Charles I. Hoover

Fluoride Varnish Efficacy in Preventing Early Childhood Caries

To determine the efficacy of fluoride varnish (5% NaF, Duraphat®, Colgate) added to caregiver counseling to prevent early childhood caries, we conducted a two-year randomized, dental-examiner-masked clinical trial. Initially, 376 caries-free children, from low-income Chinese or Hispanic San Francisco families, were enrolled (mean age ± standard deviation, 1.8 ± 0.6 yrs). All families received counseling, and children were randomized to the following groups: no fluoride varnish, fluoride varnish once/year, or fluoride varnish twice/year. An unexpected protocol deviation resulted in some children receiving less active fluoride varnish than assigned. Intent-to-treat analyses showed a fluoride varnish protective effect in caries incidence, p < 0.01. Analyzing the number of actual, active fluoride varnish applications received resulted in a dose-response effect, p < 0.01. Caries incidence was higher for ‘counseling only’ vs. ‘counseling + fluoride varnish assigned once/year’ (OR = 2.20, 95% CI 1.19–4.08) and ‘twice/year’ (OR = 3.77, 95% CI 1.88–7.58). No related adverse events were reported. Fluoride varnish added to caregiver counseling is efficacious in reducing early childhood caries incidence.

Characterization of a trypsin-like protease from the bacterium Bacteroides gingivalis isolated from human dental plaque.

A trypsin-like, membrane-bound protease from Bacteroides gingivalis was solubilized by Triton X-100 and partially purified by a combination of DEAE-Sepharose and aminophenylmercuric Sepharose chromatography, by taking advantage of the thiol group on the enzyme. The purified enzyme hydrolysed the synthetic substrates benzoyl-L-arginine-p-nitroanilide (L-BAPA), benzoyl-D,L-arginine-beta-naphthylamide (BANA) and tosyl-L-arginine methyl ester, as well as bovine serum albumin and ovalbumin, but not tosyl-L-lysine methyl ester. The enzyme activity was enhanced by SH-reagents and was inhibited to different degrees by SH-inhibitors, chelators and microbial low-molecular-weight inhibitors such as leupeptin, antipain and chymostatin. These microbial inhibitors could be of practical use as ligands for affinity chromatography for further purification. The possible involvement of the protease in periodontal diseases is also discussed.

A Randomized Clinical Trial of Anticaries Therapies Targeted according to Risk Assessment (Caries Management by Risk Assessment)

This randomized parallel group clinical trial assessed whether combined antibacterial and fluoride therapy benefits the balance between caries pathological and protective factors. Eligible, enrolled adults (n = 231), with 1–7 baseline cavitated teeth, attending a dental school clinic were randomly assigned to a control or intervention group. Salivary mutans streptococci (MS), lactobacilli (LB), fluoride (F) level, and resulting caries risk status (low or high) assays were determined at baseline and every 6 months. After baseline, all cavitated teeth were restored. An examiner masked to group conducted caries exams at baseline and 2 years after completing restorations. The intervention group used fluoride dentifrice (1,100 ppm F as NaF), 0.12% chlorhexidine gluconate rinse based upon bacterial challenge (MS and LB), and 0.05% NaF rinse based upon salivary F. For the primary outcome, mean caries increment, no statistically significant difference was observed (24% difference between control and intervention groups, p = 0.101). However, the supplemental adjusted zero-inflated Poisson caries increment (change in DMFS) model showed the intervention group had a statistically significantly 24% lower mean than the control group (p = 0.020). Overall, caries risk reduced significantly in intervention versus control over 2 years (baseline adjusted generalized linear mixed models odds ratio, aOR = 3.45; 95% CI: 1.67, 7.13). Change in MS bacterial challenge differed significantly between groups (aOR = 6.70; 95% CI: 2.96, 15.13) but not for LB or F. Targeted antibacterial and fluoride therapy based on salivary microbial and fluoride levels favorably altered the balance between pathological and protective caries risk factors.

Identification of Porphyromonas gingivalis prefimbrilin possessing a long leader peptide: possible involvement of trypsin-like protease in fimbrilin maturation

Fimbriae of Porphyromonas gingivalis have been shown to be important as one of the virulence factors for colonization on mucosal surfaces. The gene (fimA) encoding the fimbrial subunit (fimbrilin) was overexpressed in Escherichia coli by using a bacteriophage T7 promoter-polymerase expression vector system. Analysis of the resulting fimA gene product revealed that the prefimbrilin had a 46 amino acid leader peptide. This extremely long leader peptide was cleaved from the prefimbrilin by treatment with trypsin or P. gingivalis extracts containing trypsin-like protease activity, resulting in production of a mature fimbrilin. We also found that some transposon-induced trypsin-like protease deficient mutants of P. gingivalis exhibited deficiency in fimbriation and that one of the mutants accumulated a fimbrilin precursor possessing a 25 amino acid leader peptide in the cell. The presence of an extremely long leader peptide and the requirement for a leader peptidase with a substrate specificity similar to that of P. gingivalis trypsin-like protease for fimbrilin maturation indicate that P. gingivalis fimbrilin is a novel type that is different from fimbrilins of type I and IV families.

A novel approach in assessment of coronal leakage of intraorifice barriers: a saliva leakage and micro-computed tomographic evaluation.

The efficacy of amalgam, Fuji-Plus, Geristore, and mineral trioxide aggregate (MTA) as intraorifice barriers was compared in a simulated saliva leakage model. Fifty teeth were divided into 4 experimental (n = 10 each) and 2 control groups (n = 5 each). Two millimeters of the materials was placed as intraorifice barriers and brought into contact with human saliva in a coronal reservoir. Bacterial leakage into the apical reservoir was assessed daily for 3 months. Geristore barriers leaked significantly more often (5/10) than Fuji-Plus (0/10, P < .05) at 60 days. The first Fuji-Plus sample leaked after 70 days; after 90 days, 3 (Fuji-Plus), 4 (MTA, amalgam), and 6 (Geristore) samples leaked. There were no significant differences between the experimental groups at that time point. Three-dimensional gap volumes in the barrier-dentin interface and the porosity of the barrier materials were compared by using micro-computed tomography (microCT). A measurable gap was evident in only 1 specimen with an MTA barrier. MTA was significantly less porous than Fuji-Plus and Geristore (P < or = .05), whereas amalgam was too radiopaque to allow microCT measurements. In conclusion, Fuji-Plus might be an effective intraorifice barrier (up to 70 days in vitro), but all 4 materials showed leakage in some specimens at 90 days.

Clinical Trial of a Plant-Derived Antibody on Recolonization of Mutans Streptococci

Objective: This double-blinded, placebo-controlled clinical trial tested the safety and efficacy of a topical secretory IgA antibody manufactured in tobacco plants (plantibody) in preventing recolonization of mutans streptococci (MS) in human plaque as measured by whole stimulated saliva samples. Methods: Following a 9-day antimicrobial treatment with chlorhexidine (CHX), 56 eligible adults (enrollment salivary MS ≧ 104 CFU/ml; no current caries) were randomized equally to a group receiving 0, 2, 4, or 6 topical applications of plantibody followed by 6, 4, 2, or 0 applications of placebo, respectively, over a 3-week period. Results: Among the 54 subjects who completed the trial, the CHX regimen eliminated salivary MS in 69%. After 6 months, there were no significant differences in MS levels by number of applications, relative to placebo (p > 0.43). No adverse effects were observed. Conclusion: Plantibody is safe but not effective at the frequency, concentration, and number of applications used in this study.

Osteogenic and antimicrobial nanoparticulate calcium phosphate and poly-(D,L-lactide-co-glycolide) powders for the treatment of osteomyelitis.

Development of a material for simultaneous sustained and localized delivery of antibiotics and induction of spontaneous regeneration of hard tissues affected by osteomyelitis stands for an important clinical need. In this work, a comparative analysis of the bacterial and osteoblastic cell response to two different nanoparticulate carriers of clindamycin, an antibiotic commonly prescribed in the treatment of bone infection, one composed of calcium phosphate and the other comprising poly-(D,L-lactide-co-glycolide)-coated calcium phosphate, was carried out. Three different non-cytotoxic phases of calcium phosphate, exhibiting dissolution and drug release profiles in the range of one week to two months to one year, respectively, were included in the analysis: monetite, amorphous calcium phosphate and hydroxyapatite. Spherical morphologies and narrow size distribution of both types of nanopowders were confirmed in transmission and scanning electron microscopic analyses. The antibiotic-containing powders exhibited sustained drug release contingent upon the degradation rate of the carrier. Assessment of the antibacterial performance of the antibiotic-encapsulated powders against Staphylococcus aureus, the most common pathogen isolated from infected bone, yielded satisfactory results both in broths and on blood agar plates for all the analyzed powders. In contrast, no cytotoxic behavior was detected upon the incubation of the antibiotic powders with the osteoblastic MC3T3-E1 cell line for up to three weeks. The cells were shown to engage in a close contact with the antibiotic-containing particles, irrespective of their internal or surface phase composition, polymeric or mineral. At the same time, both types of particles upregulated the expression of osteogenic markers osteocalcin, osteopontin, Runx2 and protocollagen type I, suggesting their ability to promote osteogenesis and enhance remineralization of the infected site in addition to eliminating the bacterial source of infection.

Lipopolysaccharide biosynthesis-related genes are required for colony pigmentation of Porphyromonas gingivalis

The periodontopathic bacterium Porphyromonas gingivalis forms pigmented colonies when incubated on blood agar plates as a result of accumulation of mu-oxo haem dimer on the cell surface. Gingipain-adhesin complexes are responsible for production of mu-oxo haem dimer from haemoglobin. Non-pigmented mutants (Tn6-5, Tn7-1, Tn7-3 and Tn10-4) were isolated from P. gingivalis by Tn4351 transposon mutagenesis [Hoover & Yoshimura (1994), FEMS Microbiol Lett 124, 43-48]. In this study, we found that the Tn6-5, Tn7-1 and Tn7-3 mutants carried Tn4351 DNA in a gene homologous to the ugdA gene encoding UDP-glucose 6-dehydrogenase, a gene encoding a putative group 1 family glycosyltransferase and a gene homologous to the rfa gene encoding ADP heptose-LPS heptosyltransferase, respectively. The Tn10-4 mutant carried Tn4351 DNA at the same position as that for Tn7-1. Gingipain activities associated with cells of the Tn7-3 mutant (rfa) were very weak, whereas gingipain activities were detected in the culture supernatants. Immunoblot and mass spectrometry analyses also revealed that gingipains, including their precursor forms, were present in the culture supernatants. A lipopolysaccharide (LPS) fraction of the rfa deletion mutant did not show the ladder pattern that was usually seen for the LPS of the wild-type P. gingivalis. A recombinant chimera gingipain was able to bind to an LPS fraction of the wild-type P. gingivalis in a dose-dependent manner. These results suggest that the rfa gene product is associated with biosynthesis of LPS and/or cell-surface polysaccharides that can function as an anchorage for gingipain-adhesin complexes.

Immunochemical comparison of cell-wall antigens of various viridans streptococci, including strain 2A2 + 3 hot from recurrent oral aphthous ulceration in man

Several studies suggest that patients with recurrent aphthous ulceration show cell-mediated and humoral immunity to antigens of Streptococcus sanguis, particularly strain 2A2+3 HOT which is said to be antigenically similar or identical to Strep. sanguis strain ATCC 10556. However, physiological classification as well as analysis of the immunologically dominant cell-wall antigens by immunoelectrophoresis and indirect immunofluorescence showed that the strain is actually a strain of Streptococcus mitis and is antigenically more like ATCC 10557 than ATCC 10556. The findings illustrate the antigenic heterogeneity of the Strep. sanguis and Strep. mitis taxons, and demonstrate the need for antigenic analysis of viridans streptococcal strains used in immunological studies of the aetiology of disease and in antiserum production. Commercial streptococcal group and antisera were also tested.

Transposition of Tn4351 inPorphyromonas gingivalis

Abstract Genetic analysis of Porphyromonas gingivalis , an obligately anaerobic gram-negative bacterium, has been hindered by the apparent lack of naturally occurring bacteriophages, transposable elements, and plasmids. Plasmid R751:: * Ω4 has previously been used as a suicide vector to demonstrate transposition of Tn 4351 in B. uniformis . The erythromycin resistance gene on Tn 4351 functions in Bacteroides and Porphyromonas . Erythromycin-resistant transconjugants were obtained at a mean frequency of 1.6 × 10 −7 from matings between Escherichia coli HB101 containing R751:: * Ω4 and P. gingivalis 33277. Southern blot hybridization analysis indicated that about half of the erythromycin-resistant P. gingivalis transconjugants contained simple insertions of Tn 4351 and half contained both Tn 4351 and R751 sequences. The presence of R751 sequences in some P. gingivalis transconjugants most likely occurred from Tn 4351 -mediated cointegration of R751, since we were unable to detect autonomous plasmid in these P. gingivalis transconjugants. The P. gingivalis -Tn 4351 DNA junction fragments from different transconjugants varied in size. These results are consistent with transposition of Tn 4351 and with insertion at several different locations in the P. gingivalis chromosome. Tn 4351 may be useful as a mutagen to isolate well-defined mutants of P. gingivalis .