TIMP expression in IL-1Ra siRNA-transfected cells.

<p>(A) TIMP-1 and TIMP-2 mRNA expression in IL-1Ra and control siRNA-transfected cells. The cells were cultured for 1, 3, 6, 12, and 24 hours, and then mRNA levels were determined using real-time PCR. Values represent fold changes. Differences among groups were analyzed using the Student’s <i>t</i>-test. Data are expressed as the mean ± SD (n = 3). *<i>p</i> < 0.05 vs each time point control. (B) Western blot of TIMP-1 (23 kDa) and -2 (21 kDa) in cells transfected with either IL-1Ra or control siRNAs. Data are representative of three independent experiments.</p

Expression and localization of MMP-13 in periodontal tissue of IL-1Ra KO and WT mice.

<p>(A) At 4 weeks after infection, the mouse gingival epithelial layer was collected to examine MMP-13 levels. The levels of MMP-13 were measured by an ELISA. Differences among groups were analyzed by one-factor ANOVA and Bonferroni’s multiple comparison test. Data are expressed as the mean ± SD (n = 4). **<i>p</i> <0.01 vs IL-1Ra KO mice infected with <i>A</i>. <i>actinomycetemcomitans</i>. ^†<i>p</i> <0.05 vs IL-1Ra KO mice treated with PBS. (B) Localization of MMP-13 in periodontal tissue. Buccolingual sections of the second and third mandibular molars from <i>A</i>. <i>actinomycetemcomitans</i>-infected IL-1Ra KO and WT mice were stained immunohistochemically. In contrast to infected WT mice, immunohistochemical findings in periodontal tissues indicated considerable inflammatory reactions in IL-1Ra KO mice infected with <i>A</i>. <i>actinomycetemcomitans</i>.</p

Suppression of IL-1Ra expression using siRNA.

<p>(A) Determination of IL-1Ra mRNA levels using real-time PCR. At 48 hours after transfection, IL-1Ra siRNA-transfected cells showed clear knockdown of IL-1Ra mRNA compared with the control. Differences among groups were analyzed using the Student’s <i>t</i>-test. Data are expressed as the mean ± SD (n = 6). **<i>p</i> < 0.01 vs control. (B) Western blot of IL-1Ra (18 KDa) in cells transfected with either IL-1Ra or control siRNAs (n = 3). Data are representative of three independent experiments.</p

Investigation of the expression of genes involved in cell–cell and cell–matrix interactions using a PCR array.

<p>The graph shows the fold changes of gene expression in IL-1Ra and control siRNA-transfected Ca9-22 cells. MMP-13 was up-regulated in IL-1Ra siRNA-transfected cells by 4.8-fold (n = 6). The red circle (most up-regulated gene) indicates MMP-13 expression in the graph. TIMP-1 and TIMP-2 expression levels are indicated by arrows.</p

Multilevel random intercept model for changes in CAL between the baseline and after 24 months (Model 1).

<p>Multilevel random intercept model for changes in CAL between the baseline and after 24 months (Model 1).</p

CAL change patterns during the 24-month follow-up period.

<p></p><p></p><p></p><p>(A) Changes of the improved, slightly improved, stable, slightly progressed, progressed and fluctuated categories.</p><p>···▲···: Improved, ··△···: Slightly improved, ─●─: Stable.</p><p>---□---: Slightly progressed, ---■---: progressed, ─■─: Fluctuated</p><p>Differences in the CAL changes over 24 months were classified into six categories: ≤ -3 mm, improved; between -3 mm and -2 mm, slightly improved; between -1 mm to 1 mm, stable; between 1 mm and 2 mm, slightly progressed; 3mm, progressed. In addition, cases with both ≤ -3 mm and ≥ 3mm were classified as fluctuated.</p><p></p><p></p><p>(B) CAL progression patterns of the progressed category</p><p>···▲···: Cluster 1, ···△···: Cluster 2, ─●─: Cluster 3.</p><p>─□─: Cluster 4, —■—: Cluster 5</p><p>A hierarchical cluster analysis was performed for the progressed type portrayed in Fig. 2(A), and 5 clusters were generated. The slope of cluster 1 was moderate, and the slopes of the other clusters were steep. Cluster 1 may correspond to the linear-type progressed sites, and the other clusters may correspond to the burst-type progressed sites.</p><p></p><p></p><p></p> <p>(A) Changes of the improved, slightly improved, stable, slightly progressed, progressed and fluctuated categories.</p> <p>···▲···: Improved, ··△···: Slightly improved, ─●─: Stable.</p> <p>---□---: Slightly progressed, ---■---: progressed, ─■─: Fluctuated</p> <p>Differences in the CAL changes over 24 months were classified into six categories: ≤ -3 mm, improved; between -3 mm and -2 mm, slightly improved; between -1 mm to 1 mm, stable; between 1 mm and 2 mm, slightly progressed; 3mm, progressed. In addition, cases with both ≤ -3 mm and ≥ 3mm were classified as fluctuated.</p> <p>(B) CAL progression patterns of the progressed category</p> <p>···▲···: Cluster 1, ···△···: Cluster 2, ─●─: Cluster 3.</p> <p>─□─: Cluster 4, —■—: Cluster 5</p> <p>A hierarchical cluster analysis was performed for the progressed type portrayed in Fig. 2(A), and 5 clusters were generated. The slope of cluster 1 was moderate, and the slopes of the other clusters were steep. Cluster 1 may correspond to the linear-type progressed sites, and the other clusters may correspond to the burst-type progressed sites.</p

Mean values of the CAL changes during the 24-month follow-up period.

<p>CAL changes during the 24-month follow-up period are separately illustrated by the CAL at baseline and by the type of tooth surface. Baseline CAL values are divided into three groups: (A) <3mm; (B) 3 mm; and (C) > 3 mm.</p> <p>─●─: Maxillary molar, ---■---: Maxillary premolar, ···▲···: Maxillary anterior,</p> <p>─○─: Maxillary molar, ---□---: Maxillary premolar, ···△···: Maxillary anterior</p> <p>Baseline CAL values of < 3mm gradually deteriorated, while baseline CAL values of > 3 mm improved. Molars with a baseline CAL of 3 mm progressed, whereas premolars and anterior teeth were stable or improved.</p> <p>CAL: clinical attachment level.</p