Randomized Trial to Test a Chemo-Mechanical Antiplaque Regimen as Adjunct to Periodontal Therapy.

BACKGROUND: The control of dental biofilm regrowth after nonsurgical periodontal therapy is associated with better clinical outcomes. However, many patients have difficulty achieving optimal plaque control. Subjects with diabetes, in which immune and wound-healing responses are typically impaired, may benefit from intensive antiplaque control regimens after scaling and root planing (SRP). OBJECTIVES: This study aimed to evaluate the effects of an intensive, at-home, chemical, and mechanical antiplaque regimen as an adjunct to SRP for the treatment of moderate to severe periodontitis. A secondary objective was to compare responses in subjects with type 2 diabetes and nondiabetics. METHODS: This was a 6-mo, single-center, parallel-group, randomized trial. The test group received SRP and oral hygiene instructions, and subjects were instructed to use a 0.12% chlorhexidine gluconate mouthrinse twice a day for 3 mo and utilize rubber interproximal bristle cleaners twice a day for 6 mo. The control group received SRP and oral hygiene instructions. The main outcome was change in mean probing depth (PD) from baseline to 6 mo. Secondary outcomes included change in sites with deep PDs, mean clinical attachment level, bleeding on probing, plaque index, hemoglobin A1C, fasting blood glucose, C-reactive protein, and taste assessment. This study was registered at ClinicalTrials.gov as NCT04830969. RESULTS: In total, 114 subjects were randomized to either treatment. Eighty-six subjects completed the trial with no missing visits. Neither an intention-to-treat nor a per-protocol analysis showed statistically significant differences between treatment groups in mean PD at 6 mo. In a subgroup analysis, subjects with diabetes in the test group showed a statistically significant greater reduction in mean PD at 6 mo when compared to subjects with diabetes receiving the control treatment (Δ = 0.15, P = 0.04), while there were no differences within nondiabetics (Δ = 0.02, P = 0.75). CONCLUSION: Outcomes in subjects with diabetes may be improved by chemo-mechanical antiplaque measures after nonsurgical periodontal therapy. KNOWLEDGE TRANSFER STATEMENT: This study suggests diabetic subjects may benefit from an intensive, at-home, chemical, and mechanical antiplaque regimen to improve nonsurgical periodontal therapy outcomes.

Monocyte activation by Porphyromonas gingivalis LPS in aggressive periodontitis with the use of whole-blood cultures.

In this study, we re-visited the issue of hyper-responsiveness of monocytes to bacterial lipopolysaccharide (LPS) in aggressive periodontitis patients. We used whole-blood cultures to compare monocyte activation by Porphyromonas gingivalis LPS between Thai subjects with generalized aggressive periodontitis and those without periodontitis. Upon stimulation with P. gingivalis LPS, expression of co-stimulatory molecules on monocytes and expression of CD69 on NK and gamma delta T-cells were analyzed by flow cytometry, and the production of interleukin-1 beta and prostaglandin E(2) was monitored by ELISA. LPS stimulation resulted in a dose-dependent up-regulation of CD40, CD80, and CD86 on monocytes, and up-regulation of CD69 on NK cells and gamma delta T-cells in both the periodontitis and non-periodontitis groups. The levels of activation markers and the mediator production after LPS stimulation were quite similar for both groups. In conclusion, we did not observe hyper-responsiveness of monocytes to P. gingivalis LPS challenge in Thai patients with aggressive periodontitis.

Immunoglobulin G response of periodontitis patients to Porphyromonas gingivalis capsular carbohydrate and lipopolysaccharide antigens.

Porphyromonas gingivalis clonal types that participate in periodontal infections express serologically distinct surface antigens. This investigation sought to determine whether serum antibodies titers against the serotype-specific capsular carbohydrate K antigen and lipopolysaccharide antigens of P. gingivalis might reveal which serotypes are most likely to be responsible for subgingival infections in subjects with adult periodontitis. Immunoglobulin G (IgG) titers to purified K antigen and lipopolysaccharide from different P. gingivalis strains were measured by ELISA for 28 healthy controls and 51 patients with periodontal pockets known to be infected with genetically and serologically distinct P. gingivalis clonal types. Titers to purified K antigen from strains W50, HG184, A7A1-28, 49417, HG1690 and HG1691, representing serotypes K1-K6, respectively, and lipopolysaccharide from strains 381, HG1691 and W50, representing serotypes O1-O3, respectively, were measured for all subjects. Chi-square likelihood ratios, Mann-Whitney tests and receiver-operating characteristic sensitivity-specificity plots were used to compare the accuracy with which titer results for different target antigens classified subjects with or without disease. Results from assays targeting K2, K3, K4, K5, O1 and O2 generally gave poor diagnostic accuracy, whether evaluated separately or as summed titer pairs corresponding to the K/O combinations actually expressed by the target antigen parent strains. Exceptions were O3 (from W50) and K5+O2 (both from HG1690), which gave moderate accuracy in classifying subjects. In contrast, highly significant diagnostic accuracy was achieved using individual K1 (W50) and K6 (HG1691) titer data and K1+O3 (W50) and K6+O2 (HG1691) titer sum values. These observations suggest that P. gingivalis clonal types expressing K/O serotypes matching those of W50 (K1/O3) and HG1691 (K6/O2) are more likely than others to participate in periodontal infections in adult periodontitis patients and thus are more likely than others to express relevant virulence factors.

Porphyromonas gingivalis lipopolysaccharide delays human polymorphonuclear leukocyte apoptosis in vitro.

Apoptosis (programmed cell death) is a mechanism by which superfluous or damaged cells undergo changes that lead to selective removal from organ systems by phagocytic cells. Certain bacterial products delay apoptosis in neutrophils (PMNs). In this study, PMNs were incubated for up to 8 h with varying concentrations of lipopolysaccharide (LPS), lipid A or capsular polysaccharide isolated from 3 strains of Porphyromonas gingivalis (Pg) (strains HG-184, A7A1-28 and 381). Assay runs included controls containing cells and medium but no bacterial products. Fluorescence microscopy was used to evaluate apoptotic changes. PMNs exhibited a time-dependent increase in the number of apoptotic cells. When cells were cultured in the presence of LPS from any of the 3 Pg strains, apoptosis was delayed in a dose-dependent fashion (p < 0.05). The effects of these LPS preparations were similar to each other and to Escherichia coli 0111:B4 LPS. Lipid A from the 3 Pg strains also delayed apoptosis (p < 0.05), but was less potent than LPS or synthetic lipid A. Capsular polysaccharide had no significant effect on apoptosis (p > 0.05). Thus, LPS and lipid A from P. gingivalis appear to modulate the functional lifespan of PMNs. This could potentiate the inflammatory and destructive components of periodontal diseases.

Antibody reactive with Porphyromonas gingivalis serotypes K1-6 in adult and generalized early-onset periodontitis.

BACKGROUND: Six serotypes of Porphyromonas gingivalis have recently been described. We sought to test the hypothesis that serotype specific carbohydrates from these strains are important antigens that elicit potent immune responses. METHODS: Serum concentrations of IgG reactive with P. gingivalis serotypes K1-K6 were determined for 28 adult (AP) and 28 generalized early-onset (G-EOP) periodontitis patients previously determined to be seropositive for a broken cell preparation of P. gingivalis. To confirm relationships suggested for K1, K2, and K6 in the analysis of initial data, the study population was increased to 133. RESULTS: Frequency of seropositivity for the 6 serotypes ranged from 26 to 54% of subjects. IgG concentrations ranged from 0 to 453 microg/ml with many subjects seropositive to more than one serotype. Concentrations for the subset of patients who was seropositive were high (mean responses ranged from 20 to 105 microg/ml for the 6 serotypes). Significant correlations between seropositivity to serotypes K1 and K5 as well as between K5 and K6 were found. CONCLUSIONS: We examined the relationship of diagnosis, race, gender, smoking, probing depth, attachment loss, and antibody reaction with the P. gingivalis serotypes by analysis of variance. Initial findings suggested potential relationships between diagnosis, smoking, race, gender, and antibody reactive with serotypes K1, K2, and K6. A significant relationship did exist between smoking and decreased antibody reactive with P. gingivalis serotype K2. No other relationships were substantiated. We also examined the IgG subclass distribution and found that responses were almost exclusively IgG2. These data support the concept that antibody responses to all 6 serotypes are common in both AP and G-EOP and that these K serotype carbohydrates elicit potent IgG2 responses.

Capsular polysaccharide-fimbrial protein conjugate vaccine protects against Porphyromonas gingivalis infection in SCID mice reconstituted with human peripheral blood lymphocytes.

The effect of immunization with either a Porphyromonas gingivalis fimbrial protein, a capsular polysaccharide, or a capsular polysaccharide-fimbrial protein conjugate vaccine were compared in hu-PBL-SCID mice. A significantly higher human immunoglobulin G antibody response and the highest degree of in vivo protection against bacterial challenge was observed in the group immunized with the conjugate vaccine. It was concluded that capsular polysaccharide-fimbrial protein conjugate from P. gingivalis could potentially be developed as a vaccine against periodontal infection by P. gingivalis.

A clinical comparison of 2 electric toothbrush designs.

A single blind 30 day study compared the reduction of plaque and gingivitis for the Hapika Powerbrush to the Interplak ultra 10 tuft. A longitudinal parallel group design was utilized and screening evaluation was performed to determine patient eligibility prior to study enrollment, 66 subjects were entered into the study and assigned to 1 of 2 groups, each using one of the toothbrushes. At baseline, subjects received an oral soft tissue exam, a dental hard tissue exam, and were scored by the Lobene modification of the Löe and Silness gingival index (GI). Plaque was then disclosed and scored both pre and post brushing using the modified Turesky plaque/debris examination and an interproximal bleeding examination was performed post-brushing. On days 15 and 30, after an oral soft tissue and GI examination, plaque was graded by the Modified Turesky plaque/debris exam. Subjects then brushed and were graded by the Modified Turesky plaque/debris examination and an interproximal bleeding index examination. The results showed that both brushes provided a similar change in clinical indices. All produced a statistically significant reduction from baseline to day 30 for the gingival index (26.5-29.1%), the bleeding index (13.8-24.1%), and the plaque index (16.9-19.4%). A comparison of pre and post brushing scores for the plaque index at 30 showed that both brushes reduced plaque similarly with a statistically significant reduction (P < 0.05) from their pre-brushing plaque index scores at all time periods.

Effect of protoporphyrin IX limitation on porphyromonas gingivalis.

Porphyromonas gingivalis has been shown to require hemin or hemoglobin for in vitro growth. We have previously shown that protoporphyrin IX and inorganic iron can replace the hemin requirement, suggesting that the hemin requirement of this microorganism is actually a porphyrin requirement. We examined the effect of protoporphyrin IX limitation to P. gingivalis strain A7A1-28 in the presence of sufficient iron on growth characteristics, proteolytic enzyme production, virulence in a mouse abscess model, and expression of membrane proteins. Bacterial cells were grown in medium varying between 0 to 5 microM reduced growth by at least 50%. Protoporphyrin IX availability did not affect proteolytic enzyme production or virulence in a mouse abscess model. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane preparations demonstrated that protoporphyrin IX limitation induced the expression of new proteins at 42, 34, 30, 29, and 18 kDa and suppressed the production of proteins at 47, 27, 17, and 15 kDa. These studies suggest that in vivo protoporphyrin availability may modulate membrane protein expression and in turn affect host immune responses against P. gingivalis.

New directions in surgical endodontics; immediate implantation into an extraction site.

Endodontic surgical procedures may reveal compromising factors that indicate a modification of the treatment (e.g. tooth extraction, root amputation, etc.). To take advantage of the osseous height and width, as well as the natural tooth angulation, immediate placement of implants after extraction is a reasonable alternative treatment. In this study, 32 titanium alloy implants were inserted immediately after extraction of teeth diagnosed during endodontic surgery as having root fractures, perforations, or endodontic-periodontal complications. After 4 to 6 months of osseointegration, only one implant failed to integrate, and the remaining implants were prosthetically restored. Sixteen months after occlusal loading, bone loss was approximately 1.5 mm for the 31 implants remaining. It seems that the immediate placement of implants following tooth extraction due to endodontic complications is a reliable procedure.

Resistance of a Tn4351-generated polysaccharide mutant of Porphyromonas gingivalis to polymorphonuclear leukocyte killing.

In this study, we describe the development of an efficient transpositional mutagenesis system for Porphyromonas gingivalis using the Bacteroides fragilis transposon Tn4351. Using this system, we have isolated and characterized a Tn4351-generated mutant of P. gingivalis A7436, designated MSM-1, which exhibits enhanced resistance to polymorphonuclear leukocyte (PMN) phagocytosis and killing. P. gingivalis MSM-1 was initially selected based on its colony morphology; MSM-1 appeared as a mucoid, beige-pigmented colony. Analysis of P. gingivalis MSM-1 by electron microscopy and staining with ruthenium red revealed the presence of a thick ruthenium red-staining layer that was twice the thickness of this layer observed in the parent strain. P. gingivalis MSM-1 was found to be more hydrophilic than strain A7436 by hydrocarbon partitioning. Analysis of phenol-water extracts prepared from P. gingivalis A7436 and MSM-1 by Western (immunoblot) analysis and immunodiffusion with hyperimmune sera raised against A7436 and MSM-1 revealed the loss of a high-molecular-weight anionic polysaccharide component in extracts prepared from MSM-1. P. gingivalis MSM-1 was also found to be more resistant to PMN phagocytosis and intracellular killing than the parent strain, as assessed in a fluorochrome phagocytosis microassay. These differences were statistically significant (P < 0.05) when comparing PMN phagocytosis in nonimmune serum and intracellular killing in nonimmune and immune sera. P. gingivalis MSM-1 was also more resistant to killing by crude granule extracts from PMNs than was P. gingivalis A7436. These results indicate that the increased evasion of PMN phagocytosis and killing exhibited by P. gingivalis MSM-1 may result from alterations in polysaccharide-containing antigens.

Modification of experimental Porphyromonas gingivalis murine infection by immunization with a polysaccharide-protein conjugate.

To better understand the role of the capsular polysaccharide in the virulence of Porphyromonas gingivalis, the effect of immunization with a polysaccharide-protein conjugate on experimental murine infection was evaluated. The conjugate was prepared using polysaccharide isolated from P. gingivalis strain ATCC 53977 and bovine serum albumin. One group of 22 mice was immunized by intraperitoneal injection with the conjugate and a control group of 25 mice was similarly immunized with bovine serum albumin. Serum antibody reactive to the polysaccharide, as determined by enzyme-linked immunosorbent assay, was elevated in the group of mice immunized with the polysaccharide-protein conjugate but not in the mice immunized with bovine serum albumin. Both groups of mice were challenged with P. gingivalis strain ATCC 53977 (10(10) cells) administered subcutaneously on the dorsal surface. Following challenge, the mice immunized with the polysaccharide-protein conjugate appeared healthier and demonstrated less weight loss than did the control group of mice. Ulcerative lesions at secondary locations were smaller in mice immunized with the polysaccharide-protein conjugate. Thus, immunization of mice with a conjugate containing P. gingivalis polysaccharide could reduce the severity of but not prevent an invasive infection with P. gingivalis.

Activation of serum complement by polysaccharide-containing antigens of Porphyromonas gingivalis.

We previously reported that hot aqueous phenol extraction of Porphyromonas gingivalis yields a preparation containing both lipopolysaccharide (LPS) and an antigenically distinct capsular polysaccharide (PS). In the present study, we examined the capacity of phenol-water extracts from a number of strains of P. gingivalis to activate human serum complement. Anticomplementary activity of extracts from two invasive and two noninvasive strains of P. gingivalis was assessed in a sheep erythrocyte hemolytic assay and in an alternative pathway-selective rabbit erythrocyte hemolytic assay. In the sheep erythrocyte assay, extracts from noninvasive strains were found to exhibit greater anticomplementary activity than extracts derived from invasive strains. A phenol-water extract from invasive strain ATCC 53977 was further resolved into its LPS and PS fractions. Whereas isolated LPS from this strain exhibited strong anticomplementary activity, the PS fraction was only weakly active. Phenol-water extracts from three of four strains were found to be potent activators of the alternative pathway, with extracts from the two noninvasive strains being most active. The extract from the remaining strain (ATCC 53977) was a poor activator of the alternative pathway. Further analysis of this extract revealed, however, that the LPS fraction was a potent activator of the alternative pathway, although the PS fraction exhibited negligible activity. The results of this study indicate that phenol-water extracts of invasive and noninvasive strains of P. gingivalis differ in their respective anticomplementary activities, with invasive strains being less active. Although extracts from both invasive and noninvasive strains activated the alternative pathway, this activity appears to be attributable to the LPS, rather than the PS, component.

Evidence that the serotype b antigenic determinant of Actinobacillus actinomycetemcomitans Y4 resides in the polysaccharide moiety of lipopolysaccharide.

A high-molecular-weight polysaccharide-containing antigen was isolated from a phenol-water extract of Actinobacillus actinomycetemcomitans ATCC 43718 (formerly Y4) by gel permeation chromatography in lipopolysaccharide (LPS)-disaggregating buffer. The polysaccharide antigen formed a precipitin band with rabbit serotype b-specific antiserum but not with rabbit antisera to serotype a or c. Electroblotted serotype b antigen was probed with serum from a patient with localized juvenile periodontitis (LJP), resulting in a diffuse "smear" in the upper region of the lane. By utilizing an enzyme-linked immunosorbent assay, it was demonstrated that the geometric mean immunoglobulin G antibody titer to the serotype b polysaccharide was significantly higher in sera from LJP patients than in sera from periodontally healthy individuals. Moreover, LJP antibody titers to the serotype b polysaccharide exhibited age-dependent variation. Double immunodiffusion analysis revealed that the serotype b antigen formed a line of identity with low-molecular-weight LPS following reaction with serotype b-specific antiserum. Incubation of LJP serum in the presence of a lipid-free polysaccharide moiety obtained by mild acid hydrolysis of LPS from A. actinomycetemcomitans Y4 markedly reduced immunoglobulin G titer to the serotype b antigen. In contrast, solubilized lipid A was only weakly inhibitory. The results of this study indicate that the serotype b-specific determinant of A. actinomycetemcomitans resides in the polysaccharide moiety of LPS and represents a major target for immunoglobulin G antibody in serum of LJP subjects colonized by this organism.

Characterization of a polysaccharide antigen from Bacteroides gingivalis.

A polysaccharide Ag (PS) was isolated from the phenol-water extract of Bacteroides gingivalis strain A7A1-28 and separated from LPS by Sephacryl S-400 HR chromatography. The PS was composed of glucose, glucosamine, galactosamine, and galactosaminuronic acid, while the LPS contained rhamnose, mannose, galactose, glucose, glucosamine, galactosamine, phosphate, and lipid, but not galactosaminuronic acid. The PS and LPS were immunochemically distinct by immunoelectrophoresis in agarose with homologous rabbit antiserum. The phenol-water extract from strain A7A1-28 was immunoreactive by immunoelectrophoresis against antisera to three additional strains of B. gingivalis, however, the PS was only reactive with homologous serum. Immunochemical characterization of decarboxylated and deacetylated PS derivatives suggest that the acetylation of the amino sugars, but not the presence of the carboxylate residue on galactosaminuronic acid contributes to major immunodeterminant expression.

Immunochemical analysis of the types Ia and Ib group B streptococcal polysaccharides.

The types Ia and Ib group B streptococcal type-specific polysaccharides have remarkable immunologic differences despite a great deal of structural similarity. Although these two complex polysaccharides differ only by a single glycosidic linkage, they are antigenically distinct. Furthermore, terminal sialic acid residues appear to be critical to the immunodeterminant on the type Ia polysaccharide, whereas the antigenicity of the type Ib polysaccharide does not show this dependence on sialic acid. In the current investigation we defined better the immunodeterminant of these polysaccharides. With homologous rabbit antiserum, the type Ia native and core polysaccharides demonstrated partial serologic identity, whereas the type Ib native and core polysaccharides demonstrated complete serologic identity. Surprisingly, the type I degalactosylated polysaccharide, degraded structure, was capable of reacting with a population of antibodies present in type Ia antiserum similar to the complete type Ia native polysaccharide, although demonstrating a reduced level of immunodeterminant expression. Unlike the reactions of the type Ia polysaccharides with homologous rabbit antiserum, the Ib native and core polysaccharides were able to react with identical populations of antibodies in type Ib-specific antiserum. A minor population of antibodies was demonstrated in the type Ib antiserum, which was reactive with the degalactosylated polysaccharide. That a population of antibodies reactive toward the degalactosylated polysaccharide is present in both type Ia and type Ib antisera suggests that the Iabc cross-reacting determinant is due to the presence of serum antibodies reactive with this trisaccharide repeating unit, which is shared by both the type Ia and the type Ib native and core polysaccharides.