Taina Tervahartiala

The in vivo Expression of the Collagenolytic Matrix Metalloproteinases (MMP-2, -8, -13, and -14) and Matrilysin (MMP-7) in Adult and Localized Juvenile Periodontitis

Periodontal inflammation is characterized by irreversible degradation of periodontal ligament collagen fibers leading to loss of tooth attachment. Cultured gingival keratinocytes and fibroblasts express, in vitro, various matrix metalloproteinases (MMPs) which can degrade fibrillar collagens. We hypothesized that several MMPs are also synthesized in vivo by sulcular epithelium, and analyzed the collagenolytic MMPs (MMP-2, -8, -13, and -14) and matrilysin (MMP-7) in gingival tissue specimens and gingival crevicular fluid from adult and localized juvenile periodontitis patients by in situ hybridization, immunohistochemistry, and Western immunoblotting. MMP-2, -7, -8, and -13 were expressed in gingival sulcular epithelium. MMP-7 and -13 were also located in fibroblasts and macrophages, and MMP-8 in neutrophils. MMP-8- and -13-positive cells/mm2 were higher in periodontitis gingiva when compared with healthy control tissue (p < 0.01). In periodontal diseases, gingival sulcular epithelium expresses several, rather than a single, collagenolytic MMPs, and this proteolytic cascade is evidently responsible for the tissue destruction characteristic of adult and juvenile periodontitis.

Expression and induction of collagenases (MMP-8 and -13) in plasma cells associated with bone-destructive lesions

Matrix metalloproteinases (MMPs) collectively degrade extracellular matrix and basement membrane proteins in chronic inflammation and bone‐destructive lesions. This study examined the ability of immunoglobulin‐producing plasma cells, typically present in sites of chronic inflammation, to express collagenases (MMP‐8 and ‐13) in vivo and in vitro. Phorbol‐12‐myristate‐13‐acetate, interleukin‐6, and tumour necrosis factor‐α and heparin with the tumour promoter or cytokines potently enhanced (up to nine‐fold) MMP‐8 and ‐13 expression by the RPMI 8226 myeloma cell line, as evidenced by western blotting and semi‐quantitative reverse transcriptase‐polymerase chain reaction. Immunohistochemical analysis and in situ hybridization revealed that plasma cells expressed MMP‐8 and ‐13 focally in periapical granulomas, odontogenic cysts, and malignant plasmacytomas. MMP‐8 and MMP‐13 from plasma cells can participate in bone organic matrix destruction at sites of chronic inflammation and neoplastic growth. Since MMP‐13 was more frequently expressed than MMP‐8 in plasma cells of strongly recurring keratocysts and malignant plasmacytomas, it is concluded that plasma cell MMP‐13 has a particularly important role in benign and malignant bone‐destructive lesions. Copyright

Salivary MMP‐8, TIMP‐1, and ICTP as markers of advanced periodontitis

AIM Salivary matrix metalloproteinase (MMP)-8 and -14, tissue inhibitor of matrix metalloproteinase (TIMP)-1, and pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP) were analysed aiming to detect potential markers of advanced periodontitis in saliva. In addition, we compared two MMP-8 detection methods, a time-resolved immunofluorometric assay (IFMA) and an enzyme-linked immunoassay (ELISA), to differentiate periodontitis subjects from controls. MATERIAL AND METHODS Concentrations of MMP-8, MMP-14, TIMP-1, and ICTP were analysed from salivary specimens of 165 subjects, including 84 subjects having at least 14 teeth with periodontal pocket (pocket depth > or =4 mm) and 81 subjects without pocket depth as their controls. RESULTS Salivary MMP-8 detection by IFMA differentiated periodontitis subjects from controls more strongly than by ELISA. Salivary MMP-8, TIMP-1, and ICTP concentrations were higher in periodontitis subjects than those in controls. When only smokers were included in the analysis these differences were lost. The MMP-8/TIMP-1 ratio and the combination of MMP-8 and ICTP differentiated periodontitis and control groups even in smoker subjects. CONCLUSION Salivary MMP-8, TIMP-1, ICTP, and especially their ratios and combinations are potential candidates in the detection of advanced periodontitis. Differentiating periodontitis and control subjects with salivary MMP-8 detection is dependent on the selected techniques.

Determination of Matrix Metalloproteinases in Human Radicular Dentin

Matrix metalloproteinases (MMPs) are present in sound coronal dentin and may play a role in collagen network degradation in bonded restorations. We investigated whether these enzymes can also be detected in root dentin. Crown and root sections of human teeth were powderized, and dentin proteins were extracted by using guanidine-HCl and EDTA. Extracts were analyzed by zymography and Western blotting for matrix metalloproteinases detection. Zymography revealed gelatinolytic activities in both crown and root dentin samples, corresponding to MMP-2 and MMP-9. MMP-2 was more evident in demineralized root dentin matrix, whereas MMP-9 was mostly extracted from the mineralized compartment of dentin and presented overall lower levels. Western blot analysis detected MMP-8 equally distributed in crown and root dentin. Because MMPs are also present in radicular dentin, their contribution to the degradation of resin-dentin bonds should be addressed in the development of restorative strategies for the root substrate.

Salivary MMP-8, TIMP-1, and ICTP as markers of advanced periodontitis

AIM Salivary matrix metalloproteinase (MMP)-8 and -14, tissue inhibitor of matrix metalloproteinase (TIMP)-1, and pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP) were analysed aiming to detect potential markers of advanced periodontitis in saliva. In addition, we compared two MMP-8 detection methods, a time-resolved immunofluorometric assay (IFMA) and an enzyme-linked immunoassay (ELISA), to differentiate periodontitis subjects from controls. MATERIAL AND METHODS Concentrations of MMP-8, MMP-14, TIMP-1, and ICTP were analysed from salivary specimens of 165 subjects, including 84 subjects having at least 14 teeth with periodontal pocket (pocket depth > or =4 mm) and 81 subjects without pocket depth as their controls. RESULTS Salivary MMP-8 detection by IFMA differentiated periodontitis subjects from controls more strongly than by ELISA. Salivary MMP-8, TIMP-1, and ICTP concentrations were higher in periodontitis subjects than those in controls. When only smokers were included in the analysis these differences were lost. The MMP-8/TIMP-1 ratio and the combination of MMP-8 and ICTP differentiated periodontitis and control groups even in smoker subjects. CONCLUSION Salivary MMP-8, TIMP-1, ICTP, and especially their ratios and combinations are potential candidates in the detection of advanced periodontitis. Differentiating periodontitis and control subjects with salivary MMP-8 detection is dependent on the selected techniques.

Serum MMP-8, -9 and TIMP-1 in sepsis: high serum levels of MMP-8 and TIMP-1 are associated with fatal outcome in a multicentre, prospective cohort study. Hypothetical impact of tetracyclines.

Recent evidence suggests that matrix metalloproteinases (MMPs) and their endogenous inhibitors are involved in the pathogenesis of sepsis. We studied serum levels of MMP-8, MMP-9 and TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) in a multicentre, prospective cohort study of patients with sepsis treated in Intensive Care Units (ICUs). We analyzed serum samples taken on ICU admission from 248 critically ill sepsis patients. MMP-8, -9 and TIMP-1 serum levels were analyzed by enzyme-linked immunosorbent assays. Serum MMP-8, MMP-9 and TIMP-1 levels were significantly higher in patients with severe sepsis than in healthy controls. Serum MMP-8 levels among non-survivors (n=33) were significantly (p=0.006) higher than among survivors (n=215). Serum TIMP-1 but not MMP-9 levels were significantly higher among non-survivors than survivors (p<0.0001, p=0.079, respectively). Systemic MMP-8 is upregulated in sepsis suggesting that MMP-8 may contribute to the host response during sepsis. High serum MMP-8 and TIMP-1 levels at ICU admission were seen among patients with fatal outcome. With this background, clinical studies examining the ability of MMP-inhibitors (such as the non-antimicrobial properties of tetracyclines) to diminish the MMP-mediated inflammatory response are needed to develop novel therapies in order to improve the outcome of sepsis.

Matrix metalloproteinases and myeloperoxidase in gingival crevicular fluid provide site‐specific diagnostic value for chronic periodontitis

AIM To identify the diagnostic accuracy of gingival crevicular fluid (GCF) candidate biomarkers to discriminate periodontitis from the inflamed and healthy sites, and to compare the performance of two independent matrix metalloproteinase (MMP)-8 immunoassays. MATERIALS AND METHODS Cross sectional study. GCF (N = 58 sites) was collected from healthy, gingivitis and chronic periodontitis volunteers and analysed for levels of azurocidin, chemokine ligand 5, MPO, TIMP-1 MMP-13 and MMP-14 by ELISA or activity assays. MMP-8 was assayed by immunofluorometric assay (IFMA) and ELISA. Statistical analysis was performed using linear mixed-effects models and Bayesian statistics in R and Stata V11. RESULTS MMP-8, MPO, azurocidin and total MMP-13 and MMP-14 were higher in periodontitis compared to gingivitis and healthy sites (p < 0.05). A very high correlation between MPO and MMP-8 was evident in the periodontitis group (r = 0.95, p < 0.0001). MPO, azurocidin and total levels of MMP-8, MMP-13 and MMP-14 showed high diagnostic accuracy (≥0.90), but only MMP-8 and MPO were significantly higher in the periodontitis versus gingivitis sites. MMP-8 determined by IFMA correlated more strongly with periodontal status and showed higher diagnostic accuracy than ELISA. CONCLUSIONS MPO and collagenolytic MMPs are highly discriminatory biomarkers for site-specific diagnosis of periodontitis. The comparison of two quantitative MMP-8 methods demonstrated IFMA to be more accurate than ELISA.

Tumor Necrosis Factor-a and its Receptors, p55 and p75, in Gingiva of Adult Periodontitis

Tumor necrosis factor-a. (TNF-a), a pro-inflammatory cytokine, can stimulate matrix metalloproteinase synthesis and osteoclastic bone resorption. We hypothesized that elevated expression of TNF-a and its p55 and p75 receptors (TNF-R) in gingival tissue might associate with periodontitis. Immunohistochemistry was used for the study of the localization of TNF-a and its p55 and p75 TNF-R in adult periodontitis (AP) gingival tissue, in comparison with that in healthy control specimens. TNF-a and p55 TNF-R were detected in sulcular epithelial basal cells and in monocyte/macrophages, fibroblasts, and endothelial cells in the AP gingival tissue specimens, but mainly in fibroblasts and endothelial cells in control specimens. P75 TNF-R was occasionally found in monocyte/macrophage-like cells in gingival tissue specimens. The percentage of TNF-a-containing cells was not increased in AP compared with controls (13.2% ± 6.1% vs. 12.8% ± 7.6%), but, due to the increased cellularity of AP samples, the number of TNF-a positive cells/mm2 was clearly increased (1621 ± 663 vs. 664 ±191, p > 0.001). Thus, AP gingival tissue has an elevated expression of TNF-a and especially its p55 receptor, suggesting that TNF-a may contribute to tissue degradation in periodontitis.

Analysis of matrix metalloproteinases, especially MMP-8, in gingival creviclular fluid, mouthrinse and saliva for monitoring periodontal diseases.

Matrix metalloproteinase-8 is a promising candidate biomarker for oral fluid (gingival crevicular fluid, peri-implant sulcular fluid and saliva) and mouthrinse chair-side/point-of-care diagnostics to predict, diagnose and determine the progressive phases of episodic periodontitis and peri-implantitis, as well as to monitor the treatments and medications. Matrix metalloproteinase-8 can be used alone or together with interleukin-1beta and Porphyromonas gingivalis to calculate cumulative risk score at the subject level as a successful diagnostic tool, especially in large-scale public health surveys, in which a thorough periodontal examination is not feasible.

Down-regulation of trypsinogen-2 expression by chemically modified tetracyclines: association with reduced cancer cell migration.

Many types of human tumor express trypsinogen‐2, which may be a significant factor in the activation of pro‐MMPs and the invasiveness of tumors. Prevention of trypsinogen‐2 expression in cancer cells might be of benefit in cancer therapy. We describe here chemicals capable of down‐regulating the expression of trypsinogen‐2. Doxycycline (DOXY) and chemically modified tetracyclines ( CMTs ), previously known as inhibitors of the matrix metalloproteinase (MMP)–dependent proteinase cascade, down‐regulated the mRNA and protein expression of trypsinogen‐2 by COLO‐205 human colon adenocarcinoma cells at therapeutically attainable concentrations (0.1 to 1.0 μM). DOXY specifically inhibited the activation of pro‐MMP‐9 and cell migration induced by enteropeptidase, a specific activator of trypsinogen. Pro‐MMP‐9 activation and cell migration were also inhibited by tumor‐associated trypsin inhibitor (TATI), which is a highly specific inhibitor of trypsin. CMT‐3 as well as CMT‐5 also inhibited cell migration, but an effect on the enteropeptidase‐enhanced activation of pro‐MMP‐9 was not observed. Our results indicate that CMTs, DOXY and TATI inhibit cancer cell migration by down‐regulating trypsinogen‐2 expression or activity. Inhibition of trypsinogen‐2 expression may represent a mechanism contributing to the ability of CMTs to suppress the pericellular proteolytic activity of some tumors. Int. J. Cancer 86:577–581, 2000.