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Figure 2. Expression of CCR5, CXCR4, and galactosylceramide (GalCer) in human gingiva. (A) Low expression of CCR5 in healthy gingiva and increased expression in inflammation: Representative sections of (A1) healthy (n = 8) and (A2) inflamed gingiva (n = 12) obtained from surgical crown lengthening and during periodontal surgery, respectively, were subjected to single immunoenzyme labeling with CCR5 antibodies and 20x fields scanned by image analysis, as described in MATERIALS & METHODS. Final magnification shown is approximately 250X. (A3) H&E staining of healthy gingiva shown in A1. (A4) H&E staining of inflamed gingiva shown in A2. (B,C,D) Double-immunofluorescence staining of representative inflamed gingival tissues showing expression of CCR5 by T-cells (Fig. 2B ), macrophages (Fig. 2C), and dermal dendritic cells (Fig. 2D): In all fluorescent Figs., the green channel (panel 1), red channel (panel 2), and green/red merged channel (panel 3) are shown. Yellow cells in panel 3 denote co-localization of the two markers. In panel 4, the images merging from the green/red channel (panel 3) are overlaid with the image from differential interference microscopy (DIC), an optical variant of phase-contrast microscopy, for better documentation of co-localizations. Images were taken with the use of a 40X objective at a final magnification of 400X. Also included in all the images (B,C,D) is the enlarged inset image, with arrows demonstrating co-localization. (B) Several CD4+ T-cells (red channel, panel 2) in the lamina propria co-express CCR5 (panels 3 and 4). (C) Several CD68+ macrophages (red channel, panel 2) in the lamina propria co-express CCR5 (panels 3 and 4). (D) Several DC-SIGN+ dermal dendritic cells (red channel, panel 2) in the lamina propria co-express CCR5 (panels 3 and 4). (E1–4) Shown is the expression of CXCR4 and GalCer in the same healthy gingival tissue by single immunoenzyme staining: (E 1) expression of CXCR4 (arrows), (E 2) expression of GalCer (bracket), (E 3) H&E staining, (E 4) isotype control. (E 5–6) Shown is the the CXCR4 and CCR5 staining in the same inflamed gingival tissue: (E 5) expression of CXCR4 (arrows), (E 6) expression of CCR5 (arrows). (F) Shown are the mean numbers (± SE) of CCR5 cells in the healthy and inflamed gingiva. *Statistically significant difference (p < 0.05, Student’s t test). (G) Linear regression analysis was performed on the number of inflammatory cells (H&E-positive/20x field in lamina propria) (Y-axes) and the number of CCR5+cells (x-axis) per 20x field. Light and fluorescent microscopy images were acquired with a Nikon Eclipse E600 microscope equipped with a color high-resolution CCD camera and a PC running Image-Pro software (Media Cybernetics, Inc., Silver Spring, MD, USA).
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-defensins in Inflamed Human Gingiva
-defensin-1 in diseased gingival tissues. Healthy (n = 8) and inflamed (CP) (n = 8) gingival tissues were placed in RNA and, later, RNA stabilizing reagent (Quiagen, Valencia, CA, USA) and frozen at –80°C for later use. Frozen tissues were then ground, homogenized for total RNA extraction (RNeasy Kits, Quiagen), from which cDNA was synthesized (Avian RT first strand kit, Sigma, St. Louis, MO, USA). Concentration of RNA and purity of cDNA were determined by spectrophotometry. Primers used for amplification of 
10-fold) in mean copy numbers of