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Increase in HIV Receptors/Co-receptors/ -defensins in Inflamed Human Gingiva
R. Jotwani1,
M. Muthukuru1 and
C.W. Cutler1,*
1 Department of Periodontics, School of Dental Medicine, 110 Rockland Hall, Stony Brook University-SUNY, Stony Brook, NY, USA 11794-8703;
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Figure 1. Increased expression of HIV receptors in gingival inflammation. Representative serial sections of (A) healthy (n = 8) and (B) inflamed gingiva (n = 12) obtained from surgical crown lengthenings and during periodontal surgery for chronic periodontitis, respectively, were subjected to single immunoenzyme labeling for DC-SIGN (1), mannose receptor (MR [2]), and Langerin (3) and also stained for H&E (4). 20x fields were scanned by image analysis, as described in MATERIALS & METHODS, and final magnification shown (optical and digital) is approximately 250x. Positively stained cells are brown, some of which are indicated by arrows. Shown are the mean numbers (± SE) of (C) DC-SIGN+, (E) MR+, and (G) Langerin+ cells in healthy and inflamed gingiva. *Statistically significant difference (p < 0.05, Student’s t test). (D,F,H) 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 (D) DC-SIGN+, (F) MR+, and (H) Langerin+ cells (x-axis) per 20x field.
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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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Figure 3. Increased expression of  -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 -defensin-1 and β-actin were designed to give an amplified product in a range of 150 to 200 bp with the use of primer3 software. Sequence of forward and backward primers is shown in the Table in Fig. 3. We performed both conventional RT-PCR and real-time PCR to determine the purity of the amplified product and quantitative expression of -defensin-1 mRNA, respectively. We quantitated -defensin-1 with β actins as an internal control in real-time PCR. Normalized initial concentrations were then converted and represented as initial copy numbers. All quantitations were performed in triplicate. (A) Shown is the single RT-PCR amplified band of -defensin-1 (169 bp) from inflamed gingival tissue by agarose gel electrophoresis. (B) Shown is the mean copy # x 106 of -defensin-1 expression with β actins as an internal control in healthy and inflamed gingival tissues by real-time PCR. A significant (p < 0.05, Student’s t test) increase ( 10-fold) in mean copy numbers of -defensin-1 expression was observed between health and disease.
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Journal of Dental Research, Vol. 83, No. 5,
371-377 (2004)
DOI: 10.1177/154405910408300504
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