Journal: Cells

Article Title: Induction of CXCL10-Mediated Cell Migration by Different Types of Galectins

doi: 10.3390/cells10020274

Figure Lengend Snippet: Galectins induce CXCL10 in a cell type-specific manner. ( A ) SDS-polyacrylamide gel electrophoresis of recombinant human galectins (rhGal) stained with Coomassie Blue. Molecular weights of protein standards are indicated on the left. The hemagglutination assay was performed using serial two-fold dilutions of the recombinant protein. The bottom row contains protein incubated with β-lactose, a competitive carbohydrate inhibitor of galectin binding. ( B ) Cultures of human corneal fibroblasts, human corneal epithelial cells, THP-1 monocytes and human umbilical vein endothelial cells (HUVECs) were incubated with 50 µg/mL rhGal-1, -3 and -8 or 50 µg/mL IFN-γ. Quantitative real-time PCR was used to determine the levels of CXCL10 mRNA after 6 h of incubation ( n = 3 independent experiments), whereas ELISA was used to determine the levels of CXCL10 protein in cell culture supernatants after 24 h of incubation ( n = 6 independent experiments). The data represent the mean ± SEM.

Article Snippet: A mouse monoclonal antibody against CXCL10 (1.2 μg/mL; MAB266, R&D Systems) was added to the lower compartment of the chamber in neutralization studies.

Techniques: Polyacrylamide Gel Electrophoresis, Recombinant, Staining, Hemagglutination Assay, Incubation, Binding Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cells

Article Title: Induction of CXCL10-Mediated Cell Migration by Different Types of Galectins

doi: 10.3390/cells10020274

Figure Lengend Snippet: Galectins differentially dictate fibroblast-dependent immune cell chemotaxis. ( A ) Recombinant human galectin (rhGal)-1, -3 and -8 were incubated with fibroblasts for 24 h. The conditioned media were pre-cleared with β-lactose Sepharose beads three times to remove galectins prior to chemotaxis assays. The presence of galectins in pre-cleared conditioned media was evaluated by immunoblotting. ( B ) Quantification by flow cytometry of the migration of CFSE-labeled THP-1 and Jurkat cells to conditioned media from fibroblasts treated for 24 h with increasing concentrations of recombinant galectins or BSA ( n = 3 independent experiments performed in duplicate). ( C ) Migration of CFSE-labeled THP-1 and Jurkat cells to conditioned media from fibroblasts treated for 24 h with 50 μg/mL of recombinant galectins or BSA. A CXCL10 neutralizing antibody was added to the conditioned media in neutralization studies ( n = 4–5 independent experiments in duplicate). The data in ( B ) represent the mean ± SEM. The box and whisker plots show the 25 and 75 percentiles (box), the median, and the minimum and maximum data values (whiskers). Significance was determined using Student’s t test. ** p < 0.01.

Article Snippet: A mouse monoclonal antibody against CXCL10 (1.2 μg/mL; MAB266, R&D Systems) was added to the lower compartment of the chamber in neutralization studies.

Techniques: Chemotaxis Assay, Recombinant, Incubation, Western Blot, Flow Cytometry, Migration, Labeling, Neutralization, Whisker Assay

Journal: PLoS Pathogens

Article Title: Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection

doi: 10.1371/journal.ppat.1008744

Figure Lengend Snippet: Heatmap showing the significant associations by Spearman correlation between intrahepatic CXCL10, CXCR3 and HIV with liver related outcomes including fibrosis and liver enzymes (left hand panel) and between the related intrahepatic markers CXCL10, CXCR3 and IFN-γ and liver and HIV in the liver (right hand panel). P-value < 0.05 if absolute value of Spearman’s correlation is at least 0.30, < 0.01 if at least 0.44 (n = 39). HIV human immunodeficiency virus, HBV hepatitis B virus, CA cell associated, sAg surface antigen, ALP alkaline phosphatase; GGT γ-glutamyl transferase, ALT Alanine transaminase, AST Aspartate transaminase, HMGB1 high mobility group box-1, CCL-2 C-C motif chemokine 2, sCD14 soluble CD14, LPS lipopolysaccharide, CXCL10 C-X-C motif chemokine 10, CXCR3 C-X-C motif chemokine receptor 3, IFN interferon, MPO myeloperoxidase; cccDNA covalently closed circular DNA, rcDNA relaxed circular DNA; Geq genome equivalent, TE transient elastography.

Article Snippet: Briefly, slides were incubated with antibody to CXCL10 (Rabbit; 4:100, OriGene, Rockville, Maryland), LPS (Mouse; 1:100, Abcam, Cambridge UK) or myeloperoxidase (MPO) (Rabbit; 1:1000, DAKO (Agilent) Santa Clara, CA) then rabbit or mouse Polink- 2 horseradish peroxidase (HRP) and developed with Immpact DAB (3,3′-diaminobenzidine; Vector Laboratories, Burlingame, CA), or red-AP (Vector Laboratories) then counterstained and mounted in Permount (Thermo Fisher, Waltham, MA), and scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies, Vista, CA), yielding high-resolution data from the entire tissue section.

Techniques: Virus

Journal: PLoS Pathogens

Article Title: Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection

doi: 10.1371/journal.ppat.1008744

Figure Lengend Snippet: Intrahepatic and circulating parameters associated with fibrosis outcomes by regression analysis (fibrosis in kPa by TE) and odds ratio (fibrosis by Metavir on biopsy), controlled by CD4+ T cell count.

Article Snippet: Briefly, slides were incubated with antibody to CXCL10 (Rabbit; 4:100, OriGene, Rockville, Maryland), LPS (Mouse; 1:100, Abcam, Cambridge UK) or myeloperoxidase (MPO) (Rabbit; 1:1000, DAKO (Agilent) Santa Clara, CA) then rabbit or mouse Polink- 2 horseradish peroxidase (HRP) and developed with Immpact DAB (3,3′-diaminobenzidine; Vector Laboratories, Burlingame, CA), or red-AP (Vector Laboratories) then counterstained and mounted in Permount (Thermo Fisher, Waltham, MA), and scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies, Vista, CA), yielding high-resolution data from the entire tissue section.

Techniques:

Journal: PLoS Pathogens

Article Title: Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection

doi: 10.1371/journal.ppat.1008744

Figure Lengend Snippet: (A) Liver fibrosis was significantly correlated with intrahepatic HIV DNA and with mRNA for CXCL10 and CXCR3. Plots showing correlations between liver fibrosis by TE (kPa) with intrahepatic HIV DNA, CXCL10, CXCR3 (left side panels) and between liver CXCL10 levels with intrahepatic HIV DNA, CXCR3 and peripheral AST (right side panels). The lower limit of detection for HIV RNA and HIV DNA was one copy per well. If there was no HIV PCR signal, this was recorded as zero and if there was a detectable signal but <1, this was recorded as 0.5 copies. All data points were included in the Spearman correlation. r = Spearman rank correlation coefficient. HIV human immunodeficiency virus, CXCL10 C-X-C motif chemokine 10, CXCR3 C-X-C motif chemokine receptor 3, TE transient elastography, AST Aspartate transaminase. (B) Liver fibrosis (kPa) and AST were higher in participants with detectable HIV DNA in the liver. Each symbol represents values from each participant. The lines represent the median and IQR. Comparisons were made using Wilcoxon Rank Sum test (* p<0.05).

Article Snippet: Briefly, slides were incubated with antibody to CXCL10 (Rabbit; 4:100, OriGene, Rockville, Maryland), LPS (Mouse; 1:100, Abcam, Cambridge UK) or myeloperoxidase (MPO) (Rabbit; 1:1000, DAKO (Agilent) Santa Clara, CA) then rabbit or mouse Polink- 2 horseradish peroxidase (HRP) and developed with Immpact DAB (3,3′-diaminobenzidine; Vector Laboratories, Burlingame, CA), or red-AP (Vector Laboratories) then counterstained and mounted in Permount (Thermo Fisher, Waltham, MA), and scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies, Vista, CA), yielding high-resolution data from the entire tissue section.

Techniques: Virus

Journal: PLoS Pathogens

Article Title: Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection

doi: 10.1371/journal.ppat.1008744

Figure Lengend Snippet: (A) Biopsies from people living with HIV-HBV coinfection and controls who were HIV and HBV negative were examined using immunohistochemistry (IHC) for CXCL10 (red-AP). The percentage area positive for CXCL10 staining (dark pink stain) was quantified using Photoshop CS5 and Fovea tools. Comparisons were made using the Wilcoxon Rank Sum test (* p<0.05). (i) The median and IQR for percentage area positive for CXCL10 is shown as well as (ii) representative pictures. CXCL10 staining was found within inflammatory Infiltrates, close to portal regions or blood vessels. (B) Fluorescence microscopy was performed using an RNAscope approach with a probe targeting CXCL10 (red), a nuclear stain DAPI (blue or grey) and antibodies to either (i) Myeloid cells (CD68+CD163), (ii) myeloperoxidase (MPO) or (iii) Hepatocyte (green). This demonstrates CXCL10 is predominantly located in hepatocytes. Scale bars: 100𝜇m.

Article Snippet: Briefly, slides were incubated with antibody to CXCL10 (Rabbit; 4:100, OriGene, Rockville, Maryland), LPS (Mouse; 1:100, Abcam, Cambridge UK) or myeloperoxidase (MPO) (Rabbit; 1:1000, DAKO (Agilent) Santa Clara, CA) then rabbit or mouse Polink- 2 horseradish peroxidase (HRP) and developed with Immpact DAB (3,3′-diaminobenzidine; Vector Laboratories, Burlingame, CA), or red-AP (Vector Laboratories) then counterstained and mounted in Permount (Thermo Fisher, Waltham, MA), and scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies, Vista, CA), yielding high-resolution data from the entire tissue section.

Techniques: Immunohistochemistry, Staining, Fluorescence, Microscopy, RNAscope

Journal: PLoS Pathogens

Article Title: Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection

doi: 10.1371/journal.ppat.1008744

Figure Lengend Snippet: (A) A representative flow cytometry plot showing eGFP expression (indicating infection with VSV-G-pseudotyped NL4.3Δenv eGFP HIV (MOI 0.5)) in live cells after gating on singlets and live cells, in uninfected hepatocytes (left plot) and hepatocytes infected with VSV-G-pseudotyped NL4.3Δenv eGFP HIV (MOI 0.5). (B) CXCL10 production measured by ELISA following stimulation of HepG2 (open symbols), and HBV-producing AD38 (filled symbols) hepatocyte cell lines with 500ng/ml IFN-γ plus1000ng/ml P3CSK4 and infected with VSV G-pseudotyped NL4.3Δenv eGFP HIV (MOI 0.5). HIV infection is indicated by symbols having a red border. Individual symbols represent the mean of replicates from a single experiment. The median+/-SEM for each stimulus from multiple experiments is shown. Comparisons between conditions were made using Wilcoxon Rank Sum test (* p<0.05). VSV-G-pseudotyped NL4.3Δenv eGFP HIV Vesicular stomatitis virus (VSV) glycoprotein G-pseudotyped NL4.3 virus with an envelope deletion expressing green fluorescent protein, MOI multiplicity of infection, HIV human immunodeficiency virus, CXCL10 C-X-C motif chemokine 10, IFN interferon, P3CSK4 Pam3CysSerLys4.

Article Snippet: Briefly, slides were incubated with antibody to CXCL10 (Rabbit; 4:100, OriGene, Rockville, Maryland), LPS (Mouse; 1:100, Abcam, Cambridge UK) or myeloperoxidase (MPO) (Rabbit; 1:1000, DAKO (Agilent) Santa Clara, CA) then rabbit or mouse Polink- 2 horseradish peroxidase (HRP) and developed with Immpact DAB (3,3′-diaminobenzidine; Vector Laboratories, Burlingame, CA), or red-AP (Vector Laboratories) then counterstained and mounted in Permount (Thermo Fisher, Waltham, MA), and scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies, Vista, CA), yielding high-resolution data from the entire tissue section.

Techniques: Flow Cytometry, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Virus

Journal: PLoS Pathogens

Article Title: Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection

doi: 10.1371/journal.ppat.1008744

Figure Lengend Snippet: CXCL10 production was measured by ELISA (absolute and fold change, middle and lower panel) and eGFP expression measured by flow cytometry (upper panel) following stimulation of HepG2 (open symbols), and HBV-producing AD38 (filled symbols) cells, with 500ng/ml IFN-γ plus1000ng/ml P3CSK4 and infected with VSV-G-pseudotyped NL4.3Δenv eGFP HIV. The left sided panels show eGFP expression and CXCL10 production after cells were stimulated with 500ng/ml IFN-γ plus1000ng/ml P3CSK4 and infected with VSV-G-pseudotyped NL4.3Δenv eGFP HIV with a MOI of 0.0625, 0.125, 0.25 and 0.5. Increasing MOI is indicated by the triangle at the bottom of the Fig. The right sided panels show these same parameters, following infection with VSV-G-pseudotyped NL4.3Δenv eGFP HIV (red border) with MOI of 0.5 in the presence and absence of the antiretroviral agents efavirenz (EFV), raltegravir (RAL) and the fusion inhibitor T20. Individual symbols represent the mean of replicates from a single experiment. The median+/-SEM for each stimulus from multiple experiments is shown. Comparisons between conditions were made using Wilcoxon Rank Sum test (* p<0.05). HIV human immunodeficiency virus, CXCL10 C-X-C motif chemokine 10, GFP green fluorescent protein, IFN interferon, VSV-G-pseudotyped NL4.3Δenv eGFP HIV Vesicular stomatitis virus (VSV) glycoprotein G-pseudotyped NL4.3 virus with an envelope deletion expressing green fluorescent protein, MOI multiplicity of infection, P3CSK4 Pam3CysSerLys4, efavirenz (EFV), raltegravir (RAL).

Article Snippet: Briefly, slides were incubated with antibody to CXCL10 (Rabbit; 4:100, OriGene, Rockville, Maryland), LPS (Mouse; 1:100, Abcam, Cambridge UK) or myeloperoxidase (MPO) (Rabbit; 1:1000, DAKO (Agilent) Santa Clara, CA) then rabbit or mouse Polink- 2 horseradish peroxidase (HRP) and developed with Immpact DAB (3,3′-diaminobenzidine; Vector Laboratories, Burlingame, CA), or red-AP (Vector Laboratories) then counterstained and mounted in Permount (Thermo Fisher, Waltham, MA), and scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies, Vista, CA), yielding high-resolution data from the entire tissue section.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Infection, Virus

Journal: PLoS Pathogens

Article Title: Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection

doi: 10.1371/journal.ppat.1008744

Figure Lengend Snippet: Factors driving liver disease pathogenesis in HIV-HBV co-infection In HIV-HBV co-infection we propose a model where liver fibrosis is driven by an increase in production of CXCL10 by hepatocytes (left hand side of diagram) and products of microbial translocation (right hand side of diagram). Hepatocytes produce CXCL10 and production is enhanced following infection with HBV and HIV in the presence of IFN-γ. CXCL10 recruits activated T-cells and NK cells that express CXCR3 to the liver. In the absence of antiretroviral therapy, this is an ideal environment for HIV replication which produces IFN-γ and drives further CXCL10 production. In addition, altered GI tract permeability in the setting of HIV infection leads to an increase in circulating microbial products, including LPS which binds to TLR-4 and P3S4K4 which binds to TLR-2 expressed on hepatocytes, Kupffer cells and hepatic stellate cells. This cycle of CXCL10-mediated inflammation, together with elevated LPS directly activates hepatic stellate cells (HSC) driving fibrosis. HIV human immunodeficiency virus, HBV hepatitis B, CXCL10 C-X-C motif chemokine 10, IFN interferon, NK natural killer, CXCR3 C-X-C motif chemokine receptor 3, GI gastrointestinal tract, LPS lipopolysaccharide, TLR toll like receptor, HSC hepatic stellate cells.

Article Snippet: Briefly, slides were incubated with antibody to CXCL10 (Rabbit; 4:100, OriGene, Rockville, Maryland), LPS (Mouse; 1:100, Abcam, Cambridge UK) or myeloperoxidase (MPO) (Rabbit; 1:1000, DAKO (Agilent) Santa Clara, CA) then rabbit or mouse Polink- 2 horseradish peroxidase (HRP) and developed with Immpact DAB (3,3′-diaminobenzidine; Vector Laboratories, Burlingame, CA), or red-AP (Vector Laboratories) then counterstained and mounted in Permount (Thermo Fisher, Waltham, MA), and scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies, Vista, CA), yielding high-resolution data from the entire tissue section.

Techniques: Infection, Translocation Assay, Permeability, Virus

Journal: Animals : an Open Access Journal from MDPI

Article Title: Early Pregnancy Induces Expression of STAT1, OAS1 and CXCL10 in Ovine Spleen

doi: 10.3390/ani9110882

Figure Lengend Snippet: Primers used for RT-qPCR.

Article Snippet: The membranes were incubated with a goat anti-STAT1 polyclonal antibody (Abcam, Cambridge, UK, ab230428, 1:1000), a rabbit anti-OAS1 polyclonal antibody (Abcam, ab86343, 1:1000), a mouse anti-Mx1 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-166412, 1:1000), and a mouse anti-CXCL10 monoclonal antibody (Santa Cruz Biotechnology, sc-374092, 1:1000), respectively.

Techniques: Sequencing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Early Pregnancy Induces Expression of STAT1, OAS1 and CXCL10 in Ovine Spleen

doi: 10.3390/ani9110882

Figure Lengend Snippet: Relative expression values of STAT1 , OAS1 , MX1 and CXCL10 mRNA in ovine spleens measured by real-time quantitative PCR. Note: DN16 = Day 16 of the estrous cycle; DP13 = Day 13 of pregnancy; DP16 = Day 16 of pregnancy; DP25 = Day 25 of pregnancy. Significant differences ( p < 0.05) are indicated by different letters within same color column.

Article Snippet: The membranes were incubated with a goat anti-STAT1 polyclonal antibody (Abcam, Cambridge, UK, ab230428, 1:1000), a rabbit anti-OAS1 polyclonal antibody (Abcam, ab86343, 1:1000), a mouse anti-Mx1 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-166412, 1:1000), and a mouse anti-CXCL10 monoclonal antibody (Santa Cruz Biotechnology, sc-374092, 1:1000), respectively.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Animals : an Open Access Journal from MDPI

Article Title: Early Pregnancy Induces Expression of STAT1, OAS1 and CXCL10 in Ovine Spleen

doi: 10.3390/ani9110882

Figure Lengend Snippet: Expression of STAT1, OAS1, Mx1 and CXCL10 proteins in ovine spleens analyzed by western blot. Note: DN16 = Day 16 of the estrous cycle; DP13 = Day 13 of pregnancy; DP16 = Day 16 of pregnancy; DP25 = Day 25 of pregnancy. Significant differences ( p < 0.05) are indicated by different superscript letters within the same color column.

Article Snippet: The membranes were incubated with a goat anti-STAT1 polyclonal antibody (Abcam, Cambridge, UK, ab230428, 1:1000), a rabbit anti-OAS1 polyclonal antibody (Abcam, ab86343, 1:1000), a mouse anti-Mx1 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-166412, 1:1000), and a mouse anti-CXCL10 monoclonal antibody (Santa Cruz Biotechnology, sc-374092, 1:1000), respectively.

Techniques: Expressing, Western Blot