Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Human trophoblasts recruited T lymphocytes and monocytes into decidua by secretion of chemokine CXCL16 and interaction with CXCR6 in the first-trimester pregnancy.

doi: 10.4049/jimmunol.180.4.2367

Figure Lengend Snippet: FIGURE 1. The transcription and translation of chemokine CXCL16 in human first-trimester trophoblasts. The real-time quantitative PCR was used to analyze transcription of CXCL16 in trophoblasts and JAR cells with cyclophilin A (CyP) as positive control. Three independent experiments were done (including 15 placental samples) and the results were reproducible (A). The specific brown-colored stainings for CXCL16 were recognized in the cytoplasm and cytomembrane of the primary-cultured villous cytotrophoblasts (B), and extravillous cytotrophoblasts (C) by immunocytochemistry, and the positive stainings were recognized by villous cytotrophoblasts and ST (E) and by extravillous cytotrophoblasts (F) by immunohistochemistry. No background staining was observed in goat isotype controls (D and G). The experiments were repeated five times with five placenta samples, respectively. The picture is a representative one. Magnification: B, D, E, and G, 200; C and F, 400.

Article Snippet: Anti-human CXCL16 Ab (R&D Systems) were administrated to detect whether cytotrophoblasts express CXCL16.

Techniques: Real-time Polymerase Chain Reaction, Positive Control, Cell Culture, Immunocytochemistry, Immunohistochemistry, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Human trophoblasts recruited T lymphocytes and monocytes into decidua by secretion of chemokine CXCL16 and interaction with CXCR6 in the first-trimester pregnancy.

doi: 10.4049/jimmunol.180.4.2367

Figure Lengend Snippet: FIGURE 2. The accumulated concentration of CXCL16 in culture me- dium of first-trimester cytotrophoblasts was examined by ELISA. Purified cytotrophoblasts were seeded at 1 106, 5 105, 1 105, 5 104, 1 104, and 5 103 cells/ml, and then the supernatants were collected and measured after 12, 24, 36, 48, 60, 72, and 100 h of culture. The results showed CXCL16 accumulation in the medium during the course of culture. Each point represents the mean SD obtained from duplicate dishes, and the data are representative of two independent experiments.

Article Snippet: Anti-human CXCL16 Ab (R&D Systems) were administrated to detect whether cytotrophoblasts express CXCL16.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Human trophoblasts recruited T lymphocytes and monocytes into decidua by secretion of chemokine CXCL16 and interaction with CXCR6 in the first-trimester pregnancy.

doi: 10.4049/jimmunol.180.4.2367

Figure Lengend Snippet: FIGURE 6. Correlation of CXCL16 chemotaxis to CXCR6 expression level in the peripheral (A) and decidual (B) immune cells. CXCL16 che- motaxis was shown as the ratio of the number of migrated immune cells treated by CXCL16 (100 ng/ml) to the number of migrated immune cells in the control.

Article Snippet: Anti-human CXCL16 Ab (R&D Systems) were administrated to detect whether cytotrophoblasts express CXCL16.

Techniques: Chemotaxis Assay, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Expression and function of CXCL16 in cigarette smoke extract (CSE)-stimulated human umbilical arterial endothelial cells (HUAEC) and effect of CXCL16 neutralizing antibody on neutrophil and mononuclear leukocyte adhesion. HUAEC were stimulated with 1% CSE, INF-γ, or TNF-α (20 ng/ml) for 1, 4, or 24 h. Relative quantification of mRNA levels for CXCL16 and GAPDH after (A) 1 h and (B) 4 h ( n = 5–8 independent experiments). Columns show fold increase in CXCL16 mRNA expression relative to control GAPDH. Values are expressed as mean ± SEM of the 2 −ΔΔCt values. * P < 0.05 or ** P < 0.01 relative to values in the medium group. (C) Protein expression was determined by flow cytometry. Results are expressed as mean of fluorescence intensity (MFI) ( n = 7–8 independent experiments). Values are expressed as mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the medium group. (D) Following a similar protocol, CXCL16 was visualized in non-permeabilized HUAEC by immunofluorescence (green). Nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI) ( n = 4–5 independent experiments). (E,F) Endothelial cells were stimulated with 1% CSE for 24 h. Some cells were incubated with a CXCL16 neutralizing antibody (2 µg/ml) or an irrelevant isotype-matched monoclonal antibody (MOPC-21, 2 µg/ml). Subsequently, human neutrophils (E) or mononuclear cells (F) (1 × 10 6 cells/ml) incubated with or without EDTA were perfused over the monolayers for 5 min at 0.5 dyn/cm 2 and leukocyte accumulation quantified ( n = 5–7 independent experiments). Values are expressed as the mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the medium group; + P < 0.05 relative to the stimulus MOPC-21-treated group.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Quantitative Proteomics, Control, Flow Cytometry, Fluorescence, Immunofluorescence, Incubation

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Inhibition of cigarette smoke extract (CSE)-induced CXCL16 expression in arterial endothelial cells. (A) CXCL16 expression was determined by flow cytometry in human umbilical arterial endothelial cells preincubated or not with apocynin (30 µM) or allopurinol (100 µM) for 1 h and then stimulated with 1% CSE for 24 h ( n = 7 independent experiments). Results are expressed as mean of fluorescence intensity (MFI). Values are expressed as the mean ± SEM. * P < 0.05 relative to values in the medium group; ++ P < 0.01 relative to 1% CSE group. Endothelial cells were transfected with (B) Nox2, (C) Nox4, or (D) Nox5 siRNA or control siRNA. At 48 h post-transfection, cells were stimulated with 1% CSE for 24 h. CXCL16 expression was determined by flow cytometry. Results are expressed as MFI ( n = 5–11 independent experiments). Values are expressed as mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the medium group; ++ P < 0.01 relative to 1% CSE group in control siRNA transfected cells.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Inhibition, Expressing, Flow Cytometry, Fluorescence, Transfection, Control

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Cigarette smoke extract (CSE)-induced CXCL16 overexpression is decreased by RhoA, p38 mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-κB inhibition in human umbilical arterial endothelial cells (HUAEC). (A) CXCL16 expression was determined by flow cytometey in endothelial cells preincubated or not with a RhoA inhibitor (C3 transferase, 2 µg/ml) for 4 h and then stimulated with 1% CSE for 24 h. Results are expressed as mean of fluorescence intensity (MFI) ( n = 5 independent experiments). Values are expressed as mean ± SEM. * P < 0.05 relative to values in the medium group; + P < 0.05 relative to 1% CSE group. (B) HUAEC were transfected with RhoA siRNA or control siRNA. At 48 h post-transfection, cells were stimulated with 1% CSE for 24 h. CXCL16 expression was determined by flow cytometry. Results are expressed as MFI ( n = 7 independent experiments). Values are expressed as mean ± SEM. ** P < 0.01 relative to values in the medium group; + P < 0.05 relative to their respective group in control siRNA-transfected cells. (C) HUAEC were stimulated with 1% CSE for 24 h. Some cells were pretreated with PD098059 (20 µM), SB202130 (20 µM), or MOL-294 (2.5 µM) for 1 h before CSE stimulation. CXCL16 expression was determined by flow cytometry. Results are expressed as MFI ( n = 4–7 independent experiments). Values are expressed as mean ± SEM. ** P < 0.01 relative to values in the medium group; + P < 0.05 relative to values in the 1% CSE group.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Over Expression, Inhibition, Expressing, Fluorescence, Transfection, Control, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Percentage of circulating platelets expressing PAC-1, P-selectin, CXCL16, and CXCR6 from active and not active smoking chronic obstructive pulmonary disease (COPD) patients and aged-matched controls by flow cytometry. Platelets were stained with conjugated antibodies against (A) CD41 and PAC-1, (B) CD41 and P-selectin, (C) CD41 and CXCL16, and (D) CD41 and CXCR6. Results are expressed as percentage of positive cells ( n = 15 aged-matched controls, n = 17 active smokers COPD patients, n = 16 ex-smokers COPD patients). Values are expressed as mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the control group.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Flow Cytometry, Staining, Control

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Leukocyte recruitment by cigarette smoke extract (CSE)-stimulated human umbilical arterial endothelial cells (HUAEC) and CXCL16 plasma levels from whole blood of active and not active smokers patients with chronic obstructive pulmonary disease (COPD) and aged-matched controls. HUAEC were stimulated with 1% CSE for 24 h. Some cells were incubated with a CXCL16 neutralizing antibody (2 µg/ml) or an irrelevant isotype-matched monoclonal antibody (MOPC-21, 2 µg/ml). Subsequently, whole blood from patients with COPD active or not active smokers and healthy aged-matched controls incubated (A) without, or (B) with EDTA, was perfused over endothelial monolayers for 5 min at 0.5 dyn/cm 2 and leukocyte adhesion quantified ( n = 13 aged-matched controls, n = 14 active smokers COPD patients, n = 16 ex-smokers COPD patients). Values are expressed as the mean ± SEM. ** P < 0.01 relative to values in the medium group; + P < 0.05 or ++ P < 0.01 relative to 1% CSE group; Δ P < 0.05 or ΔΔ P < 0.01 relative to the values in the aged-matched control group. (C) CXCL16 plasmatic levels were measured by ELISA ( n = 17 aged-matched controls, n = 17 active smokers COPD patients, n = 18 ex-smokers COPD patients). Values are expressed as the mean ± SEM.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Clinical Proteomics, Incubation, Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Effect of cigarette smoke (CS) exposure in CXCR6-expressing and CXCRC6 knockout mice. Heterozygous (CXCR6 −/+ ) and homozygous (CXCR6 −/– ) mice were exposed or not to CS for 3 days and responses were examined 16 h later. (A) Leukocyte–arteriolar endothelium interactions was measured by intravital microscopy. Results are expressed as mean ± SEM ( n = 5–8 animals per group). * P < 0.05 or ** P < 0.01 relative to non-exposed animals; + P < 0.05 relative to CXCR6 −/+ mice. (B) Relative quantification of CXCL16 and β-actin mRNA was determined by RT-PCR. Columns show fold increase in expression of CXCL16 mRNA relative to control GAPDH values ( n = 5 independent experiments). Values are represented as mean ± SEM of the 2 −ΔΔCt values. ** P < 0.01 relative to non-exposed animals. (C) Cremaster muscle was fixed for CXCL16 and endothelium (CD31) staining. CXCL16 expression is shown in green (stained with an Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody) and vessel endothelium (red) was stained with a PE-conjugated anti-mouse CD31 monoclonal antibody. Overlapping expression of CXCL16 and CD31 is shown in yellow. Results are representative of five to six animals per group.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Knock-Out, Intravital Microscopy, Quantitative Proteomics, Reverse Transcription Polymerase Chain Reaction, Control, Staining

Journal: The Journal of investigative dermatology

Article Title: Constitutive expression and regulated release of the transmembrane chemokine CXCL16 in human and murine skin.

doi: 10.1038/sj.jid.5700751

Figure Lengend Snippet: Figure 1. Expression of human CXCL16 in normal human skin. For chromogenic staining of CXCL16 and ADAM10 in healthy human skin, acetone-fixed sections were incubated with (a, d) rabbit anti-hCXCL16 (b) rabbit anti-ADAM10 or an irrelevant (c) rabbit IgG control antibody followed by incubation with POD-coupled goat anti-rabbit IgG antibody, enzymatic staining and counterstaining with hemalumn. In (a–c) epidermal skin and in (d) dermal skin containing a blood vessel (bottom right) is shown. Bar ¼ 50 mm.

Article Snippet: Recombinant human CXCL16 extracellular domain and chemokine domain, recombinant human IFN-g, recombinant human tumor necrosis factor-a, goat anti-human CXCL16 (hCXCL16) antibody, rat anti-murine CXCL16 (mCXCL16) mAb (clone 142417), phycoery- thrin-conjugated and unconjugated mouse anti-human CXCR6 (hCXCR6) mAb (clone 56811), and rat anti-murine CXCR6 (mCXCR6) mAb (clone 221002) were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Staining, Incubation, Control

Journal: The Journal of investigative dermatology

Article Title: Constitutive expression and regulated release of the transmembrane chemokine CXCL16 in human and murine skin.

doi: 10.1038/sj.jid.5700751

Figure Lengend Snippet: Figure 2. In situ expression and in vivo-release of murine CXCL16. (a) Paraformaldehyde-fixed fresh frozen sections of murine skin were incubated with radioactively labeled antisense cRNA probes (as) for mCXCL16 and sense- controls (s), respectively. Hybridized cRNA probes were subsequently visualized by autoradiography. Bar ¼ 20mm. (b) Wound fluid from injured mice was collected every 24hours over a period of 15 days, cleared by centrifugation and subsequently analyzed for the presence of CXCL16 by an ELISA specific for mCXCL16. Data are given as mean and SD (n ¼ 5). Statistically significant release of mCXCL16 (Po0.05) is indicated by asterisks.

Article Snippet: Recombinant human CXCL16 extracellular domain and chemokine domain, recombinant human IFN-g, recombinant human tumor necrosis factor-a, goat anti-human CXCL16 (hCXCL16) antibody, rat anti-murine CXCL16 (mCXCL16) mAb (clone 142417), phycoery- thrin-conjugated and unconjugated mouse anti-human CXCR6 (hCXCR6) mAb (clone 56811), and rat anti-murine CXCR6 (mCXCR6) mAb (clone 221002) were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: In Situ, Expressing, In Vivo, Incubation, Labeling, Autoradiography, Centrifugation, Enzyme-linked Immunosorbent Assay

Journal: The Journal of investigative dermatology

Article Title: Constitutive expression and regulated release of the transmembrane chemokine CXCL16 in human and murine skin.

doi: 10.1038/sj.jid.5700751

Figure Lengend Snippet: Figure 3. Surface expression and release of CXCL16 by cultured keratinocytes. (a) HaCaT cells were harvested and analyzed for CXCL16 surface expression by flow cytometry using a purified rabbit anti-hCXCL16 antibody that was detected by a phycoerythrin-conjugated secondary antibody. The fluorescence signal of the cells stained for hCXCL16 is shown in comparison to that of unstained cells or cells receiving an isotype control antibody. (b) HaCaT cells were incubated in serum-free medium for different periods of time in the presence or absence of marimastat (5 mM). Subsequently, conditioned media were harvested and analyzed for released CXCL16 by a specific ELISA for hCXCL16. (c) PAM212 cells were incubated with a rat anti-mCXCL16 antibody, an isotype control antibody, or left unstained. After incubation with a FITC-conjugated secondary antibody cells were analyzed by flow cytometry. (d) PAM212 cells were incubated in serum-free medium for 4 hours in the presence or absence of marimastat (5 mM). Subsequently, released CXCL16 in the conditioned media was determined by a specific ELISA for mCXCL16. Data are representative for three experiments and shown as mean and SD (n ¼ 3). Asterisks indicate statistically significant differences (Po0.05) between inhibitor-treated cells and untreated cells.

Article Snippet: Recombinant human CXCL16 extracellular domain and chemokine domain, recombinant human IFN-g, recombinant human tumor necrosis factor-a, goat anti-human CXCL16 (hCXCL16) antibody, rat anti-murine CXCL16 (mCXCL16) mAb (clone 142417), phycoery- thrin-conjugated and unconjugated mouse anti-human CXCR6 (hCXCR6) mAb (clone 56811), and rat anti-murine CXCR6 (mCXCR6) mAb (clone 221002) were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Cell Culture, Flow Cytometry, Purification, Fluorescence, Staining, Comparison, Control, Incubation, Enzyme-linked Immunosorbent Assay

Journal: The Journal of investigative dermatology

Article Title: Constitutive expression and regulated release of the transmembrane chemokine CXCL16 in human and murine skin.

doi: 10.1038/sj.jid.5700751

Figure Lengend Snippet: Figure 4. Effect of ADAM10/ADAM17-inhibitors on CXCL16-release from cultured keratinocytes. (a) Cell lysate of cultured HaCaT cells was investigated for the presence of immature and processed forms of the metallproteinaseses ADAM10 and ADAM17. For Western blotting purified rabbit antibodies against the C-terminus of ADAM10 and ADAM17, respectively were used. (b–d) (b) HaCaT cells (c) human primary keratinocytes, (d) WT-ECV304 and CXCL16-ECV304 cells and were incubated for 4 hours in the presence or absence of 5 mM GI254023X or GW280264X. Subsequently, conditioned media were harvested and cell lysates were prepared. Released and cell-associated CXCL16 in media and lysates, respectively, was then quantified by ELISA. Statistically significant differences (Po0.05) between inhibitor-treated cells and untreated cells are indicated by asterisks.

Article Snippet: Recombinant human CXCL16 extracellular domain and chemokine domain, recombinant human IFN-g, recombinant human tumor necrosis factor-a, goat anti-human CXCL16 (hCXCL16) antibody, rat anti-murine CXCL16 (mCXCL16) mAb (clone 142417), phycoery- thrin-conjugated and unconjugated mouse anti-human CXCR6 (hCXCR6) mAb (clone 56811), and rat anti-murine CXCR6 (mCXCR6) mAb (clone 221002) were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Cell Culture, Western Blot, Purification, Incubation, Enzyme-linked Immunosorbent Assay

Journal: The Journal of investigative dermatology

Article Title: Constitutive expression and regulated release of the transmembrane chemokine CXCL16 in human and murine skin.

doi: 10.1038/sj.jid.5700751

Figure Lengend Snippet: Figure 5. Effect of ADAM10/ADAM17 downregulation by siRNA on CXCL16-release from kreatinocytes. Cultured keratinocytes prepared from human foreskin were transfected with different heteroduplexed siRNA oligonucleotide constructs for downregulation of ADAM10 (A10-1, 2, 3) or ADAM17 (A17-1, 2, 3), an irrelevant siRNA control or without siRNA. At 48 hours after transfection mRNA and protein was extracted. (a and b) The mRNA expression level of ADAM10 and ADAM17 was determined by real-time RT-PCR and expressed in relation to that of glyceraldehyde-3-phosphate dehydrogenase. (c) Lysates of siRNA-transfected cells were analyzed for expression of the pro- and mature form of ADAM10 and controlled for b-actin content by Western blotting. (d and e) For CXCL16 release experiments siRNA-treated cells were washed and incubated for 4 hours in fresh medium. (c and e) Subsequently, cells were harvested for analysis of ADAM10 and ADAM17 mRNA expression and media were collected for quantification of released CXCL16 by ELISA. Data are representative for three experiments and shown as mean and SD (n ¼ 3). Asterisks indicate statistically significant differences (Po0.05) between cells receiving specific siRNA and control cells treated with irrelevant siRNA.

Article Snippet: Recombinant human CXCL16 extracellular domain and chemokine domain, recombinant human IFN-g, recombinant human tumor necrosis factor-a, goat anti-human CXCL16 (hCXCL16) antibody, rat anti-murine CXCL16 (mCXCL16) mAb (clone 142417), phycoery- thrin-conjugated and unconjugated mouse anti-human CXCR6 (hCXCR6) mAb (clone 56811), and rat anti-murine CXCR6 (mCXCR6) mAb (clone 221002) were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Cell Culture, Transfection, Construct, Control, Expressing, Quantitative RT-PCR, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay

Journal: The Journal of investigative dermatology

Article Title: Constitutive expression and regulated release of the transmembrane chemokine CXCL16 in human and murine skin.

doi: 10.1038/sj.jid.5700751

Figure Lengend Snippet: Figure 8. Activity of keratinocyte-derived CXCL16 on CXCR6-transfected HEK293 cells. CXCR6-expressing HEK293 cells were treated with recombinant CXCL16 or concentrated HaCaT-conditioned (HaCaT-CM) medium for 30 minutes at either (a) 371C or (b) 41C. In a separate experiment recombinant CXCL16 or concentrated HaCaT-conditioned medium were preincubated with a neutralizing antibody to (c) CXCL16 or an (d) isotype control and then added to the CXCR6-expressing HEK293 cells for 30 minutes at 371C. Subsequently, cells were assayed for CXCR6 surface expression using a phycoerythrin-labeled mAb to CXCR6. As a control, WT-HEK293 cells expressing no CXCR6 were stained in parallel. The mean intensity of the fluorescence signal from CXCL16 or HaCaT-CM-treated cells was calculated as percentage of that from untreated cells and is shown as insets. Data are representative for three experiments.

Article Snippet: Recombinant human CXCL16 extracellular domain and chemokine domain, recombinant human IFN-g, recombinant human tumor necrosis factor-a, goat anti-human CXCL16 (hCXCL16) antibody, rat anti-murine CXCL16 (mCXCL16) mAb (clone 142417), phycoery- thrin-conjugated and unconjugated mouse anti-human CXCR6 (hCXCR6) mAb (clone 56811), and rat anti-murine CXCR6 (mCXCR6) mAb (clone 221002) were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Activity Assay, Derivative Assay, Transfection, Expressing, Recombinant, Control, Labeling, Staining, Fluorescence

Journal: International journal of oncology

Article Title: Evaluation of CXCL10 as a novel serum marker for predicting liver metastasis and prognosis in colorectal cancer.

doi: 10.3892/ijo.2011.1247

Figure Lengend Snippet: Figure 2. (a), Scattergram of serum CXCL10 in stage I-IV colorectal cancer patients and control subjects (p<0.0001). (b), Scattergram of serum CXCL10 subdivided by pathological T stage (p=0.0101). (c), Scattergram of serum CXCL10 subdivided by clinical H stage (p=0.0004). (d), The relationships between serum CXCL10 and serum CEA level (r=0.2853, p<0.0001).

Article Snippet: Primary anti-CXCL10 goat polyclonal antibody (clone AF976, R&D Systems, MN, USA) and anti-CXCR3 rabbit polyclonal antibody (clone ab8023, Abcam, Cambridge, UK) at a dilution of 1:25 were used for the labeled streptavidinbiotin method (LASB2 kit/HRP, Dako Cytomation).

Techniques: Control

Journal: International journal of oncology

Article Title: Evaluation of CXCL10 as a novel serum marker for predicting liver metastasis and prognosis in colorectal cancer.

doi: 10.3892/ijo.2011.1247

Figure Lengend Snippet: Figure 3. (a), Cancer specific survival curves, for 218 patients with colorectal cancer, according to serum CXCL10 level. Patients with higher serum CXCL10 had significantly poorer prognoses than those with lower serum CXCL10 levels (p<0.0001, log-rank test); cut-off value = 199 pg/ml. (b), Cancer specific survival curves for 163 patients in stage I-III with curative intent, according to serum CXCL10 level. Patients with higher serum CXCL10 also had significantly poorer prognoses than those with lower serum CXCL10 levels (p=0.0372, log-rank test); cut-off value = 153 pg/ml.

Article Snippet: Primary anti-CXCL10 goat polyclonal antibody (clone AF976, R&D Systems, MN, USA) and anti-CXCR3 rabbit polyclonal antibody (clone ab8023, Abcam, Cambridge, UK) at a dilution of 1:25 were used for the labeled streptavidinbiotin method (LASB2 kit/HRP, Dako Cytomation).

Techniques:

Journal: International journal of oncology

Article Title: Evaluation of CXCL10 as a novel serum marker for predicting liver metastasis and prognosis in colorectal cancer.

doi: 10.3892/ijo.2011.1247

Figure Lengend Snippet: Figure 4. CXCL10 protein expression was assessed using immunohistochemistry in normal mucosa with primary CRC (a), primary colorectal cancer (b) and liver metastasis tissue (c). Magnification, x40. Scale bar = 500 µm.

Article Snippet: Primary anti-CXCL10 goat polyclonal antibody (clone AF976, R&D Systems, MN, USA) and anti-CXCR3 rabbit polyclonal antibody (clone ab8023, Abcam, Cambridge, UK) at a dilution of 1:25 were used for the labeled streptavidinbiotin method (LASB2 kit/HRP, Dako Cytomation).

Techniques: Expressing, Immunohistochemistry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 1. VCAM-1, P-selectin, sTNFR-1, and CXCL16 are elevated in the urine in spontaneous lupus nephritis in three different mouse models of lupus. The levels of the above molecules were determined by ELISA in 24-h urine samples obtained from 6-mo-old B6.Sle1.lpr, MRL.lpr, and NZM2410 lupus mice (all of which exhibited proteinuria and glomerulonephritis), as well as 6-mo-old B6 controls (A). Indicated p values per- tain to Student’s t test or Mann-Whitney U test compar- isons of B6 urine vs the other groups (, p 0.05; , p 0.01; , p 0.001). n 5 mice/group. For each urine sample depicted in Fig. 1A, the 24-h urine level of total protein was plotted against the 24-h urine levels of the four molecules (B). Typically, healthy B6 mice do not excrete 1 mg of protein over a 24-h period (18, 19).

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 2. Urinary VCAM-1, P-selec- tin, sTNFR-1, and CXCL16 are higher than the corresponding serum levels. Se- rum and urine levels of the above four molecules were determined by ELISA, us- ing samples from 2-mo-old B6, 2-mo-old MRL.lpr, and 6-mo-old MRL.lpr mice. In- dicated p values pertain to Student’s t test or Mann-Whitney U test comparisons of B6 urine vs the other groups (indicated be- low the x-axis), or young vs old MRL.lpr samples, as indicated above each plot (, p 0.05; , p 0.01; , p 0.001). n 12 mice/group.

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 3. The urinary enrichment of VCAM-1 and CXCL16 is also noted in other strains with spontaneous lupus nephritis. Serum and urine levels of VCAM-1 and CXCL16 were determined by ELISA, using samples from 2- to 5-mo-old B6, B6.Sle1.lpr, and BXSB mice, as in- dicated. Displayed p values pertain to the Student t test or Mann-Whit- ney U test comparisons of young vs old mice (, p 0.05; , p 0.01; , p 0.001); n 6 mice/group.

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 4. VCAM-1, TNFR-1, P-selectin, and CXCL16 are hyperex- pressed within the diseased kidneys in lupus. Renal cortical lysates were prepared from B6 control, as well as BXSB and MRL.lpr mice, aged 5–6 mo. The levels of the four molecules were determined by ELISA. Indicated p values pertain to Student’s t test or Mann-Whitney U test comparisons of B6 vs other strains (, p 0.05; , p 0.01; , p 0.001). n 6 mice/group. Group means are indicated by the horizontal bars.

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques: Control, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 5. CXCL16, p-selectin, sTNFR-1, and VCAM-1 may be ex- pressed in the kidneys. Immunostaining of mouse kidneys from 2- and 7-mo-old MRL.lpr mice was performed using Abs against the above four molecules. Shown figures are representative of the histology seen in five mice per group.

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques: Immunostaining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 6. Urine VCAM-1, P-selectin, sTNFR-1, and CXCL16 are also elevated in human lupus nephritis. A, Spot urine samples from 38 SLE patients (Table I), 15 healthy adults, and 6 RA patients were assayed for the levels of VCAM-1, P-selectin, sTNFR-1, and CXCL16. Indicated p values pertain to Student’s t test or Mann-Whitney U test comparisons of the urinary levels of each molecule in patients against that in normal controls (indicated below the x-axis), or SLE vs RA, as indicated at the top (, p 0.05; , p 0.01; , p 0.001). Shown also are the correlation profiles of these urinary molecules (normalized to urine creatinine) against the corresponding urine protein-creatinine ratios (B) and SLEDAI scores (C). R: correlation coefficient.

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques: MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 7. Specificity/sensitivity ROC curves per- taining to the use of urinary VCAM-1 (A), CXCL16 (B), sTNFR-1 (C), and P-selectin (D) for discriminating SLE patients from normal controls. The data shown in Fig. 6 were used to generate the shown ROC curves, which plot the specificity and sensitivity profiles of the indicated uri- nary molecules in distinguishing SLE patients from nor- mal controls. Indicated are the corresponding AUC values.

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 8. SLE patients with active ne- phritis exhibit the highest levels of urine VCAM-1, P-selectin, sTNFR-1, and CXCL16. Urine levels of the indicated molecules were assayed in SLE patients and normal controls. The SLE patients were divided into four groups: (1) inactive SLE (SLEDAI 0); (2) mild-active, non- renal SLE (SLEDAI 1–5, renal SLE- DAI 0); (3) severe-active, nonrenal SLE (SLEDAI 6; renal SLEDAI 0); (4) ac- tive renal SLE (renal SLEDAI 0). Indi- cated p values pertain to Student’s t test or Mann-Whitney U test comparisons of each subject group vs “mild-active, nonrenal SLE” (, p 0.05; , p 0.01; , p 0.001).

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques: MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 9. Specificity/sensitivity ROC curves per- taining to the use of urinary VCAM-1 (A) and CXCL16 (B) for discriminating SLE patients with active renal dis- ease. The data shown in Fig. 8 were used to generate the shown ROC curves, which plot the specificity and sensi- tivity profiles of the indicated urinary molecules in dis- tinguishing SLE patients with active renal disease from all other SLE patients. Indicated are the corresponding AUC values.

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 10. Urine vs serum levels of VCAM-1, P-selectin, sTNFR-1, and CXCL16 in human lupus. Serum levels of the above four molecules were also assayed in SLE patients with inactive SLE (SLEDAI 0; n 5, randomly selected from the patients listed in Ta- ble I) and in patients with “active SLE” (SLEDAI 12; n 12, randomly se- lected from the patients listed in Table I). Displayed are the absolute levels (left column) as well as the albumin- normalized values (middle column) for the four molecules assayed. Displayed in the right column are the urine-serum ratios of these four molecules, after normalization against the correspond- ing albumin levels. Indicated p values pertain to Student’s t test or Mann- Whitney U test comparisons of patients vs “normal” (indicated below the x-axis), or “inactive” vs “active” pa- tients, as indicated above each plot (, p 0.05; , p 0.01; , p 0.001).

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques: MANN-WHITNEY