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: Arthritis Research & Therapy

Article Title: Expression of IL-20 in synovium and lesional skin of patients with psoriatic arthritis: differential response to alefacept treatment

doi: 10.1186/ar4038

Figure Lengend Snippet: IL-20 is expressed in PsA and RA synovial tissues . (A) Representative immunohistochemical staining of baseline IL-20 expression in synovial tissue from PsA and RA patients. Arrows indicate expression of IL-20 in both lining and sublining. (B) Quantification of IL-20 expression in RA and PsA synovial tissues. Results are shown as median IOD (integrated optical density)/mm 2 × 10 5 of 10 patients with RA and 11 patients with PsA. (C) Double-immunohistochemistry stainings of IL-20 (red) and CD68 + macrophages (blue) and CD55 + FLS (blue) in PsA synovium. A representative double immunostaining of PsA synovium from one patient is shown. Arrows indicate double-positive cells. Original magnification, ×200. CRP, C-reactive protein; DAS28, disease activity score; ESR, erythrocyte sedimentation rate; SJC, swollen-joint count; TJC, tender-joint count; VAS, visual analogue scale (100 mm).

Article Snippet: The primary antibody against human IL-20 (Zymogenetics, Seattle, WA, USA) was incubated overnight at 4ºC followed by secondary antibody affinity-purified horseradish peroxidase (HRP)-conjugated goat anti-mouse (Dako Cytomation, Glostrup, Denmark) for 30 minutes, followed by subsequent incubation with biotinylated tyramide (Perkin Elmer, Boston, MA, USA) for 30 minutes and HRP-conjugated streptavidin for 30 minutes.

Techniques: Immunohistochemical staining, Staining, Expressing, Immunohistochemistry, Double Immunostaining, Activity Assay, Sedimentation

Journal: Arthritis Research & Therapy

Article Title: Expression of IL-20 in synovium and lesional skin of patients with psoriatic arthritis: differential response to alefacept treatment

doi: 10.1186/ar4038

Figure Lengend Snippet: Alefacept treatment does not affect IL-20 expression in PsA synovium . (A) Representative immunohistochemical staining of IL-20 in the synovium of a PsA patient. (B) Quantification of IL-20 expression in PsA synovium. Results are shown as median IOD (integrated optical density)/mm 2 × 10 5 of 11 patients with PsA. (C) Correlation between the expression of IL-20 and the number of CD55 + FLSs in PsA synovium. Original magnification, ×200. Each dot represents the mean of the 18 high-power fields per patient per time point. CRP, C-reactive protein; DAS28, disease activity score; ESR, erythrocyte sedimentation rate; SJC, swollen-joint count; TJC, tender-joint count; VAS, visual analogue scale (100 mm).

Article Snippet: The primary antibody against human IL-20 (Zymogenetics, Seattle, WA, USA) was incubated overnight at 4ºC followed by secondary antibody affinity-purified horseradish peroxidase (HRP)-conjugated goat anti-mouse (Dako Cytomation, Glostrup, Denmark) for 30 minutes, followed by subsequent incubation with biotinylated tyramide (Perkin Elmer, Boston, MA, USA) for 30 minutes and HRP-conjugated streptavidin for 30 minutes.

Techniques: Expressing, Immunohistochemical staining, Staining, Activity Assay, Sedimentation

Journal: Arthritis Research & Therapy

Article Title: Expression of IL-20 in synovium and lesional skin of patients with psoriatic arthritis: differential response to alefacept treatment

doi: 10.1186/ar4038

Figure Lengend Snippet: Decreased IL-20 expression in PsA skin lesions after alefacept treatment . (A) Representative immunohistochemical staining of IL-20 expression in the lesional skin of a PsA patient. (B) Quantification of IL-20 expression in PsA lesional skin. Results are shown as median of semiquantitative score (SQ) of six patients with PsA. (C) IL-20 expression in PsA lesional skin is correlated with disease activity (PASI). All time points are depicted (each dot represents one patient per time point). *Statistical significance, P = 0.04. Original magnification, ×200. CRP, C-reactive protein; DAS28, disease activity score; ESR, erythrocyte sedimentation rate; SJC, swollen-joint count; TJC, tender-joint count; VAS, visual analogue scale (100 mm).

Article Snippet: The primary antibody against human IL-20 (Zymogenetics, Seattle, WA, USA) was incubated overnight at 4ºC followed by secondary antibody affinity-purified horseradish peroxidase (HRP)-conjugated goat anti-mouse (Dako Cytomation, Glostrup, Denmark) for 30 minutes, followed by subsequent incubation with biotinylated tyramide (Perkin Elmer, Boston, MA, USA) for 30 minutes and HRP-conjugated streptavidin for 30 minutes.

Techniques: Expressing, Immunohistochemical staining, Staining, Activity Assay, Sedimentation

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: PLoS ONE

Article Title: Lymphangiogenesis and Angiogenesis in Abdominal Aortic Aneurysm

doi: 10.1371/journal.pone.0089830

Figure Lengend Snippet: A , Elastica van Gieson staining of abdominal aortic aneurysm (AAA). Immunohistochemistry for podoplanin ( B ) and macrophages ( C ), CD19 ( E ), and myeloperoxidase (MPO) of the AAA wall. B , Lymphatic microvessels in the intima/media of the AAA (red dotted line encircling lymphatic microvessels). C , Macrophages infiltration around/within lymphatic microvessels in the intima/media or in adventitia (red square: macrophages in intima/media, black square: macrophages in adventitia). D , CD3-positive T cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. E , CD19-positive B cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. F , MPO-positive neutrophils infiltration in the intima/media or in adventitia. G–P , Double immunofluorescence staining of macrophages infiltrating the intima/media (In) and adventitia (Ad). G–P , Macrophage: green, CD11b ( G )/LYVE-1 ( H )/VEGF-C ( I )/MMP-9 ( J )/TGF-β1 ( K )/IL-4 ( L )/IL-8 ( M )/MIP-1α ( N )/IFN-γ ( O )/MCP-1 ( P ): red, DAPI: blue. LYVE-1, VEGF-C, MMP-9, TGF-β1, IL-4, IL-8, MIP-1α, and MCP-1 were expressed in the CD11b-positive macrophages in the intima/media, but not by macrophages in adventitia. Q , Double immunofluorescence staining of T-cells in intima/media and inflammatory cytokines. CD3: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. TGF-β1, IL-4, and IFN-γ were expressed in CD3-positive T lymphocytes in the intima/media. R , Double immunofluorescence staining of B lymphocytes in the intima/media and inflammatory cytokines. CD19: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. These inflammatory cytokines were not expressed in CD19-positive B lymphocytes. S , Double immunofluorescence staining of neutrophils in intima/media and inflammatory cytokines. MPO: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. These inflammatory cytokines were not expressed in MPO-positive neutrophils. Scale bars indicated 100 µm ( A–F ) and 10 µm ( G–S ).

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody against podoplanin (1∶200, DakoCytomation, Glostrup, Denmark), rabbit polyclonal antibody against Prox-1 (1∶2000, Millipore, MA, USA), rabbit polyclonal antibody against the N-terminus of human alpha smooth muscle isoform of actin (1∶25, Thermo Scientific Japan, Tokyo, Japan), mouse monoclonal antibody against human HIF-1α (1∶100, Novus Biologicals, CO, USA), rabbit monoclonal antibody against human CD11b (1∶250, Millipore, MA, USA), mouse monoclonal antibody against human macrophages (1∶100, AbD Serotec, Oxford, UK), rabbit polyclonal antibody against human LYVE-1 (1∶100, Relia Tech, Braunschweig, Germany), rabbit polyclonal antibody against human VEGF-C (1∶50, Abcam, Tokyo, Japan), rabbit polyclonal antibody against human matrix metalloproteinase (MMP)-9 (1∶100, Abnova, Taipei, Taiwan), mouse monoclonal antibody against human CD3 (1∶100, LifeSpan Biosciences, Seattle, WA), mouse monoclonal antibody against human CD19 (1∶50, Santa Cruz Biotechnology, CA, USA), mouse monoclonal antibody against human myeloperoxidase (MPO) (1∶50, Santa Cruz Biotechnology, CA, USA), rabbit polyclonal antibody against transforming growth factor beta-1 (TGF-β1) (1∶50, Abbiotec, CA, USA), rabbit polyclonal antibody against human interleukin-4 (IL-4) (1∶50, Biozol, Munich, Germany), rabbit polyclonal antibody against human interleukin-8 (IL-8) (1∶50, Abnova, Taipei, Taiwan), rabbit polyclonal antibody against human macrophage inflammatory protein-1α (MIP-1α) (1∶50, Spring bioscience, CA, USA), rabbit polyclonal antibody against human interferon-γ (IFN-γ) (1∶50, Santa Cruz Biotechnology, CA, USA), and rabbit polyclonal antibody against human monocyte chemotactic protein-1 (MCP-1) (1∶50, Abnova, Taipei, Taiwan).

Techniques: Staining, Immunohistochemistry, Double Immunofluorescence Staining

Journal: PLoS ONE

Article Title: Lymphangiogenesis and Angiogenesis in Abdominal Aortic Aneurysm

doi: 10.1371/journal.pone.0089830

Figure Lengend Snippet: A , Elastica van Gieson staining of abdominal aortic aneurysm (AAA). Immunohistochemistry for podoplanin ( B ) and macrophages ( C ), CD19 ( E ), and myeloperoxidase (MPO) of the AAA wall. B , Lymphatic microvessels in the intima/media of the AAA (red dotted line encircling lymphatic microvessels). C , Macrophages infiltration around/within lymphatic microvessels in the intima/media or in adventitia (red square: macrophages in intima/media, black square: macrophages in adventitia). D , CD3-positive T cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. E , CD19-positive B cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. F , MPO-positive neutrophils infiltration in the intima/media or in adventitia. G–P , Double immunofluorescence staining of macrophages infiltrating the intima/media (In) and adventitia (Ad). G–P , Macrophage: green, CD11b ( G )/LYVE-1 ( H )/VEGF-C ( I )/MMP-9 ( J )/TGF-β1 ( K )/IL-4 ( L )/IL-8 ( M )/MIP-1α ( N )/IFN-γ ( O )/MCP-1 ( P ): red, DAPI: blue. LYVE-1, VEGF-C, MMP-9, TGF-β1, IL-4, IL-8, MIP-1α, and MCP-1 were expressed in the CD11b-positive macrophages in the intima/media, but not by macrophages in adventitia. Q , Double immunofluorescence staining of T-cells in intima/media and inflammatory cytokines. CD3: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. TGF-β1, IL-4, and IFN-γ were expressed in CD3-positive T lymphocytes in the intima/media. R , Double immunofluorescence staining of B lymphocytes in the intima/media and inflammatory cytokines. CD19: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. These inflammatory cytokines were not expressed in CD19-positive B lymphocytes. S , Double immunofluorescence staining of neutrophils in intima/media and inflammatory cytokines. MPO: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. These inflammatory cytokines were not expressed in MPO-positive neutrophils. Scale bars indicated 100 µm ( A–F ) and 10 µm ( G–S ).

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody against podoplanin (1∶200, DakoCytomation, Glostrup, Denmark), rabbit polyclonal antibody against Prox-1 (1∶2000, Millipore, MA, USA), rabbit polyclonal antibody against the N-terminus of human alpha smooth muscle isoform of actin (1∶25, Thermo Scientific Japan, Tokyo, Japan), mouse monoclonal antibody against human HIF-1α (1∶100, Novus Biologicals, CO, USA), rabbit monoclonal antibody against human CD11b (1∶250, Millipore, MA, USA), mouse monoclonal antibody against human macrophages (1∶100, AbD Serotec, Oxford, UK), rabbit polyclonal antibody against human LYVE-1 (1∶100, Relia Tech, Braunschweig, Germany), rabbit polyclonal antibody against human VEGF-C (1∶50, Abcam, Tokyo, Japan), rabbit polyclonal antibody against human matrix metalloproteinase (MMP)-9 (1∶100, Abnova, Taipei, Taiwan), mouse monoclonal antibody against human CD3 (1∶100, LifeSpan Biosciences, Seattle, WA), mouse monoclonal antibody against human CD19 (1∶50, Santa Cruz Biotechnology, CA, USA), mouse monoclonal antibody against human myeloperoxidase (MPO) (1∶50, Santa Cruz Biotechnology, CA, USA), rabbit polyclonal antibody against transforming growth factor beta-1 (TGF-β1) (1∶50, Abbiotec, CA, USA), rabbit polyclonal antibody against human interleukin-4 (IL-4) (1∶50, Biozol, Munich, Germany), rabbit polyclonal antibody against human interleukin-8 (IL-8) (1∶50, Abnova, Taipei, Taiwan), rabbit polyclonal antibody against human macrophage inflammatory protein-1α (MIP-1α) (1∶50, Spring bioscience, CA, USA), rabbit polyclonal antibody against human interferon-γ (IFN-γ) (1∶50, Santa Cruz Biotechnology, CA, USA), and rabbit polyclonal antibody against human monocyte chemotactic protein-1 (MCP-1) (1∶50, Abnova, Taipei, Taiwan).

Techniques: Staining, Immunohistochemistry, Double Immunofluorescence Staining