The PerioGene North Study Uncovers Serum Proteins Related to Periodontitis.

The sequalae of periodontitis include irreversible degradation of tooth-supporting structures and circulatory spread of inflammatory mediators. However, the serum protein profile in periodontitis is not well described, which is partly attributable to the limited number of studies based on large and well-characterized periodontitis cohorts. This study aims to identify novel, circulating inflammation-related proteins associated with periodontitis within the PerioGene North case-control study, which includes 478 cases with severe periodontitis and 509 periodontally healthy controls. The serum concentrations of high-sensitivity C-reactive protein (hs-CRP) and a panel of 45 inflammation-related proteins were analyzed using targeted proteomics. A distinguishable serum protein profile was evident in periodontitis cases. The protein pattern could separate cases from controls with a sensitivity of 0.81 and specificity of 0.81 (area under the curve = 0.87). Adjusted levels for hs-CRP and 24 of the 45 proteins were different between cases and controls. High levels of hs-CRP and matrix metalloproteinase-12, and low levels of epidermal growth factor (EGF) and oxidized low-density lipoprotein receptor 1 (OLR-1) were detected among the cases. Furthermore, the levels of C-C motif chemokine-19, granulocyte colony-stimulating factor-3 (CSF-3), interleukin-7 (IL-7), and hs-CRP were significantly higher in cases with a high degree of gingival inflammation. The levels of CSF-3 and tumor necrosis factor ligand superfamily member-10 TNFSF-10 were higher in cases with many deep periodontal pockets. The PerioGene North study includes detailed clinical periodontal data and uncovers a distinct serum protein profile in periodontitis. The findings of lower EGF and OLR-1 among the cases are highlighted, as this has not been presented before. The role of EGF and OLR-1 in periodontitis pathogenesis and as possible future biomarkers should be further explored.

IL-1beta and TNF-alpha regulate IL-6-type cytokines in gingival fibroblasts.

UNLABELLED: Interleukin-6 (IL-6)-type cytokines are pleiotropic molecules capable of stimulating bone resorption and expressed by numerous cell types. In the present study, we tested the hypothesis that gingival fibroblasts may exert local osteotropic effects through production of IL-6 and related cytokines. IL-6-type cytokine expression and regulation by IL-1beta and tumor necrosis factor-alpha (TNF-alpha) were studied in fibroblasts from the non-inflamed gingiva of healthy individuals. Constitutive mRNA expression of IL-6, IL-11, and leukemia inhibitory factor (LIF), but not of oncostatin M (OSM), was demonstrated, as was concentration-dependent stimulation of IL-6 and LIF mRNA and of protein by IL-1beta and TNF-alpha. IL-11 mRNA and protein were concentration-dependently stimulated by IL-1beta. The signaling pathway involved in IL-6 and LIF mRNA stimulation involved MAP kinases, but not NF-kappaB. The findings support the view that resident cells may influence the pathogenesis of periodontal disease through osteotropic IL-6-type cytokine production mediated by activation of MAP kinases. ABBREVIATIONS: IL-1alpha (interleukin-1alpha); IL-1beta (interleukin-1beta); IL-6 (interleukin-6); IL-11 (interleukin-11); LIF (leukemia inhibitory factor); OSM (oncostatin M); alpha(1)-coll. I (alpha(1)-collagen I); ALP (alkaline phosphatase); BMP-2 (bone morphogenetic protein-2); OC (osteocalcin); BSP (bone sialoprotein); TNFR I (tumor necrosis factor receptor I); TNFR II (tumor necrosis factor receptor II); IL-1R1 (interleukin-1 receptor 1); GAPDH (glyceraldehyde-3-phosphate dehydrogenase); RPL13A (ribosomal protein L13A); mRNA (messenger ribonucleic acid); cDNA (complementary deoxyribonucleic acid); PCR (polymerase chain-reaction); BCA (bicinchoninic acid); ELISA (enzyme-linked immunosorbent assay); alpha-MEM (alpha modification of Minimum Essential Medium); and FCS (fetal calf serum).

Abundant secretion of bioactive interleukin-1beta by human macrophages induced by Actinobacillus actinomycetemcomitans leukotoxin.

Actinobacillus actinomycetemcomitans produces a leukotoxin that selectively kills human leukocytes. Recently, we reported that macrophages are highly sensitive to leukotoxin and that their lysis involves activation of caspase 1. In this study, we show that leukotoxin also induces the production and release of proinflammatory cytokines from human macrophages. The macrophages were challenged with leukotoxin or lipopolysaccharide (LPS) from A. actinomycetemcomitans or LPS from Escherichia coli, and the production and secretion of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) were determined at the mRNA and protein levels by reverse transcription-PCR and enzyme-linked immunosorbent assay, respectively. Leukotoxin (1 to 30 ng/ml) induced abundant production and secretion of IL-1beta, while the effects on IL-6 and TNF-alpha production were limited. Leukotoxin (1 ng/ml) caused a 10-times-higher release of IL-1beta than did LPS (100 ng/ml). The secreted IL-1beta was mainly the bioactive 17-kDa protein. At higher concentrations (>30 ng/ml), leukotoxin caused secretion of mainly inactive cytokine, the 31-kDa pro-IL-1beta. The presence of specific antibodies to IL-1beta or of a caspase 1 inhibitor blocked the secretion and production of the cytokine. Supernatants of leukotoxin-challenged macrophages stimulated bone resorption when tested in a mouse calvarial model. The activity could be blocked by an IL-1 receptor antagonist or specific antibodies to IL-1beta. We concluded that A. actinomycetemcomitans leukotoxin can trigger abundant production and secretion of bioactive IL-1beta by human macrophages, which is mediated by activation of caspase 1.

Caspase 1 involvement in human monocyte lysis induced by Actinobacillus actinomycetemcomitans leukotoxin.

Actinobacillus actinomycetemcomitans, an oral bacterium implicated in the etiology of periodontal diseases, produces a leukotoxin that selectively lyses primate neutrophils and monocytes, the major populations of defense cells in the periodontium. Though lysis requires expression of the receptor lymphocyte function-associated molecule 1 (LFA-1) on the cell surface, not all LFA-1-expressing leukocyte populations are equally susceptible to the toxin. In this study, the susceptibility of human leukocytes to leukotoxin-induced lysis is compared to their expression of LFA-1 and the activity of caspase 1. Cytolysis was determined by the activity of lactate dehydrogenase released from peripheral human leukocytes after 1-h exposure to leukotoxin. Monocytes were lysed at leukotoxin concentrations of > or = 5 ng/ml, while the corresponding values for neutrophils and lymphocytes were approximately 10 times greater. Similar LFA-1 expression was found in all susceptible cell populations irrespective of their degree of sensitivity to the toxin. Exposure of monocytes to leukotoxin increased their caspase 1 activity about fivefold within 10 to 20 min. Presence of the caspase 1 inhibitor Ac-YVAD-CMK significantly blocked the leukotoxin-induced lysis of monocytes only. At sublytic concentrations, leukotoxin induced no apoptotic activity in monocytes, as revealed by the lack of caspase 3 activation and DNA fragmentation. Monocytes are the most lysis-sensitive leukocytes for A. actinomycetemcomitans leukotoxin. Their lysis by this toxin depends on caspase 1 activation and proceeds through a process that differs from classical apoptosis.

Serum-mediated release of leukotoxin from the cell surface of the periodontal pathogen Actinobacillus actinomycetemcomitans.

The leukotoxin of the periodontopathogen Actinobacillus actinomycetemcomitans is an important virulence factor that lyses human neutrophils and monocytes and thus, it may enable the bacterium to evade the local host defense. The toxin also induces degranulation of neutrophils and cytokine release in monocytes. To trigger these biological activities, leukotoxin has to be released from the bacterium and diffuse into the periodontal tissues. To date, the conditions found to cause toxin release have been artificial and have included high ion concentration and alkaline conditions. To study the release of the toxin under conditions mimicking the natural environment of the periodontium the ability of human serum to enable leukotoxin release from the bacterial surface was examined. Suspensions of leukotoxic A. actinomycetemcomitans strains were incubated with various concentrations of human serum or serum albumin. The suspensions were centrifuged and the leukotoxin in the supernatants or the cell pellets was detected by gel electrophoresis and immunoblotting. Serum was found to cause the rapid release of leukotoxin from the bacteria in a concentration-dependent manner. Pure albumin exhibited a similar effect. The leukotoxin released was active against human neutrophils. Only a minor proportion of it was associated with membranous vesicles produced by the bacteria. The results indicate that serum, a fluid closely related to the exudate in inflamed periodontal pockets, releases leukotoxin from the cell surface of A. actinomycetemcomitans. The process may enable the diffusion of the toxin from the bacterial biofilm into the surrounding tissues, where it can exert its biological effect.

Release and activation of matrix metalloproteinase 8 from human neutrophils triggered by the leukotoxin of Actinobacillus actinomycetemcomitans.

Matrix metalloproteinase 8 (MMP 8) degrades type I collagen and may be involved in the pathogenesis of periodontitis. Latent MMP 8 is stored in neutrophil granules and can be activated when released extracellularly. The periodontitis-associated bacterium Actinobacillus actinomycetemcomitans produces an RTX-toxin, leukotoxin, that degranulates and lyses human neutrophils. This study deals with the ability of leukotoxic A. actinomycetemcomitans to trigger the release and activation of MMP 8. Whole bacteria of three A. actinomycetemcomitans strains or leukotoxin purified from the highly toxic strain HK 1519 were incubated with human neutrophils. The extracellularly released latent and active forms of MMP 8 were detected by an immunoblot technique using specific antibodies against the protease. The activity of MMP 8 was determined by a collagen degradation assay. All strains induced release and activation of MMP 8. The effect was more pronounced under aerobic than anaerobic conditions and correlated with the leukotoxicity of the strains. Pure leukotoxin also induced MMP 8 release and activation in a concentration-dependent manner. Under aerobic conditions, oxidising substances formed by the neutrophils contributed to the rapid activation of the latent enzyme. Upon anaerobic incubation, the activation was slow and mainly caused by other proteases released during neutrophil degranulation. The activation was totally abolished in the presence of serum, probably due to the serum-protease inhibitors. Compared to the calcium ionophore A 23187, a well-known stimulus of neutrophil degranulation, leukotoxin was a more powerful inducer of MMP 8 release, since it triggered the process at a 1000-fold lower concentration. The present findings reveal a specific mechanism that can be induced by A. actinomycetemcomitans leukotoxin and which may contribute to the degradation of periodontal tissues under certain conditions.

Lack of lipoprotein-dependent effects on the cytotoxic interactions of Actinobacillus actinomycetemcomitans leukotoxin with human neutrophils.

A high odds ratio has been reported for hyperlipidemia and periodontal diseases in humans, and the severity of periodontitis seems to correlate with the hyperlipidemic status of the patients. Early studies indicated that the lipoprotein-containing fraction of the serum enhances the leukotoxic activity of the periodontopathogen Actinobacillus actinomycetemcomitans against human polymorphonuclear leukocytes (PMNL). The protease inhibitors of normal serum account for this enhancement, while delipidated serum has no effect on the leukotoxin-dependent PMNL cytolysis. No information exists for the effect of serum lipoproteins or hyperlipidemic serum. The aim of this study was to evaluate the role of serum lipoproteins in the interaction of the leukotoxin of A. actinomycetemcomitans with human PMNL. Purified leukotoxin was mixed with human PMNL prepared from venous blood of healthy subjects and various varying amounts of hyperlipidemic or delipidated serum, or purified serum lipoproteins. The cytolytic activity of leukotoxin was determined by activity of the cytosol enzyme lactate dehydrogenase released from injured PMNL. The degranulating activity of the toxin was measured through the release of the granule components elastase and lactoferrin. Normal human serum without leukotoxin-neutralizing antibodies caused a 4-fold enhancement of the leukotoxic activity when present at concentrations of 5-10% in the reaction mixture. Serum lipoproteins had no effect when added at concentrations that occur normally in serum. At high concentrations, purified low density and very low-density lipoproteins increased the leukotoxicity of the mixture. Nevertheless, hyperlipidemic serum prepared from a normal serum by the addition of autologous lipoproteins had no influence on the leukotoxin-caused cytolysis compared to the normal serum. Pre-incubation of PMNL for 1 h in hyperlipidemic or delipidated serum had no effect on the leukotoxin-induced degranulation of PMNL. The results indicate that the cytotoxic interactions of A. actinomycetemcomitans leukotoxin against human PMNL are not influenced by the presence of serum lipoproteins.

Protease inhibitors, the responsible components for the serum-dependent enhancement of Actinobacillus actinomycetemcomitans leukotoxicity.

Serum enhances the leukotoxic activity of Actinobacillus actinomycetemcomitans against human polymorphonuclear leukocytes (PMNL) by a mechanism that still is unknown. Early attempts to identify the serum components responsible for this enhancement gave no conclusive results, but indicated that the lipoprotein-containing fraction of the serum was involved in the interaction. This study aimed to clarify the role of serum lipoproteins in the leukotoxin interaction, and to identify other serum components involved. The main hypothesis examined was that the leukotoxicity enhancement might depend on serum protease inhibitors that block proteolytic cleavage of leukotoxin by enzymes released from the leukocytes. PMNL were isolated from human peripheral blood and incubated with purified leukotoxin in the presence of serum or purified serum components or lipoprotein-deficient serum. Leukotoxin was also incubated with purified elastase and cathepsin G or with enzyme mixtures from degranulated PMNL. The leukotoxic activity in these mixtures was determined as the extracellular release of lactate dehydrogenase from PMNL. Cleavage of the toxin was showed by gel electrophoresis and Western blot. Morphological changes in PMNL from the above mixtures were examined by electron microscopy. Enzymes from degranulated PMNL cleaved leukotoxin to non-cytotoxic fragments. Elastase and cathepsin G were mainly responsible for the cleavage. Inhibition of leukotoxin degradation was found in the presence of whole serum or of the serum protease inhibitors alpha2-macroglobulin and alpha1-proteinase inhibitor. Under these conditions enhanced PMNL lysis was also observed. A similar enhancement of PMNL lysis was found when PMNL degranulation was blocked by EDTA. On the other hand, lipoprotein-deficient serum had no influence on the leukotoxic activity. The results indicate that the increased leukotoxicity of A. actinomycetemcomitans observed in the presence of human serum is caused by the serum protease inhibitors that counteract proteolytic degradation of leukotoxin. The degradation is caused by enzymes from degranulated PMNL triggered by leukotoxin.

The bone resorbing activity released by gingival fibroblasts isolated from patients with periodontitis is independent of interleukin-1.

Supernatants from gingival fibroblast cultures obtained from 14 patients with periodontal disease contained factor(s) capable of stimulating bone resorption in vitro, as assessed by the release of 45Ca from neonatal mouse calvariae. The possibility that the factor(s) was interleukin-1 alpha (IL-1 alpha), IL-1 beta or prostaglandin E2 (PGE2) was next investigated. The human fibroblast conditioned media (HFCM) stimulated PGE2 biosynthesis in bone. The stimulatory effect by HFCM on 45Ca release, however, was not affected by blocking prostaglandin biosynthesis with indomethacin. In contrast, 45Ca release induced by IL-1 alpha, IL-1 beta, thrombin and bradykinin was significantly reduced by indomethacin, whereas the effects of PTH and PTHrP were unaffected by indomethacin. The concentration of PGE2 in HFCM was too low to be solely responsible for the 45Ca release response. In addition, the amount of bone resorbing activity produced by the gingival fibroblasts was unaffected by cyclo-oxygenase inhibitors. Similar to IL-1 alpha and IL-1 beta, the stimulatory effect of HFCM was inhibited by gamma-interferon. HFCM did not stimulate cyclic AMP formation in the mouse calvarial bones. Antisera which specifically blocked human IL-1 alpha or IL-1 beta induced 45Ca release, and the specific IL-1 receptor antagonistic protein, did not inhibit the stimulatory effect of HFCM. These data show that gingival fibroblasts secrete bone resorbing factor(s) which is not due to IL-1 and which stimulates bone resorption by a prostaglandin- and cyclic AMP-independent mechanism.

Polymorphonuclear leukocyte degranulation induced by leukotoxin from Actinobacillus actinomycetemcomitans.

The ability of leukotoxin from Actinobacillus actinomycetemcomitans to induce release of lysosomal constituents was studied with human polymorphonuclear leukocytes (PMNL). Leukotoxin purified from A. actinomycetemcomitans or bacterial cells of a leukotoxic strain were mixed with human PMNL and the suspension was incubated under anaerobic conditions. Samples were taken at certain time intervals to examine the cell morphology of PMNL by electron microscopy and the extracellular concentrations of the granule components lactoferrin and elastase by enzyme-linked immunosorbent assay (ELISA). Electron microscopy revealed that within 10 min of exposure to leukotoxin, the number of intracellular granules was markedly reduced and the remaining granules were translocated to the periphery in PMNL. At the same time, the extracellular concentrations of lactoferrin and elastase were elevated, while that of the cytosolic enzyme lactate dehydrogenase, an indicator of cell lysis, remained low. The lysosome molecules CD63 and CD66b were also exposed on the PMNL surface, indicating fusion of lysosomes with the plasma membrane. These effects were completely abolished by the addition of anti-leukotoxin serum. Pre-incubation of PMNL with monoclonal antibodies to CD11a and CD18 that recognize alpha- and beta-chains of the LFA-1 integrin, a leukotoxin receptor on PMNL, inhibited the cytolysis, but not the release of granule components. The present results demonstrate the ability of A. actinomycetemcomitans leukotoxin to trigger a rapid release of lysosomal compounds in human PMNL. The release is due to an active process stimulated by the interaction of PMNL with the toxin or toxin-carrying bacteria.

Anaerobic neutrophil-dependent killing of Actinobacillus actinomycetemcomitans in relation to the bacterial leukotoxicity.

The effect of leukotoxin on the interaction of Actinobacillus actinomycetemcomitans with human polymorphonuclear leukocytes (PMNL) was studied under anaerobic conditions with strains able to produce high or low amounts of leukotoxin. PMNL morphology, phagocytosis and killing were examined by transmission electron microscopy and bioassays, respectively. At ratios of > or =25 bacteria/PMNL, a highly toxic A. actinomycetemcomitans strain completely destroyed the PMNL within 7 min, resulting in bacterial survival. Lowering the bacteria/PMNL-ratio enabled phagocytosis and killing of this highly toxic strain. A. actinomycetemcomitans strains with low leukotoxicity were effectively killed by PMNL under any condition. Presence of specific antibodies against A. actinomycetemcomitans or of anti-leukotoxin serum protected PMNL from being injured and allowed phagocytosis to occur. Pre-incubation of the leukotoxic strain with Porphyromonas gingivalis, a bacterium that destroys leukotoxin, abolished lysis of PMNL and inhibited phagocytosis of A. actinomycetemcomitans. The results show that leukotoxin may protect A. actinomycetemcomitans against phagocytosis by human PMNL. The protection occurs at a population level and in relation to the bacterial load. The size of the bacterial population required to counteract phagocytosis is dependent on the leukotoxin production of the strain.

Characterization of bone resorbing activity in gingival crevicular fluid from patients with periodontitis.

BACKGROUND: In attempts to elucidate factors stimulating bone resorption in patients with different inflammatory diseases in the vicinity of the skeleton, e.g., peridontal disease and rheumatoid arthritis, we are investigating the presence of bone-resorbing activity in a variety of inflammatory exudates. The aim of the present study was to characterize the bone-resorbing activity present in patients with periodontitis. METHODS: Bone-resorbing activity was assessed in gingival crevicular fluids (GCFs) collected from patients with periodontitis and from patients with no signs of gingivitis. Bone-resorbing activity was evaluated by analyzing the capacity of GCFs to stimulate the release of minerals and the breakdown of bone matrix proteins in cultured neonatal mouse calvariae. The concentrations of IL-1alpha, IL-1beta and PGE2 were determined with ELISA and RIA techniques, respectively. RESULTS: GCF eluates from 24 different healthy sites caused a 1.23+/-0.05 fold stimulation of 45Ca release, whereas GCF eluates from 45 different diseased (periodontitis) sites caused a 2.46+/-0.10 fold stimulation. The effect on 45Ca release was time- and concentration-dependent, inhibited by 3 different osteoclast inhibitors and associated with enhanced release of 3H from [3H]-proline-labelled bones. The activity in GCF causing enhanced 45Ca release was unaffected, or in some samples partially reduced, by ultrafiltration using a filter with a molecular weight cut-off of 3000 Daltons. The bone-resorbing activity was temperature sensitive (+90degrees C, 10 min). The concentrations of prostaglandin E2 (PGE2) in the diluted GCF eluates, used in the bone resorption bioassay, were too low to be responsible for the release of 45Ca. Antisera specifically neutralizing human IL-1a inhibited the stimulatory effect of GCF pooled from several diseased sites. The specific, recombinant human IL-1 receptor antagonist completely inhibited the effect of pooled GCFs. GCF eluates from diseased sites contained human IL-1alpha and IL-1beta at concentrations of 1838+/-294 pg/ml and 512+/-91 pg/ml, respectively. CONCLUSIONS: These data show that GCF contains activity(ies) stimulating osteoclastic bone resorption in vitro. The factor primarily responsible for this activity seems to be IL-1alpha, but IL-1alpha is not the sole activator of bone resorption in GCF.

Inhibition of Actinobacillus actinomycetemcomitans leukotoxicity by bacteria from the subgingival flora.

Actinobacillus actinomycetemcomitans produces a pore-forming leukotoxin that lyses human polymorphonuclear leukocytes and monocytes. Certain proteolytic bacteria may coexist with A. actinomycetemcomitans in periodontal pockets. We aimed therefore to examine whether oral bacteria can modify the leukotoxicity of A. actinomycetemcomitans. A total of 55 strains representing 45 bacterial species of the subgingival flora were tested. Each strain was incubated with the highly toxic strain of A. actinomycetemcomitans HK 1519 and the leukotoxic activity of the suspension against human polymorphonuclear leukocytes was determined from the activity of the lactate dehydrogenase released upon lysis of the leukocytes. Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Prevotella melaninogenica and Prevotella loeschii inhibited the leukotoxicity of A. actinomycetemcomitans cells as well as the activity of leukotoxin purified from the same strain. The bacterial strains without the ability to block leukotoxic activity also failed to destroy pure leukotoxin even after 5 h of incubation. The proteolytic degradation of leukotoxin by P. gingivalis was mainly dependent on the activity of the enzymes R- and K-gingipains. P. intermedia and P. nigrescens also degraded the leukotoxin by enzymes. The results imply a role of the periodontal microflora in modifying the virulence of A. actinomycetemcomitans by destroying its leukotoxin.

[The history of toothcleaning]. / Tandrengöringens historia.

A short historical review of toothcleaning with primitive and manufactured toothbrushes, toothpicks and thread of waxed silk. Mouthrinses, toothpowders or soaps and toothpastes and chewing gums composed of ingredients from nature are presented. Antibacterial and pharmacological effects are discussed.

The effect of zinc on bacterial phagocytosis, killing and cytoprotection in human polymorphonuclear leucocytes.

An in vitro study examining the effects of zinc treatment on human PMN cell phagocytosis and killing of Staphylococcus aureus and Staphylococcus epidermidis and the cytoprotection of zinc against staphylococcal toxins. Phagocytosis was studied by transmission electron microscopy using different microbiological techniques, one of which was designed to follow the kinetics of bacterial killing. No effect was found on phagocytosis and bacterial killing. The cytotoxic effects of a crude toxin and an alpha-toxin extracted from Staphylococcus aureus preparations were studied on human PMN cells using the standard 51Cr release assay. Both toxins induced a dose-dependent leakage of 51Cr, indicating cell membrane damage. These results were confirmed by electron microscopy during the phagocytosis of S. aureus, where severe PMN cellular degeneration was observed. The addition of zinc to PMN cells strongly inhibited the release of 51Cr. In conclusion, our results show that zinc in higher than physiological concentrations does not inhibit PMN cell functions such as phagocytosis and intracellular killing of S. aureus and S. epidermidis. The addition of zinc may be beneficial in certain clinical situations, such as wound healing, zinc deficiency and infections involving toxin-producing bacteria, e.g. S. aureus.

Periodontal and systemic findings in children with marginal bone loss in the primary dentition.

In a previous population-based study of 3896 7-9-year-old children living in Sweden, it was found that 32 children (0.8%) exhibited radiographic, periodontal bone loss at > or = 2 proximal surfaces of their deciduous teeth. In the present study, 26 of the 32 children were subjected to additional oral and systemic health examination. 20 other children without any radiographic evidence of bone loss in their primary dentition served as referents. None of the cases or the referents were detected to have any systemic disease. The frequency of bleeding and suppuration on probing, radiographic proximal calculus and probing attachment loss was higher among the cases than the referents. Actinobacillus actinomycetemcomitants was found subgingivally in 14 of the cases but in none of the referents. 11 of 22 cases analysed for presence of serum antibodies against A. actinomycetemcomitans leukotoxin were sero-positive compared to none of 7 referents available for analysis. Evaluation of the data from each child revealed wide variations in clinical parameters among the children in the case group. In this group, there were children with deep probing depths, probing attachment loss, suppuration on probing, proximal calculus and presence of subgingival A. actinomycetemcomitans, indicating current periodontitis. However, in the case group there were also children without positive signs of inflammatory disease, similar to the children in the reference group. In fact, the findings suggest that less than half of the number of individuals with > or = 2 proximal sites with bone loss had current periodontitis.

Neutralizing effect of zinc oxide on dehydroabietic acid-induced toxicity on human polymorphonuclear leukocytes.

The cytotoxic effect of dehydroabietic acid (DHAA), a resin acid found in rosin, was studied on human polymorphonuclear leukocytes (PMN) using leakage of 51Cr from prelabeled cells, supravital staining, and transmission electron microscopy. DHAA caused a strong dose-related release of 51Cr, a high uptake of trypan blue, and total cell necrosis as seen in transmission electron microscopy. Albumin slightly reduced the toxic effects, whereas the addition of zinc in various forms strongly inhibited these toxic effects of DHAA in the concentration range 10-500 micrograms/mL. In the presence of albumin, zinc oxide as a suspension inhibited the damage of the cell membranes more than a filtrate of zinc oxide, indicating a subsequent slow release of zinc from the zinc oxide.

Neutralizing effect of zinc oxide on dehydroabietic acid-induced toxicity on human polymorphonuclear leukocytes.

The cytotoxic effect of dehydroabietic acid (DHAA), a resin acid found in rosin, was studied on human polymorphonuclear leukocytes using leakage of 51Cr from prelabeled cells, supravital staining, and transmission electron microscopy. DHAA caused a strong dose-related release of 51Cr, a high uptake of trypan blue, and total cell necrosis, as seen in transmission electron microscopy. Albumin slightly reduced the toxic effects, whereas the addition of zinc in various forms strongly inhibited these toxic effects of DHAA in the concentration range of 10-500 micrograms/mL. In the presence of albumin, zinc oxide as a suspension inhibited the damage of the cell membranes more than a filtrate of zinc oxide, indicating a subsequent slow release of zinc from the zinc oxide.

Phagocytosis and virulence of different strains of Porphyromonas gingivalis.

In this study 17 strains of Porphyromonas gingivalis, both reference and clinical isolates, were investigated for their in vitro interaction with human polymorphonuclear leukocytes, hydrophobicity, density, and virulence in a mouse model. The results of the phagocytosis, hydrophobicity, and density experiments showed that P. gingivalis strains could be divided into two distinct groups. One group of strains were readily attached and phagocytosed when exposed to the leukocytes. These bacteria were hydrophobic and had a higher buoyant density than the other group, which were poorly phagocytosed, had a low buoyant density, and were hydrophilic. This latter group also exhibited an extracellular meshwork resembling a glycocalyx when examined by electron microscopy. There were also significant differences between strains in the mouse pathogenicity model. Two strains caused an invasive, spreading infection compared with the other 15 strains which produced small, localized abscesses. There was no clear correlation between the results of the phagocytosis assay and the virulence of the bacteria when injected subcutaneously in mice. Resistance to phagocytosis may be important for survival of these bacteria, but it does not in itself imply the ability to cause damage to the host.

Stimulation of bone resorption and cell proliferation in vitro by human gingival fibroblasts from patients with periodontal disease.

In the present communication we report that fibroblasts, isolated from human gingiva obtained from 13 different patients, secreted soluble product(s) which can promote bone resorption in vitro. Fibroblasts were isolated from explants of human gingiva, subcultured, grown to confluent monolayers, subsequently cultured in growth arrest media for 0-72 h and conditioned media harvested. Bone resorption was assessed in cultured mouse calvarial bone by quantifying the mobilization of minerals and the release of lysosomal enzymes. Human fibroblast-conditioned media (HFCM) dose-dependently stimulated the release of 45Ca from prelabelled bones and the mobilization of stable calcium and inorganic phosphate from unlabelled bones. In addition, HFCM increased the release of beta-glucuronidase and beta-N-acetylglucosaminidase from the calvaria. No effect of HFCM on the release of 45Ca from dead bones could be seen. HFCM caused a dose-dependent increased degradation of bone matrix proteins, as assessed by the release of 3H from [3H]proline-labelled calvaria. The stimulation of 45Ca release could already be seen after 3-12 h of treatment. Treatment of the bones with HFCM for 12 h was sufficient to obtain a prolonged stimulation of 45Ca release. Bones cultured in the presence of HFCM showed an increased number of osteoclasts. Calcitonin, but not indomethacin, inhibited 45Ca release stimulated by HFCM. Ultrafiltration of HFCM did not cause any loss of the 45Ca release response. The amount of bone-resorbing activity produced by the gingival cells was proportional to the number of cells. In addition, HFCM stimulated the proliferation of human fibroblasts and osteoblast-enriched mouse calvarial bone cells. It is concluded that human gingival fibroblasts secrete one or several factors that can stimulate osteoclastic bone resorption in vitro by a prostaglandin-independent pathway.