Journal: PLoS ONE

Article Title: Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

doi: 10.1371/journal.pone.0121734

Figure Lengend Snippet: Transcriptional profiling of PHH infected with HCVcc at an MOI of 0.2. (A) Relative expression of genes involved in the PAMP recognition pathway showing data from three replicates at 24 h or 48 h post-infection normalized to naive PHH at the same time points. HUGO nomenclature for the genes is to the left. Statistical analysis was performed using one-way ANOVA and P values reported in (B) Expression levels of IFNAR1 and IL28RA transcripts in naive (closed circle) and HCVcc-infected (open circle) PHH. The data points represent the RMA values from each individual analyte and horizontal bars indicate the mean levels from three biological replicates. The data shown is representative of two independent experiments.

Article Snippet: Briefly, iHLCs were infected with HCVcc at multiplicity of infection (MOI) of 0.2 and incubated for 4–6 days in presence or absence of a neutralizing monoclonal antibody directed against the extracellular domain of the IL28RA receptor subunit (ZymoGenetics).

Techniques: Infection, Expressing

Journal: PLoS ONE

Article Title: Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

doi: 10.1371/journal.pone.0121734

Figure Lengend Snippet: iHLCs were infected with HCVcc (MOI of 0.2), GT-1b HCVser or mock infected, and subsequently maintained in culture with medium replacement every 2 days. At 24 h post-infection, the NS3 PI ASV (0.5 μM) or vehicle control dimethyl sulfoxide (DMSO) was added to cell cultures during media replenishment. (A) Persistent HCVcc replication as measured by the detection of the virally-encoded core antigen and expression of type I (IFNAR1, IFNAR2) and type III IFN (IL28RA, IL10RB) co-receptor subunits in cells were monitored by Western immunoblotting at the indicated time points, with β-actin used as loading control. Right panel; Relative intensity of IFNAR1 detected in HCVcc infected cells maintained in the presence (open triangle) or absence (open square) of ASV treatment was quantified by densitometry analysis, normalized to β-actin levels in each sample, and expressed as a percentage relative to mock-infected cells. Data are representative of three independent Western immunoblot analyses. (B) Co-localization of IFNAR1 and HCV-core positive iHLCs were assessed by fluorescence microscopy. On Day 6 post-infection, naive and HCVcc-infected iHLCs were fixed, permeabilized and stained with antibodies to IFNAR1 (red) and HCV core antigen (green) as indicated. Nuclei were counterstained with Hoechst dye (blue) as shown in the overlays. (C) iHLC cultures infected in parallel with GT-1b HCVser or HCVcc were harvested at the indicated time points and IFNAR1 copy numbers estimated by quantitative RT-PCR following normalization to cellular GAPDH levels in each sample. Results are expressed as mean ± standard deviations (n = 4). Statistical analysis was performed by Bonferroni’s multiple comparison tests: **, P < 0.05; ***, P < 0.001. (D) Cell lysates were harvested at the indicated time points and protein levels of IFNAR1 and IL28RA were examined by Western immunoblotting. Susceptibility of HCVser to the NS3 PI ASV was determined using an antibody directed against NS3. Arrows indicate the presence of the processed and unprocessed forms of the HCV-encoded NS3/4A protease in infected iHLCs. Detection of β-actin served as loading control. Right panel; Relative intensity of IFNAR1 detected in HCVser infected cells maintained in the presence (open triangle) or absence (open square) of ASV treatment was quantified by densitometry analysis, normalized to β-actin levels in each sample, and expressed as a percentage relative to mock-infected cells. Data are representative of two independent Western immunoblot analyses.

Article Snippet: Briefly, iHLCs were infected with HCVcc at multiplicity of infection (MOI) of 0.2 and incubated for 4–6 days in presence or absence of a neutralizing monoclonal antibody directed against the extracellular domain of the IL28RA receptor subunit (ZymoGenetics).

Techniques: Infection, Control, Expressing, Western Blot, Fluorescence, Microscopy, Staining, Quantitative RT-PCR, Comparison

Journal: PLoS ONE

Article Title: Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

doi: 10.1371/journal.pone.0121734

Figure Lengend Snippet: (A) Naive and HCVcc infected iHLC cultures maintained in presence or absence of the IL28RA nAb were treated for 15 minutes with 10 ng/mL or 100 ng/mL of alfa or Lambda. Cell lysates were then prepared, and equal amounts of proteins subjected to Western immunoblotting to examine the levels of STAT1 phosphorylation using an antibody directed against phospho STAT1 (pSTAT1; Tyr701). Detection of total STAT1 served as loading control to ensure that equivalent amounts of protein were analyzed among samples. (B) Phosphorylation of STAT1 in iHLCs was evaluated upon stimulation using the Luminex bead-based assay. MFI values were reported as mean values of three independent cultures. Error bars show the standard deviations. Two-way ANOVA statistical analysis was performed using Bonferroni post test: ***, P < 0.001.

Article Snippet: Briefly, iHLCs were infected with HCVcc at multiplicity of infection (MOI) of 0.2 and incubated for 4–6 days in presence or absence of a neutralizing monoclonal antibody directed against the extracellular domain of the IL28RA receptor subunit (ZymoGenetics).

Techniques: Infection, Western Blot, Phospho-proteomics, Control, Luminex, Bead-based Assay

Journal: PLoS ONE

Article Title: Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

doi: 10.1371/journal.pone.0121734

Figure Lengend Snippet: (A) Expression of Mx1 protein (red) and HCV-core antigen (green) was monitored in HCVcc-infected iHLC cultures maintained in presence or absence of the IL28RA nAb (10 μg/mL). Dual immunostaining was performed as described in the legend for . Nuclei were counterstained with Hoechst dye (blue) as shown in the overlays. (B) Induction of Mx1 expression (red) was assessed in Naive iHLCs 24 h following stimulation with 10 ng/mL of alfa (a) or Lambda (b). iHLC cultures infected with HCVcc were maintained in the presence (left panels) or absence (right panels) of the IL28RA nAb (10 μg/mL). On Day 6 post-infection, cells were treated as indicated with 10 ng/mL of alfa or Lambda for 24 h, and then dual immunostaining was performed using antibodies directed against Mx1 (red) and the HCV-core antigen (green). Arrows indicate examples of different HCV-Mx1 co-localization patterns in overlaid optical field. Scale bar, 60 μm.

Article Snippet: Briefly, iHLCs were infected with HCVcc at multiplicity of infection (MOI) of 0.2 and incubated for 4–6 days in presence or absence of a neutralizing monoclonal antibody directed against the extracellular domain of the IL28RA receptor subunit (ZymoGenetics).

Techniques: Expressing, Infection, Immunostaining

Journal: BMC Veterinary Research

Article Title: Pro-inflammatory cytokines and structural biomarkers are effective to categorize osteoarthritis phenotype and progression in Standardbred racehorses over five years of racing career

doi: 10.1186/s12917-016-0873-7

Figure Lengend Snippet: Temporal course of OA biomarkers levels and corresponding clinical scores in the PTOA group. Percentual variations of OA biomarker levels and corresponding radiographic and lameness scores, in the PTOA group of racehorses ( n = 25), during 5 years of racing activity. Panel a refers to values in the sera. Panel b refers to the synovial fluid values. Data for each time point represent the mean levels of IL-1ß, IL-6, TNF-α, CTXII, COMP, Radiographic score and Lameness score. Standard deviation (SD) error bars are not plotted in these graphs for clarity. Reference value at T0 is delineated (dotted line). * p < 0.05 versus level at previous time-point. ** p < 0.001 versus level at previous time-point

Article Snippet: Interleukin-6 concentrations were measured with a commercially available sandwich ELISA kit using a mouse monoclonal antibody raised against equine IL-6 (SEA079Eq, USCN Life Science Inc., Wuhan, China).

Techniques: Biomarker Assay, Activity Assay, Standard Deviation

Journal:

Article Title: Skeletal muscle dictates the fibrinolytic state after exercise training in overweight men with characteristics of metabolic syndrome

doi: 10.1113/jphysiol.2002.036616

Figure Lengend Snippet: Affymetrix and GeneSpring expression analysis of haemostasis genes

Article Snippet: Membranes were then incubated for 2 h at room temperature with primary antibodies (t-PA and PAI-1 antibodies (Santa Cruz Biotech); β-thromboglobulin/CTAP III antibody (Affiland, Liége, Belgium); tetranectin antibody (AntibodyShop, Copenhagen, Denmark)), washed and incubated for 1 h at room temperature with peroxidase-conjugated secondary antibodies.

Techniques: Expressing

Journal:

Article Title: Skeletal muscle dictates the fibrinolytic state after exercise training in overweight men with characteristics of metabolic syndrome

doi: 10.1113/jphysiol.2002.036616

Figure Lengend Snippet: Representative acetone-fixed cryosections of skeletal muscle from entry (A) and exercise-trained (B) STRRIDE participants. Given the variable nature of tetranectin immunostaining, images were taken in regions empirically presenting the ‘strongest’ fluorescent signals in each biopsy. Bar = 50 μm.

Article Snippet: Membranes were then incubated for 2 h at room temperature with primary antibodies (t-PA and PAI-1 antibodies (Santa Cruz Biotech); β-thromboglobulin/CTAP III antibody (Affiland, Liége, Belgium); tetranectin antibody (AntibodyShop, Copenhagen, Denmark)), washed and incubated for 1 h at room temperature with peroxidase-conjugated secondary antibodies.

Techniques: Immunostaining

Journal:

Article Title: Skeletal muscle dictates the fibrinolytic state after exercise training in overweight men with characteristics of metabolic syndrome

doi: 10.1113/jphysiol.2002.036616

Figure Lengend Snippet: A, the relative expression levels of tetranectin in subject plasma as determined by ELISA. B, a representative immunoblot of subject plasma. C, quantification of plasma immunoblots. Panels A and C show the mean ±s.e.m. of results from three subjects normalized to the entry sample.

Article Snippet: Membranes were then incubated for 2 h at room temperature with primary antibodies (t-PA and PAI-1 antibodies (Santa Cruz Biotech); β-thromboglobulin/CTAP III antibody (Affiland, Liége, Belgium); tetranectin antibody (AntibodyShop, Copenhagen, Denmark)), washed and incubated for 1 h at room temperature with peroxidase-conjugated secondary antibodies.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IL-32, a proinflammatory cytokine in rheumatoid arthritis

doi: 10.1073/pnas.0511233103

Figure Lengend Snippet: IL-32 correlations with inflammation and cytokines

Article Snippet: Monoclonal antibodies against human IL-1β (IgM and AB-1) were obtained from Oncogene Science, and monoclonal antibodies against human IL-18 (IgG and AF 318) were purchased from R & D Systems.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IL-32, a proinflammatory cytokine in rheumatoid arthritis

doi: 10.1073/pnas.0511233103

Figure Lengend Snippet: Joint inflammation provoked by local IL-32γ injection. (A) Joint swelling after i.a. injection of 100 ng of recombinant IL-32γ into the right knee joints of C57/Bl6 mice, determined by the 99mTc-uptake method. IL-32 was compared with 100 ng of either murine TNFα or murine IL-1β. Polymyxin B (7 ng) was injected as control. (B) Histopathology at day 2 after i.a. injection of polymyxin B (7 ng). H&E staining was performed. (Original magnification, ×100.) (C) Joint inflammation after i.a. injection of 100 ng of IL-32γ. H&E staining was performed. (Original magnification, ×100.) (D) Severe cell influx in joint cavity and synovial tissue. H&E staining was performed. (Original magnification, ×200.) (E) Monocyte/macrophage-like cells at day 4 after IL-32γ injection. H&E staining was performed. (Original magnification, ×400.) (F) No cartilage matrix proteoglycan loss at day 4 after polymyxin B injection, visualized by Safranin O staining. (Original magnification, ×200.) (G) Depletion of cartilage proteoglycans at day 4 after IL-32γ exposure.

Article Snippet: Monoclonal antibodies against human IL-1β (IgM and AB-1) were obtained from Oncogene Science, and monoclonal antibodies against human IL-18 (IgG and AF 318) were purchased from R & D Systems.

Techniques: Injection, Recombinant, Histopathology, Staining

Journal:

Article Title: Lipoteichoic Acids from Lactobacillus johnsonii Strain La1 and Lactobacillus acidophilus Strain La10 Antagonize the Responsiveness of Human Intestinal Epithelial HT29 Cells to Lipopolysaccharide and Gram-Negative Bacteria

doi: 10.1128/IAI.70.4.2057-2064.2002

Figure Lengend Snippet: Effects of LTA from L. johnsonii strain La1 and L. acidophilus strain La10 on the release of IL-8 by HT29 cells challenged with LPS purified from E. coli. IL-8 production was measured by ELISA in supernatants of HT29 cells incubated for 24 h in medium supplemented with 2% HM in the presence of E. coli LPS at 10 ng/ml (•) or 100 ng/ml (▪) and various amounts of LTA from L. johnsonii strain La1 (A) or L. acidophilus strain La10 (B). Activation of HT29 cells by LPS plus HM alone is depicted (dashed lines). The standard deviations are indicated by the error bars. The results are representative of seven independent experiments for La1 LTA and three independent experiments for La10 LTA.

Article Snippet: Briefly, the wells of 96-well Maxisorp plates (Nunc) were coated with monoclonal antibodies against IL-8 (2 μg/ml) (ImmunoKontact; AMS Biotechnology, Lugano, Switzerland) by overnight incubation at 4°C.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Incubation, Activation Assay

Journal:

Article Title: Lipoteichoic Acids from Lactobacillus johnsonii Strain La1 and Lactobacillus acidophilus Strain La10 Antagonize the Responsiveness of Human Intestinal Epithelial HT29 Cells to Lipopolysaccharide and Gram-Negative Bacteria

doi: 10.1128/IAI.70.4.2057-2064.2002

Figure Lengend Snippet: Effect of LTA from L. johnsonii strain La1 on the release of IL-8 by HT29 cells challenged with whole E. coli bacteria. IL-8 production was measured by ELISA in supernatants of HT29 cells incubated for 24 h in medium supplemented with 2% HM and whole E. coli bacteria (2.5 × 105/ml) (▪) or E. coli LPS (100 ng/ml) (•) in the presence of various amounts of LTA from L. johnsonii strain La1. Activation of HT29 cells in the absence of LTA is depicted (dashed lines).The standard deviations are indicated by the error bars. The results are representative of two independent experiments.

Article Snippet: Briefly, the wells of 96-well Maxisorp plates (Nunc) were coated with monoclonal antibodies against IL-8 (2 μg/ml) (ImmunoKontact; AMS Biotechnology, Lugano, Switzerland) by overnight incubation at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Activation Assay

Journal:

Article Title: Lipoteichoic Acids from Lactobacillus johnsonii Strain La1 and Lactobacillus acidophilus Strain La10 Antagonize the Responsiveness of Human Intestinal Epithelial HT29 Cells to Lipopolysaccharide and Gram-Negative Bacteria

doi: 10.1128/IAI.70.4.2057-2064.2002

Figure Lengend Snippet: Effects of deacylation of the LTAs on their antagonistic activity. HT29 cells were challenged with E. coli LPS (100 ng/ml) in medium supplemented with 2% HM in the absence (dashed lines) or presence (solid lines) of various amounts of native LTA (▪) or deacylated LTA (•) purified from either L. johnsonii strain La1 (A) or L. acidophilus strain La10 (B). After 24 h, the release of IL-8 in the culture supernatants was measured by ELISA. The standard deviations are indicated by the error bars. The results are representative of two independent experiments.

Article Snippet: Briefly, the wells of 96-well Maxisorp plates (Nunc) were coated with monoclonal antibodies against IL-8 (2 μg/ml) (ImmunoKontact; AMS Biotechnology, Lugano, Switzerland) by overnight incubation at 4°C.

Techniques: Activity Assay, Purification, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Lipoteichoic Acids from Lactobacillus johnsonii Strain La1 and Lactobacillus acidophilus Strain La10 Antagonize the Responsiveness of Human Intestinal Epithelial HT29 Cells to Lipopolysaccharide and Gram-Negative Bacteria

doi: 10.1128/IAI.70.4.2057-2064.2002

Figure Lengend Snippet: Effects of different sequential treatments with LTA, LPS, and HM on IL-8 production by HT29 cells. (A) HT29 cells were preincubated for 4 h with LTA from L. johnsonii strain La1 (100 μg/ml) in the presence of 2% HM, washed twice with a serum-free medium, and then challenged for 20 h with E. coli LPS (100 ng/ml) in the presence or absence of HM. (B) HT29 cells were incubated with LTA from L. johnsonii strain La1 (50 μg/ml) in the presence of HM for 4 h before the addition of E. coli LPS (100 ng/ml). (C) HT29 cells were challenged with E. coli LPS (100 ng/ml) in the presence of HM for 4 h before the addition of LTA from L. johnsonii strain La1 (1, 10, and 50 μg/ml). IL-8 release in the supernatants after a total of 24 h of culture was measured by ELISA. The standard deviations are indicated by the error bars. The results are representative of two independent experiments.

Article Snippet: Briefly, the wells of 96-well Maxisorp plates (Nunc) were coated with monoclonal antibodies against IL-8 (2 μg/ml) (ImmunoKontact; AMS Biotechnology, Lugano, Switzerland) by overnight incubation at 4°C.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay