Effect of an alcohol-free, 1% chlorhexidine gel as an adjunct to a fluoridated dentifrice using an intraoral crown model.

BACKGROUND/AIMS: The use of chlorhexidine as a topically applied oral antiseptic is well documented; however, clinical studies examining the effects of chlorhexidine gel on in situ dental caries are limited. This study utilized an in situ caries model and a modified crossover design to examine whether the addition of a biweekly topical, alcohol-free, 1% chlorhexidine digluconate gel to a daily fluoridated dentifrice inhibited artificial caries in dental tissues better than the fluoridated dentifrice alone when compared to a nonfluoridated placebo dentifrice. METHODS: Thirty patients were recruited based on their need for a mandibular, full crown. Artificial caries lesions were created in extracted human teeth and enamel and root tissue sections 100 mum in thickness were characterized using polarized light microscopy. The sections were fixed in the crown and placed on the prepared tooth. The participants were assigned a placebo toothpaste, a toothpaste with 1,100 ppm F or a 1,100 ppm F toothpaste followed by 1 ml of 1% chlorhexidine gel at day 1 and day 14 (chlorhexidine+). Patients were instructed to brush twice daily for 4 weeks. Following each round, the sections in the crown were replaced with new sections. The sections were recharacterized and the mean changes were compared using ANOVA at alpha = 0.05. RESULTS: The chlorhexidine + Fdentifrice and the F dentifrice alone significantly reduced lesion area in enamel tissue when compared to the placebo dentifrice. Both treatments also inhibited lesion progression and initiation in root tissue better than control in this model system. Although the chlorhexidine+ group enhanced remineralization and inhibited lesion progression better than the F(-) dentifrice alone for all outcomes measured, the differences were not significant. CONCLUSIONS: The chlorhexidine, in conjunction with a fluoride dentifrice, was no more effective than the fluoride dentifrice alone. Further study is needed before this 1% alcohol-free chlorhexidine gel should be recommended as an adjunct to a fluoride dentifrice in the treatment of dental caries.

Role of C5a in the induction of tumoricidal activity in C3H/HeJ (Lpsd) and C3H/OuJ (Lpsn) macrophages.

Thioglycollate-elicited macrophages from C3H/HeJ (Lpsd) and C3H/OuJ (Lpsn) mice were cultured in a two-signal, tumoricidal assay using recombinant interferon-gamma (rIFN-gamma) as the "priming" signal and recombinant human C5a (rC5a) as the "trigger" signal. These experiments were compared directly with a well established, two-signal tumoricidal assay in which rIFN-gamma was used as the "priming" signal and protein-rich, butanol-extracted lipopolysaccharide (But-LPS) as the "trigger" signal. These studies showed that rIFN-gamma-primed macrophages can be triggered in a dose-dependent manner by rC5a to effect high levels of tumoricidal activity. Maximum levels of cytotoxicity achieved using this endogenously produced, biologically active peptide as a "trigger" signal were comparable to those obtained using But-LPS. Moreover, experiments in which anti-C5 antibody was included in macrophage cultures stimulated with rIFN-gamma and But-LPS showed a significant reduction (P less than .05) in tumoricidal activity. Because LPS has been shown to induce macrophage C5 production and enzyme release, these findings suggest that macrophage-derived C5 is locally converted to C5a (or some other biologically active C5 cleavage fragment), which functions as an autocrine trigger signal for the induction of tumoricidal activity. In summary, these data suggest 1) that rC5a can provide a "second signal" to rIFN-gamma-primed murine macrophages for the induction of tumoricidal activity and 2) that macrophage-derived C5 or C5a may represent an autocrine signal induced by exogenous "trigger signals."

Bone marrow progenitors cultured in the presence of granulocyte-macrophage colony-stimulating factor versus macrophage colony-stimulating factor differentiate into macrophages with distinct tumoricidal capacities.

Bone marrow-derived cells from C3H/HeJ mice were cultured in the presence of recombinant murine granulocyte-macrophage colony-stimulating factor (rGM-CSF) or highly purified murine macrophage colony-stimulating factor (CSF-1) for 7 days. Following this 7-day culture period, mature macrophages were harvested and replated at precise densities in the absence of exogenous rGM-CSF or CSF-1, and assayed in a two-signal tumoricidal assay. Cultures were stimulated with medium only or with combinations of recombinant interferon-gamma (rIFN-gamma) as the "priming" signal, and/or butanol-extracted lipopolysaccharide (But-LPS) as the "triggering" signal for 24 hr. At this time, 51Cr-labeled, P815 tumor target cells were added, and the percent tumor cell cytotoxicity was determined after 16 hr. Macrophages derived under the influence of rGM-CSF exhibited significant tumoricidal capacity with medium alone (16 +/- 5%). The addition of "priming" signal only (i.e., rIFN-gamma, 10.0 U/ml) significantly increased tumoricidal capacity to 31 +/- 9%. Treatment with But-LPS alone did not alter the basal tumoricidal activity of rGM-CSF-derived macrophages. Combinations of rIFN-gamma (10.0 U/ml) and But-LPS (0.5-5.0 micrograms/ml) generated highly tumoricidal macrophages (50-60% tumor cell cytotoxicity). In contrast, medium-treated CSF-1-derived macrophages exhibited a significantly lower basal level of tumor cytotoxicity (6 +/- 3%). Unlike rGM-CSF-derived macrophages, treatment of CSF-1-derived macrophages with high concentrations of rIFN-gamma alone did not increase significantly the level of cytotoxicity above that of medium-treated cultures. However, CSF-1-derived macrophages responded to the highest concentrations of But-LPS (5.0 micrograms/ml) to increase tumoricidal activity from 6 +/- 3% to 17 +/- 5%. Optimal tumoricidal activity (44 +/- 17%) was observed when CSF-1-derived macrophages were treated simultaneously with high concentrations of both rIFN-gamma and But-LPS. Thus, macrophages derived from bone marrow progenitors in either rGM-CSF or CSF-1 exhibited tumoricidal capacities that differed in basal activity as well as in their requirements for and sensitivities to "priming" and "triggering" signals.

Use of serum-free, compositionally defined medium for analysis of macrophage differentiation in vitro.

Macrophage differentiation is mediated by the action of a variety of environmental signals, such as cytokines and endotoxin. For in vitro analysis of macrophage differentiation, most studies utilize a basal tissue culture medium supplemented with fetal calf serum (FCS). As the composition of specific components within FCS varies enormously from lot to lot, one can never be certain that the differentiative effects observed in vitro are attributable solely to the exogenous signals provided to the cultures. In this study, primary macrophages were cultured in a basal medium supplemented either with FCS or a compositionally defined supplement, HL-1, and a spectrum of differentiative functions (i.e., induction of antiviral activity, Fc receptor-mediated phagocytosis, Ia antigen expression, and tumoricidal activity) were measured following stimulation with exogenous signals. The results indicate that the use of serum-free, defined media may provide an important approach to dissect and characterize the differentiative signals which are operative in macrophage activation.

Examination of macrophage cell surface antigen regulation by rIFN-gamma and IFN-alpha/beta utilizing digital imaging by a novel laser detection system. Anchored cell analysis station (ACAS) 470.

The use of a computer-controlled scanning laser instrument, the ACAS 470, for the analysis of cell-surface antigens on adherent macrophages is described. The association of specific cell surface antigens with macrophage differentiation and the modulation of these markers by environmental signals has long been recognized. However, direct analysis of individual macrophages for cell-surface antigen expression by conventional methods has been fraught with difficulties. Primary macrophage cultures form highly adherent monolayers in vitro. To be analyzed by conventional flow cytometric methods (e.g., FACS analysis of non-adherent lymphoid populations), the adherent macrophages must be detached by enzymatic or mechanical methods which can result in damage to the cell membranes and/or strip off cell surface antigens. Other conventional techniques, e.g., antibody plus complement-mediated cytotoxicity or immunofluorescent microscopy, are semiquantitative at best. Surface antigen analysis using ELISA techniques provides a total population measure of changes in cell surface antigen expression, but fails to provide the number of antigen-positive cells or the density of antigen per cell. The ACAS 470 obviates all of these technical problems and allows for an analysis of individual adherent cells for the density and topography of antigen per cell, as well as expression of an antigen within a population. Using the ACAS 470, we have examined the expression of Ia antigens (class II major histocompatibility antigens) and the Mac-1 antigen (C3bi receptor) on thioglycollate-elicited murine macrophages, basally and after treatment with interferons. In this study, the density and distribution of these antigens per cell, as well as their expression within a population, are reported.

Evaluating the effects of fluoride-releasing dental materials on adjacent interproximal caries.

BACKGROUND: The authors examined several restorative materials to evaluate their ability to inhibit demineralization and enhance remineralization of incipient carious lesions on the interproximal enamel of teeth adjacent to those restored with the materials. METHODS: Twenty-one subjects in need of a crown on a mandibular molar and a Class II inlay on an adjacent tooth took part in this six-phase study. Artificial enamel lesions were created and positioned within the interproximal portion of a crown. Lesions were photographed with polarized light microscopy and characterized before and after 30-day intraoral exposures. Each phase included the placement of a new section in the crown model and a new Class II inlay restorative material in the adjacent tooth. RESULTS: Results demonstrated that nonfluoridated resin composite, fluoridated resin composite and resin-modified glass ionomer restorative materials, when placed in subjects who brushed with a fluoridated dentifrice, demonstrated significantly (P < .05) less enamel demineralization than the nonfluoridated resin composite control placed in subjects who brushed with a nonfluoridated dentifrice. The resin-modified glass ionomer cement, however, even when brushed with a nonfluoridated dentifrice, exhibited significantly (P < .05) less demineralization than the nonfluoridated resin composite control brushed with a nonfluoridated dentifrice. CONCLUSIONS: Resin-modified glass ionomer cement appears to significantly inhibit demineralization of interproximal enamel of teeth adjacent to those restored with the material. CLINICAL IMPLICATIONS: Resin-modified glass ionomer cement restorations can enhance prevention of enamel demineralization on adjacent teeth.

De/remineralization from sodium fluoride dentifrices.

PURPOSE: To test the demineralization/remineralization effects of sodium fluoride dentifrices using an in situ single-section crown model system. MATERIALS AND METHODS: A fluoride dose response was evaluated using 0, 1100 and 2800 ppm F-, along with the effects of an enhanced fluoride delivery system (polyampholyte-NaF). The single-section crown model was employed with supervised toothbrushing twice a day. At the end of each 1-month study leg, sections were removed and replaced with new sections for the next leg. Both before and after the double-blind, crossover portion of the study, sections were evaluated by polarized light microscopy and microradiography. The change in mineral content of the enamel and root lesions was analyzed by ANOVA with a Waller-Duncan K-Ratio Test post hoc. RESULTS: The placebo dentifrice group showed a loss of mineral and was statistically different from all groups. The fluoride dentifrices showed increasing amounts of enamel mineral gain, with increasing fluoride concentration. The polyampholyte-NaF delivery system with 1100 ppm F- was equivalent to the 2800 ppm F- dentifrice. Root lesions gave similar rank-order results although all treatments showed demineralization or mineral loss.

Structural and transient components of memory.

The decomposition of free recall serial position curves into permanent and transient components (primary and secondary memory, Waugh & Norman, 1965) is widely accepted. In this paper, a hypothesis is proposed directly relating the permanent memory component to a vector quantity which reflects sequential allocation of attention during encoding. In conjunction with a previously published model for encoding and retrieval in free recall (Hogan, 1975), the hypothesis is shown to break down predicted serial position curves into permanent and transient components, in a manner analogous to but in various ways less restrictive than the decomposition proposed by Waugh and Norman.

Measurement of tumor necrosis factor alpha and beta.

Tumor necrosis factor (TNF) a and b are multifunctional cytokines elaborated primarily by monocytes and macrophages (TNF-a) or T cells (TNF-b). Some cells that bind TNF-a or -b on their surface receptors lyse as a consequence of the binding reaction. This unit presents a protocol that employs TNF-sensitive, actinomycin D-treated murine L929 fibroblasts to quantify TNF activity in supernatants derived from cell cultures, serum samples, or cerebral spinal fluid. While the assay can measure picogram concentrations of human, rat, and murine TNF-ab, it cannot distinguish between the a and b forms of any species. A describes propagation and preparation of L929 fibroblasts.

Production of tumor necrosis factor by rIFN-gamma-primed C3H/HeJ (Lpsd) macrophages requires the presence of lipid A-associated proteins.

Previous studies have shown that the activation of murine macrophages to a fully tumoricidal state requires that specific environmental signals be delivered to the macrophage in a step-wise manner: a "priming" signal first renders the macrophage stimulated, but not cytolytic. The addition of a second or "trigger" signal to the primed macrophage results in tumoricidal activity. One potent priming signal has been identified as IFN-gamma and one often used trigger signal for endotoxin-responsive (Lpsn) macrophages is LPS. In contrast to LPS-responsive macrophage, rIFN-gamma-primed C3H/HeJ (Lpsd) macrophages fail to become cytolytic in response to protein-free, phenol-water-extracted LPS preparations, but become tumoricidal when exposed in vitro to protein-rich butanol-extracted LPS or purified lipid A-associated proteins. Further characterization of the activation requirements of the C3H/HeJ macrophages revealed that for optimal elaboration of TNF in vitro, two signals were also required: rIFN-gamma and a second signal that contained LAP. C3H/HeJ macrophages macrophages primed with rIFN-gamma failed to produce TNF in response to any concentration of protein-free phenol-water extracted LPS, even when supernatants were concentrated before assaying for functional activity in a standard TNF L929 fibroblast assay. Although exposure of rIFN-gamma-primed C3H/HeJ macrophages to LAP resulted in a fully tumoricidal state equivalent to that exhibited by C3H/OuJ macrophages, the levels of TNF produced remained discrepant. Under identical conditions, C3H/OuJ macrophages produced approximately fivefold more TNF (11,776 U/ml) than C3H/HeJ macrophages (2,399 U/ml). This suggests that although C3H/HeJ macrophages can respond functionally in a "normal" manner given the correct signals, they remain quantitatively deficient in the production of certain proteins. In this system, the elaboration of TNF and macrophage-mediated tumor cell lysis were shown to be dissociable events. The tumor target used in these studies (P815) was shown to be resistant to as much as 40,000 U/ml of purified rTNF. In addition, C3H/OuJ macrophage cultures exposed to LPS only (which resulted in the production of high levels of TNF), failed to lyse these targets. Lastly, anti-mouse TNF antibody added to macrophage cultures had no effect on the induction of tumor cell lysis.

Inhibition of macrophage tumoricidal activity by glucocorticoids.

In this study, the effect of corticosteroids on the activation of macrophages to a fully tumoricidal state was examined. Thioglycolate-elicited peritoneal exudate macrophages from C3H/HeJ mice were rendered cytolytic for P815 mastocytoma cells in a two-signal tumoricidal assay that used recombinant interferon-gamma (rIFN-gamma; 1 to 10 U/ml) as a "priming" signal and butanol-extracted lipopolysaccharide (But-LPS; 0.1 to 5 micrograms/ml) as a "trigger" signal. Treatment of macrophages with either rIFN-gamma alone or But-LPS alone failed to result in significant cytolytic ability. Tumoricidal activity was markedly inhibited in a dose-dependent fashion when glucocorticoids were added simultaneously to the cultures with rIFN-gamma and But-LPS at concentrations ranging from 1 X 10(-10) to 1 X 10(-5) M. Nonglucocorticoid sex hormones failed to inhibit tumoricidal activity in this system under identical culture conditions. Inhibition was most effective if the glucocorticoids were added simultaneously with the priming and triggering signals (rIFN-gamma and But-LPS); however, if the glucocorticoids were added 24 hr after the signals were provided to the cultures, suboptimal inhibition was observed. Experiments that dissociated the priming phase of activation from the triggering phase showed that glucocorticoids inhibited both the rIFN-gamma-induced priming stage as well as the But-LPS-induced triggering stage of activation. These observations provide evidence that glucocorticoids, but not other steroid hormones, inhibit the activation of macrophages to a fully tumoricidal state by interfering with either the priming or triggering signals in this two-signal model of macrophage activation.

Lipid A-associated proteins provide an alternate "second signal" in the activation of recombinant interferon-gamma-primed, C3H/HeJ macrophages to a fully tumoricidal state.

Previous studies have shown that the activation of murine macrophages to a fully tumoricidal state requires that specific environmental signals be delivered to the macrophage in a stepwise manner: a "priming" signal first renders the macrophage responsive to a second or "trigger" signal. One potent "priming" signal has been identified as the T cell-derived lymphokine, interferon-gamma (IFN-gamma) and one often used "trigger" signal is lipopolysaccharide (LPS), the endotoxin derived from Gram-negative bacteria. In these studies, endotoxin-responsive C3H/OuJ (Lps(n)) and endotoxin-hyporesponsive C3H/HeJ (Lps(d)) macrophages were exposed in vitro to recombinant IFN-gamma (rIFN-gamma) and various preparations of endotoxin or purified lipid A-associated proteins (LAP). The resultant tumoricidal responses were evaluated to define the activation requirements of murine macrophages and to examine further the LPS defect exhibited by C3H/HeJ mice. The findings presented herein demonstrate that C3H/OuJ macrophages primed by rIFN-gamma respond to protein-free LPS (phenol-water extracted LPS), protein-rich LPS (butanol-extracted LPS), or purified LAP. In contrast, rIFN-gamma-primed C3H/HeJ macrophages failed to become cytolytic with phenol-water extracted LPS, but could be rendered fully tumoricidal if either butanol-extracted LPS or LAP were used as "second signals." These data indicate that C3H/HeJ macrophages are fully responsive to the priming effects of IFN-gamma, but remain restricted in their capacity to recognize protein-free LPS as a second signal. Alternate second signals, such as LAP, may provide a compensatory pathway by which these macrophages are rendered fully tumoricidal.

Measurement of antiviral activity induced by interferons alpha, beta, and gamma.

The Basic Protocol in this unit describes an assay for murine IFN-induced antiviral activity and employs vesicular stomatitis virus (VSV) and IFN-sensitive fibroblasts. Support Protocol describes the preparation of VSV cultures and the calculation of multiplicity of infection (MOI; i.e., concentration of viral particles required to infect cells). Support Protocol describes the antibody neutralization assay, which can be used to identify the bioactive species of IFN in a sample, or to test the potency of an antibody preparation against a particular species of IFN. Alternate Protocol 1 covers measurement of human IFN-induced antiviral activity; while the steps are quite similar to Basic Protocol, different viral and cell cultures are described.

Test of the health promotion model as a causal model of construction workers' use of hearing protection.

The health promotion model (HPM) was tested as a causal model of construction workers' use of hearing protection (N = 359). Theoretical and exploratory models fit well, with the theoretical model accounting for 36.3% of variance and the exploratory model accounting for 50.6% of variance in hearing protection use. Value of use (benefits of using hearing protection), barriers to use, and self-efficacy were significant predictors in both the theoretical and exploratory models, but perceived health status was a predictor only in the theoretical model. In the exploratory model, where modifying factors were allowed direct relationships with use of hearing protection, two modifying factors--noise exposure and interpersonal influences-modeling--were significant predictors. Results of this test of the HPM are consistent with the revised HPM (Pender, 1996). There were significant direct paths from modifying factors to behaviour. Use of hearing protection was best predicted by behavior-specific predictors, such as perceived barriers to use of hearing protection. Results support the use of the HPM to predict use of hearing protection.

The fall of zinc protoporphyrin levels in workers treated for chronic lead intoxication.

A temporal fall of zinc protoporphyrin (ZPP) levels in whole blood was observed in 51 patients with occupational chronic lead intoxication who were removed from exposure, treated with intravenous calcium disodium edetate (EDTA), and followed for periods up to 2,273 days. ZPP levels fell, with a mean half-life of 68 days, to a mean baseline level of 36 micrograms/dl of whole blood. The baseline ZPP level was positively associated with the length of exposure (p less than .01) and the blood lead half-life (p less than .001). The amount of EDTA received had no apparent effect on ZPP levels. These data suggest that the fall of ZPP levels is largely a function of red blood cell turnover. The baseline ZPP level appears to be a useful biologic index of the biologically active pool of lead for at least two years after removal from exposure.

Urinary tract infections in childhood: the place of the nitrite test.

The effectiveness of the nitrite test available on the N-Multistix (Ames Co.) was compared with that of the microscopic examination of urine as a screening test for the detection of urinary tract infections in children presenting to a general paediatric clinic. The nitrite test gave a positive result in 59% of children with urinary infections, while microscopic pyuria (more than 50 white blood cells per cubic millimetre of uncentrifuged urine) was found in 72% of the same group. Of children who did not have a urinary tract infection, 2.8% had microscopic pyuria compared with only 0.2% who showed a false-positive nitrite test result. The ease and rapidity of the nitrite test make it a useful screening test for the presumptive diagnosis of urinary tract infections, and in certain circumstances it is preferable to the conventional microscopic examination of urine. However, as in the case of microscopic examination, urine culture must always be performed to avoid missing the urinary infections that are not detected by the screening test.

Effect of fluoridated milk on progression of root surface lesions in vitro under pH cycling conditions.

The aim of this study was to assess the effect of milk with 0, 2.5 or 5 ppm F on progression and remineralization of caries-like root surface lesions using a pH cycling model. The root surface lesions were created utilizing a partially saturated lactic acid buffer at pH 4.6. Longitudinal sections were cut through the lesion and analyzed using polarized light microscopy (PLM) and microradiography (MRG). The sections were then coated with an acid resistant varnish, except the outer natural surface that would be exposed to water, milk or fluoridated milk and cycled in a de- and remineralizing system for 2 weeks. The lesions were characterized again by PLM and MRG after treatment. A significant reduction in lesion progression was found by PLM and MRG after treatment with either non-fluoridated or fluoridated milk when compared to the control group. Using quantitative MRG, mineral change and distribution in the lesions were recorded. A possible protective effect of fluoridated milk on root surface caries was supported by a reduction in the progression of the lesions and an increase in the mineral within the lesion.