Kenneth Markowitz

Aggregatibacter actinomycetemcomitans and Its Relationship to Initiation of Localized Aggressive Periodontitis : Longitudinal Cohort Study of Initially Healthy Adolescents

ABSTRACT Aggregatibacter actinomycetemcomitans is frequently associated with localized aggressive periodontitis (LAP); however, longitudinal cohort studies relating A. actinomycetemcomitans to initiation of LAP have not been reported. A periodontal assessment was performed on 1,075 primarily African-American and Hispanic schoolchildren, ages 11 to 17 years. Samples were taken from each child for A. actinomycetemcomitans. A cohort of 96 students was established that included a test group of 38 A. actinomycetemcomitans-positive students (36 periodontally healthy and 2 with periodontal pockets) and 58 healthy A. actinomycetemcomitans-negative controls. All clinical and microbiological procedures were repeated at 6-month intervals. Bitewing radiographs were taken annually for definitive diagnosis of LAP. At the initial examination, clinical probing attachment measurements indicated that 1.2% of students had LAP, while 13.7% carried A. actinomycetemcomitans, including 16.7% of African-American and 11% of Hispanic students (P = 0.001, chi-square test). A. actinomycetemcomitans serotypes a, b, and c were equally distributed among African-Americans; Hispanic students harbored predominantly serotype c (P = 0.05, chi-square test). In the longitudinal phase, survival analysis was performed to determine whether A. actinomycetemcomitans-positive as compared to A. actinomycetemcomitans-negative students remained healthy (“survived”) or progressed to disease with attachment loss of >2 mm or bone loss (failed to “survive”). Students without A. actinomycetemcomitans at baseline had a significantly greater chance to remain healthy (survive) compared to the A. actinomycetemcomitans-positive test group (P = 0.0001). Eight of 38 A. actinomycetemcomitans-positive and none of 58 A. actinomycetemcomitans-negative students showed bone loss (P = 0.01). A. actinomycetemcomitans serotype did not appear to influence survival. These findings suggest that detection of A. actinomycetemcomitans in periodontally healthy children can serve as a risk marker for initiation of LAP.

Pretty painful: Why does tooth bleaching hurt?

Vital bleaching procedures are a popular means of improving the appearance of discolored teeth. There is a wide array of whitening products for home and dental office use; all involves placing peroxide containing gels or solutions in contact with the teeth. In order to whiten teeth peroxide has to be able to penetrate tooth structure and oxidize colored compounds in the dentin. Unfortunately beauty comes with a price; many patients undergoing peroxide based whitening procedures complain of bleaching sensitivity (BS) arising in the treated teeth. In BS, pain can occur in healthy intact teeth without any provoking stimulus. Currently the mechanism of nociceptors activation in BS is unknown. A more common form of dental pain-dentin sensitivity (DS) occurs when stimuli such as cold or tactile stimulation contact areas of exposed dentin in otherwise healthy teeth. In DS, stimulation of the dentin results in fluid shifts in the dentinal tubules, these fluid shifts activate mechanosensitive nerve endings in the deep dentin and pulp. Since many aspects of BS and DS symptoms differ, it is hypothesized that that the mechanism of pain generation differs for these two conditions. Recently the functional properties of a chemosensitive ion channel-TRPA1 have been described. This channel is activated by a variety of oxidizer compounds including hydrogen peroxide. Pulpal sensory afferents express TRPA1. It is hypothesized that direct activation of intradental nerve activity via TRPA1 is the mechanism of BS pain. If this theory were correct, tooth sensitivity treatments that reduce the excitability of the intradental nerves such as potassium salts, would be the treatment of choice for BS.

A Consortium of Aggregatibacter actinomycetemcomitans, Streptococcus parasanguinis, and Filifactor alocis Is Present in Sites Prior to Bone Loss in a Longitudinal Study of Localized Aggressive Periodontitis

ABSTRACT Aggregatibacter actinomycetemcomitans-induced localized aggressive periodontitis (LAP) in African-American adolescents has been documented but is poorly understood. Two thousand fifty-eight adolescents aged 11 to 17 years were screened for their periodontal status and the presence of A. actinomycetemcomitans in their oral cavity. Seventy-one A. actinomycetemcomitans-negative and 63 A. actinomycetemcomitans-positive periodontally healthy subjects were enrolled, sampled, examined, and radiographed yearly for 3 years. Gingival and periodontal pocket depth and attachment levels were recorded. Disease presentation was characterized by bone loss (BL). Subgingival sites were sampled every 6 months to assess (i) the role of A. actinomycetemcomitans in BL and (ii) the association of A. actinomycetemcomitans and other microbes in their relationships to BL. Sixteen of 63 subjects with A. actinomycetemcomitans developed BL (the other 47 subjects with A. actinomycetemcomitans had no BL). No A. actinomycetemcomitans-negative subjects developed BL. Human oral microbe identification microarray (HOMIM) was used for subgingival microbial assessment. On a subject level, pooled data from A. actinomycetemcomitans-positive subjects who remained healthy had higher prevalences of Streptococcus and Actinomyces species, while A. actinomycetemcomitans-positive subjects with BL had higher prevalences of Parvimonas micra, Filifactor alocis, A. actinomycetemcomitans, and Peptostreptococcus sp. human oral taxon 113 (HOT-113). At vulnerable sites, A. actinomycetemcomitans, Streptococcus parasanguinis, and F. alocis levels were elevated prior to BL. In cases where the three-organism consortium (versus A. actinomycetemcomitans alone) was detected, the specificity for detecting sites of future BL increased from 62% to 99%, with a sensitivity of 89%. We conclude that detecting the presence of A. actinomycetemcomitans, S. parasanguinis, and F. alocis together indicates sites of future BL in LAP. A synergistic interaction of this consortium in LAP causation is possible and is the subject of ongoing research.

Mechanotransducers in Rat Pulpal Afferents

The hydrodynamic theory suggests that pain associated with stimulation of a sensitive tooth ultimately involves mechanotransduction as a consequence of fluid movement within exposed dentinal tubules. To determine whether putative mechanotransducers could underlie mechanotransduction in pulpal afferents, we used a single-cell PCR approach to screen retrogradely labeled pulpal afferents. The presence of mRNA encoding BNC-1, ASIC3, TRPV4, TRPA1, the α, β, and γ subunits of ENaC, and the two pore K+ channels (TREK1, TREK2) and TRAAK were screened in pulpal neurons from rats with and without pulpal inflammation. ASIC3, TRPA1, TREK1, and TREK2 were present in ~67%, 64%, 14%, and 10% of pulpal neurons, respectively. There was no detectable influence of inflammation on the proportion of neurons expressing these mechanotransducers. Given that the majority of pulpal afferents express ASIC3 and TRPA1, our results raise the possibility that these channels may be novel targets for the treatment of dentin sensitivity.

Activity of antimicrobial peptide mimetics in the oral cavity: I. Activity against biofilms of Candida albicans

Naturally occurring antimicrobial peptides hold promise as therapeutic agents against oral pathogens such as Candida albicans but numerous difficulties have slowed their development. Synthetic, non-peptidic analogs that mimic the properties of these peptides have many advantages and exhibit potent, selective antimicrobial activity. Several series of mimetics (with molecular weight < 1000) were developed and screened against oral Candida strains as a proof-of-principle for their antifungal properties. One phenylalkyne and several arylamide compounds with reduced mammalian cytotoxicities were found to be active against C. albicans. These compounds demonstrated rapid fungicidal activity in liquid culture even in the presence of saliva, and demonstrated synergy with standard antifungal agents. When assayed against biofilms grown on denture acrylic, the compounds exhibited potent fungicidal activity as measured by metabolic and fluorescent viability assays. Repeated passages in sub-minimum inhibitory concentration levels did not lead to resistant Candida, in contrast to fluconazole. Our results demonstrate the proof-of principle for the use of these compounds as anti-Candida agents, and their further testing is warranted as novel anti-Candida therapies.

Aggregatibacter actinomycetemcomitans as an Early Colonizer of Oral Tissues: Epithelium as a Reservoir?

ABSTRACT This study examined in vivo and in vitro colonization by Aggregatibacter actinomycetemcomitans, an organism highly associated with aggressive periodontitis. Thirteen volunteers (5 were A. actinomycetemcomitans positive for buccal epithelial cells [BECs] and teeth, 5 were A. actinomycetemcomitans positive for teeth only, and 3 were A. actinomycetemcomitans-negative controls) had two mandibular stents fabricated. Each stent contained 3 removable hydroxyapatite (HA) tooth surrogates. One HA square was removed from a stent at 5 time points over 7 h to assess the transfer of A. actinomycetemcomitans from teeth or BECs to HA. Streptococcus, Actinomyces, A. actinomycetemcomitans, and total anaerobic counts were evaluated on each square over time. In vitro experiments evaluated binding, desorption, transfer, and reattachment of A. actinomycetemcomitans wild-type and mutant strains to BECs and saliva-coated HA (SHA). Streptococcus and Actinomyces formed 80% of the cultivable flora on HA in all subjects. Transfer of A. actinomycetemcomitans to HA was not seen in subjects with A. actinomycetemcomitans on teeth only. All 5 subjects with A. actinomycetemcomitans on BECs showed transfer of A. actinomycetemcomitans to HA. In vitro, A. actinomycetemcomitans desorbed from BECs and transferred to SHA. A. actinomycetemcomitans binding to SHA was irreversible and did not transfer to BECs. The adhesin Aae showed specificity for BECs. Fimbrial mutants showed the greatest reduction in binding to SHA. A. actinomycetemcomitans migrated from BECs to HA in vivo and to SHA in vitro; however, A. actinomycetemcomitans movement from teeth and SHA to BECs did not occur. In vivo, A. actinomycetemcomitans colonized HA within 6 h and thus can be considered an early colonizer. BECs are a likely reservoir for A. actinomycetemcomitans tooth colonization.

Lactoferrin Knockout Mice Demonstrates Greater Susceptibility to Aggregatibacter actinomycetemcomitans–Induced Periodontal Disease

BACKGROUND Among the innate defense mechanisms in the oral cavity, lactoferrin (LF) is a vital antimicrobial that can modify the host response against periodontopathogens. Aggregatibacter actinomycetemcomitans is the main periodontopathogen of localized aggressive periodontitis. The aim of this study is to evaluate the role of LF during A. actinomycetemcomitans-induced periodontitis. METHODS Differences in the expression levels of cytokines, chemokines, chemokine receptors, and bone loss markers between wild-type (WT) and LF knockout mice (LFKO(-/-)) were evaluated by real time-PCR. Serum IgG and LF levels were quantified by ELISA. Alveolar bone loss among the groups was estimated by measuring the distance from cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) at 20 molar sites. RESULTS Oral infection with A. actinomycetemcomitans increased LF levels in periodontal tissue (P = 0.01) and saliva (P = 0.0004) of wild-type infected (WTI) mice compared to wild-type control mice. Pro-inflammatory cytokines such as interferon-γ, tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-12 were increased in the infected LF knockout (LFKO(-/-)I) mice compared to the WTI mice, whereas the anti-inflammatory cytokines IL-4 and IL-10 were decreased. Chemokines and chemokine receptors showed different expression patterns between WTI and LFKO(-/-)I mice. The LFKO(-/-)I mice developed increased bone loss (P = 0.002), in conjunction with increased expression of receptor activator of nuclear factor-κB ligand and decrease in osteoprotegerin, compared to WTI mice. CONCLUSIONS These results demonstrate that the infected LFKO(-/-) mice were more susceptible to A. actinomycetemcomitans-induced alveolar bone loss, with different patterns of immune responses compared to those of WTI mice.

Aggregatibacter actinomycetemcomitans-induced bone loss and antibody response in three rat strains.

BACKGROUND The aim of this study is to compare the colonization, immunoglobulin (Ig) G response, and alveolar bone loss in Aggregatibacter actinomycetemcomitans (Aa)-inoculated Fawn Hooded Hypertensive (FHH), Dahl Salt-Sensitive (DSS), and Brown Norway (BN) rats. METHODS Each rat strain was divided into wild-type Aa-inoculated and non-inoculated control groups. Blood taken at 12 weeks after inoculation was assessed for Aa-specific IgG antibodies by an enzyme-linked immunosorbent assay. Colonization was assessed 12 weeks postinoculation. Bone loss was estimated by measuring the distance from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) at 20 molar sites. Colonization and antibody levels were compared by using the Student t test. Diseased rats were defined as having two sites per quadrant with CEJ-ABC distances that were significantly greater than the control CEJ-ABC distances. RESULTS The Aa colonization of FHH rats was significantly higher than in other strains (P <0.05). The Aa-specific IgG levels in the DSS Aa-inoculated group were significantly higher than in its control group (P <0.05). Only FHH rats showed Aa disease-associated bone loss (P = 0.0021). CONCLUSIONS Aa colonized and caused more disease in FHH rats than in the other rat strains. The rat strains each responded differently to the same Aa strain.

Activity of Potent and Selective Host Defense Peptide Mimetics in Mouse Models of Oral Candidiasis

ABSTRACT There is a strong need for new broadly active antifungal agents for the treatment of oral candidiasis that not only are active against many species of Candida, including drug-resistant strains, but also evade microbial countermeasures which may lead to resistance. Host defense peptides (HDPs) can provide a foundation for the development of such agents. Toward this end, we have developed fully synthetic, small-molecule, nonpeptide mimetics of the HDPs that improve safety and other pharmaceutical properties. Here we describe the identification of several HDP mimetics that are broadly active against C. albicans and other species of Candida, rapidly fungicidal, and active against yeast and hyphal cultures and that exhibit low cytotoxicity for mammalian cells. Importantly, specificity for Candida over commensal bacteria was also evident, thereby minimizing potential damage to the endogenous microbiome which otherwise could favor fungal overgrowth. Three compounds were tested as topical agents in two different mouse models of oral candidiasis and were found to be highly active. Following single-dose administrations, total Candida burdens in tongues of infected animals were reduced up to three logs. These studies highlight the potential of HDP mimetics as a new tool in the antifungal arsenal for the treatment of oral candidiasis.

Macrophage inflammatory protein-1α shows predictive value as a risk marker for subjects and sites vulnerable to bone loss in a longitudinal model of aggressive periodontitis.

Improved diagnostics remains a fundamental goal of biomedical research. This study was designed to assess cytokine biomarkers that could predict bone loss (BL) in localized aggressive periodontitis. 2,058 adolescents were screened. Two groups of 50 periodontally healthy adolescents were enrolled in the longitudinal study. One group had Aggregatibacter actinomycetemcomitans (Aa), the putative pathogen, while the matched cohort did not. Cytokine levels were assessed in saliva and gingival crevicular fluid (GCF). Participants were sampled, examined, and radiographed every 6 months for 2–3 years. Disease was defined as radiographic evidence of BL. Saliva and GCF was collected at each visit, frozen, and then tested retrospectively after detection of BL. Sixteen subjects with Aa developed BL. Saliva from Aa-positive and Aa-negative healthy subjects was compared to subjects who developed BL. GCF was collected from 16 subjects with BL and from another 38 subjects who remained healthy. GCF from BL sites in the 16 subjects was compared to healthy sites in these same subjects and to healthy sites in subjects who remained healthy. Results showed that cytokines in saliva associated with acute inflammation were elevated in subjects who developed BL (i.e., MIP-1α MIP-1β IL-α, IL-1β and IL-8; p<0.01). MIP-1α was elevated 13-fold, 6 months prior to BL. When MIP-1α levels were set at 40 pg/ml, 98% of healthy sites were below that level (Specificity); whereas, 93% of sites with BL were higher (Sensitivity), with comparable Predictive Values of 98%; p<0.0001; 95% C.I. = 42.5–52.7). MIP-1α consistently showed elevated levels as a biomarker for BL in both saliva and GCF, 6 months prior to BL. MIP-1α continues to demonstrate its strong candidacy as a diagnostic biomarker for both subject and site vulnerability to BL.