Porphyromonas gingivalis produces proteases capable of degrading cytokines, host heme proteins, and some antimicrobial peptides. In this work, I show that P. gingivalis culture supernatants fully or partially degrade human neutrophil peptide alpha-defensins and human beta-defensins after 30 minutes. This observation suggests that proteases from P. gingivalis degrade defensins and this activity could abrogate defensin-related innate immune functions.

Antimicrobial peptides are among the repertoire of host innate immune defenses. In mucosal immunity, the health-disease balance can be greatly modulated by the interplay between host immune factors and colonized microflora. Microbial ecology within dental plaque is constantly shaped by environmental factors present within the oral cavity. Several antimicrobial peptides are detected in saliva and their bactericidal activities against oral bacteria, including Streptococcus mutans, the primary etiologic agent of dental caries, have been clearly demonstrated. However, the role of these antimicrobial peptides in S .mutans ecology and host caries experience is not well-defined. We hypothesized that various strains of S. mutans possess different inherent susceptibility/resistance profiles to host salivary antimicrobial peptides and that host-specific quantities of these peptides may influence plaque colonization by particular S. mutans strains. S. mutans strains from subjects with variable caries experience were tested for susceptibility to a panel of antimicrobial peptides, including HNP-1-3, HBD-2-3 and LL-37, revealing that the susceptibilities of S. mutans to these peptides were strain-specific. S. mutans strains from high caries subjects showed greater resistance to these peptides at varying concentrations than those from caries-free subjects. In addition, when combinations of these peptides were tested, they showed either additive or synergistic interaction against S. mutans. Determinations of the salivary levels of these peptides showed that their concentrations were highly variable among subjects with no correlation to host caries experience. However, positive relationships between the salivary concentrations of HNP-1-3 and MS in dental plaque were found. Additionally, the levels of a number of these peptides in saliva appeared to be positively correlated within an individual. An analysis of the salivary peptide concentrations and the susceptibility profiles of S. mutans strains showed that S. mutans strains obtained from subjects with higher concentration of HNP-1-3 in saliva appeared to be more resistant to HNP-1. Collectively, our findings showed that salivary antimicrobial peptides affect S. mutans ecology by restricting the overall growth of this bacterium within the oral cavity and that their activity may help select resistant strains of S. mutans to colonize within dental plaque. The relative ability of S. mutans to resist host salivary antimicrobial peptides may be considered a potential virulence factor for this species.

The objective was to determine if a correlation exists between Streptococcus mutans and DMFT in the oral cavity. This study examined the feasibility of microbial screenings as an additional caries predictor tool. The sample included 108 participants (ages 18-25) in low, moderate, and high socioeconomic groups. Subjects were selected from one dental clinic and one college in Virginia. Subjects were assessed for DMFT, salivary and plaque bacterial load, and CRA. Salivary load positively correlated with the DMFT. Plaque bacterial load and CRA negatively correlated with DMFT. Comparison of salivary bacterial load among economic levels showed higher bacterial loads with lower economic level only. Twice the observed values were found in the low socioeconomic level with CRA. DMFT and economic level showed differences between economic levels. Microbial screenings may be a useful, additional tool in determining caries risk in oral hygiene programs for all income populations.

Aim: The aim of this study is to determine the varying levels of salivary statherin production in patients with varying levels of risk for diabetes. The goal is to identify a causal relationship and thus, statherin could be used as a preliminary biomarker for identifying patients with diabetes. Materials and Methods: Saliva from 47 participants were collected in order to quantify the levels of statherin production via western blot analysis. Participants were also asked to fill out self-reported questionnaires regarding risk factors for type 2 diabetes. The questionnaire consisted of 7 questions regarding age, sex, history of diabetes, hypertension, level of physical activity, and weight class. Each individual factor as well as total risk for type two diabetes was compared to levels of salivary statherin levels via the unpaired t-test and one way ANOVA testing. Results: Risk factors for type two diabetes such as; age, sex, history of diabetes, hypertension, level of physical activity, and weight class showed no correlation to levels of salivary statherin secretion. All risk factors combined as a total risk level for type two diabetes also did not show a correlation to levels of salivary statherin secretion. Conclusions: It can be concluded in this study that salivary statherin protein does not show a correlation for risk or status of type two diabetes. Salivary statherin does not act as a useful biomarker for detection of type two diabetes.

Objectives: Tooth replacement therapy using endosseous implants has become an essential component of contemporary dental practice. While a plethora of factors determine clinical success, the bucco-lingual and apico-coronal dimensions of the peri-implant mucosa play an important role in both esthetics and the maintenance of peri-implant health. Studies, most of which treat mucogingival defects in the natural dentition, comparing acellular dermal matrix (ADM) and autologous subepithelial connective tissue grafts (sCTG) have shown similar clinical outcomes. The purpose of this non-inferiority trial is to determine the clinical efficacy of ADM in the augmentation of peri-implant mucosa thickness (PMT) as compared to an autologous sCTG in human adults. Methods: Twenty healthy adults treatment planned for a single tooth implant restoration in need of simultaneous peri-implant mucosa augmentation at the time of implant placement were recruited on the basis of an eligibility criteria. Patients were randomly assigned to the control group (autologous sCTG), or the experimental group (ADM allograft). Clinical measurements of mucosal thickness at the site were made with a periodontal probe and an endodontic spreader at baseline and 16 weeks post-op. These measurements were made by a masked, calibrated examiner. Gingival health, oral hygiene, wound healing and patient reported outcomes were also obtained. Mann-Whitney U tests were used to compare the mean mucosal thickness changes between the groups. Results: The mean gain in PMT was approximately 1.5mm in the control group and 0.8mm in the experimental group. When measured at 1, 3 and 5mm apical from the CEJ, only the 3mm site exhibited a difference between the groups that approached statistical significance (control: 2.08 ± 0.80mm, test: 0.83 ± 1.37mm, Mann Whitney U = 10.00, p=0.05). Changes in keratinized mucosa width, healing index and patient reported outcomes were generally similar between the two groups. Conclusions: Within the limitations of this study, both autologous sCTG and ADM appear to be adequate materials to augment PMT without sacrificing other relevant clinical parameters and/or patient related outcomes.

The treatment of deep carious lesions (DCLs) represents a challenge for dentists, as these lesions require removal of dentin in proximity to the pulp that represents a higher risk of ending in pulp exposure. There is increasing evidence supporting minimally invasive caries removal methods, such as stepwise excavation procedure (SWP), instead of non-selective (complete) dentin caries removal for the treatment of DCLs. However, recent research has indicated that dentists have not completely adopted less invasive caries removal methods for the management of DCLs. These studies have been primarily conducted in Europe or South America; thus, few studies have been conducted in the United States to understand dentists’ management options of deep carious lesions in permanent teeth. Additionally, the proportion of dentists who would be willing to adopt less invasive carious tissue removal is unknown, especially in the United States. This dissertation consists of three studies that analyzed the effectiveness of SWP, the factors associated with success, an assessment of its costs and the acceptability of this treatment among U.S dentists. Study I assessed the patient factors predicting a successful SWP by retrospectively investigating a sample from the University of Iowa College of Dentistry from January 2004 through December 2012. Study I showed that SWPs showed a 75% success rate when evaluated within 36 months of the initial treatment. Patients who had a successful SWP treatment were slightly younger than patients whose SWP treatment was not successful. Study II performed a cost-effectiveness analysis by comparing SWP vs. the standard caries treatment (complete caries removal). Study II showed that SWP significantly reduced cost, resulting in an average savings of 64% compared to TCR over a 5-year period. Study III consisted of a statewide survey that contacted 1,434 Iowa dentists with the aim of identifying the most important factors that influence dentists’ decisions for DCLs treatment using conjoint analysis. From the total, 36.4% (n=522) clinicians answered the survey. One of the main study findings was that among the three factors (hardness of dentin, depth of the lesion and patient age) depth of the lesion was the most important factor influencing dentists’s decision when selecting a less invasive caries removal method. Study III did not find any significant association between years of practicing dentistry, type of practice and practice setting regarding being willing to select a less invasive caries removal method. In conclusion, this dissertation has demonstrated that the stepwise caries removal is a cost-saving and highly successful treatment for the management of deep caries lesions. Studies I and II have shown that this method has a high clinical success rate and less long-term treatment costs. Regarding the acceptability of SWP among clinicians, this dissertation has shown that most dentists in Iowa would consider less invasive caries methods for a DCL; however, these techniques have not been completely adopted and accepted among American dentists. Thus, dentists should be trained in less invasive caries methods, given instruction about which criteria they should use to assess a DCL, and provide them with the best evidence-based strategies for managing deep lesions. The findings of this dissertation should engage stakeholders to provide dentists with professional incentives for using these techniques. Specifically, our results support the idea that SWP minimizes costs while providing the appropriate patient care.

The TRUShape 3D Conforming File (TRUShape), a novel, S-shaped nickel-titanium (NiTi) rotary file, was developed to facilitate cleaning and shaping of irregular-shaped root canals. The purpose of this study was to evaluate the shaping ability of TRUShape compared to Vortex Blue (VB) when used in non-round, oval-shaped root canals by micro-computed tomography (MCT). Thirty single-rooted human mandibular premolar teeth with radiographically similar root canal size and curvature were randomly allocated to two groups (N=15), and mechanically prepared with TRUShape or VB. Each tooth was submitted to MCT at 20 μm resolution at three time intervals: before shaping, and after shaping to an intermediate apical size 30 and a final apical size 40. Three-dimensional data sets were superimposed and evaluated for root canal volume, surface area, and treated surface. Matched axial slices in the apical, middle, and coronal root thirds were evaluated for area, roundness, and canal transportation expressed as center of mass shift (CMS). Data were statistically analyzed using parametric and non-parametric tests. Root canal volumes increased similarly and significantly overall (p< 0.001; from an initial volume of 7.3±3.5 mm3 to an intermediate volume of 8.7±3.1 mm3 and a final volume of 9.9±3.0 mm3). Treated canal surface was significantly larger in the TRUShape group at both apical sizes 30 and 40 with 72±15% vs. 55±23% and 85±12% vs. 71±20% non-static voxels for TRUShape and VB, respectively (p< 0.05). Canal transportation was less than 100 μm in all but 8 out of 90 cross sections and was not significantly different between groups. This MCT study demonstrated the TRUShape 3D Conforming File to be effective in the mechanical preparation, specifically, the surface treatment, of single-rooted premolars with non-round, oval-shaped root canals. Funding was provided by the American Association of Endodontists (AAE) Foundation and the Department of Endodontics at the University of Iowa, College of Dentistry.

Background: The role of cytokines in the pathogenesis of periodontal disease and the effect of smoking on these mediators has been reported. However, findings have been variable and simultaneous measurement of multiple cytokines has been limited. This study utilized a quantitative multiplex assay to measure a comprehensive panel of Th1, Th2, and pro-inflammatory cytokines and chemokines (including several novel cytokines) in gingival crevicular fluid (GCF) in chronic periodontitis subjects. The impact of cigarette smoking on these GCF mediators was also assessed. Methods: Forty subjects (age 40-75 years) with generalized severe chronic periodontitis (20 smokers and 20 non-smokers) and 12 periodontally healthy controls participated in the study. GCF was collected from four sites in the diseased groups: 2 diseased sites [(probing depth (PD) and clinical attachment level (CAL) ≥5mm with bleeding on probing (BOP)] and 2 healthy sites (PD and CAL ≤3mm, no BOP); 2 healthy sites were sampled in the healthy controls. The volumes of the GCF samples were measured and the GCF mediators assessed in duplicate utilizing a multiplex immunoassay (Luminex). Intragroup, intergroup and pooled comparisons were performed using non-parametric tests including the Mann-Whitney and the Wilcoxon matched-pairs signed-rank test. Results: GCF in diseased sites (vs. controls) contained significantly (p<0.05) higher amounts of IL-2, IFN-γ, IL-3, IL-4 (Th1/Th2 cytokines); IL-1α, IL-1β, IL-6, IL-12 (p40) (pro-inflammatory cytokines); IL-8, MIP-1, RANTES (chemokines); and IL-15 (regulator of T-cells and NK cells). IP-10 was the only mediator exhibiting lower levels (p<0.0005) in diseased sites compared to controls. Smoking had an inhibitory effect (p<0.05) on pro-inflammatory cytokines (IL-1α, IL-6, IL-12 (p40)); chemokines (IL-8, IP-10, MCP-1, MIP-1, RANTES) and regulators of T-cells and NK cells (IL-7, IL-15) in comparison to sites within non-smokers. Interestingly, smokers had elevated GCF levels (p<0.05) of IL-1α, IL-1β and IL-3 relative to sites in healthy controls. Conclusions: Similar to previous reports, periodontitis subjects had significantly elevated cytokines and chemokines compared to healthy controls. Smokers exhibited a decrease in several pro-inflammatory cytokines, chemokines and regulators of T-cells and NK-cells as compared to nonsmokers however; little influence was observed on Th1/Th2 cytokines. Interestingly, smokers exhibited decreased amounts of GCF IL-7, IL-12(p40), IL-15, IP-10, MCP-1, MIP-1 and RANTES, which calls for future investigation. The multiplex comprehensive assay used in this study to assess cytokines in a single GCF sample is a significant advancement. This technology can be used to compare serum and GCF cytokine profiles in periodontitis and correlate systemic and localized immune responses. This should provide insight into the impact of smoking, as well as other host modifiers, on important systemic and periodontal interactions.

Porphyromonas gingivalis, a Gram negative anaerobe implicated in the progression of periodontal disease, is capable of surviving and causing infection despite high levels of reactive nitrogen species found in the oral cavity due to its efficient nitrosative stress response. HcpR is an important sensor-regulator that plays a vital step in the initiation of the nitrosative stress response in many Gram negative anaerobic bacteria. We employ a combination of X-ray crystallography, SAXS, resonance Raman spectroscopy, UV-Vis spectroscopy, and molecular biology techniques to better understand this key regulator. Knockout of the hcpR gene in W83 P. gingivalis results in the inability of the bacteria to grow in physiological concentrations of nitrite and complementation of hcpR using the novel plasmid Pg108 rescues this phenotype. HcpR causes a drastic, dose dependent upregulation of PG0893, a gene coding for a putative NO reductase, when exposed to nitrite or nitric oxide. Full transcriptome sequencing reveals that hcp is the only significantly upregulated gene when P. gingivalis is exposed to nitrite and knockout of hcp resulted in a phenotype that is similar to that of the hcpR deficient strain. HcpR directly regulates the expression of hcp via direct binding to an inverted repeat sequence in the promoter region of the hcp gene. We present a 2.6 Å crystal structure of the N-terminal sensing domain of HcpR and show that it is FNR-CRP regulator. A putative hydrophobic heme binding pocket was identified in the junction between the N-terminal domain and the dimerization helix. Mutation of two methionine residues (Met68 and Met145) in this pocket abrogates activation of HcpR thus verifying the binding site. Heme bound to HcpR exhibits heme iron as a hexa-coordinate system in the absence of nitric oxide (NO) and upon nitrosylation transitions to a penta-coordinated system. Finally, Small Angle X-ray Scattering experiments of the full length HcpR reveal that the C-terminal DNA binding domain of HcpR has a high degree of interdomain flexibility.

Streptococcus sanguinis, an abundant and benign inhabitant of the oral cavity, is an important etiologic agent of infective endocarditis, particularly in people with pre-disposing cardiac valvular damage. Although commonly isolated from patients with IE, little is known about the factors that make any particular S. sanguinis isolate more virulent than another or, indeed, whether significant differences in virulence exist among isolates. To investigate the virulence of multiple isolates, a variation of the Bar-seq (barcode sequencing) method was employed. A conserved chromosomal site was identified for subsequent insertion of a barcode identifier, unique for each strain. Barcode insertion did not affect growth in vitro or in a rabbit model of endocarditis. Pooling of these strains and inoculation into rabbits demonstrated that all strains were capable of causing disease; however, virulence varied widely among strains. Genomic comparisons of the more virulent strains versus less virulent strains failed to conclusively identify any single gene responsible for virulence. Given this result, we continued our examination of the manganese transport system SsaACB, which is present in every strain of S. sanguinis examined. Although its contribution to virulence has not been confirmed in any strain other than SK36, it has been shown to be required for virulence in multiple species of streptococci, making it a candidate for emerging targeted therapies. In S. sanguinis strain SK36, previous studies have confirmed that loss of the manganese transport protein SsaB is tantamount to loss of virulence. Moreover, ssaB-deficient mutants are deficient for serum growth—a phenotype we have previously found to be associated with virulence. Our in vitro studies of manganese transporter-deficient strain SK36 supported this, but also revealed the emergence of suppressor mutants. In each suppressor mutant that was isolated, mutations were identified that mapped to a common gene, SSA_0696. Deletion of SSA_0696 resulted in restored in vitro growth in the ssaACB-deficient background, unearthing a novel mechanism for bacterial growth under manganese limitation. Fortunately, the suppressor mutant phenotype was not maintained in vivo; however, the combined results of these experiments suggest the efficacy of future therapeutics may require consideration of virulence at the species level and the incorporation of multiple targets.