1, 1∶2, 1∶4. Lymphocyte proliferation was measured with the cell proliferation ELISA BrdU-colorimetric assay. Figure 7A: TNF- α and IL-10 cytokines produced by LPS-stimulated CDs; Figure 7B: Lymphocyte proliferation. Data are expressed as means ± SD of duplicate measures determined in two independent experiments. Statistically significant differences of data are established at P

    Article Snippet: Media were changed before stimulation and, then, cells were incubated in the presence of 100 µl of a cell suspension (1×107 cfu/ml) of each Bacteroides strain for 24 h. Purified LPS from Salmonella enterica serotype Typhimurium (Sigma Chemical Co, Madrid, Spain) was used at a concentration of 1 µg/ml as a positive control.

    Techniques: Activation Assay, Generated, Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, Produced

    CD14 bright /CD56+ monocyte subset is expanded in older healthy controls and produces more cytokines in response to lipopolysaccharide. (a) Representative dot plot of CD14 and CD56 expression on monocytes. Three relevant subpopulations are separated by the quadrants and marked by the arrows in the right panel. APC, allophycocyanin; FITC, fluorescein isothiocyanate; FL1-H, fluorescence intensity on channel that detects emissions from fluorescein isothiocyanate. (b) Scatterplot showing the correlation between age and the peripheral blood frequencies of CD14 bright /CD56+ monocytes in healthy controls. (c) Bar graphs depict the frequency of tumor necrosis factor-positive (TNF+) ( n = 5), interleukin 10-positive (IL-10+) ( n = 8) and IL-23+ ( n = 7) cells and the mean intracellular IL-1β content ( n = 7) in CD56+ and CD56– monocytes of healthy controls in response to lipopolysaccharide. (d) Spontaneous reactive oxygen intermediate (ROI) production of CD56– and CD56+ monocytes ( n = 4).

    Journal: Arthritis Research & Therapy

    Article Title: CD56+ monocytes have a dysregulated cytokine response to lipopolysaccharide and accumulate in rheumatoid arthritis and immunosenescence

    doi: 10.1186/ar4321

    Figure Lengend Snippet: CD14 bright /CD56+ monocyte subset is expanded in older healthy controls and produces more cytokines in response to lipopolysaccharide. (a) Representative dot plot of CD14 and CD56 expression on monocytes. Three relevant subpopulations are separated by the quadrants and marked by the arrows in the right panel. APC, allophycocyanin; FITC, fluorescein isothiocyanate; FL1-H, fluorescence intensity on channel that detects emissions from fluorescein isothiocyanate. (b) Scatterplot showing the correlation between age and the peripheral blood frequencies of CD14 bright /CD56+ monocytes in healthy controls. (c) Bar graphs depict the frequency of tumor necrosis factor-positive (TNF+) ( n = 5), interleukin 10-positive (IL-10+) ( n = 8) and IL-23+ ( n = 7) cells and the mean intracellular IL-1β content ( n = 7) in CD56+ and CD56– monocytes of healthy controls in response to lipopolysaccharide. (d) Spontaneous reactive oxygen intermediate (ROI) production of CD56– and CD56+ monocytes ( n = 4).

    Article Snippet: Measurement of cytokine production PBMCs were stimulated for four hours (to measure TNF production) or sixteen hours (to measure IL-10, IL-23 and IL-1β production) with 100 ng/ml ultrapure lipopolysaccharide (LPS) (InvivoGen, Toulouse, France) or were left untreated.

    Techniques: Expressing, Fluorescence

    OSM producing neutrophils did not express a classical N1 phenotype (A) OSM + neutrophils contained a distinct population of Arg1 + MPO hi cells, representative data. (B) 3.7±1.3% of cells in the CD45 gate were OSM + ; within the OSM + gate, 56.0±8.2% of the cells were CD16 + IL-5R− neutrophils. Within the neutrophil gate 72.56± 11.9% of the cells were Arg1 + MPO hi , and 22.1± 8.0% were Arg1 − MPO lo , n=5. (C) Blood neutrophils were either left untreated, or treated under N1 polarizing conditions (LPS/IFNγ), N2 polarizing conditions (IL-4/IL-13) or with GM-CSF. GM-CSF treated neutrophils secreted elevated levels of OSM into the cell culture supernatants, while N1 and N2 polarizing conditions did not induce OSM, (n=4–7, p

    Journal: The Journal of allergy and clinical immunology

    Article Title: Neutrophils are a major source of the epithelial barrier disrupting cytokine Oncostatin M in mucosal airways disease

    doi: 10.1016/j.jaci.2016.10.039

    Figure Lengend Snippet: OSM producing neutrophils did not express a classical N1 phenotype (A) OSM + neutrophils contained a distinct population of Arg1 + MPO hi cells, representative data. (B) 3.7±1.3% of cells in the CD45 gate were OSM + ; within the OSM + gate, 56.0±8.2% of the cells were CD16 + IL-5R− neutrophils. Within the neutrophil gate 72.56± 11.9% of the cells were Arg1 + MPO hi , and 22.1± 8.0% were Arg1 − MPO lo , n=5. (C) Blood neutrophils were either left untreated, or treated under N1 polarizing conditions (LPS/IFNγ), N2 polarizing conditions (IL-4/IL-13) or with GM-CSF. GM-CSF treated neutrophils secreted elevated levels of OSM into the cell culture supernatants, while N1 and N2 polarizing conditions did not induce OSM, (n=4–7, p

    Article Snippet: Neutrophil cultures were treated with GM-CSF (1–25 ng/mL, R & D Systems, Minneapolis, MN), FSTL1 (100–1000 ng/mL, R & D Systems), Interferon-γ (IFNγ) (10 ng/mL, R & D Systems), lipopolysaccharide (LPS) (1 μg/mL, Enzo Life Sciences, Farmingdale, NY), Interleukin 4 (IL-4) (20 ng/mL, R & D Systems), Interleukin 13 (IL-13) (20 ng/mL, R & D Systems), Interleukin 25 (IL-25) (10–100ng/mL, R & D Systems), Interleukin 33 (IL-33) (10–100 ng/mL, R & D Systems), thymic stromal lymphopoietin (TSLP) (10–100 ng/mL, R & D Systems) or Leukotriene C4 (LTC4 ) (10−6 –10−7 Cayman Chemical, Ann Arbor, MI) for 20 hours, cell culture supernatants were collected for protein analysis and cell lysates were collected to isolate RNA.

    Techniques: Cell Culture