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Notes: Abstract Matrix metalloproteinases (MMPs) and serine proteinases seem to be related to tissue destruction in periodontitis. The presence of MMPs in gingival crevicular fluid (GCF) and saliva, however, has not been studied comprehensively with the enzyme-linked immunosorbent assay (ELISA)-technique, We therefore examined the levels of MMP-1,-3.-8 and -9, and their endogenous inhibitor, tissue inhibitor of matrix metalloproteinases (TIMP-1). in GCF and saliva of patients with adult periodontitiss (AP) and localized juvenile periodontitis (LJP). Elevated levels of MMP-1 were detected in LJP GCF compared to AP and control GCF. Elevated levels of TIMP-1 were also detected in LJP GCF in comparison to AP and control GCF Higher MMP-8 levels were detected in AP GCF compared to LJP and control GCF. The relative low levels of MMP-3 were present in all studied GCF samples. Elevated levels of MMP-8 were further detected in saliva of AP compared to LJP and the controls. Both MMP-1 and TIMP-1 were detected in all studied saliva samples, but not significant differences were detected between the studied groups. Our ELISA-results confirm that (i) PMN MMP-8 and MMP-9 are the main collagenase and gelatinase in AP GCF, whereas GCF collagenase in LJP seems to be of the MMP-1-type; (ii) only low levels of TIMP-1. endogenous MMP-inhibitor. are present in AP GCF. which emphasises the importance of doxycycline as a possible adjunctive drug in the treatment of AP patients; (iii) tests based on specific antibodies against PMN MMPs. especially MMP-8, might serve as a reliable method of measuring and monitoring enzyme levels in GCF from different periodontitis patients.

Notes: Abstract We previously reported that low-dose doxycycline (DOXY) therapy reduces host-derived collagenase activity in gingival tissue of adult periodontitis (AP) patients. However, it was not clear whether this in vivo effect was direct or indirect. In the present study, inflamed human gingival tissue, obtained from AP patients during periodontal surgery, was extracted and the extracts partially purified by (NH4)2SO4 precipitation. The extracts were then analyzed for collagenase activity using SDS-PAGE/fluorography/laser densitometry, and for gelatinase activity using type I gelatin zymography as well as a new quantitative assay using biotinylated type I gelatin as substrate. DOXY was added to the incubation mixture at a final concentration of 0–1000 μM. The concentration of DOXY required to inhibit 50% of the gingival tissue collagenase (IC50) was found to be 16–18 μM in the presence or absence of 1.2 mM APMA (an optimal organomercurial activator of latent procollagenases); this IC50 for DOXY was similar to that exhibited for collagenase or matrix metalloproteinase (MMP)-8 from polymorphonuclear leukocytes (PMNs) and from gingival crevicular fluid (GCF) of AP patients. Of interest, Porphyromonas gingivalis collagenase was also inhibited by similar DOXY levels (IC50= 15 μM), however the collagenase activity observed in the gingival tissue extracts was found to be of mammalian not bacterial origin based on the production of the specific αA (3/4) and αB (1/4) collagen degradation fragments. In contrast, the inhibition of collagenase purified from culture media of human gingival fibroblasts (MMP-1) required much greater DOXY levels (IC50=280 μM). The predominant molecular forms of gelatinolytic activity present in the AP patients gingival tissue extracts were found to closely correspond to the 92 kD PMN-type gelatinase (MMP-9) although small quantities of 72 kD fibroblast-type gelatinase (MMP-2), and some other low molecular weight gelatinases, were also detected. The IC50 of DOXY versus gingival tissue gelatinolytic activity was estimated at 30–50 μM measured using either type I gelatin zymography or the biotinylated type I gelatin assay. We conclude that MMPs in inflamed gingival tissue of AP patients, like those in GCF, originate primarily from infiltrating PMNs rather than resident gingival cells (fibroblasts and epithelial cells) or monocyte/macrophages, and that their pathologically-elevated tissue-degrading activities can be directly inhibited by pharmacologic levels of doxycycline.

Notes: Abstract Accelerated periodontal tissue destruction in patients with labile insulin-dependent diabetes mellitus (DM) and localized juvenile periodontitis (LJP) has been suggested to be related to functional abnormalities of neutrophils. We have recently found that collagenase in gingival crevicular fluid (GCF) of adult periodontitis patients is primarily derived from neutrophils and that neutrophil collagenase activity is more sensitive to inhibition by tetracyclines than collagenase produced by fibroblasts. This study is to characterize the cellular sources, activation and inhibition of collagenase in GCF of DM patients and to compare it with collagenase in LJP GCF. We found differences which may have therapeutic implications. Specific doxycycline inhibition tests revealed that GCF collagenase in DM is derived from neutrophils, whereas the enzyme in LJP originates primarily from fibroblasts. Oxidant, sodium hypochlorite activated efficiently GCF collagenase of DM but not LJP patients. In contrast, plasmin activated LJP GCF collagenase but not that of DM patients. In GCF of DM patients 50–60% of collagenase existed in an active form, whereas in LJP GCF, the enzyme was almost completely in a latent form. The results suggest that collagenase in GCF of periodontitis patients with labile DM is primarily derived from neutrophils and that tetracycline therapy may be an effective adjunct in treatment aimed at controlling the periodontal breakdown in these patients. On the other hand, in LJP the anti-collagenase property of tetracyclines may be less important for control of periodontal tissue destruction because of the tetracycline-resistance of fibroblast collagenase.

Notes: Abstract Proteinases play a key rôle in the physiological degradation and remodelling of the periodontal tissues. The rôle of these enzymes in tissue remodelling in connection with the insertion of dental endosseous implants has not been elucidated. Therefore, the aim of the present study was to identify the eventual presence of collagenase, gelatinase and elastase activities in periimplant sulcus fluid (PTSF) of osseointegrated implants. Gelatinolytic activity in the samples was studied with gelatin-zymograms. Collagenase activity and its susceptibility to tetracycline-inhibition were monitored with SDS-polyacrylamide gel electrophoresis and laser densitometry, and elastase activity with synthetic substrate. Low activities of elastase and collagenase were detected in both PISF of osseointegrated implant patients and gingival crevicular fluid (GCF) of the control patients whereas significantly higher activities were delected in GCF of adult periodontitis patients. Also the profiles of gelatinases were similar in PISF of osseointegrated implant patients and GCF of the controls, but differed from the profile of active gelatinases present in GCF of adult periodontitis patients. The similar activities/characteristics of these proteinases in both periimplant sulcus fluid of healthy dental implants and GCF of healthy natural teeth suggest that they comprise the major proteinases for both periodontal and periimplant tissue remodelling or destruction.

Notes: Abstract The aim of the present study was to characterize the eventual presence and molecular forms of gelatinase/type IV collagenase activities in gingival crevicular fluid (GCF) and saliva in different forms of periodontitis: patients with clinically healthy periodontiura served as controls. Enzyme activities were monitored electrophoretically by zymography using gelatin and type IV collagen as substrates and analyzed visually and/or densitometrically. Both saliva and GCF collected from adult periodontitis, localized juvenile periodontitis and type II diabetes mellitus periodontitis patients contained species moving identically with gelatinase isolated from human neutrophils or MMP-9 (mean 98 kD), and species with mobility similar to gelatinase in fibroblast cell culture supernatants or MMP-2 (mean 76 kD). Hitherto, undescribed high molecular weight forms (mean 128 kD), were found, possibly representing polymerized or complexed enzyme active/activated in situ in the gel matrix. Small molecular forms of gelatinases (mean 51 kD and 46 kD), unable to cleave type IV collagen, were also found, most likely representing in vivo proteolytically activated, truncated enzymes. Although multiple forms of gelatinases/type IV collagenases in saliva and GCF may take part in the tissue destruction in periodontitis, their profile judged according to molecular weights does not differentiate between different forms of periodontitis.

Notes: Abstract. Dental plaque is the major aetiological factor in periodontal diseases and contains several proteolytic enzymes. The origin of these proteinases is, however, poorly studied. This study was undertaken to characterize collagenase present in dental plaque of adult periodontitis patients. Vertebrate-type rather than bacterial-derived collagenase activity was detected in extracts of both supra- and subgingival dental plaque extracts of adult periodontitis patients. Dental plaque collagenase was found to exist predominantly in autoactive form. Dental plaque collagenase from periodontally healthy individuals existed in latent form. Latent dental plaque collagenase from periodontitis lesions could be activated by a 95 kD chymotrypsin-like proteinase from Treponema denticola and human leukocyte cathepsin G but not by human plasmin. Incubation of purified latent leukocyte collagenase with whole cells of Fusobacterium nucleatum, Eubacterium saburreum, Prevotella buccae and Porphyromonas gingivalis, however, did not result to the activation of the enzyme. Doxycycline in vitro inhibited dental plaque collagenase with an IC50-value of 20μM. Dental plaque collagenase degraded more efficiently type I and 11 collagens than type III collagen. Western-blot analysis with specific anti-human neutrophil collagenase-antibody revealed that both in supra-and subgingival dental plaque extracts dental plaque collagenase had undergone proteolytic conversion from an 80 kD proform to a 58 kD active form which is associated with catalytic autoactivity as measured by functional collagenase assay. This reflects proteolytic activation of leukocyte collagenase in dental plaque probably by other proteases derived from potent periodontopathogenic bacteria such as T. denticola or other PMN proteases such as cathepsin G. Multiple different molecular weight gelatinases (20-200 kD) including fragmented low molecular weight human neutrophil 92 kD gelatinase species were detected in both supra- and subgingival dental plaque extracts. Leukocyte collagenase, previously found to be the main type of collagenase present in adult periodontitis gingiva, gingiva crevicular fluid and saliva, is also the predominant type of collagenase in the plaque of periodontitis patients. Fragmented but catalytically active neutrophil gelatinase species are also present in dental plaque. The dental plaque has potential to serve as a reservoir and site of activation of neutrophil (PMN)-derived matrix metalloproteinases in the periodontal inflammation.

Notes: Allergic contact dermatitis caused by gold is rare, and only isolated cases have been reported. Patch Jesting with gold may cause a long-lasing reaction. The purpose of this study is to describe a well-studied case of gold allergy caused by denial gold crowns. A gold-sensitized patient and a non-sensitized control subject were examined using patch tests, immunohistochemistry. electron microscopy and blast transformation reactions. Sodium thiosulfate, auranofin and sodium thiomalate gave positive patch test reactions. Immunohistochemistry and electron microscopy were performed from biopsies taken from allergic patch lest reactions caused by gold sodium thiosulfate 1 day and 17 days after applying the patches, from normal skin and from a 17-day-old allergic patch test reaction caused by ammonium persulfate. Down-regulation had taken place by 17 days in the allergic ammonium persulfate reaction, but not in the 17-day allergic gold lest reaction. The patient reacted to all but one of the gold-induced blast transformation tests, sodium chloroaurate being non-inductive. The non-sensitized control subject did not exhibit any reactions. In conclusion, gold sodium thiosulfate, gold sodium thiomalate and auranofin can be used as patch test substances for gold allergy, though long-lasting allergic patch test reactions may develop. In vitro gold salt induced blast transformation is an alternative test for gold allergy. The slow down-regulation of the allergic patch test reactions needs to be studied further.

Notes: Abstract: The aim of this study was to clear whether gelatinase B is associated with peri-implant bone loss (PBL). Peri-implant sulcus fluid was collected from 46 implant sites in 12 patients. These sites were also characterized using modified Gingival Index (mGI). Activated and total gelatinase B levels, measured using a modified urokinase assay, showed correlation with PBL (n=46, Spearman's rank correlation test). Activated and total gelatinase B values were significantly higher in PBL〉3 mm group (n=6) compared to PBL〈1 mm (n=29) and 1 〈PBL〈3 mm (n=11) groups (rank sum test). Activated gelatinase B level in mGI〉0.5 group (n=24) was clearly higher compared to mGI=0 (n=13) and p〈inlineGraphic alt="leqslant R: less-than-or-eq, slant" extraInfo="nonStandardEntity" href="urn:x-wiley:09057161:CLR951:les" location="les.gif"/〉0.5 (n=9) groups (Rank sum test). We conclude that gelatinase B is associated with PBL. Activation of gelatinase B together with elevated mGI eventually reflect active phases of peri-implantitis and may prove to be diagnostically useful.

Notes: When activated under physiologic or pathologic conditions leukocytes adhere to one another or to other cell types. Adhesion receptors mediate these interactions. In the study reported here, the distribution of the adhesion receptors LFA-1 (CD1la/CD18), ICAM-1 (CD54), CD2 and LFA-3 (CD58) in recurrent oral ulcers (ROU) were studied. Nine tissue specimens from five female patients (mean age 33 yr, age range 21–40 yr) with ROU were studied using the avidin-biotin-peroxidase complex (ABC) method. The main mononuclear cell infiltrations were in lamina propria (LP) and the epithelium next to the basement membrane (BM), laterally to the ulcers. In this area, ICAM-1 was strongly expressed in capillaries and in postcapillary venules. LFA-1, LFA-3 and CD2 were expressed in 65±11%, 70±16% and 80±1%, respectively, of all mononuclear cells. The findings indicate that LFA-1/ICAM-1 and CD2/LFA-3 interactions may play roles in cell to cell adhesion events in ROU.