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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 1. Semiquantitative RT-PCR analysis for expression of CCL28, CCR10, and CCR3 in various mouse tissues. Total RNA samples were prepared from salivary glands, colon, appendix, small intestine with- out Payer’s patches, and Payer’s patches obtained from 12- to 16-wk-old BALB/c mice. RT-PCR was conducted as described in Materials and Methods. Representative results from three separate experiments are shown. Relative signal intensities obtained by normalization with G3PDH are shown in the lower panels as mean SD.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 2. Surface expression of CCR10 and CCR3 and migration to CCL28 of CD3B220low cells isolated from mouse parotid glands. A, Flow cytometric analysis. Single cells were prepared from parotid glands excised from 12- to 16-wk-old BALB/c mice. For detection of cells expressing CCR10, cells were first incubated with CCL27-Fc or control Fc. After washing, cells were incubated with biotin-labeled goat anti- human IgG. After washing, cells were stained with a cocktail of APC-labeled streptavidin, CyChrome-labeled anti-B220, and PE-labeled anti-CD3. For detection of cells expressing CCR3, cells first were incubated with rabbit polyclonal anti-mCCR3 or control rabbit IgG. After washing, cells were incubated with a cocktail of FITC-labeled anti-rabbit IgG, CyChrome-labeled anti-B220, and PE-labeled anti-CD3. Finally, cells were analyzed on FACSCalibur. Representative results from six independent experiments are shown. B, Chemotaxis assay. Single cells prepared from parotid glands were added to the inserts of Transwell plates, with lower wells containing medium without or with indicated concentrations of mCCL2, mCCL28, or mCCL27. After 4 h at 37°C, cells migrated into lower wells were harvested. Original cells and cells migrated into lower wells were stained with PE-labeled anti-CD3 and CyChrome-labeled anti-B220 and were analyzed by flow cytometry in the presence of a known number of counting beads. Filled bars, CD3B220low cells; open bars, CD3B220 cells. The data are mean SD from three separate experiments. C, Plasma cell morphology of CCR10-expressing cells. Single cells prepared from mouse parotid glands were incubated with CCL27-Fc or control Fc. After washing, cells were stained with FITC-labeled goat anti-human IgG. Cells were then placed on glass slides, fixed with methanol, and further stained with May-Gru¨nwald-Giemsa. Cells in the same fields were observed on a fluorescent microscope under UV (Ca) and visible (Cb) lights. No FITC-staining cells were seen by control Fc (data not shown). Magnification, 1000.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Expressing, Migration, Isolation, Incubation, Control, Labeling, Staining, Chemotaxis Assay, Cytometry, Clinical Proteomics, Microscopy

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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 3. Immunohistochemistry and immunoelectron microscopy of CCL28 in human and mouse salivary glands. Periodate-lysine-4% parafor- maldehyde-prefixed frozen sections of human submandibular gland (A–C, J, and K), mouse parotid gland (D and G), mouse submandibular gland (E and F), and mouse sublingual gland (F and I) were stained with goat anti- human CCL28 (A, B, and J), goat anti-mouse CCL28 (D–F), or normal goat IgG (C, G–I, and K). Immunohistochemistry (A–I): scale bar, 100 m (A–C) and 20 m (D–I); , acinus; , duct. Immunoelectron microscopy (J and K): scale bar, 1 m; arrow, immunoreactive granule; Lu, lumen.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Immunohistochemistry, Immuno-Electron Microscopy, Staining

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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 4. Exocrine secretion of CCL28. A, Measurement of CCL28 in whole and parotid saliva by ELISA. Whole saliva and parotid secretions were obtained from healthy adult donors. All assays were done in triplicate and mean values were calculated. For details, see Materials and Methods. Whole and parotid saliva samples obtained from the same donors were connected by lines. B, Immunoblot analysis for CCL28. Recombinant CCL28, parotid saliva samples (10 l), and mature milk samples (20 l) were loaded as indicated. For details, see Materials and Methods. Repre- sentative results from two separate experiments are shown.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant

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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 5. Analysis of the amino acid sequence of human CCL28. A, Amino acid comparison of histatin-5 and CCL28-C. The zinc-binding mo- tifs HExxH and HExxxH are underlined. B, Hydrophobicity plot of hista- tin-5 and CCL28-C. C, A phylogenic tree of chemokines and antimicrobial peptides. MEC/CCL28 and CCL28-C are boxed.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Sequencing, Comparison, Binding Assay

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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 6. Antimicrobial assay. CCL28, mCCL28, CCL27, CCL28-C, and histatin-5 were examined for antimicrobial activity against C. albicans, P. aeruginosa, and Streptococcus mutans by using the CFU assay. For details, see Materials and Methods. All assays were done in triplicate. Vertical bars indicate SD. Representative results from three separate experiments are shown.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Activity Assay, Colony-forming Unit Assay

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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 8. Flow cytometric analysis on CCL28 antimicrobial activity. C. albicans were treated with CCL28 as indicated. After washing, cells were stained with FITC-labeled annexin V for 10 min and were washed again. After addition of PI at 2 g/ml, cells were immediately analyzed on FACSCalibur. The representative results of three independent experiments are shown.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Activity Assay, Staining, Labeling

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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 9. Antimicrobial activity of CCL28 analyzed by scanning electron microscopy. C. albicans (A–F) was mock treated (A and D) or treated with 10 M CCL28 for 30 min (B and E) or 60 min (C and F). P. aeruginosa (G and H) and Streptococcus mutans (I and J) were mock treated (G and I) or treated with 10 M CCL28 for 2 h (H and J). Microbes were immobilized, dried, coated with 3-nm thick platinum-paradium, and observed by a scanning electron microscope. Scale bars: A, 2 m; D, 200 nm; and G, 300 nm.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Activity Assay, Electron Microscopy, Microscopy

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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 7. Salt-sensitive antimicrobial activity of CCL28. The effects of NaCl concentrations on the antimicrobial activity of CCL28 against C. albicans and P. aeruginosa were examined by using CFU assay. For de- tails, see Materials and Methods. All assays were done in triplicate. Ver- tical bars indicate SD. Representative results from two separate experi- ments are shown.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Activity Assay, Colony-forming Unit Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma.

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Fig. 2 Tumor-derived CCL28 recruits pericytes to promote vascular normalization in the tumor microenvironment

Article Snippet: Chromatin Immunoprecipitation (ChIP) Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Derivative Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma.

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Fig. 3 Tumor-derived CCL28 promotes the expression of angiopoietin-1 via CCR3 in pericytes

Article Snippet: Chromatin Immunoprecipitation (ChIP) Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Derivative Assay, Expressing

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma.

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Fig. 6 CCL28 is involved in bevacizumab-mediated vascular normalization

Article Snippet: Chromatin Immunoprecipitation (ChIP) Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques:

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma.

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Fig. 7 A schematic diagram of tumor microenvironment modulation effects of CCL28

Article Snippet: Chromatin Immunoprecipitation (ChIP) Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques:

Journal: Biology

Article Title: CCL28: A Promising Biomarker for Assessing Salivary Gland Functionality and Maintaining Healthy Oral Environments

doi: 10.3390/biology13030147

Figure Lengend Snippet: CCL28-deficient mice have reduced acidic mucin production in SMG and SLG, with bacterial invasion occurring in their SMG. ( A – F ) SMG and SLG were obtained from WT or CCL28-deficient (CCL28-KO) mice. After preparing the sections, H&E staining ( A , B ) and AB staining ( C , D ) were performed, respectively. Representative images are shown. Scale bars: 100 μm. Relative intensities of AB staining are shown as the means ± SD of results from three independent experiments. *, p < 0.05 ( E , F ). ( G ) Sections of the SMG from WT or CCL28-KO mice were stained with Gram-Hucker’s solution. Representative images are shown. Scale bar: 100 μm (upper panel). The lower panel shows an enlarged image of the dotted area in the upper panel. Scale bar: 10 μm (lower panel). The arrow indicates the gram-positive bacteria.

Article Snippet: The primary antibodies used for immunofluorescence analyses were as follows: Rabbit polyclonal anti-MUC2 (PA5-79702, Invitrogen, Carlsbad, CA, USA) and anti-α-SMA (PA5-87638, Invitrogen); Goat polyclonal anti-CK18 (sc-31700, Santa Cruz Biotechnology, Dallas, TX, USA), anti-mouse CCL28 (AF533, R&D Systems, Minneapolis, MN, USA), and anti-mouse CCR10 (AAM69, Bio-Rad Laboratories, Hercules, CA, USA); Rat monoclonal anti-EpCAM (clone G8.8, BioLegend, San Diego, CA, USA), anti-c-Kit (clone 2B8, BioLegend), and anti-CD31 (clone MEC13.3, BioLegend) antibodies.

Techniques: Staining, Bacteria

Journal: Biology

Article Title: CCL28: A Promising Biomarker for Assessing Salivary Gland Functionality and Maintaining Healthy Oral Environments

doi: 10.3390/biology13030147

Figure Lengend Snippet: CCL28-deficient mice have altered expression of salivary gland markers in the SMG. ( A ) Sections of SMG from WT or CCL28-KO mice were stained for UEA-1 (green) and DNA (blue). Representative images are shown. Scale bar: 40 μm. The right panel shows the same image as the left panel, with the blue signal of the nuclear stain omitted and only the green signal of UEA-1 shown in white. ( B ) Relative intensities of UEA-1 staining are shown as the means ± SD of results from three independent experiments. *, p < 0.05. ( C ) Sections of SMG from WT or CCL28-KO mice were stained for MUC2 (red) and DNA (blue). Representative images are shown. Scale bar: 40 μm. The right panel shows the same image as the left panel, with the blue signal of the nuclear stain omitted and only the red signal of MUC2 shown in white. ( D ) Relative intensities of MUC2 staining are shown as the means ± SD of results from three independent experiments. *, p < 0.05. ( E ) Semi-quantitative RT-PCR for mRNA expression of MUC2, AQP5, α-Amylase, and GAPDH was performed using cDNA prepared from the SMG derived from each of the four WT or CCL28-KO mice. ( F ) The relative expression level of each indicated gene, analyzed using the values obtained by measuring the bands in ( E ), is shown as the means ± SD of results from four independent mice. *, p < 0.05, **, p < 0.01.

Article Snippet: The primary antibodies used for immunofluorescence analyses were as follows: Rabbit polyclonal anti-MUC2 (PA5-79702, Invitrogen, Carlsbad, CA, USA) and anti-α-SMA (PA5-87638, Invitrogen); Goat polyclonal anti-CK18 (sc-31700, Santa Cruz Biotechnology, Dallas, TX, USA), anti-mouse CCL28 (AF533, R&D Systems, Minneapolis, MN, USA), and anti-mouse CCR10 (AAM69, Bio-Rad Laboratories, Hercules, CA, USA); Rat monoclonal anti-EpCAM (clone G8.8, BioLegend, San Diego, CA, USA), anti-c-Kit (clone 2B8, BioLegend), and anti-CD31 (clone MEC13.3, BioLegend) antibodies.

Techniques: Expressing, Staining, Quantitative RT-PCR, Derivative Assay

Journal: Biology

Article Title: CCL28: A Promising Biomarker for Assessing Salivary Gland Functionality and Maintaining Healthy Oral Environments

doi: 10.3390/biology13030147

Figure Lengend Snippet: CCL28-deficient mice lead to structurally defective SMG formation. ( A ) Sections of SMG from WT or CCL28-KO mice were stained with PAS reagent. Representative images are shown. Scale bar: 100 μm. The arrows in the image indicate the duct in which the entire structure was recognized. ( B ) The number of ducts in which the entire structure was recognized, per PAS-stained image of SMG sections from WT or CCL28-KO mice, respectively, was counted as the means ± SD of results from three independent experiments. **, p < 0.01. ( C ) Sections of the SMG from WT or CCL28-KO mice were stained for Phalloidin (F-actin; green) and DNA (blue). Representative images are shown. Scale bar: 40 μm (left panel). The right panel shows an enlarged image of the dotted area in the left panel. Scale bar: 20 μm (right panel). ( D ) Sections of the SMG from WT or CCL28-KO mice were stained for MUC2 (red) and DNA (blue). Representative images are shown. Scale bar: 40 μm (left panel). The right panel shows an enlarged image of the dotted area in the left panel. Scale bar: 10 μm (right panel). ( E ) Sections of the SMG from WT or CCL28-KO mice were stained for CK18 (red) and DNA (blue). Representative images are shown. Scale bar: 40 μm (left panel). The right panel shows an enlarged image of the dotted area in the left panel. Scale bar: 10 μm (right panel). ( F ) Sections of the SMG from WT or CCL28-KO mice were stained for α-SMA (green) and DNA (blue). Representative images are shown. Scale bar: 40 μm. The right panel shows the same image as the left panel, with the blue signal of the nuclear stain omitted and only the green signal of α-SMA shown in white. ( G ) Semi-quantitative RT-PCR for mRNA expression of CK18, α-SMA, and GAPDH was performed using cDNA prepared from the SMG derived from each of the four WT or CCL28-KO mice. ( H ) The relative expression level of each indicated gene, analyzed using the values obtained by measuring the bands in ( G ), is shown as the means ± SD of results from four independent mice. *, p < 0.05.

Article Snippet: The primary antibodies used for immunofluorescence analyses were as follows: Rabbit polyclonal anti-MUC2 (PA5-79702, Invitrogen, Carlsbad, CA, USA) and anti-α-SMA (PA5-87638, Invitrogen); Goat polyclonal anti-CK18 (sc-31700, Santa Cruz Biotechnology, Dallas, TX, USA), anti-mouse CCL28 (AF533, R&D Systems, Minneapolis, MN, USA), and anti-mouse CCR10 (AAM69, Bio-Rad Laboratories, Hercules, CA, USA); Rat monoclonal anti-EpCAM (clone G8.8, BioLegend, San Diego, CA, USA), anti-c-Kit (clone 2B8, BioLegend), and anti-CD31 (clone MEC13.3, BioLegend) antibodies.

Techniques: Staining, Quantitative RT-PCR, Expressing, Derivative Assay

Journal: Biology

Article Title: CCL28: A Promising Biomarker for Assessing Salivary Gland Functionality and Maintaining Healthy Oral Environments

doi: 10.3390/biology13030147

Figure Lengend Snippet: CCL28-deficient mice have reduced expression of stem cell markers in SMG. ( A ) Sections of the SMG from WT or CCL28-KO mice were stained for c-Kit (green) and DNA (blue). Representative images are shown. Scale bar: 40 μm (left panel). The right panel shows an enlarged image of the dotted area in the left panel. Scale bar: 10 μm (right panel). ( B ) Sections of the SMG from WT or CCL28-KO mice were stained for EpCAM (green) and DNA (blue). Representative images are shown. Scale bar: 40 μm (left panel). The right panel shows an enlarged image of the dotted area in the left panel. Scale bar: 10 μm (right panel). ( C ) Semi-quantitative RT-PCR for mRNA expression of c-Kit, EpCAM, and GAPDH was performed using cDNA prepared from the SMG derived from each of the four WT or CCL28-KO mice.

Article Snippet: The primary antibodies used for immunofluorescence analyses were as follows: Rabbit polyclonal anti-MUC2 (PA5-79702, Invitrogen, Carlsbad, CA, USA) and anti-α-SMA (PA5-87638, Invitrogen); Goat polyclonal anti-CK18 (sc-31700, Santa Cruz Biotechnology, Dallas, TX, USA), anti-mouse CCL28 (AF533, R&D Systems, Minneapolis, MN, USA), and anti-mouse CCR10 (AAM69, Bio-Rad Laboratories, Hercules, CA, USA); Rat monoclonal anti-EpCAM (clone G8.8, BioLegend, San Diego, CA, USA), anti-c-Kit (clone 2B8, BioLegend), and anti-CD31 (clone MEC13.3, BioLegend) antibodies.

Techniques: Expressing, Staining, Quantitative RT-PCR, Derivative Assay

Journal: Biology

Article Title: CCL28: A Promising Biomarker for Assessing Salivary Gland Functionality and Maintaining Healthy Oral Environments

doi: 10.3390/biology13030147

Figure Lengend Snippet: CCL28-deficient mice have reduced expression of CD31, a vascular endothelial marker in SMG. ( A ) Sections of the SMG from WT or CCL28-KO mice were stained for CD31 (green) and DNA (blue). Representative images are shown. Scale bar: 40 μm. The right panel shows the same image as the left panel, with the blue signal of the nuclear stain omitted and only the green signal of CD31 shown in white. ( B ) Semi-quantitative RT-PCR for mRNA expression of CD31 and GAPDH was performed using cDNA prepared from the SMG derived from each of the four WT or CCL28-KO mice.

Article Snippet: The primary antibodies used for immunofluorescence analyses were as follows: Rabbit polyclonal anti-MUC2 (PA5-79702, Invitrogen, Carlsbad, CA, USA) and anti-α-SMA (PA5-87638, Invitrogen); Goat polyclonal anti-CK18 (sc-31700, Santa Cruz Biotechnology, Dallas, TX, USA), anti-mouse CCL28 (AF533, R&D Systems, Minneapolis, MN, USA), and anti-mouse CCR10 (AAM69, Bio-Rad Laboratories, Hercules, CA, USA); Rat monoclonal anti-EpCAM (clone G8.8, BioLegend, San Diego, CA, USA), anti-c-Kit (clone 2B8, BioLegend), and anti-CD31 (clone MEC13.3, BioLegend) antibodies.

Techniques: Expressing, Marker, Staining, Quantitative RT-PCR, Derivative Assay

Journal: Cell Death & Disease

Article Title: The chemokine receptor CCR10 promotes inflammation-driven hepatocarcinogenesis via PI3K/Akt pathway activation

doi: 10.1038/s41419-018-0267-9

Figure Lengend Snippet: CCR10-transfected HepG2 and LO2 cells were treated with either the CCR10 agonist-ligand CCL28 or the Akt inhibitor A6730. a Activation of the CCL28-CCR10 axis by CCL28 significantly increased Akt phosphorylation, PCNA protein expression, and b relative cell proliferation in both cell lines, while Akt inhibition produced the opposite effects. Relative cell proliferation is defined as the fold-change in proliferation relative to the untreated parent cell line. * P < 0.05 vs. CCR10 group. All values are reported as means ± standard errors of the mean (SEMs)

Article Snippet: For some in vitro experiments, HepG2 and LO2 cell lines were pre-treated with the pro-inflammatory cytokine TNF (concentrations as indicated; R&D Systems, Minneapolis, MN, USA) for 4 h, recombinant human CC chemokine ligand 28 (CCL28) (400 nM; R&D Systems) for 2 h, or the allosteric Akt inhibitor A6730 (10 μM; Sigma, St. Louis, MO, USA) for 2 h . Human CCL28 is a natural ligand-agonist for human CCR10 .

Techniques: Transfection, Activation Assay, Phospho-proteomics, Expressing, Inhibition, Produced

Journal: Cell Death & Disease

Article Title: The chemokine receptor CCR10 promotes inflammation-driven hepatocarcinogenesis via PI3K/Akt pathway activation

doi: 10.1038/s41419-018-0267-9

Figure Lengend Snippet: Following short-term DEN-induced inflammation (10 days after i.p. DEN injection), ( a ) Western blotting analysis showed significantly enhanced TNF protein expression, CCR10 protein expression, PI3K protein expression, Akt phosphorylation, and PCNA protein expression. Knocking-out CCR10 significantly opposed these inflammation-induced effects but did not significantly affect TNF or PI3K protein expression.* P < 0.05 vs. vehicle WT group, † P < 0.05 vs. DEN-treated WT group. b Western blotting analysis of CCR10 protein expression, PI3K protein expression, Akt phosphorylation, and PCNA protein expression in murine liver tissue 6 h after intraperitoneal (i.p.) injection of TNF, which produced significant increases in CCR10 protein expression, PI3K protein expression, Akt phosphorylation, and PCNA protein expression. Pretreatment with the CCR10 agonist-ligand CCL28 significantly increased Akt phosphorylation and PCNA expression levels, while pretreatment with the Akt inhibitor A6730 produced the opposite effects. Neither CCL28 nor A6730 had any significant effect upon CCR10 or PI3K expression. * P < 0.05 vs. vehicle group, † P < 0.05 vs. TNF group. All values are reported as means ± standard errors of the mean (SEMs). n = 12 mice in each group

Article Snippet: For some in vitro experiments, HepG2 and LO2 cell lines were pre-treated with the pro-inflammatory cytokine TNF (concentrations as indicated; R&D Systems, Minneapolis, MN, USA) for 4 h, recombinant human CC chemokine ligand 28 (CCL28) (400 nM; R&D Systems) for 2 h, or the allosteric Akt inhibitor A6730 (10 μM; Sigma, St. Louis, MO, USA) for 2 h . Human CCL28 is a natural ligand-agonist for human CCR10 .

Techniques: Injection, Western Blot, Expressing, Phospho-proteomics, Produced

Journal: Inflammatory Bowel Diseases

Article Title: B Cells Secrete Eotaxin-1 in Human Inflammatory Bowel Disease

doi: 10.1097/mib.0b013e3182802950

Figure Lengend Snippet: FIGURE 5. TLR4 ligands induce CCR9 and CCR10 expression by eosinophils. A, Histogram depicting levels of homing receptors in eosinophils. Gray fill, isotype control; thick black line, anti-CCR10; thin black line, anti-CCR9; gray line, anti-a4b7. Eosinophils from a patient with UC. B. CCR9 expression levels on peripheral blood eosinophils from healthy subjects (n ¼ 9), patients with CD (n ¼ 33), and patients with UC (n ¼ 16) and CCR10 expression levels on eosinophils from healthy subjects (n ¼ 9), patients with CD (n ¼ 22), and patients with UC (n ¼ 10), **P , 0.001, *P , 0.01, compared with healthy subjects. C, TLR4 expression by eosinophils correlates with CCR10 expression in patients with UC (n ¼ 22). D, Plasma concentrations of LPS correlate with percentages of eosinophils that express CCR10 in patients with UC (n ¼ 39). E, E. coli LPS increases CCR9 expression by purified eosinophils from patients with IBD. Gray fill, media alone; black line, 1 mg/mL E. coli LPS. Representative data from 5 blood samples (CD, n ¼ 3; UC, n ¼ 2). F, Eosinophils isolated from patients with IBD actively chemotaxis in response to CCL25 and CCL28. Treatment with E. coli LPS for 18 hours increased responses to CCL28 compared with untreated cells (P ¼ 0.015). Eosinophils from 2 patients with CD and 1 patient with UC.

Article Snippet: Bottom chambers contained recombinant human chemokines, CCL25 and CCL28 (R&D Systems), at 1000 ng/mL in R10.

Techniques: Expressing, Control, Clinical Proteomics, Isolation, Chemotaxis Assay

Journal: Inflammatory Bowel Diseases

Article Title: B Cells Secrete Eotaxin-1 in Human Inflammatory Bowel Disease

doi: 10.1097/mib.0b013e3182802950

Figure Lengend Snippet: FIGURE 6. Model depicting the possible effect of systemic LPS on eosinophil migration patterns in IBD. During decreased disease activity, hypo-acylated LPS induces eotaxin-1 release from TLR4+ B cells. Eotaxin-1 may direct circulating eosinophils to traffic to certain regions of the gastrointestinal tract or induce emigration of nascent eosinophils from the bone marrow. During increased disease activity, hexa-acylated LPS stimulates CCR9 and CCR10 expression by TLR4+ eosinophils. CCL25 and CCL28 are tissue expressed and may thus direct eosinophils to specific areas of the gastrointestinal tract other than those directed by eotaxin-1.

Article Snippet: Bottom chambers contained recombinant human chemokines, CCL25 and CCL28 (R&D Systems), at 1000 ng/mL in R10.

Techniques: Migration, Activity Assay, Expressing

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

Article Title: CCL28-Deficient Mice Have Reduced IgA Antibody-Secreting Cells and an Altered Microbiota in the Colon.

doi: 10.4049/jimmunol.1700037

Figure Lengend Snippet: FIGURE 1. Generation of CCL28-deficient mice. CCL28-deficient mice (CCL28-knockout/EGFP–knock-in mice) were generated on the C57BL/6 background by replacing exon 1 of the CCL28 gene with a gene cassette encoding EGFP and Neor. (A) Targeting strategy: the murine genomic CCL28 locus, the targeting vector construct, and the targeted allele with a cassette encoding EGFP and Neor are shown. (B) Genomic PCR analysis. PCR was performed for CCL28 using genomic DNA prepared from the tails of WT mice, heterozygous CCL28-deficient mice (CCL28+/2), and homozygous CCL28-deficient mice (CCL282/2). Representative results from two separate experiments are shown. (C) RT-PCR analysis. CCL28 expression was determined by RT-PCR using cDNA samples derived from the indicated tissues. GAPDH was used as an internal control. Represen- tative results from two separate experiments are shown.

Article Snippet: In brief, microbial cells in midlogarithmic growth phase were resuspended at 105 cells per milliliter in 1 mM potassium phosphate buffer (PPB; a mixture of 1 mM K2HPO4 and 1 mM KH2PO4; pH 7.2), mixed with an equal volume of 1 mM PPB, with or without recombinant mouse CCL28 (R&D Systems) in 2-fold serial dilutions, and incubated in a U-bottom 96-well microtest plate at room temperature for 2 h with brief mixing every 15 min. After appropriate dilutions with 1 mM PPB, cells were plated on agar plates and grown at 37 ̊C overnight.

Techniques: Knock-Out, Knock-In, Generated, Plasmid Preparation, Construct, Reverse Transcription Polymerase Chain Reaction, Expressing, Derivative Assay, Control

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

Article Title: CCL28-Deficient Mice Have Reduced IgA Antibody-Secreting Cells and an Altered Microbiota in the Colon.

doi: 10.4049/jimmunol.1700037

Figure Lengend Snippet: FIGURE 2. Immunofluorescence staining of CCL28 and IgA in the colon and sublingual gland. (A) Immunofluorescence staining of CCL28. Tissue sections of the colon and sublingual gland from WT and CCL28- deficient (CCL28-KO) mice were stained for CCL28 (green) and DAPI (blue). Scale bars, 100 mm. Representative results from three separate experiments are shown. (B) Double immunofluorescence staining of CCL28 and CD31. Tissue sections of the colon (Figure legend continues)

Article Snippet: In brief, microbial cells in midlogarithmic growth phase were resuspended at 105 cells per milliliter in 1 mM potassium phosphate buffer (PPB; a mixture of 1 mM K2HPO4 and 1 mM KH2PO4; pH 7.2), mixed with an equal volume of 1 mM PPB, with or without recombinant mouse CCL28 (R&D Systems) in 2-fold serial dilutions, and incubated in a U-bottom 96-well microtest plate at room temperature for 2 h with brief mixing every 15 min. After appropriate dilutions with 1 mM PPB, cells were plated on agar plates and grown at 37 ̊C overnight.

Techniques: Staining

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

Article Title: CCL28-Deficient Mice Have Reduced IgA Antibody-Secreting Cells and an Altered Microbiota in the Colon.

doi: 10.4049/jimmunol.1700037

Figure Lengend Snippet: FIGURE 7. Severe DSS-induced colitis of CCL28-deficient mice. Co- litis was induced in WT and CCL28-deficient (CCL28-KO) mice by ad- ministration of 2% DSS in the drinking water for 7 d, followed by regular drinking water. (A) Body weight. Mice were weighed daily. (B) Macroscopic images of the colon on day 7. (C) The average length of the colon on day 7 (left panel). The length ratio of DSS(+)/DSS(2) is also shown (right panel). The data are shown as mean 6 SE of six mice. (Figure legend continues)

Article Snippet: In brief, microbial cells in midlogarithmic growth phase were resuspended at 105 cells per milliliter in 1 mM potassium phosphate buffer (PPB; a mixture of 1 mM K2HPO4 and 1 mM KH2PO4; pH 7.2), mixed with an equal volume of 1 mM PPB, with or without recombinant mouse CCL28 (R&D Systems) in 2-fold serial dilutions, and incubated in a U-bottom 96-well microtest plate at room temperature for 2 h with brief mixing every 15 min. After appropriate dilutions with 1 mM PPB, cells were plated on agar plates and grown at 37 ̊C overnight.

Techniques:

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/elife.78206

Figure Lengend Snippet: Figure 2. Absence of CCL28 confers protection in a lethal Acinetobacter pneumonia 959

Article Snippet: At UC San Diego, we generated a new colony of Ccl28-/- mice with Cyagen 467 Biosciences (Santa Clara, California), using CRISPR/CAS9 technology.

Techniques:

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/elife.78206

Figure Lengend Snippet: Figure 3. Surface expression of the CCL28 receptors CCR3 and CCR10 on neutrophils 991

Article Snippet: At UC San Diego, we generated a new colony of Ccl28-/- mice with Cyagen 467 Biosciences (Santa Clara, California), using CRISPR/CAS9 technology.

Techniques: Expressing

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/elife.78206

Figure Lengend Snippet: Figure 5. CCL28 enhances neutrophil antimicrobial activity. (A) Murine bone marrow 1040

Article Snippet: At UC San Diego, we generated a new colony of Ccl28-/- mice with Cyagen 467 Biosciences (Santa Clara, California), using CRISPR/CAS9 technology.

Techniques: Activity Assay

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/elife.78206

Figure Lengend Snippet: Figure 1-figure supplement 2. CCL28 does not confer protection in a Salmonella 1092

Article Snippet: At UC San Diego, we generated a new colony of Ccl28-/- mice with Cyagen 467 Biosciences (Santa Clara, California), using CRISPR/CAS9 technology.

Techniques:

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/elife.78206

Figure Lengend Snippet: Figure 1-figure supplement 4. Wild-type and Ccl28-/- mice exhibit similar numbers of B 1122

Article Snippet: At UC San Diego, we generated a new colony of Ccl28-/- mice with Cyagen 467 Biosciences (Santa Clara, California), using CRISPR/CAS9 technology.

Techniques: