bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of cytokines in the modulation of neutrophil chemotaxis in localized juvenile periodontitis
Authors: Agarwal S. , Suzuki J. B. , Riccelli A. E.
Published: July 2006

Decreased neutrophil chemotaxis has been implicated in the pathophysiology of the disease, localized juvenile periodontitis (LJP). The bioloigcal basis for the altered neutrophil function in LJP has been suggested to be an intrinsic cellular defect, involving a decrease in the number of N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) receptors on the cell surface. We have investigated the relative contribution of serum‐borne factors in the modulation of neutrophil functions in LJP, in a large population of LJP patients and healthy control subjects (HS). Treatment of HS‐neutrophils with LJP‐sera, resulted in a decreased neutrophil chemotactic response, and down regulation of FMLP receptors on the cell surface. Pretreatment of LJP‐sera with anti‐TNF and anti‐IL‐1 antibodies effectively, although incompletely, neutralized the ability of LJP‐sera to modulate chemotaxis and FMLP receptor levels in HS‐neutrophils. The changes induced by LJP sera were specific and sustained and could not be reversed by placing LJP‐serum treated neutrophils in HS‐serum. Sera obtained from HS and patients with adult periodontitis (AP), both of which exhibit normal chemotaxis, and patients with clinically diagnosed LJP with normal neutrophil chemotaxis (LJP‐nctx) did not modulate HS neutrophil chemotaxis or FMLP receptors. Furthermore, recombinant human TNF‐a, rhIL‐lα, and rhIL‐lα and rhIL‐1β, at very low concentrations (15 pg/ml to 150 pg/ml), modulated the chemotactic response as well as FMLP receptor numbers on HS‐neutrophils, in a manner similar to those observed in LJP. The present findings demonstrate that the biologic basis for the altered neutrophil function may not be an intrinsic cellular defect in neutrophils, but at least in part due to quantitatively small but biologically significant elevations in the levels of TNF‐α and 1L‐1 in the serum.
Improvement of gingival and alveolar bone status in periodontitis‐affected hamsters treated with 15‐methyl prostaglandin E1
Authors: Caillon P. , Saffar J. L.
Published: July 2006

Exogenous PGE1 at pharmacological doses suppresses acute and chronic inflammation manifestations. As periodontitis possesses features of both acute and chronic inflammation, attenuation of periodontal destruction in hamsters was attempted by using 15‐M‐PGE1, a stable PGE1 analog. The agent was tested at 2 different doses (100 and 150 μg/kg/d) and its effects were matched against disease‐free and periodontitis‐affected animals. No effect was found with the 100 μg regimen. In contrast, in the 150 μg group, in the gingiva around the first right mandibular molar the pocket epithelium and infiltrated connective tissue (ICT) areas, the mean vascular lumen, the number of PMNLs adherent to blood vessels and infiltrating the ICT were significantly reduced. The number of osteoclasts was markedly diminished as well, but their intrinsic activity was enhanced. Bone formation was totally inhibited in this treatment group. These results indicated that 15‐M‐PGE1 effectively improved gingival inflammation mostly by reducing edema and PMNL recruitment and controlled alveolar bone destruction by reducing the osteoclast recruitment. From a therapeutic point of view the complete inhibition of formation seems to contraindicate its use.
Acute‐phase proteins in gingival crevicular fluid during experimentally induced gingivitis
Authors: Adonogianaki E. , Moughal N. A. , Mooney J. , Stirrups D. R. , Kinane D. F.
Published: July 2006

The dynamics of four acute‐phase proteins were investigated in gingival crevicular fluid (GCF) during the course of a 21 day experimental gingivitis study. These acute‐phase proteins were the protease inhibitors α2‐macroglobulin (α2‐M) and α1‐antitrypsin (α1‐AT) and the iron‐binding proteins transferrin (TF) and lactoferrin (LF). 6 healthy volunteers ceased all oral hygiene procedures for 3 weeks. GCF was sampled at seven day intervals from two sites per subject by paper strips for 30 s during the experimental gingivitis period and for two additional weeks after the reinstitution of oral hygiene. The mainly serum derived α2‐M, αl‐AT and TF exhibited very similar dynamics which reflects their common origin in GCF. Their levels increased significantly from baseline and remained high for at least one week after the reinstitution of oral hygiene measures (repeated measures MANOVA; α2‐M: p=0.015; α1‐AT: p=0.012; TF: p=0.02). This probably reflects increased vascular permeability in the gingivae and, to a lesser degree, local production by gingival inflammatory cells. In contrast to the serum derived acute‐phase proteins, the neutrophil derived LF rose significantly from baseline (repeated measures MANOVA; p=0.001) but dropped rapidly after the reinstitution of oral hygiene measures. This could be because dental plaque was removed and thus neutrophil chemotactic agents in the crevice were decreased.
Comparative histochemical, biochemical and immunocytochemical studies of cathepsin B in human gingival
Authors: Kennett C. N. , Cox S. W. , Eley B. M.
Published: July 2006

Cathepsin B activity was demonstrated histochemically in unfixed cryostat sections of inflamed human gingiva using the 2‐methoxy‐4‐naphthylamide (MNA) substrates Z‐Val‐Lys‐Lys‐Arg‐MNA and Z‐Ala‐Arg‐Arg‐MNA with a post‐azo‐coupling technique. Enzyme localisation was confirmed by immunocyto‐chemistry with polyclonal sheep anti‐human cathepsin B. In both cases, staining was found in connective tissue fibroblasts and also in cells varying in shape from rounded to more irregular forms. The latter were present both in areas of cellular infiltration and in the oral and pocket epithelium. Examination of adjacent sections with monoclonal antibodies directed against leukocyte differentiation antigens showed that the rounded to irregular cells were CD68 positive macrophages and monocytes. The histochemical staining had the form of fine cytoplasmic particles consistent with the known lysosomal occurrence of cathepsin B. Cells stained by the post‐coupling method using the tryptase substrates Z‐Ala‐Ala‐Lys‐MNA and D‐Val‐Leu‐Arg‐MNA showed a different distribution and morphology, with reaction product confined to mast cell granules. The differences between the cathepsin B and tryptase staining patterns were confirmed by differential extraction from cryostat sections with salt‐free and high‐salt buffers respectively. Biochemical characterisation of activities in the extracts with the 7‐amino‐4‐trifluoromethyl coumarin (AFC) substrates Z‐Val‐Lys‐Lys‐Arg‐AFC and Z‐Ala‐Ala‐Lys‐AFC and protease inhibitors confirmed the identity of the two enzymes. Selective inhibitors could also be used in histochemical incubations to distinguish between cathepsin B and tryptase staining.
Periodontal regeneration: potential role of bone morphogenetic proteins
Authors: Ripamonti U. , Reddi A. H.
Published: July 2006

Initiation of osteogenesis and cementogenesis is a problem central to periodontal regeneration. A major advance in the understanding of bone formation has been the identification of an entirely new family of protein initiators, the bone morphogenetic proteins, that regulate cartilage and bone differentiation in vivo. The purification, genetic cloning and expression of recombinant human bone morphogenetic proteins (BMPs) have laid the foundation for the cellular and molecular dissection of bone development and regeneration. The striking evolutionary conservation of the BMP genes indicates that they are critical in the normal development and function of animals. In addition to postfetal osteogenesis, the BMPs may play multiple roles in embryonic development and organogenesis, including skeletogenesis and the development of craniofacial and dental tissues. The availability of recombinant human BMPs provides several challenges and opportunities to gain insights into the mechanisms regulating the regeneration of bone and cementum for optimal outcome in the periodontal patient.
Tetracyclines inhibit Porphyromonas gingivalis‐induced alveolar bone loss in rats by a non‐antimicrobial mechanism
Authors: Chang K. M. , Ramamurthy N. S. , McNamara T. F. , Evans R. T. , Klausen B. , Murray P. A. , Golub L. M.
Published: July 2006

Tetracyclines have been widely used as adjuncts in periodontal therapy due to the antimicrobial efficacy of these drugs. Recently, their ability to inhibit host‐derived matrix metalloproteinases (collagenase and gelatinase) and bone resorption in organ culture has also been invoked as a therapeutic rationale. The current study was undertaken to determine whether tetracyclines can inhibit alveolar bone loss in vivo due to a non‐antimicrobial action of these drugs. Experimental periodontitis was induced by inoculating adult, male Sprague‐Dawley rats with P. gingivalis (strain 381) following kanamycin/ampicillin pretreatment. Doxycycline, non‐antimicrobial chemically‐modified tetracycline (CMT‐1) and vehicle alone were administered daily to 3 infected groups of rats (n=6 rats per group; each group housed in a sterilized inflatable isolator) beginning 10 days after P. gingivalis inoculation. The control group (n = 6; non‐infected rats) received only vehicle. After 5 weeks of daily drug administration by gastric intubation, the experiment was terminated and blood samples were taken from each animal to determine antibody levels against P. gingivalis. Plaque samples were collected from each group of animals before and after P. gingivalis inoculation and at the end of the experiment for microbiological examination. The jaws were removed from each rat, defleshed and then analyzed morphometrically and radiographically to assess bone loss. Serum antibody levels against P. gingivalis were significantly elevated in the 3 infected groups compared to the non‐infected controls. This, together with the microbiologic findings, indicated that these groups of rats were infected with P. gingivalis. Significantly greater bone loss was observed in the untreated P. gingivalis‐infected group compared to the controls based on both morphometric and radiographic measurements. Oral administration of doxycycline or CMT‐1 to the P. gingivalis‐infected rats completely inhibited this bone loss. As expected, CMT‐1 did not exhibit an antimicrobial effect against P. gingivalis. Therefore, this study indicates that tetracyclines can inhibit alveolar bone loss in vivo by a mechanism which is independent of the antimicrobial efficacy of these drugs.
Ultrastructure of cementogenesis as affected by growth hormone in the molar periodontium of the hypophysectomized rat
Authors: Clayden A. M. , Young W. G. , Zhang C. Z. , Harbrow D. , Romaniuk K. , Waters M. J.
Published: July 2006

To document the effect of hypophysectomy and growth hormone replacement on the ultrastructure of cementogenesis in the developing rat third molar, 12 female Wistar rats were randomly allocated to normal control, hypophysectomized or hypophysectomized plus human growth hormone (for 10 days) treatment groups. The results of this study by electron and light microscopy and morphometry have shown that qualitative and quantitative changes occur in the organelles of cementoblasts forming cellular cementum as a result of hypophysectomy and growth hormone replacement. After hypophysectomy, the changes of less prominent nucleoli and nuclear pores, less prominent Golgi apparatuses and decreased endoplasmic reticulum can be interpreted as diminished cementum matrix biosynthesis – an interpretation that can be confirmed morphometrically by less cellular cementum formation. Growth hormone replacement for 10 days reactivates protein synthesis and cementogenesis as evidenced by ultrastructural changes in cementoblasts and a greater production of cementum.
Elastase and lactoferrin in gingival crevicular fluid: possible indicators of a granulocyte‐associated specific host response
Authors: Gustafsson A. , Åsman B. , Bergström K.
Published: July 2006

Periodontitis affects a limited number of susceptible humans. The aim of this study was to determine whether there is a difference in the inflammatory reaction between patients with gingivitis and those with periodontitis. For this purpose the levels of elastase and lactoferrin were measured in gingival crevicular fluid (GCF) from three types of sites: i) inflamed sites in patients with gingivitis alone, inflamed sites both ii) with and iii) without tissue destruction in patients with periodontitis. Elastase activity, measured with a chromogenic substrate was significantly higher in the two types of sites in periodontitis patients. Lactoferrin levels, measured with ELISA were the same in the three types of sites. in vitro activation of granulocytes from healthy volunteers with Fc‐receptor stimulation showed that the entire release of lactoferrin occurred immediately. In contrast, elastase release was time‐dependent and continued throughout the experiment. Thus, the degranulation of the specific (lactoferrin) and azurophil granule (elastase) are under separate control and the two parameters can be combined in a ratio in order to characterize the granulocytes of a given patient. Assuming an immediate release of lactoferrin from the activated granulocytes in vivo, similar amounts of lactoferrin in the three types of sites can be regarded as reflecting similar numbers of granulocytes in the three types of sites. Consequently, a higher elastase activity in GCF from patients with periodontitis indicates a higher rate of release from the cells per se and a granulocyte‐associated specific host response.
Characteristics of older adults experiencing periodontal attachment loss as gingival recession or probing depth
Authors: Beck J. D. , Koch G. G.
Published: July 2006

While attachment loss currently is the best field examination indicator that periodontal disease has progressed, this measure is not disaggregated enough to differentiate between attachment loss that results in deeper pockets and attachment loss that results primarily in recession of the gingiva. If these two conditions have different etiologies, then efforts at predicting who will experience the condition from a single, general risk assessment model will be difficult. The purpose of this paper is to determine whether the characteristics of people who experience attachment loss primarily as increased probing depths are different from those people who experience attachment loss primarily as gingival recession. Baseline and 18‐month follow‐up periodontal measures were gathered on a representative sample of community‐dwelling adults aged 65+. Based on measurement errors obtained one week apart on a reliability subsample, a threshold level of 3+mm was set to define attachment loss. Attachment loss was further characterized as resulting from mostly increased probing depth or from mostly increased gingival recession. In mesio‐buccal sites, 58% of the people had attachment loss mostly due to increased probing depth with 42% having attachment loss mostly due to gingival recession. For buccal sites, the figures were 24% and 76% respectively. The logistic regression models for both sites indicated that people who had their attachment loss in the form of increased probing depths had different characteristics than people who had most of their attachment loss in the form of gingival recession. These epidemiologic data indicate that different processes may be involved in people who experience attachment loss due to deeper probing depths as compared to people whose attachment loss is due more to gingival recession and not separating the two types of attachment loss may be a reason for inacuracies in risk prediction.
Recommended revisions to American Dental Association guidelines for acceptance of chemotherapeutic products for gingivitis control
Authors: Imrey P. B. , Chilton N. W. , Pihlstrom B. L. , Proskin H. M. , Kingman A. , Listgarten M. A. , Zimmerman S. O. , Ciancio S. G. , Cohen M. E. , D'Agostino R. B. , Fischman S. L. , Fleiss J. L. , Gunsolley J. C. , Kent R. L. , Killoy W. J. , Laster L. L. , Marks R. G. , Varma A. O.
Published: July 2006

This paper presents suggested revisions to the American Dental Association's 1985 guidelines for acceptance of anti‐gingivitis chemotherapeutic agents. The areas of study design, choice and quality control of clinical gingivitis measurements, statistical analysis, and minimum strength of effect, are addressed. The revisions articulate certain aspects of study design which were implicit in the 1985 guidelines, clarify language on cross‐over designs and independence of studies, and recommend use of a United States population in at least one trial supporting a product. Separate recording and analysis of a product's effect on gingival bleeding is proposed, and quality control of clinical measurements receives enhanced emphasis. Modestly elaborated statistical reporting guidelines and strengthened approval criteria, based on size of estimated effect as well as statistical significance, are advocated.
Diacylglycerol in peripheral blood neutrophils from patients with localized juvenile periodontitis
Authors: Leino L. , Hurttia H. , Peltonen E.
Published: July 2006

Neutrophils from patients with localized juvenile periodontitis (LJP) show several functional abnormalities. Recently, it has become increasingly apparent that the reason for these changes lies in part at the post receptor level of cellular metabolism. In this study we have analyzed intracellular diacylglycerol (DAG), a second messenger and an endogenous activator of protein kinase C, in un‐stimulated and agonist‐stimulated neutrophils. from five LJP patients showing a chemotaxis defect and matched normal individuals. No difference was observed in the basal cellular DAG between the two groups. In neutrophils from LJP patients the DAG levels increased by 67% and 111% from the basal level following stimulation with N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) and unopsonized zymosan particles, respectively, while in control cells the mean increases were 36% and 65%, respectively. Incubation with serum‐opsonized zymosan particles produced an identical rise in DAG in both groups. These data indicate that the stimulation of receptors for FMLP and unopsonized zymosan may produce an enhanced accumulation of DAG in neutrophils from LJP patients. In addition to DAG mass analysis, we determined the effect of R59022, a DAG‐kinase inhibitor, on zymosan‐stimulated luminol‐amplified chemiluminescence (CL) of neutrophils. In control cells R59022 significantly enhanced unopsonized zymosan induced CL, but it had no effect on cells from LJP patients, suggesting a possible change in the regulation of DAG‐kinase in LJP.
Evidence for the existence of intraepithelial nerve endings in the junctional epithelium of rat molars: an immunohistochemical study using protein gene product 9.5 (PGP 9.5) antibody
Authors: Maeda Takeyasu , Sodeyama Takao , Hara Kohji , Takano Yoshiro
Published: July 2006

Innervation of the junctional epithelium was investigated in rat molars by means of immunohistochemistry for protein gene product 9.5 (PGP 9.5) at light and electron microscopic levels. In comparison with our previous study on same tissues using neurofilament protein (NFP)‐antibody, the PGP 9.5‐immuno‐staining further disclosed numerous nerve fibers in the gingiva of rat molars and revealed the existence of a well‐developed plexus of PGP 9.5‐positive nerve fibers. The interproximal portion also contained numerous nerve fibers.

Observation of horizontal sections revealed a denser innervation toward the inner junctional gingival epithelium than the outer marginal epithelium. The nerve fibers, beaded in appearance and extending from the nerve bundles in the lamina propria, penetrated into the junctional epithelial layer and were distributed throughout the junctional epithelium, with some nerves being located near the epithelial surface. Non‐neuronal cells showing PGP 9.5‐immunoreactivity were absent in the junctional epithelium. In immunoelectron microscopy, the axoplasm of nerves in the gingiva was filled with electron‐dense reaction products of PGP 9.5, except for the cell organellae. The nerve fibers were devoid of Schwann cell investment and terminated among the epithelial cells in the junctional epithelium, frequently beneath the epithelial surface. The intraepithelial nerve endings contained various kinds of vesicles including large‐cored ones, supporting the presence of peptidergic innervation shown by previous studies. These findings confirmed the usefulness of PGP 9.5‐immunohisto‐chemistry for the identification of delicated nerve fibers in dental tissue, and suggested the dense network of nerve fibers that may serve as sensory receptor and other functions in the junctional epithelium.
Vascular adhesion molecules and initial development of inflammation in clinically healthy human keratinized mucosa around teeth and osseointegrated implants
Authors: Tonetti M. S. , Gerber L. , Lang N. P.
Published: July 2006

Expression of vascular cell adhesion molecules (HEV‐CAMs) by capillary loops represents an early step necessary for leukocyte extravasation and subsequent migration to sites of inflammation. The purpose of this investigation was to compare, the presence and distribution of ICAM‐1, ELAM‐1, VCAM‐1 and PECAM‐1 expression in the microvasculature of healthy gingiva and periimplant keratinized mucosa. The selected HEV‐CAMs were detected by a three stage immunoperoxidase technique in serial sections from clinically characterized sites. Biopsies were qualitatively and quantitatively analyzed. All biopsies displayed a small inflammatory infiltrate subjacent to the most coronal extension of the junctional epithelium. HEV‐CAMs were expressed both in the sulcular and oral aspects of biopsies. Intensity of ICAM‐1, ELAM‐1 and VCAM‐1 staining, however, was consistently higher in the region of the infiltrated connective tissue subjacent to the most coronal extension of the junctional epithelium. Only a fraction of microvascular loops were positive for ICAM‐1, ELAM‐1 or VCAM‐1 when compared to the constitutively expressed PECAM‐1. No significant differences were observed between gingiva and keratinized peri‐implant mucosa. It was concluded that in healthy gingiva or peri‐implant mucosa 1. HEV‐CAM expressing capillary loops were in close topographic association with the inflammatory infiltrate; 2. only a fraction of capillary loops are activated to express ICAM‐1 ELAM‐1 and VCAM‐1 at any given time; and 3. HEV‐CAM expression in the periodontium may be influenced by local factors.
Effect of long‐term methotrexate‐induced neutropenia on experimental periodontal lesion in rats
Authors: Yoshinari N. , Kameyama Y , Aoyama Y. , Nishiyama H. , Noguchi T.
Published: July 2006

The effects of long‐term methotrexate (MTX)‐induced neutropenia on the periodontal lesion in rats were investigated histologically, histometrically and bacteriologically. A nylon thread was inserted into the interdental gingiva between the 1st and 2nd right maxillary molars of the animals 3 weeks before an application of MTX. The animals were then divided into Groups A and B. Group B were injected intraperitoneally with 1.0 mg/kg of MTX 3 times per week for 9 weeks. Group A received saline as a control. Five animals were killed at the 1st, 3rd, 5th, 7th, and 9th week. In Group A, the neutrophils did not decrease during these 9 weeks. In Group B, however, the neutrophils decreased during the 3rd to 9th week. Whereas the experimental side of Group A showed only moderate alveolar bone resorption between the 7th and 9th week, [in Group B] a marked alveolar bone resorption occurred in Group B occurred in the same period. Alveolar bone loss in the experimental side of Group B was significantly greater (p < 0.01) than in Group A in the 7th to 9th weeks. The percentage of gram‐negative rods increased in both control and experimental sides of Group B at the 9th week. The results of the present study indicate that neutropenia is induced by a long‐term application of MTX in rats and that alveolar bone destruction increases as time goes by in the area where a nylon thread was inserted.
Scanning electron microscopy of Eikenella corrodens colony morphology variants
Authors: Cobb C. M. , Helber J. T. , Hirschberg R.
Published: July 2006

Eikenella corrodens is a gram‐negative, human pathogen which exhibits colony morphology variation. Scanning electron microscopy (SEM) was used to examine large (non‐corroding) and small (corroding) colony variants from the type strain (ATCC 23834) and two clinical isolates (strains VA1 and CM1), Large colonies were large, flatter, and appeared relatively featureless compared to small colonies and had even, smooth colony margins. Small colonies were more raised from the medium surface, and often had a central raised region surrounded by flatter border. Cells on the surface of large colonies were more regularly arranged at the colony edge, and end‐to‐end rows of cells around the colony were seen in some strains. Cells in the center of the upper surface of small colonies were usually randomly arranged. Within cross sections of small colonies, cells were arranged randomly or perpendicular to the medium; in large colonies, cells were random or arranged horizontally. Amorphous, “slime” material was often seen covering groups of cells in large and small variants. An unusual variant, possibly a mutant, which combined features of both colony types was isolated.
Interleukin‐1β stimulates collagenase production by cultured human periodontal ligament fibroblasts
Authors: Ohshima M. , Otsuka K. , Suzuki K.
Published: July 2006

To examine the effects of interleukin‐lβ (IL‐1β) on collagenase production by human periodontal ligament fibroblasts (PLF) and gingival fibroblasts (GF) in Culture, collagenase activity in conditioned media was determined using a novel procedure that circumvented interference by enzyme inhibitors. Fibroblasts obtained from five paired periodontal ligament and gingival tissues were cultured for two weeks, and then incubated for a further 72 h in α‐MEM supplemented with various concentrations of IL‐1β (0 to 1250 pg/ml). The conditioned media from individual cultures were harvested and treated with dithiothreitol to inactivate TIMPs, and then with APMA, to activate the latent collagenase. Collagenase activity was measured fluorometrically using FITC‐collagen as a substrate. IL‐lβ induced a ∼2.4 to 5.2‐fold increase in collagenase activity in PLF compared to a ∼1.4 to 2.2‐fold increase in GF. These results are in contrast to previous studies in which collagenase activity was measured in the presence of TIMPs, and indicate that PLF are more sensitive to IL‐1β than GF. Since both PLF and GF are present in periodontal lesions, it is possible that collagenase secretion stimulated by exposure to inflammatory cell products such as IL‐lβ may participate in the destruction of collagen fibers involved in periodontal attachment.
Human antibody responses to outer envelope antigens of Porphyromonas gingivalis serotypes
Authors: Ebersole J. L. , Steffen M. J.
Published: July 2006

Immunological studies examining the homogeneity of the major antigenic components of P. gingivalis have suggested 3 serotypes and have indicated a limited distribution of the serotypes in an individual patient. These studies prompted us to define the immunodominant antigens and distribution of immune responses to P. gingivalis serotypes. Serum IgG antibody levels in periodontitis patients in the present study were most frequently elevated above the normal subjects when tested against P. gingivalis serotype A (i.e., 33277). Nearly 1/3 of the patients showed significantly elevated antibody to multiple serotypes of the P. gingivalis apparently resulting from cross‐reacting antigens. We determined distinctive differences among outer envelope protein and antigen patterns obtained from the three serotypes. Moreover, the results identified considerable similarities in the qualitative and quantitative antigen response patterns among patients to a particular serotype. There was a strong positive correlation between IgG antibody levels (ELISA) and the total level of reactivity determined in the immunoblots, as well as a positive correlation to the proportion of antibody to particular antigens. These findings suggest that responses to these antigens comprised a major portion of the response to the intact microorganism. Additionally, the detection of antibody to particular antigen bands was indicative of early responses to each of the P. gingivalis serotypes. The results of our study indicate that a subpopulation of periodontitis patients develop an extensive serum antibody response often to multiple serotypes of P. gingivalis and may define a patient population with a P. gingivalis disease. Finally, our results indicate a more consistent antigenic composition for P. gingivalis which may enhance the potential for strategies to immunologically interfere with disease caused by this microorganism.
Evidence of a direct relationship between neutrophil collagenase activity and periodontal tissue destruction in vivo: role of active enzyme in human periodontitis
Authors: Lee W. , Aitken S. , Sodek J. , McCulloch C. A. G.
Published: July 2006

To assess the temporal relationship between periodontal tissue destruction and the activity of collagenase, exudate from inflamed periodontal tissues was collected and latent and active collagenase activities were measured by a functional assay in a longitudinal cohort study. Comparisons were made between human subjects with either: 1) inflammation with a previous history of progressive loss of connective tissue and bone support (n=14); 2) inflammation and previous history of bone loss but now clinically stable (n=27); or 3) inflammation and no loss of bone support (n=17). Experiments using specific enzyme inhibitors, blocking antibodies and SDS‐PAGE fluorograph to identify the pattern of collagen substrate degradation demonstrated that the collagenase activity was derived from neutrophils and not from bacteria or other host cells. Active collagenase activity pooled from 6 sites per subject was respectively 5 and 6‐fold higher in the group with progressive loss of connective tissue compared to the groups with either inflamed tissues alone or with inflammation and previous bone loss. In contrast, latent collagenase was increased up to 2 fold higher in the group with inflammation but no bone loss compared to the group with progressive lesions. Moreover, the ratio of active to total collagenase activity was 50% higher in the group with progressive lesions. Although in all subjects successive measurements of site‐specific active collagenase 1 month apart demonstrated wide variation (r<0.50), only in sites with progressive periodontal destruction was there significant increase of active collagenase with time (1.28×l0−4 collagenase units per day). There were also sharp elevations in active enzyme level at the time of detection of loss of connective tissue attachment in specific sites of 8 subjects. At the time of detection of connective tissue attachment loss, there was an overall 40% increase of pooled active collagenase activity in all subjects with progressive loss of connective tissue compared to pre‐breakdown sampling times. These data provide strong in vivo evidence for a direct role of active neutrophil collagenase in the pathological destruction of periodontal connective tissue.
Osteopontin adhesion receptors on gingival fibroblasts
Authors: D'Errico J. A. , Sauk J. J. , Prince C. W. , Somerman M. J.
Published: July 2006

Osteopontin (OPN) promotes attachment and spreading of cells in an RGD dependent fashion, suggesting that OPN interacts with integrins on cell surfaces. Here in, we show that LM‐609, a monoclonal antibody to the αvβ3 integrin (a vitronectin receptor), inhibited OPN‐mediated attachment of gingival fibroblasts. To characterize the cell surface receptors responsible for this interaction, we performed OPN‐sepharose affinity chromatography using detergent extracts of 35S‐methionine or l25I‐surface labeled gingival fibroblasts. Proteins bound to the OPN‐matrix were eluted with EDTA and subjected to SDS‐PAGE under reducing conditions. EDTA eluates from both 125I‐surface labeled and 35Smethionine labeled extracts demonstrated prominent bands in the 90kDa and 50kDa regions, by both autoradiography and fluorography, respectively. These studies suggest that OPN is associated with other cell surface molecules in addition to αvβ3. Furthermore, these as yet to be characterized proteins, may prove to have a stronger affinity for OPN than αvβ3.
Increased intracellular levels of lysosomal β‐glucuronidase in peripheral blood PMNs from humans with rapidly progressive periodontitis
Authors: Pippin D. J. , Cobb C. M. , Feil P.
Published: July 2006

Release of potent lysosomal enzymes by degranulation of polymorphonuclear leukocytes (PMNs) in host gingiva may contribute significantly to tissue destruction and the pathogenesis of periodontal disease. A pilot study established that peripheral blood PMNs from humans with rapidly progressive periodontitis (RPP) contained significantly increased amounts of intracellular lysosomal β‐glucuronidase as compared to healthy controls. This investigation gained insight into the question: are the increased levels of β‐glucuronidase in persons with RPP an a priori genetically determined PMN characteristic, or a reactive phenomenon induced by the periodontal disease process during granulopoiesis? Twelve healthy controls and twelve otherwise healthy individuals with RPP participated in a repeated measures design of T0 (initial, baseline), T1 (four weeks after disease control therapy), and T2 (two months later). At each visit clinical indices (GI, pocket depths, GCF flow, plaque index) were performed and peripheral blood obtained. PMNs were isolated and suspended as 5×106 cells in 2.0 ml of HBSS. PMN suspensions were tested for total intracellular β‐glucuronidase, degranulation induced by 1×10−6 M and 5×10−7 M FMLP challenges, and unchallenged for non‐specific enzyme release. PMNs from individuals with RPP contained significantly higher absolute amounts of β‐glucuronidase and released greater absolute amounts at FMLP challenge at T0, T1, and T2 compared to controls. No relationship was found between any of the clinical indices and β‐glucuronidase levels and no pattern was discovered relating to the repeated measures over time. We conclude that RPP peripheral blood PMNs contain elevated levels of β‐glucuronidase that are not induced by the periodontal disease process.
Calibration and reliability of the Periotron 6000 for individual gingival crevicular fluid samples
Authors: Chappie I.L.C. , Cross I. A. , Glenwright H.D. , Matthews J.B.
Published: July 2006

The Periotron 6000® fluid analyser has become widely used as a diagnostic tool for a variety of oral diseases and recent work has questioned its reliability. This paper investigates for the first time, the detailed calibration curves of 2 Periotron 6000 machines across a range of 23 different fluid volumes. Within and between machine reliability is analyzed and the shape of the calibration line determined. The measurement errors incurred by using a single fluid sample, as opposed to mean values of triplicate samples are also determined. We conclude that there are 3 sections to the calibration line, 2 linear and a quadrilateral zone, and that 2 separate regression equations should be used; from 0–0.1 μl and from 0.1–1.0 μl. Within machine calibration errors were only 3.2±7.5%, but values for volumes below 0.2 μl were as high as 18.7%. Using a single fluid sample rather than mean values of multiple samples, incurred a further 4±4% error, which was as high as 7% for volumes lower than 0.12 μl. Whilst significant differences in volume reading existed between different machines (p<0.0004) and between the same volumes of different fluids (p<0.00001), individual Periotron calibrations were extremely reproducible and reliable. We conclude that the Periotron 6000 is a reliable and convenient instrument for measuring fluid volumes greater than 0.2 μl. For volumes lower than 0.2 μl errors in measurement may be too high for some investigations, but this is likely to be due to problems with evaporation and with measurement technique, rather than errors directly due to the Periotron itself. Finally, for optimum accuracy, the digital display should be re‐set to zero after each sample is measured.
Succession of putative peri‐implant pathogens after root‐form and plate‐form implant placement in partially dentate adult monkeys
Authors: Eke P. I. , Braswell L. , Fritz M. , Eke Paul I.
Published: July 2006

This report describes the succession of putative peri‐implant pathogens in partially dentate monkeys after dental implantation and prosthetic reconstruction. Tooth and implant (6 root‐end form, 4 blade‐vent implants) sites in eight monkeys were monitored microbiologically and clinically during the pre‐implant stage, abutment connection stage, bridge placement stage, and three and six months after the bridge placement stage. Tooth and implant sites were cleaned monthly post‐extraction. Microbiological studies included dark field microscopy, selective and non‐selective culture, and primary phenotypic characterization of culture isolates. After implant surgery, the median proportion of several putative peri‐implant pathogens studied were significantly elevated. Following fixture placement, P. intermedia replaced P. melaninogenica as the predominant Black Pigmented Anaerobic Bacilli (BPAB) in the mouth. After abutment connection stage, levels of P. intermedia, A. actinomycetemcomitans, F. nucleatun, Haemophilus sp. and spirochetes were significantly elevated at implant and tooth sites. Three months after bridge installations, P. intermedia and A. actinomycetemcomitans remained significantly elevated at implant sites. At six months after bridge installation, levels of P. intermedia, F. nucleatum and A. actinomycetemcomitans declined significantly relative to levels at three months. Porphyromonas sp. and spirochetes were not significantly elevated although their levels correlated with gingival redness. P. intermedia, Porphyromonas sp. and spirochetes levels correlated significantly with probing depth. Correlation was detected between P. gingivalis and spirochetes; and between A. actinomycetemcomitans and F. nucleatum. Our studies show a transitional increase in levels of several organisms resembling putative pathogens of human peri‐implant infection, associated with implant placements in partially edentulous mouths and supports early prophylactic interventions to control their levels.
Multilayer culture of periodontal ligament epithelial cells: a model for junctional epithelium
Authors: Pan Y.‐M. , Firth J.D. , Salonen J.I. , Uitto V.‐J.
Published: July 2006

The unique features of junctional epithelium involve lack of keratinization, limited differentiation and a relatively permeable structure. In order to study the relationship between differentiation and permeability of stratified epithelium a model system was developed. Porcine periodontal ligament epithelial cells were cultured on the polycarbonate nucleopore membrane of the Transwell® two‐compartment culture system. Within 5 days of culture the cells formed a confluent multilayered structure. Subsequently, maturation of the structure and differentiation of surface cells took place. Transmission electron microscopy showed that the cells were arranged into basal and suprabasal layers with sparse desmosomal attachments and wide intercellular spaces resembling the organization of junctional epithelium. The basal cells attached to a subepithelial basal lamina through numerous hemidesmosomes. The cytokeratin profile of the cultured epithelium (K5, 6, 14, 16, 19) resembled that of the cells of junctional epithelium attached to the tooth surface. The older cultures expressed differentiation markers, K4, K13 and involucrin, theraby resembling sulcular epithelium. The epithelial permeability, measured by diffusion of phenol red, radioactive dextran or methionine tracers, and as transepithelial electrical resistance, decreased with the increased cell number and maturation of the cultures. The new model provides an organotypic culture system which allows to control differentiation of a multilayered periodontal epithelium. It thus may serve as a valuable new tool for studies on the permeability and behaviour of periodontal epithelium under the influence of exogenous and endogenous factors.
Cytotoxic effect of vesicles produced by Porphyromonas gingivalis on fibroblasts in culture
Authors: Gulgand M. , Pallen P. , Mouton C. , Bonnaure‐Mallet M.
Published: July 2006

It has been shown that the vesicles produced by Porphyromonas gingivalis under certain growth conditions contribute to its pathogenicity. In this study, we demonstrate the cytotoxic effect of the vesicles using two methods: on quantitative (the colorimetric cytotoxicity test using sulforhodamine B) and the other qualitative (flow cytometry).
Determination of small sample volumes in the analysis of drugs at specific sites in the oral cavity
Authors: Medlicott N.J. , Tucker I.G. , Rathbone M.J. , Holborow D.W.
Published: July 2006

Two methods for the determination of sample volumes between 0.2 and 0.6 μl were compared by preparing standard curves for volumes over this range. The first method used a Periotron and the second the sample mass. A linear model was fitted and 95% confidence limits for volumes estimated by each method were calculated. This showed that use of either the maximum Periotron reading or the sample mass allowed estimation of volumes to within ±0.056 mUl and ±0.047 mUl respectively. It is proposed that measurement of sample mass provides a simple and accurate method to determine sample volume when analysing drug concentrations at specific sites in the oral cavity.
Presence of activated eosinophils, high IgE and sCD23 titers in gingival crevicular fluid of patients with adult periodontitis
Authors: Suzuki T. , Sugitam N. , Yoshie H. , Hara K.
Published: July 2006

Our previous studies showed that the expression of CD23 on polymorphonuclear leukocytes (PMNLs) in gingival crevicular fluid (GCF) from adult periodontitis (AP) patients was higher than in autologous peripheral blood (PB). Percentages of eosinophils in GCF PMNLs ranged between 6 and 14%. The purpose of the present studies was to increase understanding of the potential role of eosinophils and their products, including CD23, in periodontal disease. We analysed the eosinophil fraction in GCF and PB by flow cytometry using monoclonal antibodies to CD23b (BB10), eosinophil cationic protein (ECP) in stored and secretory forms (EG1 and EG2), and CD67 (80H3). Simultaneously, we measured IgE and soluble CD23 titer and GCF and serum by ELISA. Flow cytometric analysis of BB10, EG2 and 80H3 binding showed that GCF eosinophils from AP were activated. A large BB10+ EG2+ cellular fraction was detected in GCF from AP whereas it was very low in autologous serum (9.30±2.460 vs 0.16±0.10, p>0.001). GCF from gingivitis patients exhibited no flow cytometric evidence for the presence of BB10+ EG2+ cells. BB10+ EG1+ cells, or inactivated eosinophils rated lower in GCF than in PB both in gingivitis and periodontitis patients (0.45±0.63 vs 1.83±0.96 and 0.15±0.30 vs 1.30±0.20, p>0.05. respectively). IgE titer in AP patients reached 1208.1 ±421.2 IU/ml in GCF while only 49.1 ±50.4 in sera. Soluble CD23 in GCF reached 236.1 ±81.3 ng/ml in GCF and 5.6±1.8 ng/ml in sera. GCF of gingivitis patients, however, contained no detectable sCD23. Thus. GCF from AP patients displayed a high rate of activated eosinophils secreting ECP. while GCF of gingivitis patients did not. These results suggest that ECP‐secreting activated eosinophils are relevant to the pathology of adult periodontitis.
Effect of substance P administration on vascular permeability in the rat oral mucosa and sublingual gland
Authors: Györfi A. , Fazekas Á , Lrmes F. , Rosivall L.
Published: July 2006

Neuropeptides, including substance P (SP) may play a role in neurogenic inflammation. Although SP‐immunoreactive (SP‐IR) axons are known to be present within the oral mucosa (OM) and salivary glands, the functional significance of SP in oral mucosa and sublingual salivary gland (SLG) is not fully understood. The present experiments were carried out to study the effects of SP infused into the left common carotid artery on vascular permeability in the OM and in the SLG of male rats. Vascular permeability was assessed on the basis of Evans Blue extravasation. Separate groups of animals received histamine (H1) receptor antagonist (chloropyramine, 10 mg kg−1 i.v.) or prostaglandin synthesis inhibitor (indomethacin, 4 mg kg−1 i.v.) prior to SP infusions. Infusion of SP in doses of 30 and 74 pmol min−1 increased the vascular permeability of OM by 162.3±16.3% (n=8, p>0.05) and 482.7±46.7% (n=8, p>0.001), respectively. SP in a dose of 15 pmol min−1 did not increase Evans Blue extravasation in OM (38.3±4.0 μ g−1, n=8, compared to the control: 44.0±7.9 μg g−1, n = 8, NS). Although SP increased plasma extravasation in OM, it failed to affect vascular permeability in SLG (15 pmol min−1 SP: 46.9±6.9 μg g–1, n = 6, NS; 30 pmol min−1 SP: 54.1 ±6.2 μg g−1, n= 11 NS; 74 pmol min−1 SP: 49.7±2.3 μg g−1, n = 7, NS; compared to the control: 48.9±5.8 μg g−1, n = 8). After chloropyramine administration the SP effect on vascular permeability decreased in OM by 41.5±5.9% (n=10, p>0.05). Indomethacin pretreatment similarly diminished the effect of SP on the dye extravasation in OM by 43.9±6.1% (n = 8, p>0.01). Our results suggest that the effect of SP on plasma extravasation in the oral mucosa is partly elicited via the release of vasoactive agents (histamine, prostaglandins), and the microvasculature of SLG has lower sensitivity of SP than that of the oral mucosa.
Influence of sE‐selectin and L‐selectin on the regulation of cell migration during chronic periodontitis
Authors: Krugluger W. , Nell A. , Solar P. , Matejka M. , Boltz‐Nitulescu G.
Published: July 2006

Soluble endothelial leukocyte adhesion molecule‐1 (sE‐selectin) levels in peripheral blood (PB) and gingival capillary blood (GCB) of both healthy donors (HD) and patients with adult periodontitis (AP) were assayed by ELISA. Binding of sE‐selectin to polymorphonuclear neutrophils (PMN) from PB, GCB and crevicular fluid (GF), and expression of L‐selectin and sialyl‐Lewisx (sLex) on these cells were analyzed by immunofluorescence and flow cytometry. No significantly enhanced serum levels of sE‐selectin in patients with AP. compared to HD (28±5 ng/ml vs 19±3 ng/ml, respectively), and no differences in the concentration of sE‐selectin in GCB (16±1 ng/ml vs 16±2 ng/ml, respectively) were observed. On PB‐PMN no significant differences in the expression of L‐selectin and sLex were found and binding of sE‐selectin to PB‐PMN was comparable between HD and patients with AP. Binding of sE‐selectin to GCB‐PMN was significantly higher in patients with AP compared to HD (mean channel fluorescence (MCF)=88.5± 13.2 vs MCF=24.2±5.3, respectively). The expression of sLex on GCB‐PMN did not differ significantly between the two groups. A significant decrease in the expression of the adhesion molecule L‐selectin on GCB‐PMNs compared to PB‐PMN was found in patients with AP but not in HD. CF‐PMN showed decreased expression of both L‐selectin and sLex compared to PMN from PB and GCB, both in HD and patients with AP. Taken together, these data suggest that PMN from patients with AP had reduced selectin‐mediated adhesive capabilities to inflamed gingival endothelium.