Focus on primary care: periodontal disease: implications for women's health.

A definite relationship is emerging between periodontal infections and systemic conditions. The objective of this review is to address this relationship as it pertains to cardiovascular disease and diabetes mellitus. Furthermore, because recent reports link the presence of periodontal disease to preterm delivery, the possible relationship between the development and progression of periodontal disease and certain hormonal states in women such as puberty, oral contraceptive use, menopause, and pregnancy will also be discussed. Although the current literature suggests a strong association between periodontal disease and a number of the discussed systemic conditions, causality can only be established with prospective studies. Intervention studies are needed to address how treatment effects the incidence and/or severity of periodontal disease-related systemic illness.

Components of a randomized clinical trial.

The purpose of this paper is to discuss a number of components associated with the design, development and conduct of a randomized clinical trial (RCT). In a RCT, a patient is entered into a protocol in which therapy is randomly assigned. Thus, neither the patient nor the care provider have active roles in deciding treatment. The elements of informed consent were developed to ensure that the patient's interests are protected. The informed consent document serves as the cornerstone of a clinical study by introducing patients to the research protocol, informing them of their role as participants in the study, and educating them of their rights. Questions regarding ethics arise during all phases of a study. A study designed with insufficient sample size will provide inconclusive data, thus making participation in the study of limited to no value. Conversely, having patients enrolled in a RCT after data has proved superiority of an arm of the study postpones beneficial therapy for patients and society. Other ethical conflicts arise when subjects are recruited for clinical trials. Special care must be taken to avoid coercion in protected populations such as students, employees, the mentally challenged, and the indigent. Careful planning with adequate attention to consent from development can minimize problems and optimize patient care and research integrity.

Randomized clinical trials in periodontology: ethical considerations.

Ethical justification for starting a clinical trial requires at the outset an accurate statement of "no difference" regarding the two or more agents to be compared. This may be expressed as "theoretical equipoise" (no data to support the superiority of one of the agents) or, preferably, as "clinical equipoise" (there are insufficient data to resolve controversy among experts as to which is superior). This presents a problem when a placebo control is proposed, particularly when the outcome measure entails irreversible loss of function; preliminary data often suggest the superiority of the "active agent." Informed consent should ordinarily include the fact that treatment assignments will be accomplished by a process of randomization and, in the case of double-blind designs, that neither the subject nor the investigator will know the subject's treatment assignment until the end of the trial. In general, clinical trials should be monitored by data and safety monitoring boards that have access to unblinded data; they should be guided by stopping rules that should be agreed upon by all concerned before the trial is begun. Women and minorities must be included unless there is strong justification for their exclusion. Care must be taken to balance the competing objectives of validity (the results will be correct), generalizability (the results will be broadly applicable), and efficiency (the study will not be unduly expensive).

The antiviral spectrum of Listerine antiseptic.

The mechanism of activity and the antiviral spectrum of Listerine antiseptic have not been examined thoroughly. We therefore tested its effect on laboratory strains of herpes simplex type 1 and type 2 (enveloped DNA viruses), influenza A virus (enveloped RNA virus), rotavirus (nonenveloped RNA virus), and adenovirus type 5 (nonenveloped DNA virus). Each virus was mixed with an equal volume of Listerine for 30 seconds to 5 minutes, and the residual infectivity of the virus was assessed. An antiviral effect was defined as greater than 95% reduction of infectivity. Exposure to Listerine for 30 seconds had an antiviral effect against herpes simplex type-1 and type-2 (96.3% and 100% reduction in infectious virus, respectively) and influenza A (100% reduction). In contrast, rotavirus-induced plaque formation was reduced by 12.2% after 30 seconds of exposure to Listerine, whereas 5 minutes of exposure to Listerine resulted in a 21.5% increase in plaque formation. Exposure of adenovirus to Listerine had a minimal effect on the cytopathocity of the virus, with a 33.4% reduction in virus levels after 5 minutes. The antiviral activity of Listerine is thus not related to the viral genome but is probably directed to the viral envelope.

Contaminated implant surfaces: an in vitro comparison of implant surface coating and treatment modalities for decontamination.

The relationship between implant surfaces and decontamination treatments was studied in vitro to determine which implant surfaces were most effectively decontaminated, and which treatment was most effective for treating a particular implant surface. The implants used in the study were press fit cylindrical titanium units with machined, plasma sprayed, and hydroxyapatite-coated surfaces. Radioactive endotoxin (125I-LPS) was prepared from Porphyromonas gingivalis (ATCC 33277). Implants were coated with 125I-LPS and treated by burnishing with a cotton pellet soaked in water, citric acid solution (CA), or 0.12% chlorhexidine (CHX); or treated with an air-powder abrasive (AIR). Radioactivity was determined after each of two treatment cycles. The results for each implant surface were analyzed using ANOVA to determine differences between treatments. The remaining 125I-LPS after two treatment cycles were: for machined implants AIR < CA, with AIR = water = CHX and water = CHX = CA; for plasma sprayed implants AIR < water = CHX = CA; for hydroxyapatite implants AIR = CA < water < CHX. In evaluating treatment modalities, it was found that machined implants were decontaminated more effectively than the other surfaces by all treatments; the exception was citric acid treatment which was equally effective on either machined or hydroxyapatite surfaces. These results indicate that machined implants (without surface coating) are most readily decontaminated by a variety of methods; this characteristic should be considered, since long-term success of implants may involve treating periimplantitis. Further, the results indicate that air abrasives are effective for decontaminating implant surface, with the exception that hydroxyapatite coated surfaces can be treated equally with air abrasives or citric acid.

Differential effect of TGF-beta 1 and PDGF on proliferation of periodontal ligament cells and gingival fibroblasts.

Regeneration of periodontal tissues requires orchestration of several cell types. Two cell types, gingival fibroblastic cells (gingival fibroblasts) and cells from the periodontal ligament (PDL cells), were studied to compare the effects of supplemental addition of TGF-beta 1 and PDGF on proliferation. Cells obtained from healthy donors were cultured in 10% FBS supplemented with either 10 ng/ml TGF-beta 1, 20 ng/ml PDGF, or both. Thymidine incorporation was measured after 24, 48, or 72 hours. Data from PDL (analyzed by ANOVA) showed the following relations: at 24 hours TGF beta 1/PDGF = PDGF > TGF-beta 1 = control; at 48 hours TGF beta 1/PDGF > TGF-beta 1 > PDGF > control; at 72 hours TGF beta 1/PDGF > TGF-beta 1 > PDGF = control. Gingival fibroblast cultures showed the following relations: at 24 and 48 hours TGF beta 1/PDGF = PDGF > TGF-beta 1 = control; at 72 hours, TGF beta 1/PDGF = PDGF > control with TGF beta 1 not different from control or factor combinations. Both TGF-beta 1 and TGF-beta 1/PDGF showed a significantly greater increase in proliferation of PDL cells than in gingival fibroblasts at 48 and 72 hours (Student t test P < 0.05). In contrast, PDGF stimulated proliferation of gingival fibroblasts was significantly greater than PDL cells at 72 hours (P < 0.05). Thus, supplementation of complete cultures (containing 10% FBS) with TGF-beta 1 alone or combined with PDGF stimulates proliferation of PDL cells to a significantly greater extent than proliferation of gingival fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between functional Na+ pumps and mitogenesis in cultured coronary artery smooth muscle cells.

An increase in functional sarcolemmal Na(+)-K(+)-ATPase (Na+ pump) precedes proliferation in vascular smooth muscle cells (VSMCs) seeded in 10% fetal bovine serum (FBS), but its role in mitogenesis is unresolved. Enzymatically dispersed canine coronary artery VSMCs were seeded in FBS and studied through confluence. Before a shift in cell cycle (G1-->S, G2 + M) and appearance of the nonmuscle isoform of myosin (MHCnm), intracellular Na+ content (Na+i) and cell volume (CV) increased (day 0 through day 3). Na+ pump number ([3H]-ouabain binding) increased at day 4 followed by a decrease in Na+i and CV. When Na+ pumps were inhibited by the addition of ouabain to FBS, VSMCs were arrested in G1, and MHCnm was not upregulated. Na+i increased similarly to that in FBS but failed to correct to day 0 levels. Withdrawal of ouabain at day 4 in culture led to an increase in Na+ pump number, a decrease in Na+i, entry of cells into S and G2 + M, and upregulation of MHCnm. These data suggest that Na+i, phenotypic modulation, and entry of cells into the cell cycle are temporally related, with Na+ pump-mediated correction of increased Na+i as a key event in the VSMC mitogenic process.

Neutrophil modulation by Actinobacillus actinomycetemcomitans. II. Phagocytosis and development of respiratory burst.

Compromised neutrophil function has been found in a number of patients with localized juvenile periodontitis (LJP), although the pathogenic mechanism is unknown. Since infection with Actinobacillus actinomycetemcomitans is frequently found in patients with LJP, we have evaluated in vitro the effect of a bacterial extract of A. actinomycetemcomitans on the development of the respiratory burst by neutrophils. Pre-incubation of neutrophils with bacterial extract increased H2O2 induced by FMLP and zymosan in a dose-dependent fashion. Substitution of FMLP for bacterial extract produced similar results. Moreover, FMLP and bacterial extract had an additive effect on superoxide production following phagocytosis of zymosan. In contrast, bacterial extract significantly decreased PMA-stimulated H2O2, but pre-incubation with FMLP instead of bacterial extract failed to decrease PMA-stimulated H2O2. Bacterial extract did not change the percentage of cells activated by FMLP, opsonized zymosan, or PMA. Heat-treated bacterial extract induced effects similar to non-treated extract. Bacterial extract treated with proteinase K or phenol extraction increased FMLP or zymosan stimulated H2O2 equivalent to non-treated bacterial extract. In contrast, proteinase K or phenol extraction abolished the inhibitory effect of bacterial extract on PMA-stimulated H2O2 production. The bacterial extract component(s) that inhibits PMA-stimulated H2O2 is therefore a protein(s), resistant to 56 degrees C, and is not endotoxin. The partially activated state of PMNs exposed to A. actinomycetemcomitans extract, combined with their reduced ability to respond to a protein kinase C-dependent stimulus, may partially explain the abnormalities noted in LJP patients.

Neutrophil modulation by Actinobacillus actinomycetemcomitans. I. Chemotaxis, surface receptor expression and F-actin polymerization.

Localized juvenile periodontitis is an early onset periodontitis, usually localized to molars and incisors. Patients usually present with decreased chemotaxis of systemic neutrophils (PMNs) and infection with Actinobacillus actinomycetemcomitans. The pathogenic mechanisms involved have not been clarified. The purpose of this study was to determine if an extract of A. actinomycetemcomitans could induce changes in PMN chemotaxis similar to those reported in LJP patients. It was demonstrated that the bacterial extract was chemotactic for neutrophils. When neutrophils were pre-incubated with the bacterial extract, chemotaxis toward zymosan-activated serum, FMLP and the bacterial extract was inhibited in two different chemotaxis assays (Boyden chamber and under-agarose). Bacterial extract had no effect on random migration in either assay. Pre-incubation with the extract induced increased expression of CD11b/CD18 (Mac-1), Gp110, and FMLP receptors and increased F-actin polymerization following FMLP or PMA stimulation compared to cells not treated with the extract. Treatment of the bacterial extract with proteinase K or phenol extraction reversed the PMN chemotaxis inhibition activity, but increased significantly the random migration of PMNs. Heating the bacterial extract to 56 degrees C had no effect on its activity. The component(s) in the bacterial extract that inhibits chemotaxis is therefore a protein(s), not sensitive to 56 degrees C, and is not endotoxin. This study suggests that A. actinomycetemcomitans may contribute to the pathogenesis of localized juvenile periodontitis by inhibiting chemotaxis. Interference with chemotaxis by A. actinomycetemcomitans, however, occurs through a mechanism other than inhibition of actin assembly, reduction of CD11b/CD18 or Gp110 expression, or blockage/downregulation of FMLP receptors.

Induction of acetyl-LDL receptor activity by phorbol ester in human monocyte cell line THP-1.

Human monocytic cell line THP-1 incubated with as little as 10 ng/ml of phorbol myristate acetate bound and metabolized 1-2 micrograms of Ac-LDL over a 5-h period. In the absence of phorbol treatment, no specific metabolism of Ac-LDL occurred. Optimal levels of receptor were reached after 72 h of exposure. Induction of receptor was dependent on protein and RNA synthesis and was partially reversed upon removal of the phorbol. Induction of receptor required activation of the protein kinase C pathway. Metabolism of Ac-LDL by THP-1 cells at 37 degrees C was saturated at 25 micrograms/ml. Binding at 4 degrees C was saturable with an average Kd of 8.0 x 10(-9) M. Cell population studies by fluorescent activated cell sorting indicated that approximately 87% of the THP-1 population was expressing scavenger receptor activity 96 h after phorbol treatment as compared to 99% for murine macrophage cell line P388D1. Uptake of Ac-LDL by THP-1 resulted in an 11-fold increase in the rate of cholesterol esterification which was saturable at 50 micrograms/ml. Incubation of cells for 48 h with 50 micrograms/ml of Ac-LDL resulted in a 60% increase in free cholesterol and a 10-fold increase in the cholesteryl ester content of the cells. Lipid accumulation in THP-1 cells after Ac-LDL uptake was readily visible by Oil Red-O staining. Solubilization of THP-1 cells, before and after phorbol treatment, followed by ligand blotting with Ac-LDL detected the presence of a 250-kDa protein only in cells treated with phorbol. The protein comigrated with the scavenger receptor derived from mouse macrophage cell line P388D1.

A new method for isolation of salivary neutrophils and determination of their functional activity.

The purposes of this study were to develop a new method for isolating salivary neutrophils (SPMNs), and to determine their functional activity. Studies of neutrophils in the oral cavity have been largely limited to crevicular PMNs, because of difficulties in obtaining viable SPMNs free of epithelial cells. A method to obtain SPMNs is presented. Donors performed rapid sequential rinsings by placing in their mouths 15 mL of Hanks' balanced salt solution [free of calcium or magnesium ions (HBSS-CMF)], which contained 0.1% gelatin, then swishing the solution for 30 s, and expectorating into a polypropylene receptacle containing 400 mL 4 degrees C HBSS-CMF. This sequence was repeated for 20 min. The collected solution was stirred for 10 min, the cells were washed, and the re-suspended pellet was passed sequentially through a 20-microns and a 10-microns nylon mesh. The cells consisted of 97.7 +/- 1.7% SPMNs, only 2.3% epithelial cells, and were almost free of oral debris. The SPMNs were studied for CD11b expression, H2O2 production, and F-actin polymerization. SPMNs had significantly higher resting values for CD11b, H2O2 production, and F-actin polymerization compared with blood neutrophils (BPMNs). SPMNs responded to stimulation by chemotactic peptide or phorbol ester in a dose-dependent fashion, with levels of CD11b, H2O2, and F-actin comparable with BPMNs at optimal stimulant concentrations. The elevated resting levels of CD11b, H2O2, and F-actin for SPMNs were probably caused by exposure to gingival and oral bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular Na+ regulation of Na+ pump sites in cultured vascular smooth muscle cells.

Enzymatically dispersed cells from canine saphenous vein and femoral artery were grown in fetal calf serum and studied at day 0 (freshly dispersed) through confluence in primary culture. Intracellular Na levels (Nai), but not intracellular K (Ki), were increased after 24 h in culture and then decreased to a steady state by 4 days. Na+ pump site number [( 3H] ouabain binding) increased through day 3 and remained elevated. Nai was still elevated at 2 days when the Na+ pump site number began to increase. Total pump turnover (maximum ouabain-inhibited 86Rb uptake) reflected the increase in Na+ pump site number. These key events precede the observed increases in both protein production and cellular proliferation. If the same cells are maintained in defined medium, without fetal calf serum, Nai, Ki, and the number of [3H]ouabain binding sites do not change with time. These data are consistent with the suggestion that the initial mitogenic response of vascular smooth muscle cells to fetal calf serum involves an increased Na+ influx, and a Nai accumulation, caused by low Na+ pump density. The synthesis of new pump sites effects a decrease in the accumulated Nai, which may be related to cell proliferation.