Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: Contribution of HSV-2 infection-induced CXCR3 ligands to CD4 + T cell infiltration into mouse vagina. Seven days prior to HSV-2 challenge, BALB/c mice were injected with progesterone in multiple sites. One day prior to HSV-2 challenge, CXCL9, CXCL10, and CXCL11 neutralizing antibodies were delivered to the vagina of mice, alone or in combination, while isotype matched control IgG was used as the control. Mice were then anesthetized with pentobarbital sodium and challenged intravaginally with 10 μL/ mouse HSV-2 at a concentration of 6 × 10 7 PFU/ml or mock- challenged. Vaginal lavage fluids and cervical-vaginal tissues were collected at day 7 after challenge. (A) HSV-2 infection induces the production of mouse CXCR3 ligands. The protein levels of CXCL9 and CXCL10 ligands in vaginal lavage fluids were measured by CBA, and the protein level of CXCL11 was detected by ELISA. (B) CXCL9 mediates the migration of CD4 + T cells to the vaginal foci of infected mice. CD4 + T cells in infection foci were detected using anti-CD4 Ab by IHC. The scale bar indicates 100 μm. Data shown are mean ± S.D. ( n = 5 mice/group) of three independent experiments (A) . *** p < 0.001. One representative out of three independent experiments is shown (B) .

Article Snippet: Abs against mouse CD4, CXCL9, CXCL10, and CXCL11 were purchased from R&D Systems (MAB554, AF-492-NA, AF-466-NA, and AF-572, USA).

Techniques: Infection, Injection, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Migration

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: HSV-2 infection induces the production of CXCR3 ligands in human cervical epithelial cells. (A) HSV-2 infection activates the promoters of human CXCR3 ligands. ME180 cells in 24-well plates were co-transfected with 150 ng CXCL9-Luc, CXCL10-Luc or CXCL11-Luc, and 15 ng internal control plasmid phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 or ultraviolet-inactivated HSV-2 (UV-HSV-2) at an MOI of 1 for 24 h. DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (B) HSV-2 infection induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the mock-infected control. (C) HSV-2 infection induces the production of CXCR3 ligands. As depicted in (B) , cell supernatants were collected, and the protein level of CXCR3 ligands was measured by CBA. Data shown are mean ± S.D. of three independent experiments (A, B, and C). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Abs against mouse CD4, CXCL9, CXCL10, and CXCL11 were purchased from R&D Systems (MAB554, AF-492-NA, AF-466-NA, and AF-572, USA).

Techniques: Infection, Transfection, Control, Plasmid Preparation, Luciferase, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: HSV-2 infection-induced CXCL9 plays a predominant role in mediating CD4 + T cell migration. (A) The concentrations of CXCR3 ligands in the supernatants of ME180 cells infected with HSV-2 or mock-infected with DMEM were detected by CBA. (B,C) CXCL9 induced by HSV-2 recruits the migration of PBMCs (B) and CD4 + T cells (C) . ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1 h. (D) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by HSV-2 infection. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. (E) Recombinant CXCL9 significantly induces the migration of CD4 + T cells. DMEM containing recombinant CXCL9, CXCL10, or CXCL11 (48 pg/mL, 55 pg/mL and 175 pg/mL, respectively; the lowest concentration induced by HSV-2 infection) was added to the lower chamber of transwell plates. (F,G) Recombinant CXCL10 or CXCL11 mediates the migration of CD4 + T cells in a dose-dependent manner. DMEM containing recombinant CXCL10 or CXCL11 was added to the lower chamber of transwell plates. CXCL10 or CXCL11 was started from 55 pg/mL and 175 pg/mL, respectively, at a concentration gradient of two times. The activated CD4 + T cells were placed in the upper chamber. After 2 h incubation, cells migrated to lower chambers were collected and counted using an automatic cell counter. Cells migration was expressed as percentage of input. Input cells in the upper chamber were 5 × 10 5 . Data shown are mean ± S.D. of three independent experiments (A–G) . ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Abs against mouse CD4, CXCL9, CXCL10, and CXCL11 were purchased from R&D Systems (MAB554, AF-492-NA, AF-466-NA, and AF-572, USA).

Techniques: Infection, Migration, Control, Neutralization, Incubation, Recombinant, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: HSV-2 ICP4 promotes the production of human CXCR3 ligands. (A) ICP4 induces the activation of CXCR3 ligand promoters. ME180 cells in 24-well plates were transfected with 300 ng expression plasmid of HSV-2 gene or empty vector together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 24 h post-transfection, DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (B) The expression of HSV-2 genes was detected using anti-Flag Ab by Western Blot. ME180 cells were transfected with 3 μg HSV-2 gene expression plasmid for 24 h. The proteins were collected and detected using mouse anti-Flag Ab. (C) ICP4 induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were transfected with 3 μg ICP4 expression plasmid for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH gene was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the empty vector-transfected control. (D) ICP4 induces the production of CXCR3 ligands. As depicted in (C) , cell supernatants were collected, and the protein levels of CXCR3 ligands were measured by CBA. (E,F) CXCL9 induced by ICP4 recruits the migration of PBMCs (E) and CD4 + T cells (F) . ME180 cells in 6-well plates were transfected with 3 μg ICP4 expression plasmid for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1h. (G) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by ICP4. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. As depicted in Figure , cells migrated to lower chambers were counted. Cells migration was expressed as percentage of input. One representative out of three independent experiments is shown (B) . Data shown are mean ± S.D. of three independent experiments (A,C–G) . ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Abs against mouse CD4, CXCL9, CXCL10, and CXCL11 were purchased from R&D Systems (MAB554, AF-492-NA, AF-466-NA, and AF-572, USA).

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Western Blot, Gene Expression, Real-time Polymerase Chain Reaction, Control, Migration, Neutralization, Infection, Incubation

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: HSV-2 ICP4 regulates the expression of CXCR3 ligands via the p38 MAPK signaling pathway. (A–C) HSV-2 regulates the expression of CXCL9 (A) , CXCL10 (B) , and CXCL11 (C) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 at an MOI of 1 and supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (D–F) HSV-2 ICP4 regulates the expression of CXCL9 (D) , CXCL10 (E) , and CXCL11 (F) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 300 ng empty vector or ICP4 expression plasmid together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were cultured in complete DMEM supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (G) ICP4 activates p38 MAPK signaling pathway. ME180 cells were transfected with 3 μg ICP4 expression plasmid. The protein level of p38, phospho-p38 (p-p38) or phospho-C/EBP-β (p-C/EBP-β) was detected by Western Blot. Data shown are mean ± S.D. of three independent experiments (A–F) . ns, not significant, *** p < 0.001. One representative out of three independent experiments is shown (G) .

Article Snippet: Abs against mouse CD4, CXCL9, CXCL10, and CXCL11 were purchased from R&D Systems (MAB554, AF-492-NA, AF-466-NA, and AF-572, USA).

Techniques: Expressing, Transfection, Infection, Luciferase, Plasmid Preparation, Cell Culture, Western Blot

Journal: Journal of reproductive immunology

Article Title: Evaluation of immunological markers of ovine vaginal irritation: Implications for preclinical assessment of non-vaccine HIV preventive agents

doi: 10.1016/j.jri.2017.09.014

Figure Lengend Snippet: Ovine-specific reagents used in these studies

Article Snippet: 2.2 Reagents The ovine-specific reagents used in cytokine ELISA and flow cytometry assays are shown in . table ft1 table-wrap mode="anchored" t5 caption a7 Specificity Vendor Vendor Location Ovine ELISA Reagents Cytokine Capture Antibodies IL-6, IL-8 EMD Millipore Corporation Temecula, CA, USA IL-17α, CXCL10 Kingfisher Biotech, Inc. Saint Paul, MN, USA TNFα LifeSpan BioSciences, Inc. Seattle, WA, USA IL-1β United States Biological Salem, MA, USA Cytokine Detection Antibodies IL-6, IL-8 EMD Millipore Corp. Temecula, CA, USA IL-17α, CXCL10 (biotinylated) Kingfisher Biotech, Inc. Saint Paul, MN, USA TNFα, IL-1β (biotinylated) United States Biological Salem, MA, USA Rabbit IgG (HRP) EMD Millipore Corp. Temecula, CA, USA Recombinant Protein Standards IL-6, IL-8, IL-17α, CXCL10, IL-1β Kingfisher Biotech, Inc. Saint Paul, MN, USA TNFα United States Biological Salem, MA, USA Ovine Flow Cytometry Reagents Ovine CD4, CD8, CD14 Accurate Chemical & Scientific Corp. Westbury, NY, USA Ovine CD11b Research Technology Innovation (RTI) Brookings, SD, USA Mouse IgG1 isotype control, Anti-mouse IgG1--PE, Anti-mouse IgG1--FITC BD Pharmingen San Diego, CA, USA Open in a separate window Ovine-specific reagents used in these studies 2.3 Characterization of CVL leukocytes After allowing debris to settle, viable CVL cells were quantified by trypan blue exclusion.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Flow Cytometry, Control

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

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: Clinical and Vaccine Immunology

Article Title: Increased CXC Ligand 10 Levels and Gene Expression in Type 1 Leprosy Reactions

doi: 10.1128/cvi.00042-11

Figure Lengend Snippet: FIG. 1. Circulating CXCL10 in leprosy patients and healthy con- trols. Each point represents one sample from a different individual. The groups were healthy controls, borderline tuberculoid without re- action (BT), borderline lepromatous without reaction (BL), polar lep- romatous without reaction (LL), BT with reaction (BT plus RR), BL with reaction (BL plus RR), and LL patients with T2R (erythema nodosum leprosum). For patients with reaction (BT plus RR, BL plus RR, and T2R), the sample was taken at the time of reaction, prior to treatment. For LL, n 6; for all other groups, n 10. The horizontal lines indicate median values. The median level of CXCL10 in LL patients with type 2 reaction was not significantly elevated (P 0.9).

Article Snippet: Tissue sections were stained by an indirect immunoperoxidase method using goat anti-human CXCL10 (R&D Systems, Inc.; 1:40) or rabbit anti-human CXCR3 (Sigma) (1:75) for 60 min, followed by biotinylated donkey anti-goat IgG or goat anti-rabbit IgG (both 1:40; Jackson ImmunoResearch Laboratories, Inc.) for 30 min. Peroxidase-conjugated avidin-biotin complexes (Vectastain Elite ABC Reagent kit) were applied for 30 min. All incubations were followed by three washes with phosphate-buffered saline isotonic buffer, pH 7.4.

Techniques:

Journal: Clinical and Vaccine Immunology

Article Title: Increased CXC Ligand 10 Levels and Gene Expression in Type 1 Leprosy Reactions

doi: 10.1128/cvi.00042-11

Figure Lengend Snippet: FIG. 2. Circulating CXCL10 in repeated samples from leprosy patients who developed T1R. Each graph depicts the measurements made from serum samples obtained at 1 month intervals in borderline tuberculoid (A, no. 1 to 10) and borderline lepromatous (B, no. 11 to 20) patients. All patients with clinical T1R received standard multidrug treatment starting at the first visit. The arrows indicate the visits at which T1R was diagnosed. The number above each time point indicates the dose of prednisone started on that day, tapered monthly as indicated; in some cases, corticosteroids were not used initially but were started 1 to 2 months after the initial clinical diagnosis of T1R. Each serum specimen was obtained before corticosteroid treatment was initiated. CXCL10 was significantly elevated during T1R in the 10 BT patients (A) (P 0.0006) and the 10 BL patients (B) (P 0.0001). In patients 12, 16, and 17 (B), T1R was diagnosed by histopathological criteria only; when they were excluded from the analysis, the association of T1R with CXCL10 remained significant (P 0.05).

Article Snippet: Tissue sections were stained by an indirect immunoperoxidase method using goat anti-human CXCL10 (R&D Systems, Inc.; 1:40) or rabbit anti-human CXCR3 (Sigma) (1:75) for 60 min, followed by biotinylated donkey anti-goat IgG or goat anti-rabbit IgG (both 1:40; Jackson ImmunoResearch Laboratories, Inc.) for 30 min. Peroxidase-conjugated avidin-biotin complexes (Vectastain Elite ABC Reagent kit) were applied for 30 min. All incubations were followed by three washes with phosphate-buffered saline isotonic buffer, pH 7.4.

Techniques: Biomarker Discovery

Journal: Clinical and Vaccine Immunology

Article Title: Increased CXC Ligand 10 Levels and Gene Expression in Type 1 Leprosy Reactions

doi: 10.1128/cvi.00042-11

Figure Lengend Snippet: FIG. 3. Expression levels of CXCL10 and IFN- genes in skin biopsy specimens from leprosy patients. (A to H) Real-time quantitative RT-PCR was performed on cDNA obtained from sequential skin biopsy specimens from individual leprosy patients who were placed on MDT, none of whom had T1R at the time of the first biopsy. Five patients (A to E) had clinical symptoms of T1R at the time of the second biopsy; two of these (C and D) had a third biopsy after the reaction had resolved. Three patients (F to H) had no clinical symptoms of T1R at the time of the initial or second biopsy. Data were obtained using the standard-curve method and were normalized using 18S rRNA values, repeated three times for all determinations. The results are presented as the mean standard deviation.

Article Snippet: Tissue sections were stained by an indirect immunoperoxidase method using goat anti-human CXCL10 (R&D Systems, Inc.; 1:40) or rabbit anti-human CXCR3 (Sigma) (1:75) for 60 min, followed by biotinylated donkey anti-goat IgG or goat anti-rabbit IgG (both 1:40; Jackson ImmunoResearch Laboratories, Inc.) for 30 min. Peroxidase-conjugated avidin-biotin complexes (Vectastain Elite ABC Reagent kit) were applied for 30 min. All incubations were followed by three washes with phosphate-buffered saline isotonic buffer, pH 7.4.

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation