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: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Regulated production of the chemokine CCL28 in human colon epithelium.

doi: 10.1152/ajpgi.00162.2004

Figure Lengend Snippet: Fig. 1. CCL28 immunostaining of normal and inflamed human colon. A–C: sections of normal human colon. D–F: inflamed colon from a patient with ulcerative colitis. G–I: inflamed colon adjacent to a colon cancer. A, D, and G were immunostained for CCL28, and C, F, and I are adjacent sections stained with hematoxylin and eosin (H&E). CCL28 immunostaining was most marked in the inflamed colon (D, G) and restricted to epithelial cells. No immunostaining was seen in sections stained with the isotype-matched control antibody at same con- centration (B, E, I). Original magnification, 200. Sim- ilar results with no or minimal immunostaining for CCL28 were noted in normal colon from 3 additional healthy subjects. Ulcerative colitis is shown as a model of intestinal inflammation, and each of 5 patients exam- ined with that disease had a marked increase in colon epithelial CCL28 similar to that shown, although such increases are not specific for ulcerative colitis and were also seen in other causes of colon inflammation as shown and in Crohn’s colitis.

Article Snippet: Mouse IgG1 and sodium butyrate were from Sigma (St. Louis, MO). rhCCL28, biotinconjugated affinity-purified goat anti-human CCL28, and murine MAb to human CCL28 (clone 62705) were from R&D Systems (Minneapolis, MN).

Techniques: Immunostaining, Staining, Control

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Regulated production of the chemokine CCL28 in human colon epithelium.

doi: 10.1152/ajpgi.00162.2004

Figure Lengend Snippet: Fig. 2. Expression of CCL28 mRNA in human intestinal xenografts. A: mice carrying human colon or small intestinal (S.I.) xenografts were injected with PBS or IL-1 (1 g); the xenografts were harvested 5 h later, and total RNA was isolated from the colon xenografts. CCL28, IL-8, and -actin transcripts were amplified by RT-PCR. B: CCL28 and IL-8 mRNA levels were assessed by real-time PCR in human colon and small intestinal xenografts from the PBS or IL-1-injected mice. Values are means SD of 3 or more xenografts under each set of conditions. C: sections of human colon xenografts from the severe combined immunodeficient mice injected with human IL-1 (D–F) or PBS (A–C) were immunostained for CCL28 (A, D), stained with an isotype- matched control mouse IgG1 (B, E), or stained with H&E (C, F). Original magnification, 200.

Article Snippet: Mouse IgG1 and sodium butyrate were from Sigma (St. Louis, MO). rhCCL28, biotinconjugated affinity-purified goat anti-human CCL28, and murine MAb to human CCL28 (clone 62705) were from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Injection, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Staining, Control

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Regulated production of the chemokine CCL28 in human colon epithelium.

doi: 10.1152/ajpgi.00162.2004

Figure Lengend Snippet: Fig. 3. Constitutive and regulated CCL28 mRNA expression by human intestinal epithelial cell lines. A: total RNA isolated from the indicated cell lines was amplified by RT-PCR. RNA from peripheral blood mononuclear lymphocytes (PBMC) was used as a positive control. Negative control reac- tions had no added RNA. B: HCA-7 cells were left unstimulated (control) or were stimulated with IFN- (40 ng/ml), TNF- (20 ng/ml), or IL-1 (20 ng/ml). RNA isolation and amplification methods were as in A.

Article Snippet: Mouse IgG1 and sodium butyrate were from Sigma (St. Louis, MO). rhCCL28, biotinconjugated affinity-purified goat anti-human CCL28, and murine MAb to human CCL28 (clone 62705) were from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Control

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Regulated production of the chemokine CCL28 in human colon epithelium.

doi: 10.1152/ajpgi.00162.2004

Figure Lengend Snippet: Fig. 4. Regulated expression of CCL28 mRNA. A: HCA-7 cells were stimu- lated with IL-1 (20 ng/ml; ) or infected with S. dublin (I). Cells were harvested at the indicated times. CCL28 mRNA levels were assessed by real-time PCR and normalized to -actin, which was nearly constant in all groups throughout the experiments. Data are expressed as the fold change in mRNA levels in stimulated or infected cultures relative to control cultures incubated for the same duration. Values are means SE of 3 repeated experiments. *P 0.05 compared with control cultures. B: HCA-7 cells were left unstimulated (control) or stimulated with IL-1 (20 ng/ml) for 18 h or with 2 mM butyrate for 24 h or were preincubated with 2 mM butyrate for 6 h, after which IL-1 (20 ng/ml) was added and cultures were incubated for an additional 18 h. CCL28 mRNA expression assessed by real-time PCR is expressed as in A. Values are means SD from a representative experiment. Similar data were obtained in 2 repeated experiments. *P 0.05 compared with control; **P 0.05 compared with butyrate or IL-1. C: HCA-7 cells were left unstimulated (control), stimulated with IL-1 (20 ng/ml) or bacterial flagellin (100 ng/ml) for 18 h, or preincubated with 2 mM butyrate for 6 h, after which bacterial flagellin (100 ng/ml) was added and cultures were incubated for an additional 18 h. CCL28 mRNA expression was assessed as in A. Values are means SD from a representative experiment.

Article Snippet: Mouse IgG1 and sodium butyrate were from Sigma (St. Louis, MO). rhCCL28, biotinconjugated affinity-purified goat anti-human CCL28, and murine MAb to human CCL28 (clone 62705) were from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Control, Incubation

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Regulated production of the chemokine CCL28 in human colon epithelium.

doi: 10.1152/ajpgi.00162.2004

Figure Lengend Snippet: Fig. 5. CCL28 secretion by agonist stimulated or S. dublin infected HCA-7 cells. A: HCA-7 cells were left unstimulated or stimulated with IL-1 (20 ng/ ml; ) or infected with S. dublin (I). Culture super- natants were harvested at the indicated times and assayed for CCL28 by ELISA. Values are means SD from a representative experiment. Similar results were obtained in 2 repeated experiments. *P 0.05 compared with unstimulated or uninfected control cultures. CCL28 secretion in unstimulated cultures was 17 pg/ml at 6 h and 25 pg/ml at 24 h (not shown). B and C: HCA-7 cells were left unstimu- lated (control) or were stimulated with IL-1 (20 ng/ml) for 18 h or 2 mM butyrate for 24 h or were preincubated with 2 mM butyrate for 6 h, after which IL-1 (20 ng/ml) was added and cultures were incubated for an additional 18 h. CCL28 levels in supernatants were assayed by ELISA (B), and production of CCL28 by individual cells was deter- mined by enzyme-linked immunospot assay (C). *P 0.05 compared with control; **P 0.05 compared with butyrate or IL-1.

Article Snippet: Mouse IgG1 and sodium butyrate were from Sigma (St. Louis, MO). rhCCL28, biotinconjugated affinity-purified goat anti-human CCL28, and murine MAb to human CCL28 (clone 62705) were from R&D Systems (Minneapolis, MN).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Control, Incubation, Enzyme-linked Immunospot

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Regulated production of the chemokine CCL28 in human colon epithelium.

doi: 10.1152/ajpgi.00162.2004

Figure Lengend Snippet: Fig. 6. Pharmacological inhibitors of NF-B activation abrogate IL-1-stimu- lated upregulation of CCL28. HCA-7 cells were incubated in the presence or absence of BAY 11–7082 (10 M) or N-acetyl-leucinyl-leucinyl-norleucinal (ALLN; 10 M) after which IL-1 was added. Growth medium containing BAY 11–7082 and IL-1 was changed every 3 h. Cells were harvested after 12 h and CCL28 mRNA levels were determined by real-time PCR. Values are means SD from a representative experiment. *P 0.05 compared with IL-1-stimulated cultures without inhibitors. Similar results were obtained in 2 repeated experiments.

Article Snippet: Mouse IgG1 and sodium butyrate were from Sigma (St. Louis, MO). rhCCL28, biotinconjugated affinity-purified goat anti-human CCL28, and murine MAb to human CCL28 (clone 62705) were from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Incubation, Real-time Polymerase Chain Reaction

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: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

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: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

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: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

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: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

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: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

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: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

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: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

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: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

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: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

Techniques: Activity Assay, Colony-forming Unit Assay