Journal: The Journal of infectious diseases

Article Title: CXCL16 contributes to neutrophil recruitment to cerebrospinal fluid in pneumococcal meningitis.

doi: 10.1086/656532

Figure Lengend Snippet: Figure 1. In vitro and in vivo expression analysis of CXCL16. A, Levels of soluble CXCL16 (expressed as nanograms per milliliter) in cerebrospinal fluid samples of patients with pneumococcal meningitis (right) ( ) and control patients with noninflammatory central nervous system diseases n p 20 (left) ( diagnoses: migraine [ ], tension-type headache [ ], exertional headache [ ], normal pressure hydrocephalus [ ], facial n p 16 n p 8 n p 1 n p 1 n p 1 paresis [ ], amyotrophic lateral sclerosis [ ], and herniated disc [ ]. Data are the mean value standard deviation ( ). B, CXCL16 n p 4 n p 1 n p 1 P ! .05 levels (expressed as picograms per milliliter) in the supernatant of cell cultures (RAW264.7 macrophages, bEnd5 brain microvascular endothelial cells, and Hoxb8 neutrophils; in each experiment) stimulated with ethanol-killed Streptococcus pneumoniae (EKP) or left unstimulated (medium). Data n p 6 are the mean value standard deviation ( ). C–E, Immunohistochemical staining of CXCL16 in formalin-fixed and paraffin-embedded brains: P ! .05 uninfected control mice (C), infected mice at 24 h after infection (D), and infected mice at 48 h after infection (E ). Insets show microvascular endothelial cells and parenchymal cells. The scale bar denotes 100 mm. F and G, Expression of L-selectin and CXCL16 in the brains of uninfected control mice, infected wild-type mice, MyD88/ mice, and Toll-like receptor (TLR) 2/TLR4/ mice determined by protein array analysis (F ). Optical densities of CXCL16 expression are expressed in the percentage of positive controls (G).

Article Snippet: Slides were then incubated overnight at 4 C with a rat anti–mouse CXCL16 antibody (R&D Systems) or the appropriate isotype control immunoglobulin.

Techniques: In Vitro, In Vivo, Expressing, Control, Standard Deviation, Immunohistochemical staining, Staining, Infection, Protein Array

Journal: The Journal of infectious diseases

Article Title: CXCL16 contributes to neutrophil recruitment to cerebrospinal fluid in pneumococcal meningitis.

doi: 10.1086/656532

Figure Lengend Snippet: Figure 2. Functional analysis of CXCL16. A and B, Cerebrospinal fluid (CSF) pleocytosis (CSF white blood cells) (A) and bacterial cerebellar titers (expressed as colony-forming units, presented in log titers) (B) in mice infected with Streptococcus pneumoniae and treated intraperitoneally either with a monoclonal antibody to murine CXCL16 (CXCL16-Ab) or with an IgG2A isotype control (isotype) before (left) (7 animals per group) or 24 h after (right) (11 animals per group) infection. Evaluation was performed 24 h after administration of the antibody to each group. C–F, Fluoresence-activated cell sorter analyses on Hoxb8 neutrophils. C, Expression of the neutrophil marker Mac-1. D, Expression of the CXCL16 receptor CXCR6 in unstimulated neutrophils (31%). E, Expression of the CXCL16 receptor CXCR6 in neutrophils stimulated with tumor necrosis factor (TNF)–a (56%). F, Expression of the CXCL16 receptor CXCR6 in neutrophils stimulated with ethanol-killed Sreptococcus pneumoniae (EKP) (66%). G, Chemotactic activities of CXCL16 on Hoxb8 neutrophils using a microchemotaxis chamber. Cell migration is expressed as the mean number of leukocytes that migrated per field. MF, microscopic field; rCXCL16, diluted recombinant CXCL16. H, Expression of the typical neutrophil-attracting chemokines CXCL1 (KC), CXCL2 (MIP-2), and CXCL5 (LIX) in mouse brain homogenates, as determined using a mouse cytokine antibody array. Optical densities of all 3 chemokines in uninfected controls, infected isotype (isoAb)–, and CXCL16-antibody (CXCL16-Ab)–treated animals (4 per group, done twice) are expressed in a percentage of positive controls.

Article Snippet: Slides were then incubated overnight at 4 C with a rat anti–mouse CXCL16 antibody (R&D Systems) or the appropriate isotype control immunoglobulin.

Techniques: Functional Assay, Infection, Control, Expressing, Marker, Migration, Recombinant, Ab Array

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: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: (A) CXCL16 mRNA expression in the kidney during MuPyV infection. Expression is shown as fold change relative to sham infected samples. Data are from three independent experiments (n = 10-11). (B) Expression of NKCC2 [marks the ascending loop of Henle ], CD8, and CXCL16 in epithelia in sham-infected and 8 dpi kidneys; bottom right photomicrographs are merged images. Representative of two independent experiments. (C) CXCL16 expression in sham-infected, 4 dpi, and 8 dpi kidney lysates (left). Western blot image is representative of two independent experiments with each lane indicating protein lysate from kidneys of individual mice. Protein band intensity quantification for sCXCL16 was normalized to β-actin and analyzed by ImageLab and normalized to the loading control (right). Data are combined from two independent experiments (n = 3-5). (D) Experimental design of in vivo CXCL16 mAb administration. Mice were administered 250 µg of CXCL16 mAb or control rat IgG every two days from days 4-14 post infection and euthanized at 15 dpi. Numbers of CD45 mAb i.v.-negative, CD8 + CD44 + D b -LT359 tetramer + T cells and CD4 + CD44 + T cells in kidneys and spleens of infected mice given anti-CXCL16 or control rat IgG. Data were analyzed by one-way ANOVA (A and C) and by multiple Mann-Whitney tests (D).

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Infection, Western Blot, Control, In Vivo, MANN-WHITNEY

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: (A) Dense lymphoplasmacytic infiltrate surrounding and infiltrating cortical tubules (H&E, x400). (B) Immunohistochemical staining for Large T antigen in nuclei of tubular epithelium (x250). (C) Expression of CXCR6 on infiltrating CD8 + cells. Photomicrographs (right) are enlarged images in the white squares (left); bottom right are merged images. No 1 o , no primary antibody. (D) Log-fold change of CXCL16 and CXCR6 across four independent studies, each comparing KTx biopsies from patients with PVAN and stable graft function. The error bars indicate the 95% confidence interval of log-fold change, with horizontal dashed grey line indicating log-fold change = 0, or no difference. GSE120495 is an RNA-seq study, while remaining studies are microarray based. Given the heterogeneity of the data, a Cauchy combination method with uniform weights was employed to integrate the p-values across these studies. The combined p-values for CXCL16 = 0.000337 and CXCR6 = 2.72x10 -05 . ns, non-significant at adjusted p-value > 0.05; *, statistical significance at adjusted p-value ≤ 0.05; **, statistical significance at adjusted p-value ≤ 0.01; ***, statistical significance at adjusted p-value ≤ 0.001.

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Immunohistochemical staining, Staining, Expressing, RNA Sequencing, Microarray

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: Summary of differential gene expression of CXCL16 and CXCR6 in four studies of KTx biopsies with PVAN. Because of the heterogeneity of the data (i.e., one RNA-seq, three microarray), a Cauchy combination method with uniform weights was employed to integrate the p-values across these studies. P-values significant after false discover rate (FDR) correction are bolded and italicized in the Adj. P-value column. Cauchy combined p-values < 0.05 are bolded and italicized.

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Gene Expression, Microarray