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

Journal: Cellular and molecular biology (Noisy-le-Grand, France)

Article Title: Homocysteine modulates CXCL10/CXCR3 axis activity to induce endothelial dysfunction.

doi: 10.14715/cmb/2024.70.2.28

Figure Lengend Snippet: Fig. 3. (A, B) qRT-PCR and Western blot analysis of CXCL10 and CXCR3 expression in HAECs treated with different concentrations of Hcy. (C) IHC analysis of the aorta in HHcy mice showing CXCL10 and CXCR3 expression. (D) qRT-PCR analysis of CXCL10 and CXCR3 expression in arterial endothelial cells.

Article Snippet: Additionally, HAECs were exposed to specific agents including Anti-CXCL10 antibodies (701225, Invitrogen, USA), Anti-CXCR3 antibodies (ab71864, Abcam, UK), IgG control antibodies (31154, Thermo Fisher, USA), NBI-74330 (a CXCR3 inhibitor, 4528, Tocris Bioscience, UK), and CXCL10 agonist (266-IP, R&D Systems, USA).

Techniques: Quantitative RT-PCR, Western Blot, Expressing

Journal: Cells

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

doi: 10.3390/cells10020274

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

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

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

Journal: Cells

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

doi: 10.3390/cells10020274

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

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

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

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Trophoblast-Induced Changes in C-X-C Motif Chemokine 10 Expression Contribute to Vascular Smooth Muscle Cell Dedifferentiation During Spiral Artery Remodeling

doi: 10.1161/atvbaha.112.300354

Figure Lengend Snippet: Figure 2. C-X-C motif chemokine 10 (CXCL10), stanniocalcin-1, and placental growth factor mRNA expression is significantly increased by trophoblast conditioned media (CM). Messenger RNA expression in vascular spheroids was measured by quantitative real-time PCR after stimulation with trophoblast CM for 24 hours (n=4). Data are expressed as mRNA expression relative to an external calibrator. A, CXCL10. B, Stanniocalcin-1. C, Placental growth factor. All data are presented as mean±SEM. *P<0.05.

Article Snippet: CXCL10 recombinant protein was purchased from R & D Systems (Abingdon, UK).

Techniques: Expressing, RNA Expression, Real-time Polymerase Chain Reaction

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Trophoblast-Induced Changes in C-X-C Motif Chemokine 10 Expression Contribute to Vascular Smooth Muscle Cell Dedifferentiation During Spiral Artery Remodeling

doi: 10.1161/atvbaha.112.300354

Figure Lengend Snippet: Figure 3. C-X-C motif chemokine 10 (CXCL10) protein is expressed by vascular spheroids stimulated with trophoblast con- ditioned media. A, Vascular spheroid lysates were examined by Western blot analysis for the presence of CXCL10. Tubulin was used as an internal loading control. The image shown is represen- tative of 3 independent experiments. Densitometric analysis of Western blots (n=3) with mean±SEM CXCL10/tubulin presented as a fold-change over control-treated spheroids. *P<0.05. Vascu- lar spheroids treated with control media (B) or trophoblast condi- tioned media (C) were cryosectioned and sections were examined by immunostaining for the presence of CXCL10. Sections were taken through approximately the center of the spheroid. Negative control incubated with rabbit IgG in place of primary antibody is inset. Scale bar,100 µm.

Article Snippet: CXCL10 recombinant protein was purchased from R & D Systems (Abingdon, UK).

Techniques: Western Blot, Control, Immunostaining, Negative Control, Incubation

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Trophoblast-Induced Changes in C-X-C Motif Chemokine 10 Expression Contribute to Vascular Smooth Muscle Cell Dedifferentiation During Spiral Artery Remodeling

doi: 10.1161/atvbaha.112.300354

Figure Lengend Snippet: Figure 4. C-X-C motif chemokine 10 (CXCL10) protein is expressed by first trimester decidua and dissected spiral arteries stimulated with trophoblast conditioned media. CXCL10 (A and C) and α-smooth muscle actin protein (D) expression was examined by immunohis- tochemistry in serially sectioned first trimester decidua (n=5; representative image shown). CXCL10 and α-smooth muscle actin protein colocalized to the same cells (indicated by arrowheads). Trophoblasts (labeled with CK7, B) were present at this stage of remodeling. Negative control (inset) was incubated with nonimmune IgG in place of primary antibody. Scale bar represents 100 µm or 50 µm in zoom. (E) Expression of CXCL10 (green) and (F) VWF, an endothelial cell marker (red), was examined in a dissected spiral artery treated with extravillous trophoblast (EVT) conditioned media (G, merge). EC indicates endothelial cells; and VSM, vascular smooth muscle. Negative control (inset) was incubated with nonimmune IgG in place of primary antibody. Scale bar, 50 µm.

Article Snippet: CXCL10 recombinant protein was purchased from R & D Systems (Abingdon, UK).

Techniques: Expressing, Labeling, Negative Control, Incubation, Marker

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Trophoblast-Induced Changes in C-X-C Motif Chemokine 10 Expression Contribute to Vascular Smooth Muscle Cell Dedifferentiation During Spiral Artery Remodeling

doi: 10.1161/atvbaha.112.300354

Figure Lengend Snippet: Figure 5. The role of IFN-γ in C-X-C motif chemokine 10 (CXCL10) expression. A, Recombinant IFN-γ induces CXCL10 expression in vascular spheroids. Control or rhIFN-γ was added to spheroids made of endothelial cell (EC) alone, vascular smooth muscle cell (VSMC) alone, or cocultured EC/VSMC. CXCL10 expression was measured by Western blot analysis (n=4). B, Neutralizing IFN-γ in extravillous trophoblast (EVT) conditioned media (CM) decreased vascular spheroid CXCL10 expression. Control media or EVT CM was incubated with IFN-γ–neutralizing antibody or corresponding IgG control and added to EC/VSMC spheroids. CXCL10 expression was determined by Western blot analysis. *P<0.05 (n=5).

Article Snippet: CXCL10 recombinant protein was purchased from R & D Systems (Abingdon, UK).

Techniques: Expressing, Recombinant, Control, Western Blot, Incubation

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Trophoblast-Induced Changes in C-X-C Motif Chemokine 10 Expression Contribute to Vascular Smooth Muscle Cell Dedifferentiation During Spiral Artery Remodeling

doi: 10.1161/atvbaha.112.300354

Figure Lengend Snippet: Figure 6. The effects of extravillous tro- phoblast (EVT) conditioned media (CM) and C-X-C motif chemokine 10 (CXCL10) on vascular smooth muscle cells (VSMCs). VSMCs were incubated for 72 hours in media containing 0.5% FCS, then a further 72 hours with indicated concentrations of EVT CM and recombi- nant human CXCL10 (n=at least 5 inde- pendent experiments). Expression of (A) α-smooth muscle actin and (B) calponin was examined by Western blot analysis. Time-lapse microscopy was used to analyze the effects of rhCXCL10 (C) and EVT CM (D) on VSMC motility during a 24-hour incubation. Data are displayed as the mean±SEM of a minimum of 3 pooled experiments. *P<0.05; **P<0.01.

Article Snippet: CXCL10 recombinant protein was purchased from R & D Systems (Abingdon, UK).

Techniques: Incubation, Expressing, Western Blot, Time-lapse Microscopy