bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin‐6 production in human fibroblasts derived from periodontal tissues is differentially regulated by cytokines and a glucocorticoid
Authors: Okada N. , Kobayashi M. , Mugikura K. , Okamatsu Y. , Hanazawa S. , Kitano S. , Hasegawa K.
Published: July 2006

Interleukin‐6 (IL‐6) is thought to be a major mediator of the host's defense against infection, and it regulates immune responses in inflamed tissue. In this study, we investigated the regulation of IL‐6 production in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). Pro‐inflammatory cytokines including interleukin (IL)‐lα, IL‐1β and tumor necrosis factor (TNF)‐α stimulated IL‐6 production in HGF and HPLF in a time‐ and dose‐dependent manner. This IL‐lα, IL‐lβ, or TNF‐α‐induced IL‐6 production was enhanced, but the cAMP accumulation they induced was inhibited by the addition of indomethacin. This result suggests that endogenous prostaglandin E2 (PGE2) partially inhibits IL‐l or TNF‐α‐induced IL‐6 production, and that the enhancement of IL‐6 production by IL‐l or TNF‐α may not be caused through endogenous PGE2‐induced cAMP‐dependent pathway. Dexamethasone (DEX), a glucocorticoid which is a inhibitor of nuclear factor kappa B (NF‐kB) activation, markedly inhibited IL‐l (α or β) or TNF‐α‐induced IL‐6 production; so this production may be partially mediated through NF‐kB. IL‐l (α or β) and TNF‐α enhanced IL‐6 production synergistically. IL‐6 production in HGF or HPLF stimulated with IL‐lβ was augmented by the addition of interferon (IFN)‐(gama), but was slightly suppressed by the addition of IL‐4. Endogenous IL‐6 enhanced IL‐l (α or β)‐induced IL‐6 production in the presence of IL‐6 soluble receptor (IL‐6sR). Accordingly, in inflamed periodontal tissues, gingival fibroblasts and periodontal ligament fibroblasts stimulated with pro‐inflammatory cytokines such as IL‐l or TNF‐α, may produce IL‐6, and this production can be differentially modulated by endogenous PGE2, IL‐6sR, T cell‐derived cytokines such as IFN‐(gama) or IL‐4, and glucocorticoids.
Cystatin A in gingival crevicular fluid of periodontal patients
Authors: Blankenvoorde M. F. J. , Henskens Y. M. C. , van der Weijden G. A. , van den Keijbus P. A. M. , Veerman E. C. I. , Amerongen A. V. Nieuw
Published: July 2006

Cystatins are physiological inhibitors of cysteine proteinases which are widely distributed in human tissues and fluids. In the present study we analysed both the cystatin activity and the different cystatin isoforms in gingival crevicular fluid and saliva samples of nine periodontitis patients. All crevicular fluid samples, which were collected with filter paper points, showed cystatin activity ranging from 7–67 units/ing protein. The mean cystatin activity (24 units/mg protein) was significantly lower (p < 0.05) than that of the saliva samples (mean 93 units/mg protein). The cystatin isoforms in the crevicular fluid were further characterized by immunoblotting with specific antibodies against cystatin C, S, SN and A. While they were clearly present in saliva, cystatin C, cystatin S and cystatin SN could not be detected in any of the crevicular fluid samples. Remarkably, cystatin A was found in all the crevicular fluids as well as in the saliva samples. It is concluded that the cystatin activity found in crevicular fluid is caused, at least partially, by cystatin A. Furthermore, the gingival crevicular fluid is not a major contributor of cystatin C, S and SN activity in saliva.
Polyclonal B cell activators and in vitro induction of auto‐antibody reactive with collagen
Authors: Hahn Chin‐Lo , Schenkein Harvey A. , Tew John G.
Published: July 2006

Cells producing autoantibodies are known to be present in chronically inflamed periodontal tissues. In sites of chronic inflammation, polyclonal B cell activators (PBA) are known to exhibit adjuvant activity when combined with foreign antigens. These results prompted an examination of PBA in eliciting an antibody response to an autoantigen (i.e. collagen type I). Rat lymphocytes were stimulated with rat collagen (type I), microbial PBA (LPS) or the combination of LPS plus rat collagen in vitro. Anti‐collagen antibody‐forming cells (AFC) were enumerated using an ELISPOT assay. Collagen or LPS alone elicited few anti‐collagen AFC but the addition of LPS to collagen resulted in a substantial adjuvant effect and yielded maximal responses to collagen. Comparisons of anti‐collagen AFC from short‐term immunized (2‐6 wk after booster), non‐immunized and long‐term immunized (3‐4 months after booster) animals were performed. It revealed that cells from recently immunized rats werc significantly easier to activate than the other 2 groups. The adjuvant effect of microbial PBA may be important in anti‐collagen antibody production and thus the localization of PBA in periodontal pockets may explain why anti‐ collagen AFC are restricted to the chronically inflamed periodontal tissues.
Maintenance of new cementum formed during cyclosporin A administration after suspension of the treatment
Authors: Ayanoglou C. M. , Lesty C.
Published: July 2006

The aim of the present investigation was to examine if new cementum (NC) formed during cyclosporin A (CsA) administration was maintained after supension of the treatment. Thirty mg/kg/d of CsA were given to 3 male Sprague‐Dawley rats. Three control rats received oil‐based vehicle solution. Nine wk later the drug and vehicle administration were stopped and the rats continued to be fed with the same standard laboratory diet and water ad libitum for 5 months. The rats were anaesthetized, the tissues fixed by intracardiac perfusion of fixative solution and the mandibles processed for Epon inclusion. Histological, histomorphometric and ultrastructural analysis revealed that (a) NC covered extensive areas of the root surfaces; its structural characteristics were identical to those observed in the rats killed during CsA administration, (b) collagen fibres of the adjacent connective tissue were functionally inserted into the NC. (c) In the presence of cervical NC spurs the extent of the apical downgrowth of the junctional epithelium, measured parallel to the cemento–dentinal junction, was decreased (up to 64%) compared to the one occurring in areas devoid of NC deposits. These results suggest that (a) NC deposition and its functional relations with the adjacent connective tissue are not reversible after cessation of CsA treatment and (b) in the presence of cervical NC spurs the amount of connective tissue attachment on the root surfaces is increased.
α1‐Proteinase inhibitor in gingival crevicular fluid of humans with adult periodontitis: serpinolytic inhibition by doxycycline
Authors: Lee H. M. , Golub L. M. , Chan D. , Leung M. , Schroeder K. , Wolff M. , Simon S. , Crout R.
Published: July 2006

The serum protein, α1‐proteinase inhibitor (α1PI), defends the host against serine proteinases, e.g. PMN elastase. Using a rabbit anti‐serum against human α1‐PI, this protein in GCF was quantified from a standard curve constructed from dot‐blot analysis and characterized by Western blot. GCF was collected on filter paper strips from healthy (H), gingivitis (G) and adult periodontitis (AP) patients, then extracted with Tris/NaCl/CaCl2 buffer, pH 7.6. α1‐PI concentration increased with G and was highest in AP subjects. H sites only showed intact α1‐PI (52 kDa); no degradation fragments (48 kDa) were detected. In G and AP subjects, α1‐PI degradation fragments were seen in 17% and 71% of GCF samples, respectively. Both collagenase and α1‐Pl‐degrading activities in GCF increased with severity of inflammation (GCF flow). Moreover, the α1‐PI degrading (or serpinolytic) activity was characterized as a matrix metalloproteinase, probably collagenase, based on its in vitro response to a panel of different proteinase inhibitors including doxycycline. We propose: (1) that collagenase promotes periodontal breakdown not only by degrading collagen, but also by depleting α1‐PI regulation of elastase and other serine‐proteinases, thereby favoring a broader attack on extracellular matrix (ECM) constituents, and (2) based on a recent longitudinal double‐blind study using the techniques described above for α1‐PI analysis, that low‐dose doxycycline administration to humans with adult periodontitis can inhibit this broad cascade of ECM degradation.
A comparison of 2 polytetrafluoroethylene membranes in guided tissue regeneration in sheep
Authors: Danesh‐Meyer M. J. , Pack A. R. C. , McMillan M. D.
Published: July 2006

The purpose of this study was to evaluate the sheep as a suitable animal model in which to compare periodontal wound healing after surgically induced Class II furcation defects were treated with different guided tissue regeneration (GTR) membranes. 22 mandibular second premolar sites in 11 sheep were used. The experimental design included untreated control teeth, sites treated by surgery alone, and others where surgery included placement of either Gore‐Tex Periodontal Material (GTPM) or Gore‐Tex Soft Tissue Patch (GTSTP). Sheep were killed 7 wk after surgery. Mesiodistal sections through decalcified second mandibular premolars were evaluated histologically. Histomorphometric analysis of digitized images allowed both area and linear measurements of newly regenerated furcal tissues. Percentage areas of cementum, bone, epithelium and connective tissue filling the furcation region were calculated. Coronal regeneration of alveolar bone and cementum were expressed as a percentage of the original height of the defect. Mean values for each group were subsequently analysed by 1‐way ANOVA. Significantly greater regeneration was achieved with both of the GTR membranes, by comparison with the non‐GTR surgical control group, with respect to percentage areas of cementum, bone and connective tissue, and the linear measurement of cementum (p>0.05). This study demonstrates successfully the use of the sheep animal model for GTR research, and shows that few differences exist between GTR healing when either GTPM or GTSTP is used in the treatment of class II furcation defects.
Increased titre and avidity of IgG antibodies to Porphyromonas gingivalis whole cells and a cell surface protein in subjects with adult periodontitis
Authors: Benjamin P. A. , Rogers P. A. , U. S. , Johnson N. W. , Cole M. F. , Curtis M. A.
Published: July 2006

The titre and avidity of IgG antibodies to Porphyromonas gingivalis whole cells and a 47 kDa cell surface protein were determined in scrum samples taken from 20 subjects with adult periodontitis and 20 controls, matched for age, gender, ethnic origin and oral hygiene status. Antibody litres were measured by ELISA and antibody avidity was determined by a chaotrope‐dissociation ELISA. Avidity was defined as the molarity of chaotrope required to reduce absorbance by 50% (ID50). The mean IgG antibody log titre to whole cells (8. 29 vs. 6. 92; p<0.01) and to the 47 kDa antigen (7. 61 vs. 6. 77; p<0.05) were higher in cases than in controls. Mean IgG antibody avidity to whole cells (4. 59 vs. 2. 47; p<0.001) and to the surface protein (2. 54 vs. 1. 67; p<0.001) were also higher in cases than in controls. In cases, IgG antibody titre was highly correlated with avidity for both whole cells (r=0.878; p= <0.001) and the 47 kDa protein (r=0.683; p<0.001). There was a weaker positive correlation between the titre and the avidity of antibody to whole cells (r=0.591; p<0.01) in the control population but antibody titre and avidity for the 47 kDa sonicate antigen were not correlated in the controls (r=0.104). We conclude that many patients with adult periodontitis have effective humoral immunity to P. gingivalis. However, in up to half the patients with adult periodontitis, antibody titres and avidities were low and similar to control values, indicating either susceptibility due to poor host response or that disease is not associated with this particular pathogen.
A longitudinal study of Actinobacillus actinomycetemcomitans in army recruits
Authors: Müller H.‐P. , Eger T. , Lobinsky D. , Hoffmann S. , Zöller L.
Published: July 2006

During recruiting examinations 201 recruits, 18‐25 yr old, were examined for subgingival and extracrevicular Actinobacillus actinomycetemcomitans. The organism was isolated in 55 subjects, most often at low levels. Cluster analysis revealed 3 clusters with no (A, n = 86) or minor (B, n = 92) periodontal disease and low DMF‐S, as well as established periodontitis, increased DS and high DMF‐S (C, n = 22). When leaving the 12‐months' service, 105 recruits were re‐examined (54 cluster A, 41 cluster B, 9 cluster C subjects, 1 recruit who was not clustered). An increase of periodontal probing depth (PPD) of 3 mm at 1 or more sites occurred in 33 subjects: 9 (17%) in cluster A, 16 (39%) in cluster B, 7 (78%) in cluster C and in the not‐clustered recruit. Considerable variation in frequency distributions of PPD alterations was observed, therefore significant (p<0.1) mean increase (1‐sample t‐test) and skew g1 (S‐statistic) were additionally considered to define an “active” case. A total of 7 recruits (6.7%) met the criteria. Logistic regression analysis revealed a significant influence of self‐reported smoking habits on activity status. Thus, heavy smokers (>20 cigarettes/d) had a 14‐fold higher risk (p = 0.030) for developing or progressing periodontitis compared to non‐ or light smokers (< 10 cigarettes/d). In particular, cluster B recruits appeared to have a lower risk (p = 0.11) for developing periodontitis than cluster C recruits (established periodontitis, high DMF‐S). A. actinomycetemcomitans was isolated in 29 recruits (27.6%) at baseline and 30 recruits (28.6%) after 12 months. Presence of the organism was not a risk factor for periodontitis. However, in active subjects, significantly more samples were only A. actinomycetemcomitans‐positive at re‐examination compared to inactive recruits. It was concluded that smoking is a significant risk factor for periodontitis. Subjects with established periodontitis tend to deteriorate further. A. actinomycetemcomitans seems not to increase the risk for developing or progressing periodontitis in this age group. Longer studies involving larger populations are needed to confirm these observations.
Genetic studies of syndromes with severe periodontitis and palmoplantar hyperkeratosis
Authors: Hart T. C. , Stabholz A. , Meyle J. , Shapira L. , Van Dyke T. E. , Cutler C. W. , Soskolne W. A.
Published: July 2006

The Papillon‐Lefevre and Haim Munk syndromes are characterized by the presence of both palmoplantar hyperkeratosis (PPK) and severe early onset periodontitis. It is the early onset periodontal disease component that distinguishes these from other more common forms of PPK. It has been proposed that the periodontal disease component may be a casual association in individuals with PPK. Genetic syndromes with palmoplantar keratosis and severe early onset periodontitis may be due to specific bacterial infections in individuals with PPK. Recently, keratin gene mutations have been identified in several conditions typified by palmoplantar keratosis. The present study sought to test the hypothesis that a keratin gene defect similar to those previously identified in other PPK conditions is responsible for the Haim Munk and the Papillon‐Lefevre syndromes. We have performed genetic linkage studies to test for linkage between polymorphic DNA loci within 2 cytokeratin gene families and the disease phenotype in Haim Munk syndrome and Papillon‐Lefevre syndrome. Families with individuals segregating for the Haim Munk syndrome and the Papillon‐Lefevre syndrome were examined to determine disease status, and genotyped for microsatellite DNA markers closely linked to the acidic (type I) and the basic (type II) cytokeratin genes on chromosomes 12 and 17. Genotype data were evaluated for microsatellite allele homozygosity in affected individuals. Results of these preliminary genetic studies suggest that the gene defect in Haim Munk syndrome is not due to a gene defect in either the type I or the type II keratin gene clusters. These findings suggest that Haim Munk syndrome may be genetically distinct from other more common forms of PPK that have been linked to the cytokeratin gene families, and suggest that mutations in genes other than keratin genes are responsible. Additional family studies are needed to confirm these preliminary findings.
Evidence for genetic control of changes in f‐actin polymerization caused by pathogenic microorganisms: in vitro assessment using gingival fibroblasts from human twins
Authors: Hassell T. M. , Baehni P. , Harris E. L. , Walker C. , Gabbiani G. , Geinoz A.
Published: July 2006

Attachment to and migration upon a substratum, as well as other functions of connective tissue cells, are regulated mainly by cytoplasmic structural proteins, particularly filamentous actin (f‐actin). Pathogenic microorganisms exert negative effects on cytoskeletal proteins. In the present study, normal gingival fibroblasts from 10 sets of human twins (6 fraternal, DZ; 4 identical, MZ) were exposed to soluble extracts from Porphyromonas gingivalis or Fusobacterium nucleatum, then f‐actin was stained using FITC‐labeled phalloidin. Cells were examined under fluorescence, and a computer‐assisted image analyzer quantitated f‐actin polymerization as fluorescence intensity on a per‐cell basis. Intraclass correlation coefficients for f‐actin in MZ/MZ vis‐à‐vis DZ/DZ paired cell cultures were determined to assess the possible heritability of responses to the microorganism preparations. F‐actin labeling was significantly different between control cultures and those exposed to the extracts. Both F. nucleatum and P. gingivalis effected f‐actin and fibroblast morphology. When the data were adjusted for gender and age effects, and for differences in control f‐actin levels, fibroblasts from MZ twin pairs were moderately similar in both absolute and relative responses to bacterial challenges; cells from DZ twins showed little similarity when response was measured on the absolute scale, and moderate similarity using the relative scale.
Molecular factors associated with compartmentalization of gingival immune responses and transepithelial neutrophil migration
Authors: Tonetti M. S.
Published: July 2006

PMN migration into the gingival sulcus is a tightly regulated process aimed at selectively increasing leukocyte availability at the site of bacterial plaque aggression, i.e. the superficial portion of the junctional epithelium. The evidence reviewed in this paper indicates that, besides the action of complement fragments, arachidonic acid metabolites, formyl peptides and other bacterial products, the establishment of a gradient of ICAM‐1 expression across the junctional epithelium and the expression of IL‐8 in its superficial layers probably represent important regulatory mechanisms leading to PMN migration into the gingival sulcus. Such mechanisms can be regulated by the autocrine and paracrine action of some pro‐inflammatory cytokines and could possibly be initiated by specific bacteria‐keratinocyte interactions. The advantage of such a redundant regulatory mechanism leading to PMN transepithelial migration is probably related to the key role of the neutrophil in the maintenance of a local host‐parasite equilibrium on one side, and on the tissue injury associated with PMN persistence or random migration within periodontal tissues on the other. Several investigations are in progress aimed at identifying the initial environmental stimuli leading to PMN recruitment into the gingival sulcus and at further exploring the important regulatory events.
Immunoregulatory roles of adhesive interactions between lymphocytes and gingival fibroblasts
Authors: Murakami S. , Shimabukuro Y. , Saho T. , Hino E. , Kasai D. , Hashikawa T. , Hirano H. , Okada H.
Published: July 2006

Chronic adult periodontitis is usually characterized by inflammatory cell accumulation in the extravascular periodontal connective tissue. In order to reveal how the lymphocyte migration and retention in periodontal lesions is regulated, we have focused on the molecular basis for the adhesive interactions between lymphocytes and human gingival fibroblasts (HGF). In this study, we investigated the involvement of cell adhesion molecules in adhesive interactions between lymphocytes and HGF. We found that activated lymphocytes bound strongly to HGF and VLA integrins, extracellular matrix receptors, play crucial roles in the binding. Interestingly, we first revealed that CD44 molecules (hyaluronate receptor) on lymphocytes also participated in lymphocyte‐HGF interactions and that hyaluronate anchored on the surface of HGF functioned as the ligand for CD44. In addition. when HGF were stimulated with inflammatory cytokines such as IL‐1. TNFα and IFNγ, the binding avidity between lymphocytes and HGF was significantly increased and the adhesion was mainly mediated by LFA‐l/ICAM‐1 pathway. We then examined the possibility whether lymphocyte‐HGF interaction may cause activation of HGF. When HGF directly interacted with lymphocytes for 3 h, IL‐lβ mRNA expression was clearly increased in HGF. These findings suggested that the adhesive interactions between lymphocytes and HGF was mediated at least by VLA integrins, LFA‐l/ICAM‐1 and CD44/hyaluronate and that the heterotypic cell‐cell interactions could mutually cause intracellular signal transduction.
Molecular and cellular mechanisms for periodontal diseases: role of Th1 and Th2 type cytokines in induction of mucosal inflammation
Authors: Yamamoto M. , Fujihashi K. , Hiroi T. , McGhee J. R. , Dyke T. E. Van , Kiyono H.
Published: July 2006

An accumulation of elevated numbers of macrophages (Mø) and Ig producing cells is associated with localized and chronically inflamed gingiva of patients with adult periodontitis. When gingival lymphocytes were isolated from inflamed tissues and examined by flow cytometry, approximately 20–30% of lymphocytes were CD4+ T cells. For the analysis of Thl and Th2 cytokine expression by these CD4+ T cells, RNA was extracted and reverse transcriptase polymerase chain reaction (RT‐PCR) was performed by using specific 5′ and 3′primers for IFN‐γ and IL‐2 (Thl), IL‐4, IL‐5, IL‐6, IL‐10 and IL‐13 (Th2) and β‐actin (housekeeping gene). Two distinct cytokine profiles were noted based on the expression of selected Thl and Th2 cytokines. Thus, one pattern was represented by the expression of mRNA for IFN‐γ, IL‐6. IL‐10 and IL‐13. while the other case consisted of mRNA for IFN‐γ, IL‐6 and IL‐13. Except for a few cases, messages for IL‐2, IL‐4 and IL‐5 were not detected by cytokinespecific RT‐PCR. The predominant expression of Th2 cytokines (e.g. IL‐6. IL‐10 and IL‐13) may contribute to the induction of high B cell responses in local disease sites. On the other hand, lack of IL‐4 may be responsible for the accumulation of Mφ in diseased periodontium. We also investigated whether a relationship exists between IL‐4 receptor (IL‐4R) expression and Mø persistence in the absence of exogenous IL‐4. Gingival Mγ, when compared with monocytes (MN)/Mø from peripheral blood mononuclear cells (PBMC), expressed high levels of IL‐4R mRNA. When gingival Mø were incubated with recombinant IL‐4 (rIL‐4). the cell viability was dramatically reduced by apoptosis. These findings clearly show that the lack of IL‐4 may contribute to the persistant occurrence of Mø at the disease site and addition of exogenous rIL‐4 to gingival Mø cultures leads to cell death by apoptosis.
Porphyromonas gingivalis proteinases as virulence factors in the development of periodontitis
Authors: Travis J. , Pike R. , Imamura T. , Potempa J.
Published: July 2006

Porphyromonas gingivalis contains exceedingly high concentrations of cysteine proteinases with trypsin‐like activity which have been implicated as virulence factors in adult‐onset periodontitis. These enzymes, referred to as gingipains, cleave protein and peptide substrates after arginine (gingipain R) and lysine residues (gingipain K), and it has been found that neither is easily inhibited by host proteinase inhibitors. Examination of the properties of each proteinase clearly indicates a role(s) for both in the dysregulation of a number of normally tightly controlled pathways. The effects of such uncontrolled proteolysis are the development of edema (kallikrein/kinin pathway activation by gingipain R), neutrophil infiltration (complement pathway activation by gingipain R), and bleeding (degradation of fibrinogen by gingipain K). Since three of the major hallmarks of periodontitis involve increased crevicular flow, neutrophil accumulation at infected sites and bleeding on probing, it seems likely that both P. gingivalis‐derived proteinases are important virulence factors in the development of periodontal disease.
Analysis of the protease and adhesin domains of the PrpRI of Porphyromonas gingivalis
Authors: Curtis M. A.
Published: July 2006

The production of extracellular proteolytic enzymes is a widely used strategy by human parasites including bacteria, protozoa and helminths in order to ensure survival in the colonized host. The potential benefits to the organism arise through modifications to the external environment of the cell and include the release of essential nutrients, the disablement/deregulation of the host defences and the exposure of previously shielded substrata as new sites for colonization. Damage to the host may arise through direct proteolysis of structural proteins, deregulation of the inflammatory response or the compromising of the local host defences below the threshold necessary for effective defence. In order to examine these interactions and how they may be regulated in the periodontal diseases, we are examining the properties of proteases of the oral anaerobe Porphyromonas gingivalis with specificity for arginyl peptide bonds (ArgI, ArgIA and ArgIB): a family of enzymes which has been shown to exert effects on a variety of host proteins with roles in the control of inflammation and tissue homeostasis. Analysis of the gene for ArgI (protease polyprotein for ArgI ‐ prpRl) together with structural and immunochemical studies of these 3 interrelated forms indicates that they may be regarded as critical determinants in multiple aspects of the life cycle of the organism via both proteolysis and binding processes. Together with the highly conserved nature of the gene, the data suggest that the PrpRI of P. gingivalis is an essential colonization determinant which may play an important role in the periodontal disease process.
Multiple colonization defects in a cysteine protease mutant of Porphyromonas gingivalis
Authors: Kuramitsu H , Tokuda M , Yoneda M , Duncan M , Clio M.
Published: July 2006

A cysteine protease mutant, G‐102, of Porphyromonas gingivalis 381 defective in the rgp‐l gene has been recently constructed in this laboratory. In order to evaluate the role of the protease in the virulence properties of P. gingivalis, a number of putative periodontopathic properties of the mutant were evaluated. Relative to the parental strain, mutant G‐102 was demonstrated to be defective in interacting with Gram‐positive bacteria as well as cultured epithelial cells. In addition, the mutant was altered in attaching to the protein components of extracellular matrix as well as to type I collagen. Some of these alterations could result from the decreased autoaggregation displayed by mutant G‐102 relative to strain 381. However, since the epithelial cell attachment assays were carried out at very low bacterial densities, it is unlikely that reduced autoaggregation of the mutant is responsible for its decreased ability to attach to these eucaryotic cells. Electron microscopic examination of the cells also revealed that mutant G‐102 was altered in normal fimbrae expression. In addition, reduced expression of the 43 kDa fimbrial subunit in the mutant was detected with both Western and Northern blotting. These results indicated that the rgp‐l gene product can play either a direct or indirect role in the colonization properties of P. gingivalis.
Biological effects of drug‐loaded biodegradable membranes for guided bone regeneration
Authors: Chung C.‐P. , Ki D.‐K. , Park Y.‐J. , Nam K.‐H. , Lee S.‐J.
Published: July 2006

The biological effects of drug‐loaded biodegradable novel membrane for guided bone regeneration (GBR) was evaluated. The membranes were polyglycolic acid mesh coated with poly‐L‐lactic acid containing flurbiprofen, tetracycline or PDGF‐BB. Porous structure was generated in the membranes by using a phase inversion method. The membrane was less toxic, nicely biodegradable and biocompatible for 8 wk after implantation in the dorsal skin of the rat. The drugs released from the membranes were shown to be effective for new bone formation. Tetracycline, flurbiprofen or PDGF‐BB loaded membrane was markedly effective for osteoid tissue and new bone formation in the bony defect prepared in rat calvaria to compare with that by unloaded membrane. These results suggested that drug‐loaded biodegradable barrier membrane might be a potential tool for GBR in periodontal therapy.
Interactions between periodontopathogenic bacteria and cytokines
Authors: Fletcher J. , Reddi K. , Poole S. , Nair S. , Henderson B. , Tabona P. , Wilson M.
Published: July 2006

Cytokines produced in response to plaque bacteria clearly play a key role in the periodontal diseases. However, we know very little about the interactions between cytokines and periodontopathogenic bacteria. The aims of this study were to determine whether the key pro‐inflammatory cytokines interleukin‐1β (IL‐1β) and IL‐6 could affect the growth of Actinobacillus actinomycetemcomitans or Porphyromonas gingivalis and to determine whether these organisms could hydrolyse IL‐1β, IL‐6 or the anti‐inflammatory IL‐1 receptor antagonist (IL‐1ra). Culture medium containing up to 100 ng/ml of IL‐1β or IL‐6 was inoculated with A. actinomycetemcomitans (serotypes a, b and c) or P. gingivalis and growth was monitored by measuring changes in electrical conductivity every 3 min for up to 48 h. IL‐1β, IL‐6 or IL‐1ra were added to culture supernatants and incubated for up to 24 h. Samples were taken at various times, analysed by SDS‐PAGE and the separated proteins transferred by Western blotting to PVDF membranes and probed with anticytokine antibodies. None of the cytokines tested had any effect on the rate of growth or yield of A. actinomycetemcomitans or P. gingivalis. Supernatants from P. gingivalis cultures, but not those from A. actinomycetemcomitans, hydrolysed IL‐1β, IL‐6 and IL‐1ra. The hydrolysate from the P. gingivalis supernatant‐treated IL‐1β was unable to stimulate the release of IL‐6 from human gingival fibroblasts showing that it had lost biological activity. These results suggest that P. gingivalis can perturb the cytokine network, not only by stimulating the release of cytokines from host cells, but also by removing them from its local environment.
Clonal analysis by ribotyping of Fusobacterium nucleatum isolates obtained from healthy young adults with optimal plaque control
Authors: Suchett‐Kaye G. , Décoret D. , Barsotti O.
Published: July 2006

Fusobacterium nucleatum is a Gram‐negative anaerobic rod implicated in the pathogenesis of periodontal disease. However, this organism has also been frequently identified in high numbers in healthy adults. These observations suggest that the species may comprise different clonal types, some of which may participate in disease. The purpose of the present investigation was to use restriction endonuclease analysis (REA) and ribotyping to characterize F. nucleatum clonal types isolated from healthy young adults with optimal plaque control and investigate the stability of some of these clonal types. A group comprising 11 dental students and 11 dental outpatients with optimal plaque control was sampled. Clonal stability was investigated by sampling the dental student group at baseline and at 16 months. One hundred and thirty‐two clinical isolates of F. nucleatum were successfully recovered from 15/22 individuals. For the positive subjects, 29 different clonal types were identified by REA and ribotyping, each subject and site being colonized by 1–4 clonal types. For the dental students, 9 and 15 different clonal types were identified at baseline and 16 months, respectively. None of the students harboured identical clonal types at both sampling times. Our results show that ribotyping is a useful technique for monitoring the distributions of F. nucleatum clonal types and indicate that healthy individuals with optimal plaque control can be colonized by more than one F. nucleatum clonal type and that these clonal types appear to be unstable.
Inhibitory effects of YM175, a bisphosphonate, on the progression of experimental periodontitis in beagle dogs
Authors: O'uchi Naoto , Nishikawa Haruko , Yoshino Taiji , Kanoh Hiroyuki , Motole Hiroyuki , Nishimori Emi , Shimaoka Toshio , Abe Tetsushi , Shikama Hisataka , Fujikura Takashi , Matsue Miyoko , Matsue Ichiro
Published: July 2006

This study examined the efficacy of YM175 [disodium dihydrogen (cycloheptylamino) methylene‐1,1 ‐bisphosphonate] in reducing alveolar bone loss caused by experimental periodontitis in beagle dogs. Thirty‐six dogs were used and divided into 6 groups. Periodontitis was induced in 30 dogs (groups 2–6) by ligating the bilateral mandibular third and fourth premolar teeth with silk ligatures and by feeding a soft diet. Six dogs were sham‐operated (group 1). Saline (placebo), flurbiprofen (0.02mg/kg) and YM175 (0.01, 0.1 and 1.0 mg/kg) were administered to the dogs (groups 2–6) 5 d/wk for 25 wk. Radiographic and morphometric analyses were performed. In placebo‐treated animals (group 2), the ligation caused a significant decrease in the alveolar bone height by 0.57 and 1.91 mm at 2 and 25 wk, respectively. YM175 (1.0 mg/kg) prevented the decrease in bone height by 47 and 31% at 2 and 25 wk. YM175 (0.1 mg/kg) and flurbiprofen tended to prevent bone loss after 15 wk. Although the ligation elicited no significant change in bone mineral density, it significantly decreased bone volume. YM175 (1.0 mg/kg) and flurbiprofen tended to increase the bone volume. The number of formative or resorptive Haversian canals and the bone turnover through the periosteal bone surface were increased by the ligation, indicating the increased turnover of the cortical bone. YM175 (1.0 mg/kg) reduced the increased bone turnover. The gingival index was maximally increased at 2 wk and was suppressed by YM175. These results suggest that YM175 prevents alveolar bone loss by reducing the increased alveolar bone turnover in dogs with periodontitis.
Protease‐activated receptors and their role in IL‐6 and NF‐IL‐6 expression in human gingival fibroblasts
Authors: Hou L. , Ravenall S. , Macey M. G. , Harriott P. , Kapas S. , Howells G. L.
Published: July 2006

The serine protease thrombin is formed at sites of coagulation and inflammation and has been shown to have important proinflammatory cellular effects relevant to the pathogenesis of periodontal disease. Thrombin acts via specific cell surface receptors termed protease‐activated receptor‐1 (PAR‐1) and PAR‐3, which have a distinctive method of activation. Proteolytic cleavage of the extracellular domain by thrombin reveals a hidden amino terminus which then acts as a “tethered ligand”. A short synthetic peptide (SFLLRN) can also mimic the tethered ligand and activate PAR‐1 but not PAR‐3. Also, a trypsin‐sensitive receptor termed PAR‐2 has been described which is activated by the PAR‐1 activating peptide SFLLRN. Here we show conclusively by flow cytometric and Northern blot analysis that human gingival fibroblasts (HGF) express PAR‐1 but not PAR‐2. In functional studies we also show that thrombin and SFLLRN stimulated increased expression of mRNA encoding nuclear transcription factor NF‐IL‐6 and IL‐6 in vitro. At optimal concentrations, thrombin (10−7 M) induced 7.6±0.01 ng/ml immunoactive IL‐6 and PAR‐1 activating peptide (5 × 10−5 M) induced 2.2 ± 0.2 ng/ml (mean ± standard error of mean). A proteolytically inactive recombinant thrombin (serine 195 to alanine) was without activity. These data show that HGF express PAR‐1 and suggest that PAR‐1 activation stimulates increased NF‐IL‐6 and IL‐6 gene expression and IL‐6 secretion by HGF in vitro. Whether HGF express PAR‐3 is unknown, but the fact that SFLLRN was not a complete replacement for thrombin raises the possibility that HGF may express additional thrombin receptors. These findings add weight to the importance of the cytokine‐like role played by thrombin and raise the possibility that protease‐activated receptors may play a role in the pathogenesis of inflammatory periodontal disease.
Effect of two delivery systems for recombinant human bone morphogenetic protein‐2 on periodontal regeneration in vivo
Authors: King Gaston N. , King Nicola , Hughes Francis J.
Published: July 2006

Resorbable collagen membranes for guided tissue regeneration in periodontal therapy have shown promise but are not osteoinductive. As recombinant human bone morphogenetic protein‐2 (rhBMP‐2) is known to have an affinity for collagen, the use of this osteoinductive agent incorporated into a collagen vehicle may act as a suitable carrier to promote periodontal regeneration. The aim of this study was to investigate the effects of two different collagen delivery systems for rhBMP‐2 in rat periodontal fenestration defects. Using the collagen membrane delivery system, 3 groups of adult Wistar rats which had surgical defects created on the right side of the mandible involving the removal of bone and exposure of the molar roots were treated with either rhBMP‐2 in collagen membrane (BMPm) (n = 12 animals), or collagen membrane only (COLm) (n = 12), or were left untreated (UN) (n = 14). Using the collagen gel delivery system, surgical defects were treated with either rhBMP‐2 incorporated in a collagen gel carrier (BMPg) (n = 5) or had collagen gel only (COLg) (n = 6). Animals were killed 10 d postoperatively and tissues processed for histology. New bone formation was significantly greater in BMPg compared with both BMPm and controls (p<0.05). However, new cementum formation was significantly greater in BMPm (721 ± 166 μm2, mean ± SE) compared with COLm, COLg and UN (p<0.02) (190 ± 44 μm2, 327±114 μm2 and 172 ± 33 μm2, respectively) and more than 1.5 times BMPg (451 ± 158 μm2). In conclusion, both carrier systems for rhBMP‐2 significantly increased new bone formation compared with controls during the early stages of periodontal wound healing. However, the more slowly dissolving collagen membrane carrier system for rhBMP‐2 produced significantly greater new cementum compared with the collagen gel carrier, suggesting that a more prolonged exposure of rhBMP‐2 is required to increase cementogenesis.
PAF levels in saliva are regulated by inflammatory cells
Authors: Ribaldi E. , Guerre M. , Mezzasoma A. M. , Staffolani N. , Goracci G. , Gresele P.
Published: July 2006

Platelet activating factor (PAF), a powerful inflammatory phospholipid mediator, has been detected in normal human saliva and found to be increased in periodontitis. The cellular source of PAF in saliva is controversial although several data suggest an origin related to the presence of inflammatory cells. PAF levels in biological fluids are regulated by PAF‐producing cells and by the PAF‐degrading acetylhydrolase. Although in normal human saliva acetylhydrolase activity is very low, no information is available on the levels of this enzyme in inflammatory conditions of the mouth. The aim of our study was to assess the contribution of inflammatory cells to the levels of PAF in saliva in normal subjects and in patients with periodontitis. PAF was measured by radioimmunoassay (RIA) in mixed uncentrifuged saliva and in cell‐free saliva from healthy subjects, before and after tooth brushing, and in patients with periodontitis. In healthy subjects PAF levels were significantly higher in whole saliva than in centifuged saliva (1.51 ± 0.22 vs. 0.92 ± 0.04 ng/ml, p<0.0039). A significant increase in the amount of PAF was detected in whole saliva, but not in centifuged saliva, 2 h after tooth brushing. In patients with periodontitis PAF levels were not different from those of healthy individuals when using centrifuged saliva but were significantly higher when using whole, uncentrifuged saliva. Exogenous radiolabelled PAF was degraded much more rapidly by the saliva of periodontitis patients than by that of normal subjects. In conclusion, our study shows that inflammatory cells regulate the levels of PAF in saliva contributing to its production and degradation. The differential degradation of PAF in normal and inflammatory saliva highlights the absolute need of a series of methodological precautions when performing studies on salivary PAF.
Human cementum tumor cells have different features from human osteoblastic cells in vitro
Authors: Arzate Higinio , Alvarez‐Pérez Marco A. , Aguilar‐Mendoza Ma. Eugenia , Alvarez‐Fregoso Octavio
Published: July 2006

Cells obtained from human cementoblastoma and alveolar bone were isolated and cultured. Initial and late stages of mineralization were assessed by using atomic force microscopy, scanning electron microscopy and X‐ray microanalysis. In cultures of cementoblastoma‐derived cells the initial stages of mineralization showed well‐defined sphericai‐shaped structures, while the osteoblastic cells showed plaque‐like deposits. These morphological patterns of mineral deposition could serve as nucleation centers for hydroxyapatite crystals. Late stages of mineralization at 28 and 35 d maintained those morphological differences established in initial cultures. The material deposited by cemetoblastoma and osteoblastic cells, analyzed by EDX spectra, revealed similar Ca/P ratios for both cell types. These values were similar to those reported for hydroxyapatite in enamel and bone. Alkaline phosphatase specific activity (A1P), of osteoblastic cells at 3, 7 and 11 d, showed an increase of 27.9, 50.9 and 37.0% (p< 0.001), respectively. However, at 15 and 19 d there was an increase of AIP activity of cementoblastoma cells by 39.4 and 34.5% over osteoblastic cells (p<0.001). Immunostaining of cementoblastoma and osteoblastic cells using a specific mAb against a cementum‐derived attachment protein revealed strong immunostaining of cementoblastoma cells which was localized to the cell membrane and fibril‐like structures (96.2 ± 1.3). A few osteoblastic cells also stained weakly with the anti‐CAP mAb (6.4 ± 0.6). Sections of decalcified paraffin embedded cementoblastoma specimens, when immunostained with anti‐CAP mAb, showed strong immunostaining of the cells surrounding the regular and irregularly‐shaped calcified masses of the tumor. Putative cementocytes also stained positively. Immunostaining with a polyclonal antibody against osteopontin strongly stained the osteoblastic cells (89.0 ± 3.6). Cementoblastoma cells showed weaker staining (54.2 ± 2.4). The results suggest that cementoblastoma cells could be a major source of specific cementum proteins. These cells could provide the opportunity to elucidate the regulation of the cementogenesis process.
Periotron 8000: calibration characteristics and reliability
Authors: Ciantar M. , Caniana D. J.
Published: July 2006

The Periotron is an instrument designed to quantify submicrolitre volumes of fluid sampled on a filter paper strip. To date 3 models have been manufactured: the Periotron 600 (1976), the Periotron 6000 (1983) and more recently the Periotron 8000 (1995). This paper investigated for the first time the calibration characteristics and reliability of the Periotron 8000. The fluids under investigation were: de‐ionised water‐ human serum, fetal bovine serum and an ultrafiltrate of fetal bovine serum. Quantitative analysis was studied by recording a series of Periotron readings over a volume range of 0–1.0 μl for each fluid. The average of 5 Periotron values for each particular fluid was then plotted versus the respective fluid volume. Qualitative changes in fluid composition versus Periotron Scores were also analysed. Volume conversion for Periotron scores using both Periotron MLCONVRT software and a best fit equation selected from Table Curve 2D software compared well. The results of this study revealed that: 1) differences in calibration fluid composition (e.g. protein content) are reflected in the Periotron scores; 2) positioning of filter paper strip between the jaws of the Periotron should be standardised, 3) calibration of the Periotron 8000 seems to be consistent over a 1‐wk interval.
Histological alterations following short‐term smokeless tobacco exposure in humans
Authors: Payne Jeffrey B. , Johnson Georgia K. , Reinhardt Richard A. , Schmid Marian
Published: July 2006

The purpose of this study was to investigate the histological changes following short‐term smokeless tobacco application in humans. Sixteen smokeless tobacco‐using subjects participated in this trial. Each subject had used at least 3 cans of snuff per week for the previous 2 yr and had an existing lesion at the site of habitual snuff placement. The experimental design included subject placement of moist snuff (University of Kentucky reference tobacco brand 1S3) at a new site in the mandibular arch. At either 2 or 7 d, biopsies were taken from the new lesions and from a non‐placement site in the opposing arch. The volume density of inflammatory cells was determined by point counting. Keratin and epithelial thickness were evaluated by digitizing morphometry. Data were analyzed by repeated measures analysis of variance. In 7‐d lesions, increased keratin thickness was observed at the new sites compared to the non‐placement sites (p = 0.05). Increased volume density of fibroblasts (p=0.027) and decreased volume densities of macrophages (p=0.0083) and mast cells (p=0.05) were observed at 2 d in new versus non‐placement sites. Clinically, the new sites showed erythema, erythema plus ulceration, or white striations. This study demonstrated histological and clinical changes at new snuff placement sites in as few as 2–7 d, underscoring the rapidity of tissue alterations following snuff use.
A study of the proliferative activity of the long junctional epithelium using argyrophilic nucleolar organizer region (AgNORs) staining
Authors: Uno T. , Hashimoto S. , Shimono M.
Published: July 2006

The proliferative activity of the long junctional epithelium (LJE) in rats was examined using stains for argyrophilic proteins of the nucleolar organizer region (AgNORs protein). The LJE was experimentally produced by insertion of a rubber piece between maxillary molars for 1 wk. After removal of the rubber, the length and AgNORs parameters of the LJE were measured and analyzed statistically. The LJE widely covered the apical side of the exposed root surface 4 wk after the removal. Its length was longest after 4 and 8 wk; it became shorter subsequently. The AgNORs were visible as black dots of various sizes and numbers on the sections. A high potential for proliferation was obvious in the LJE after 4 wk and was maintained until 12 wk after the removal. The AgNORs ratio on the connective tissue interface of the LJE was about twice of that of normal junctional epithelium after 4–12 wk. The proliferative activity on the root surface side was slightly increased after 4 wk. There was no significant difference in proliferative activity between the coronal and apical sides. These results suggest that the proliferative activity of the LJE is maintained continuously at a high level on the connective tissue interface supplying the epithelial cells. Basal cells proliferate at the connective tissue interface of the LJE, migrate directly to the root surface or via the apical portion and finally desquamate from the surface of the epithelium.