Martin Levine

Susceptibility to Dental Caries and the Salivary Proline-Rich Proteins

Early childhood caries affects 28% of children aged 2–6 in the US and is not decreasing. There is a well-recognized need to identify susceptible children at birth. Caries-free adults neutralize bacterial acids in dental biofilms better than adults with severe caries. Saliva contains acidic and basic proline-rich proteins (PRPs) which attach to oral streptococci. The PRPs are encoded within a small region of chromosome 12. An acidic PRP allele (Db) protects Caucasian children from caries but is more common in African Americans. Some basic PRP allelic phenotypes have a three-fold greater frequency in caries-free adults than in those with severe caries. Early childhood caries may associate with an absence of certain basic PRP alleles which bind oral streptococci, neutralize biofilm acids, and are in linkage disequilibrium with Db in Caucasians. The encoding of basic PRP alleles is updated and a new technology for genotyping them is described.

Antibody Response to Actinomyces Antigen and Dental Caries Experience: Implications for Caries Susceptibility

ABSTRACT Fluoridated dentifrices reduce dental caries in subjects who perform effective oral hygiene. Actinomyces naeslundii increases in teeth-adherent microbial biofilms (plaques) in these subjects, and a well-characterized serum immunoglobulin G (IgG) antibody response (Actinomyces antibody [A-Ab]) is also increased. Other studies suggest that a serum IgG antibody response to streptococcal d-alanyl poly(glycerophosphate) (S-Ab) may indicate caries experience associated strongly with gingival health and exposure to fluoridated water. The aim of this study was to investigate relationships between A-Ab response, oral hygiene, S-Ab response, and caries experience. Measurements were made of A-Ab and S-Ab concentrations, caries experience (number of decayed, missing, and filled teeth [DMFT], number of teeth surfaces [DMFS], and number of decayed teeth needing treated [DT]), exposure to fluoridated water (Flu), mean clinical pocket depth (PD; in millimeters), and extent of plaque (PL) and gingival bleeding on probing (BOP). A-Ab concentration, the dependent variable in a multiple regression analysis, increased with S-Ab concentration and decreased with PL and DMFT adjusted for Flu (R2 = 0.51, P < 0.002). Residual associations with age, DMFS, DT, and BOP were not significant. In addition, an elevated A-Ab response, defined from immunoprecipitation and immunoassay measurements, indicated a significant, 30% reduction in DMFT after adjustment for significant age and Flu covariance (analysis of variance with covariance F statistic = 10.6, P < 0.003; S-Ab response and interactions not significant). Thus, an elevated A-Ab response indicates less caries in subjects performing effective oral hygiene using fluoridated dentifrices. Conversely, a low A-Ab response is suggestive of decreased A. naeslundii binding to saliva-coated apatite and greater caries experience, as reported by others.

Fast ELISA for measuring serum antibody responses

A method which speeds up the enzyme-linked immunosorbent assay (ELISA) is described. The procedure uses a modified Falcon fast assay screening system (Becton Dickinson Labware, Lincoln Park, NJ) and Falcon round-bottom 96-well plates. Antigen is adsorbed onto beads which extend from a lid and fit into 96-well plates. The beads are washed in a trough and reacted to antibody in the round-bottom plate. The labor required to wash the plates after coating with antigen, antibody or conjugate is thereby reduced. Greater flexibility and accuracy result, especially with the use of more than one 96-well plate. In this study, naturally occurring human IgG antibody responses to two isolated bacterial antigens were measured in over 200 subjects. It was found that numerical taxonomy could be used to split out the high IgG responders. The IgM response to one of the antigens was less variable and not significantly related to the IgG response. The fast ELISA is as useful to operate as the standard ELISA, but less stressful on the operator and more rapid.

Naturally occurring human serum precipitins specific for D-alanyl esters of glycerol teichoic acid.

Abstract An antigen common to various oral bacteria and to which precipitating IgG is present in about 30% of young adult human sera was obtained from culture filtrates of Streptococcus mutans GS-5 and detected by electroimmunoassay as described elsewhere. The antigen was isolated by gel filtration, affinity chromatography with Sephacryl and finally passed through a strong anion exchange gel (AGMPI) to give a major peak (S 1 ) and a long tail (S 2 and S 3 ). These three preparations, yielding about 10% of the starting antigen activity, were composed of protein, fatty acids, sugar, glycerol and phosphate. Glycerol and phosphate accounted for 26% of the dry weight of the S 1 -preparation and in all preparations were in an approximately 1:1 molar ratio typical of glycerol teichoic acid. The S 1 -fraction had a greater sugar and fatty acid content and a different amino acid composition compared to the S 2 - and S 3 -preparations which were similar in these respects. On exposure to pH 8.0 buffer, the precipitin activity disappeared and this was accompanied by a loss of alanine. Shorter exposure to the pH 8.0 buffer caused a slightly increased electrophoretic mobility compared to non-alkali-exposed antigen and a lesser loss of alanine. Hapten precipitin inhibition assays indicated that d -alanine and d -alanyl ethyl ester inhibited the reaction much more effectively than l -alanyl ethyl ester, l -alanine, or d - or l -glutamine. These human sera are thus specific for d -alanyl esters of glycerol teichoic acid. Two possible structures bearing the antigenic determinant are described.

Bacterial Lysine Decarboxylase Influences Human Dental Biofilm Lysine Content, Biofilm Accumulation, and Subclinical Gingival Inflammation

BACKGROUND Dental biofilms contain a protein that inhibits mammalian cell growth, possibly lysine decarboxylase from Eikenella corrodens. This enzyme decarboxylates lysine, an essential amino acid for dentally attached cell turnover in gingival sulci. Lysine depletion may stop this turnover, impairing the barrier to bacterial compounds. The aims of this study are to determine biofilm lysine and cadaverine contents before oral hygiene restriction (OHR) and their association with plaque index (PI) and gingival crevicular fluid (GCF) after OHR for 1 week. METHODS Laser-induced fluorescence after capillary electrophoresis was used to determine lysine and cadaverine contents in dental biofilm, tongue biofilm, and saliva before OHR and in dental biofilm after OHR. RESULTS Before OHR, lysine and cadaverine contents of dental biofilm were similar and 10-fold greater than in saliva or tongue biofilm. After 1 week of OHR, the biofilm content of cadaverine increased and that of lysine decreased, consistent with greater biofilm lysine decarboxylase activity. Regression indicated that PI and GCF exudation were positively related to biofilm lysine after OHR, unless biofilm lysine exceeded the minimal blood plasma content, in which case PI was further increased but GCF exudation was reduced. CONCLUSIONS After OHR, lysine decarboxylase activity seems to determine biofilm lysine content and biofilm accumulation. When biofilm lysine exceeds minimal blood plasma content after OHR, less GCF appeared despite more biofilm. Lysine appears important for biofilm accumulation and the epithelial barrier to bacterial proinflammatory agents. Inhibiting lysine decarboxylase may retard the increased GCF exudation required for microbial development and gingivitis.

Effects of immunization with natural and recombinant lysine decarboxylase on canine gingivitis development

Periodontal disease, gingival inflammation (gingivitis) and periodontal attachment loss (periodontitis), causes tooth loss and susceptibility to chronic inflammation. Professionally scaling and cleaning the teeth regularly controls the disease, but is expensive in companion animals. Eikenella corrodens is common in canine oral cavities where it is a source of lysine decarboxylase (LDC). In human dental biofilms (plaques), LDC converts lysine to cadaverine and impairs the gingival epithelial barrier to bacteria. LDC vaccination may therefore retard gingivitis development. Year-old beagle dogs provided blood samples, and had weight and clinical measurements (biofilm and gingivitis) recorded. After scaling and cleaning, two dogs were immunized subcutaneously with 0.2mg native LDC from E. corrodens and 2 sets of four dogs with 0.2mg recombinant LDC purified from Escherichia coli. A third set of 4 dogs was immunized intranasally. Rehydragel(®), Emulsigen(®), Polygen™ or Carbigen™ were used as adjuvant. Four additional pairs of dogs were sham-immunized with each adjuvant alone (controls). Immunizations were repeated twice, 3 weeks apart, and clinical measurements were obtained after another 2 weeks, when the teeth were scaled and cleaned again. Tooth brushing was then stopped and the diet was changed from hard to soft chow. Clinical measurements were repeated after 1, 2, 3, 4, 6 and 8 weeks. Compared with sham-immunized dogs, gingivitis was reduced over all 8 weeks of soft diet after subcutaneous immunization with native LDC, or after intranasal immunization with recombinant LDC in Carbigen™, but for only 6 of the 8 weeks after subcutaneous immunization with recombinant LDC in Emulsigen(®) (repeated measures ANOVA). Subcutaneous vaccination induced a strong serum IgG antibody response that decreased during the soft diet period, whereas intranasal immunization induced a weak serum IgA antibody response that did not decrease. Immunization with recombinant LDC may provide protection from gingivitis if procedures are optimized.

Analysis of the specificity of natural human antibody reactive to Actinomyces

The specificity of a frequently-occurring precipitin response to soluble antigens from cell-walls and culture filtrates of A. viscosus ATCC 19246 was examined. After precipitation with isopropanol (50-75% v/v), antigen fractions of different charge and molecular weight were isolated by ion exchange and gel filtration. When heated in mineral acid or alkali above 0.15 M, each of the purified antigens lost precipitating activity, but now inhibited the precipitin reaction between serum and exogenous unheated antigen. The inhibitor was isolated over Biogel P30 and characterized as a peptide fragment (mol. wt about 2 kd) containing approximately 50 moles of ornithine and 6-12 moles, respectively, of aspartate, serine, threonine, glutamate, glycine, alanine and histidine per 100 moles amino acids. The inhibitor was totally destroyed by heating for 1.0 hr in 2.0 M HCl. Variability in the number of fragments and differences in the non-antigenic portions probably accounted for the complexity of the antigens. Ornithine, putrescine, N-acetyl putrescine and various sugars had little or no effect on the precipitin reaction with intact antigen at high concentrations (200 mM), whereas the fragment inhibited completely at 0.4 mM. This indicates that neither ornithine nor its side-chain amides are exclusively recognized by antibody. However, ornithine may be part of a larger sequence and/or important in forming the configuration recognized by the human antibodies.

Biofilm Lysine Decarboxylase, a New Therapeutic Target for Periodontal Inflammation

BACKGROUND Lysine, a nutritionally essential amino acid, enters the oral cavity in gingival crevicular fluid (GCF). During oral hygiene restriction (OHR), lysine decarboxylase (LDC) in dento-gingival biofilms converts lysine to cadaverine. Lysine depletion impairs the dental epithelial barrier to bacterial proinflammatory products. Antibodies to LDC from Eikenella corrodens (Ecor-LDC) inhibit LDC activity and retard gingival inflammation in beagle dogs. Whether E. corrodens is the major source of LDC in dental biofilms and whether the lysine analog tranexamic acid (TA) inhibits LDC activity, biofilm accumulation, and GCF exudation in a human gingivitis model were examined. METHODS Antibodies raised in goats to LDC-rich extracts from E. corrodens cell surfaces were used to inhibit Ecor-LDC and detect it in biofilm extracts using Western blots. Ecor-LDC activity was measured at pH 4.0 to 11.0 and its TA dissociation constant (Ki) at pH 7.0. Young adults used a 5% or 10% TA mouthwash three times daily during OHR for 1 week. RESULTS Ecor-LDC antibodies and TA inhibited biofilm LDC. Ki of TA for Ecor-LDC was 940 μM. TA reduced plaque index (PI) by downshifting the PI correlation with biofilm lysine content after OHR without TA. GCF was correspondingly suppressed. However, greater TA retention in saliva partially relieved GCF suppression but not biofilm lysine depletion. CONCLUSIONS TA slightly inhibits LDC but strongly reduces biofilm by inhibiting bacterial lysine uptake. Unfortunately, TA may impair dental epithelial attachments by also inhibiting lysine transporter uptake. Ecor-LDC inhibitors other than lysine analogs may maintain sufficient lysine levels and attachment integrity to prevent periodontal inflammation.

Salivary Proteins May Be Useful for Determining Caries Susceptibility

ARTICLE TITLE AND BIBLIOGRAPHIC INFORMATION Salivary proteins as a biomarker for dental caries-A systematic review. Martins C, Buczynski AK, Maia LC, Siqueira WL, Castro GF. J Dent 2013; 41(1): 2-8. REVIEWER Martin Levine, BDS, BSc, PhD PURPOSE/QUESTION: What is the evidence for an association between individual salivary protein composition or content and dental caries experience? SOURCE OF FUNDING Canadian Government Institutes of Health Research TYPE OF STUDY/DESIGN Systematic review LEVEL OF EVIDENCE Level 2: Limited-quality, patient-oriented evidence STRENGTH OF RECOMMENDATION GRADE Grade B: Inconsistent or limited-quality patient-oriented evidence.

Elevated antibody to D-alanyl lipoteichoic acid indicates caries experience associated with fluoride and gingival health

BackgroundAcidogenic, acid-tolerant bacteria induce dental caries and require D-alanyl glycerol lipoteichoic acid (D-alanyl LTA) on their cell surface. Because fluoride inhibits acid-mediated enamel demineralization, an elevated antibody response to D-alanyl LTA may indicate subjects with more acidogenic bacteria and, therefore, an association of DMFT with fluoride exposure and gingival health not apparent in low responders.MethodsCluster analysis was used to identify low antibody content. Within low and high responders (control and test subjects), the number of teeth that were decayed missing and filled (DMFT), or decayed only (DT) were regressed against fluoride exposure in the water supply and from dentrifice use. The latter was determined from gingival health: prevalences of plaque (PL) and bleeding on probing (BOP), and mean pocket depth (PD). Age was measured as a possible confounding cofactor.ResultsIn 35 high responders, DMFT associated with length of exposure to fluoridated water (F score), PL and BOP (R2 = 0.51, p < 0.001), whereas in 67 low D-ala-IgG responders, DMFT associated with PL, age, and PD (R2 = 0.26, p < 0.001). BOP correlated strongly with number of 7 7 decayed teeth (DT) in 54 high responders (R2 = 0.57, p < 0.001), but poorly in 97 low responders (R2 = 0.12, p < 0.001). The strength of the PD association with DMFT, or of BOP with DT, in high responders significantly differed from that in low responders (p < 0.05).ConclusionCaries associates with gingival health and fluoridated water exposure in high D-alanyl LTA antibody responders.