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: PLoS ONE

Article Title: Human Primary Adipocytes Exhibit Immune Cell Function: Adipocytes Prime Inflammation Independent of Macrophages

doi: 10.1371/journal.pone.0017154

Figure Lengend Snippet: Panel A shows the analysis of CD4+ cells migration caused by adipocytes-produced chemokines after 10 h incubation. The lower compartment (insert) contained attached adipocytes treated with LPS (200 ng/ml) or untreated, as well as antibodies against MCP-1 and IP-10 (2 µg/ml) to neutralize the effect of these two chemo-attractants. The upper chamber (insert) contained purified and rested CD4+ T cells. The cells were incubated at 37°C for 10 h. The inserts were then removed and the cells migrated toward the adipocytes were collected in culture medium without serum. The number of migrated cells was obtained by cell counting using a Bürker-Türk chamber. This figure represents two independent experiments with similar results (only differ in the number of migrated cells). Each experiment was done in triplicate. Error bars show standard deviations of means for triplicate cultures per condition. The p-value obtained from an independent two-tailed test samples (T-test). P<0.05 was accepted as statistically significant. The controls in the migration assay were 1- culture medium without serum, 2- culture medium serum free with LPS, and culture medium serum free with IgG isotype (this is not shown in Figure 10 A and 10 B , because this was executed separately and the obtained results were the same as culture medium serum free with LPS). Panel B shows the analysis of CD4+ cells migration caused by adipocyte-produced chemokines after 30 h incubation. All cell migration conditions were similar to the conditions described in panel A , except that the cells were incubated at 37°C for 30 h.

Article Snippet: One migration experiment was performed with the plated CD4+ cells (7×10 5 ) suspension on top of a culture insert in the absence of serum and incubated at 37°C for 30 h. Monoclonal antibodies against human MCP-1 and IP-10 (R & D system) were used to neutralize the effect of these two chemoattractants in migration assay.

Techniques: Migration, Produced, Incubation, Purification, Cell Counting, Two Tailed Test

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: Cell death and differentiation

Article Title: RIG-I-like helicases induce immunogenic cell death of pancreatic cancer cells and sensitize tumors toward killing by CD8(+) T cells.

doi: 10.1038/cdd.2014.96

Figure Lengend Snippet: Figure 1 RLH activation induces secretion of proinflammatory cytokines and induction of apoptosis in murine pancreatic cancer cells. (a) Panc02 cells were stimulated with indicated amounts of ppp-RNA, poly(I:C) or left untreated. OH-RNA served as transfection control. IFN-b levels were analyzed with qRT-PCR relative to HPRT and secretion of CXCL10 or IL-6 was measured with ELISA; (b) Panc02 cells were stimulated with RNA (24 h for poly(I:C) and 48 h for ppp-RNA) and viability was assessed by FACS analysis using annexin V/PI staining; (c) Panc02 cells were incubated with siRNA specific for RIG-I or MDA5 for 24 h and subsequently stimulated with ppp-RNA or poly(I:C). Induction of apoptosis was measured by annexin V/PI staining. Silencing efficacy, as assessed by western blot, is shown; (d) activated caspase-9 (green) was visualized using green FLICA caspase-9 assay kit. Cell membranes were costained with cholera toxin B subunit (red) and nuclei with DAPI (blue); (e and f) Panc02 cells were treated as indicated for 48 h. Full length PARP-1 (116 kDa) and the cleaved large fragment of PARP-1 (89 kDa) (e) as well as the autophagy markers LC3B-I and LC3B-II (f) were analyzed by western blot. Results are representative of at least three independent experiments

Article Snippet: After fixation with paraformaldehyde and permeabilization with saponin (both Sigma), DCs were stained for IL-6 (PE, clone MP5-20F3; BioLegend) or goat anti-mouse CXCL10 (R&D Systems).

Techniques: Activation Assay, Transfection, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Incubation, Western Blot

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 2. Upregulation of CXCL10 and MHC class II in human neutrophils in urine during BCG infusion therapy. (a,b) Comprehensive analysis of mRNA expression in urine-derived neutrophils compared to peripheral blood neutrophils. Blood and urine were collected from three patients after one week from the 6th BCG infusion. Comprehensive analysis of mRNA in neutrophils was performed using a DNA tip microarray. (a) Cluster analysis after adjustment and standardiza- tion. The mRNA expression in neutrophils obtained from urine samples (vertical axis) or periph- eral blood (horizontal axis) was analyzed. White lines indicate the thresholds for genes that are upregulated or downregulated > 2-fold between urine- and blood-derived neutrophils. A relatively higher expression in urine-derived neutrophils is indicated using arrows, including expression for CXCR3 ligands (CXCL9 and CXCL10) and MHC class II (HLA-DRB1, HLA-DPA1, and HLA-DQA1). (b) Volcano plot depicting the differentially expressed genes between peripheral blood-derived and urine-derived neutrophils after the 6th BCG infusion. The horizontal axis denotes the fold change in mRNA expression in neutrophils from the urine and blood, while the vertical axis represents the –log10 (p-value) for a t-test of differences in neutrophils from the blood and urine. These data represent the top 6000 genes of the –log10 (p-value). The gene expressions of CXCR3 ligands (CXCL9, CXCL10, and CXCL11) and MHC class II (HLA-DQA2, HLA-DPA1, and HLA-DQA1) were also detected as characteristic features of urine-derived neutrophils (arrows). (c,d) Representative data of intracellular- stained neutrophilic cells obtained via flow cytometric analysis. The CD33+CD15+ neutrophilic cells in the blood (c) or urine (d) samples were obtained from the same patient who was treated with 4th BCG infusions and are presented as CXCL10 MFI (upper panels) and HLA-DR MFI (lower panels). Gray-closed histograms indicate each background staining, and light blue line histograms denote the staining of CXCL10 or HLA-DR. (e–g) Comparison of intracellular expression of (e,f) CXCL10 and (g) HLA-DR in neutrophilic cells from the blood (open circle) and urine (closed circle) samples. These samples were collected after one week from the 2nd to the 6th BCG infusions (after each infusion). (e) ∆CXCL10 MFI was calculated as follows: ∆CXCL10 MFI = (MFI of PE-conjugated anti-CXCL10 mAb staining) −(MFI of PE-conjugated control IgG staining). (f) The neutrophilic cells

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Expressing, Derivative Assay, Microarray, Staining, Comparison, Control

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 3. Effect of BCG on CXCL10 and MHC-II expression in human or mice neutrophilic cells in vitro. Human (a,d) or mouse (b,e) peripheral blood was diluted ten-fold in 10% FCS RPMI1640, or mouse bone marrow cells (4 × 106/mL; c,f) were incubated with or without 4 µg/mL of BCG for 20 h. Following incubation, the expression levels of CXCL10 (a–c) and MHC class II (d–f) in human (CD33+CD15+) or mouse neutrophils (CD45+Ly6G+) were analyzed, as described in Figure S2. Statistical significance was calculated with the paired t-test, * p < 0.05 (n = 3). Abbreviations: BCG, Bacillus Calmette–Guérin; CXCL10, chemokine (C-X-C motif) ligand 10; HLA-DR, human major histocompatibility complex class II cell surface receptor; MFI, mean fluorescence intensity; and I-A/I-E, mouse major histocompatibility complex class II cell surface receptor.

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Expressing, In Vitro, Incubation, Immunopeptidomics, Cell Surface Receptor Assay

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 4. Upregulation of CXCL10 and MHC class II in monocytes and neutrophils in peritoneal effusion cells after BCG injections. Mice were injected with B16F10 cells (5 × 104 cells/100 µL/head), and the PECs were collected after two weeks. The PECs induced after one injection of BCG (40 µg/head) after 16 h and the PECs induced after five repeated injections of BCG (40 µg/head) after 16 h from the final injection are presented as “1-shot” and “5-shots”, respectively. These PECs were intracellularly stained with each antibody, and the relative expression (MFI) of CXCL10 and I-A/I-E was analyzed in CD45+Ly6C+ cells and CD45+Ly6G+ cells, respectively. (a–i) Representative flow cytometric analysis of mouse monocytes (Ly6C+ cells) and neutrophils (Ly6G+ cells) via flow cytometry. The (a–c) panels present flow cytometric analysis of the PECs induced 2 weeks after B16F10 cell injection (presented as “Tumor”). The (d–f) panels show representative flow cytometric analysis of the PECs induced 16 h after the administration of BCG (presented as “1-shot). The (g–i) panels indicate representative flow cytometric analyses of the PECs induced via five repeated BCG injections at one-week intervals. The PECs were collected 16 h after the final BCG admin- istration (presented as “5-shots”). The left panels (a,d,g) show CD45+ leukocytes presented with the gates of Ly6C+ cells (monocytic cells) and Ly6G+ cells (neutrophilic cells). (j–l) The number and proportion of myeloid cells (Ly6C+ and Ly6G+ cells) of the PECs. The peritoneal effusion cells obtained after injection of B16F10 cells are presented as “tumor” (open circles). The cells induced 16 h after a single administration of BCG are presented in the group “1-shot” (closed circles). The cells induced via five repeated injections of BCG are presented in the group “5-shots” (closed triangles). The (j) number of the cells in peritoneal fluid were counted using a hemocytometer, and the proportions of (k) Ly6C+ cells and (l) Ly6G+ cells in CD45+ leukocytes were analyzed via flow cytometry. (m–p) The intracellular expression levels of CXCL10 and MHC-II (I-A/I-E) in mouse monocytes (Ly6C+ cells) and neutrophils (Ly6G+ cells) after BCG injection. These PECs were intracellularly stained with each anti- body, and the relative expression (MFI) of (m,n) CXCL10 and (o,p) I-A/I-E was analyzed in (m,o) CD45+Ly6C+ cells and (n,p) CD45+Ly6G+ cells, respectively. Statistical analyses were per- formed using the Kruskal–Wallis test with the Dunn’s post-hoc test. Each bar is presented as the mean of data. * p < 0.05; ** p < 0.01; and ns, not significant. Abbreviations: PECs, peritoneal exudate cells; CXCL10, C-X-C motif chemokine ligand 10; BCG, Bacillus Calmette–Guérin; and MFI, mean fluorescence intensity.

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Injection, Staining, Expressing, Cytometry

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 7. CXCL10 and MHC class II expression in neutrophils induced via BCG was inhibited via partial neutrophil depletion using anti-Ly6G mAbs. BCG (40 µg/100 µL/head) was injected into the peritoneal cavity, following which the antibodies (100 µg/50 µL/head; control mAb, open circle; or anti-Ly6G mAb, closed circle) were injected into the

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Expressing, Injection, Control

Journal: Journal of Virology

Article Title: Human Nasal Turbinate Tissues in Organ Culture as a Model for Human Cytomegalovirus Infection at the Mucosal Entry Site

doi: 10.1128/JVI.01258-20

Figure Lengend Snippet: Nasal mucosal tissue transcriptional response to HCMV infection. RNA was extracted from mock- and HCMV-infected tissues at 24 hpi and subjected to transcriptome analysis. To account for the potential donors’ tissue-to-tissue variability, two pools (each representing a mixture of 5 independent donor tissues) for each experimental condition were used, together representing 10 tissues from different individuals. (A) MA plot (log ratio versus abundance) comparing gene expression profiles in mock- and HCMV-infected tissues for all 26,340 queried genes. Genes of higher abundance in infected tissues are indicated by positive changes, and those of lower abundance in infected tissues are indicated by negative changes. Red dots denote significant genes (adjusted P [Padj] < 0.1); orange dots denote added nonsignificant genes (see Materials and Methods). (B) Heat map representation of all differentially expressed and added genes (red and orange dots in panel A), comparing the two pools of mock- and HCMV-infected tissues. Normalized expression values were scaled at gene level (scale is shown at top-right), then hierarchically clustered and drawn as a heat map. Representative upregulated innate immunity genes further analyzed by qRT-PCR as shown in panel F and downregulated epithelial-cell related genes are indicated. (C) Selected antiviral and proinflammatory innate immunity genes with a strong (>3-fold) upregulation in HCMV-infected versus mock-infected tissues. (D) AQP5 and MUC2 mRNA expression levels in HCMV infected tissues relative to the normalized levels (normalized to a value of 1) in mock-infected tissues. (E and F) The indicated viral/cellular mRNA levels were analyzed by qRT-PCR and normalized by cellular β-actin. The fold change between HCMV-infected and mock-infected tissues (F) is shown for each cellular gene in 6 independent nasal turbinate tissues (T1 to T6) obtained from different individuals. (G and H) Functional analysis of conditioned medium (CM) recovered at 1 (E) and 5 (F) dpi from mock- and HCMV-infected nasal turbinate tissues (prepared as described in Materials and Methods). (G) Human foreskin fibroblasts (HFF) and human retinal pigmented epithelial cells (ARPE) cultures were pretreated overnight with conditioned medium (CM) and infected with HCMV. HCMV IE1 mRNA levels were analyzed by qRT-PCR at 24 hpi and normalized by cellular β-actin. (H) Peripheral blood leukocytes (PBL) transwell migration toward CM from mock- or HCMV-infected nasal turbinate cultures (treated with ganciclovir when indicated). CM samples were preincubated with no IgG (control) or with α-CXCL10 antibodies, as indicated. The number of migrated cells was determined by flow cytometry. The data shown are representative of at least three independent experiments. Significant changes are indicated as *, P < 0.05; **, P < 0.01; ***, P < 0.001. NT, nontreated; n.s., nonsignificant.

Article Snippet: In pretreatment experiments, when indicated, CM was incubated with 0.2 μg/ml mouse anti-human CXCL10 monoclonal antibodies (MAB266; R&D Systems) for 1 h at 37°C and 5% CO 2 , before the transwell migration assay.

Techniques: Infection, Gene Expression, Expressing, Quantitative RT-PCR, Functional Assay, Migration, Control, Flow Cytometry

Journal: Journal of Virology

Article Title: Human Nasal Turbinate Tissues in Organ Culture as a Model for Human Cytomegalovirus Infection at the Mucosal Entry Site

doi: 10.1128/JVI.01258-20

Figure Lengend Snippet: Primers and probes for real-time PCR analysis

Article Snippet: In pretreatment experiments, when indicated, CM was incubated with 0.2 μg/ml mouse anti-human CXCL10 monoclonal antibodies (MAB266; R&D Systems) for 1 h at 37°C and 5% CO 2 , before the transwell migration assay.

Techniques: Real-time Polymerase Chain Reaction, Sequencing, SYBR Green Assay