Preliminary assessment of safety and effectiveness in humans of ProBiora3, a probiotic mouthwash.

AIMS: To conduct a pilot human clinical trial to assess the safety and to test the ability of a probiotic mouthwash, ProBiora(3), to affect the levels of Streptococcus mutans and certain known periodontal pathogens in the mouth when administered twice daily over a period of 4 weeks. METHODS AND RESULTS: The mouthwash contained three specific strains of naturally occurring oral bacteria and was tested at two dose levels: 10(6) and 10(8) colony forming units each of Strep. oralis strain KJ3sm, Strep. uberis strain KJ2sm, and the spontaneous lactic acid-deficient variant of Strep. rattus, strain JH145. Substantial decreases in the levels of the marker bacteria were observed. No safety issues were noted with the twice daily application of this mouthwash. CONCLUSIONS: Despite the small number of subjects and the use of young, orally healthy adults, along with the inherent variability in the microbiological measurements, the probiotic mouthwash was able to substantially affect the levels of dental pathogens in saliva and periodontal pathogens in subgingival plaque. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this pilot human study suggest that the probiotic mouthwash product may be safe for daily use as an aid in maintaining both dental and periodontal health.

A spontaneous lactate dehydrogenase deficient mutant of Streptococcus rattus for use as a probiotic in the prevention of dental caries.

AIMS: To study the ability of daily applications of Streptococcus rattus strain JH145 to affect the numbers of an implanted Streptococcus mutans strain in a rat model. METHODS AND RESULTS: A spontaneous L(+)-lactate dehydrogenase (LDH)-deficient mutant of Streptococcus rattus, JH146, was isolated by screening on selective medium and compared with a previously isolated spontaneous LDH deficient strain, JH145. Both strains were shown to have single base pair deletion mutations in the structural gene (ldh) for LDH, and reversion frequencies were approximately the same. Animals treated once daily with >or=10(6) CFU (colony forming units) of JH145 showed a statistically significant decrease in the proportion of implanted S. mutans to total cultivable bacteria in oral swab samples. The rate of decrease in S. mutans levels was dose-dependent. No adverse effects were observed by in-life observation of treated animals, and histopathological, haematological and blood chemistry analyses were unremarkable. CONCLUSIONS: The results presented indicate that daily application of JH145, a naturally occurring LDH-deficient variant of S. rattus, can compete with S. mutans for its habitat on the tooth surface. SIGNIFICANCE AND IMPACT OF THE STUDY: S. rattus JH145 has potential as a probiotic for use in the prevention of dental caries.

Parathyroid hormone fragments inhibit active hormone and hypocalcemia-induced 1,25(OH)2D synthesis.

Carboxyl (C)-terminal fragments of parathyroid hormone (PTH) oppose the calcemic, phosphaturic, and bone-resorbing effects of active hormone. To study the action of these fragments on 1,25(OH)(2)D (1,25-dihydroxyvitamin D) synthesis, we infused parathyroidectomized rats with human or rat active 1-34 or 1-84 PTH at doses selected to produce similar calcemic responses. Human active PTH influenced neither phosphate nor 1,25(OH)(2)D concentrations. However, active 1-34 rat PTH decreased phosphate to the same level as vehicle-treated rats and increased 1,25(OH)(2)D to very high levels, whereas active 1-84 PTH decreased phosphate but maintained 1,25(OH)(2)D. As the latter effect could have been due to C-terminal fragment generation during its metabolic breakdown, we infused a mixture of rat C-terminal fragments alone or with rat 1-34. The C-terminal fragments decreased 1,25(OH)(2)D and prevented hypocalcemic-induced 1,25(OH)(2)D synthesis. When infused with active rat 1-34, they lowered the 1,25(OH)(2)D level to that seen with intact rat 1-84. The C-terminal fragments did not influence either basal or rat 1-34- or 1-84-induced CYP27B1 mRNA levels, suggesting that their inhibitory effects on 1,25(OH)(2)D synthesis appears to be post-transcriptional.

Stimulation of calcitonin secretion by calcium receptor activators: evaluation using a new, highly sensitive, homologous immunoradiometric assay for rat calcitonin.

Current rat calcitonin immunoassays use human calcitonin antisera, and suffer from poor sensitivity, long incubation periods, nonspecific interferences, and unreliability. The homologous immunoradiometric assay (IRMA) for rat calcitonin described here overcomes these problems. Overnight incubation yields a detection limit of 0.4 pg/mL, a standard curve that is linear to >1800 pg/mL, and intra- and interassay coefficients of variation of <7%. Gel filtration chromatography of rat plasma and rat medullary thyroid carcinoma 44-2 cell media showed that the vast majority of immunoreactivity coeluted with calcitonin standard. In 44-2 cells, increasing extracellular Ca2+ concentration or incubation with the calcimimetic compound NPS R-467 markedly increased calcitonin secretion. Plasma calcitonin levels were elevated in rats anesthetized with ketamine/xylazine and in conscious rats with chronic renal insufficiency. Calcitonin levels decreased following EGTA-induced hypocalcemia and were undetectable after thyroparathyroidectomy. In normal conscious rats, plasma calcitonin levels averaged 3-5 pg/mL and increased up to 100-fold following calcium (Ca) infusion or NPS R-467 administration. The assay also quantified calcitonin in plasma of normal and Ca-injected mice. This assay has revealed that plasma calcitonin levels in normal rats are much lower than the detection limits of most existing assays, but can increase by 100-fold on activation of the C-cell Ca2+ receptor.

Clinical and laboratory evaluation of powered electric toothbrushes: in vivo determination of average force for use of manual and powered toothbrushes.

Mechanical oral hygiene instruments are intended to aid in the removal of stain and dental plaque from tooth surfaces. Certain home hygiene procedures, however, can lead to soft and hard tissue trauma. Power assisted brushing instruments are gaining in popularity, yet there is limited information on the interaction of these home care instruments with commercial dentifrices, and the resultant impact on oral tissues. In this study, the average forces applied during in vivo toothbrushing were determined for three powered brushing instruments (Rota-dent, Interplak and Braun Oral-B Plaque Remover) and a manual toothbrush (Oral-B P40). The Rota-dent instrument was found to be used with the lowest brushing pressure followed, in order, by the Braun Oral-B Plaque Remover. Interplak and the manual toothbrush. The average amount of dentifrice applied to the three powered brush heads was directly related to the size of the head, with Rota-dent typically receiving the least and Interplak the most applied dentifrice.

[Fluoride ions and apatite recrystallization of apatite in enamel]. / Fluoridové ionty a rekrystalizace apatitu ve sklovine.

The authors discuss the basic process on which the principle of fluoridation in based--i.e. the mutual exchange of F- ions and the OH- group in hydroxyapatite of enamel. Model investigations assessing the energy difference between hydroxyapatite and F- and fluorapatite and the OH- group indicate that fluorapatite tends to release steadily fluoride ions and to change into hydroxyapatite. This tendency, apparently inopportune from the aspects of caries prevention, ensures, however, a permanent low F- level above the enamel surface. The presence of F- ions facilitates recrystalization of apatite in the initial caries stage. The investigation tries to throw new light, from the point of view of theoretical chemistry at a molecular level, on the mechanism of remineralization, one of the most important ways which implements the preventive action of fluoride.

Immunoradiometric assay of corticotropin with use of avidin-biotin separation.

We have developed a "sandwich"-type immunoradiometric assay for corticotropin (ACTH), with a detection limit of 2 ng/L. Two antibodies are used: a mouse monoclonal antibody directed against ACTH[1-17] and labeled with 125I; and a purified polyclonal goat antibody directed against ACTH[34-39] and conjugated to biotin. We could separate 125I-labeled antibody bound to ACTH from 125I-labeled antibody not bound to ACTH by using an avidin-biotin bridge, with avidin bound to a polystyrene ball. This assay reacts with ACTH[1-39] but shows no reaction with ACTH fragments [1-24], [1-17], or [34-39], or with melanotropin, endorphins, or lipotropin. This assay is sensitive enough to detect ACTH in plasma of all normal adults. Concentrations measured in 94 adults between 0800 and 1000 hours were normally distributed on a log scale, with a mean of 19.5 ng/L and a 95% range of 7.1 to 53.8 ng/L. Dexamethasone given at 2300 hours to 14 adults suppressed ACTH to less than 4 ng/L in 13 of the subjects and to 8 ng/L in the 14th. Metyrapone given to 13 adults at 2300 hours increased ACTH to 245.3 ng/L (95% range, 90.1 to 667.7 ng/L). This assay accurately classified patients with disorders of the adrenal system.

Evaluation of a chairside gingival protease test for use in periodontal diagnosis.

Collagen degradation is a major factor in the destruction of diseased periodontal tissues. Evidence from several investigations indicates that this connective tissue breakdown process may be associated with elevated levels of proteolytic activity in gingival crevicular fluid. A rapid, chairside assay for neutral proteases in biological fluids was evaluated clinically for reproducibility and an indicator of periodontal disease status. This colorimetric test is based on the use of an insoluble, covalently linked dye-collagen substrate. Enzymatic activity is monitored by estimating the production of soluble dye-labeled collagen fragments resulting from the action of sample fluid proteases on the test substrate. Gingival fluid samples were collected on three successive days from 366 interdental sites in 34 volunteers, with and without periodontal disease. Results demonstrate that the assay is reproducible with 98.6% of sampled sites remaining stable throughout the 3-day study. Furthermore, the highest percentage of elevated neutral protease activity (NPA) values and the highest NPA values were found in patients clinically diagnosed as having advanced periodontal disease.

Highly sensitive two-site immunoradiometric assay of parathyrin, and its clinical utility in evaluating patients with hypercalcemia.

We have developed a highly sensitive, two-site immunoradiometric assay (IRMA) for human parathyrin (PTH) that is specific for the intact, secreted, biologically active 84-amino-acid peptide. This assay has several technical advantages: it does not detect even high concentrations of inactive carboxyl-terminal fragments, results are available within 24 h, and the detection limit for intact hormone is low (1 ng/L). The assay readily measures concentrations of PTH in all healthy subjects and distinguishes these values from low or undetectable PTH values observed in clinical situations in which PTH secretion is expected to be suppressed. We found complete separation of results from 37 patients with surgically proven hyperparathyroidism and those from 23 patients with hypercalcemia associated with malignancy, the latter having PTH values at or below the lower limits of normal for this assay. The sensitivity, specificity, and rapid turnaround time of this two-site IRMA should advance the laboratory evaluation of patients with disorders of calcium metabolism.

Two-monoclonal-antibody sandwich-type assay for thyrotropin, with use of an avidin-biotin separation technique.

We have developed a sensitive, specific, noncompetitive, sandwich-type radioimmunoassay for human thyrotropin (hTSH), which can be performed in 30 min. The assay involves two monoclonal antibodies, selected for high affinity and specificity and also for reaction against antigenic sites on hTSH that are distal from each other. One of these antibodies is labeled with 125I; the other is conjugated covalently to biotin. Polystyrene beads were also conjugated covalently to biotin. After conjugation, the beads were incubated with avidin. These beads represent a rapid, simple method for separating hTSH-bound antibody from free antibody. The biotin-antibody-hTSH-125I-labeled antibody complexes bind to the beads and hTSH concentration is directly related to counts per minute. This assay can detect hTSH at a concentration of 0.06 milli-unit/L in serum.

Modification by salivary pellicles of in vitro enamel remineralization.

The effectiveness of inorganic calcifying solutions to remineralize enamel with subsurface demineralization decreased with saliva pre-treatments conducive to the formation of enamel pellicles. Greater reductions in remineralization rates occurred with longer pellicle formation times. It is suggested that enamel pellicles may act to control surface deposition and favor subsurface precipitation.

Demineralization and remineralization of dental enamel.

Experimentation in vitro using organic acid buffers as demineralizing media shows that caries-like lesions can be obtained which are very similar in morphology and developmental stages to early lesions formed naturally under oral conditions. The use of these chemical systems and of mechanistic models advanced to explain the unique histological features of incipient caries have yielded a good understanding of the processes involved in caries formation. The study of natural and induced factors influencing the demineralization process has been greatly facilitated by the use of bacteriological systems in which demineralization is produced by direct colonization of cariogenic microorganisms on the surfaces of extracted teeth. Comparison of results obtained with these latter systems and with chemical systems has allowed us, for example, to elucidate the mechanism by which acquired salivary pellicles and fluoride topical solutions decrease the rate of enamel demineralization. The pellicle retards transport of matter across the enamel surface, whereas the fluoride topical solutions decrease the cariogenicity of the colonizing bacteria.

Effect of salivary pellicle formation time on in vitro attachment and demineralization by Streptococcus mutans.

Enamel subsurface demineralization induced by Streptococcus mutans was significantly reduced by seven-day saliva pre-treatments conducive to the formation of enamel pellicles. A two-hour saliva pre-treatment was ineffective. Results suggest that the protection provided by long-term pellicles may relate to changes in ionic transport rates rather than cell attachment.