Expanding assessments of translational research programs: supplementing metrics with value judgments.

The University of Washington (UW) Institute for Translational Health Sciences (ITHS), funded by a Clinical and Translational Sciences Award program, has supplemented its initial Kellogg Logic Model-based program evaluation with the eight judgment-based evaluative elements of the World Health Organization's (WHO) Health Services Assessment Model. This article describes the relationship between the two models, the rationale for the decision to supplement the evaluation with WHO evaluative elements, the value-added results of the WHO evaluative elements, and plans for further developing the WHO assessments.

Strategies for mentor matching: lessons learned.

Mentoring serves to guide early stage researchers toward opportunities which can further their careers. The most beneficial mentoring experience occurs when both the mentor and mentee share a common background and have appropriate expectations. Our CTSA serves individuals in a five state region with widely disparate needs and we have often struggled to provide appropriate guidance for those requesting mentoring services. Here we present an overview of our past mentor identification strategy along with a proposed new direction to increase flexibility, sustainability and better serve researchers in our region.

PAR1- and PAR2-induced innate immune markers are negatively regulated by PI3K/Akt signaling pathway in oral keratinocytes.

BACKGROUND: Protease-Activated Receptors (PARs), members of G-protein-coupled receptors, are activated by proteolytic activity of various proteases. Activation of PAR1 and PAR2 triggers innate immune responses in human oral keratinocytes (HOKs), but the signaling pathways downstream of PAR activation in HOKs have not been clearly defined. In this study, we aimed to determine if PAR1- and PAR2-mediated signaling differs in the induction of innate immune markers CXCL3, CXCL5 and CCL20 via ERK, p38 and PI3K/Akt. RESULTS: Our data show the induction of innate immunity by PAR1 requires both p38 and ERK MAP kinases, while PAR2 prominently signals via p38. However, inhibition of PI3K enhances expression of innate immune markers predominantly via suppressing p38 phosphorylation signaled by PAR activation. CONCLUSION: Our data indicate that proteases mediating PAR1 and PAR2 activation differentially signal via MAP kinase cascades. In addition, the production of chemokines induced by PAR1 and PAR2 is suppressed by PI3K/Akt, thus keeping the innate immune responses of HOK in balance. The results of our study provide a novel insight into signaling pathways involved in PAR activation.

Effect of implant surface and grafting on implants placed into simulated extraction sockets: a histologic study in dogs.

PURPOSE: To study bone healing at implant sites in simulated extraction sockets with 1-mm marginal defects and compare healing around a turned surface (T) to that around a porous oxide surface prepared by anodic oxidation (AO) with or without the use of an autogenous bone graft. MATERIALS AND METHODS: All mandibular premolars and first molars were extracted from 10 mongrel dogs. After 9 weeks, four sites were prepared on both sides of all mandibles. Each osteotomy was widened in the coronal 5 mm to create a marginal defect of 1 mm around the implants. Autogenous bone was collected during the drilling procedure. The sites were randomized to receive implants with a T or an AO surface, with or without bone grafting. The animals were sacrificed 4 months after implant placement for histologic analysis. RESULTS: Clinically, all sites healed with complete bone fill. The combination of an AO implant and a bone graft resulted in a significantly greater percentage of bone-to-implant contact (BIC) (P < .05) versus all other groups. The highest point of BIC was achieved with the AO group, which was significantly greater than the lowest group (T). No significant differences between groups were found when the apical 4 mm (non-gap areas) were compared (P = .65). CONCLUSIONS: Studies have demonstrated that bone can fill in a marginal defect around a titanium implant with varied histologic BIC, depending on implant surface type and defect dimensions. Based upon this animal study using 10 mongrel dogs, marginal circumferential defects of 1 mm showed significantly higher BIC values for implants that were prepared by AO compared to implants with a turned surface. The addition of autogenous bone grafts further enhanced the degree of BIC.

Differential and coordinated expression of defensins and cytokines by gingival epithelial cells and dendritic cells in response to oral bacteria.

BACKGROUND: Epithelial cells and dendritic cells (DCs) both initiate and contribute to innate immune responses to bacteria. However, much less is known about the coordinated regulation of innate immune responses between GECs and immune cells, particularly DCs in the oral cavity. The present study was conducted to investigate whether their responses are coordinated and are bacteria-specific in the oral cavity. RESULTS: The beta-defensin antimicrobial peptides hBD1, hBD2 and hBD3 were expressed by immature DCs as well as gingival epithelial cells (GECs). HBD1, hBD2 and hBD3 are upregulated in DCs while hBD2 and hBD3 are upregulated in GECs in response to bacterial stimulation. Responses of both cell types were bacteria-specific, as demonstrated by distinctive profiles of hBDs mRNA expression and secreted cytokines and chemokines in response to cell wall preparations of various bacteria of different pathogenicity: Fusobacterium nucleatum, Actinomyces naeslundii and Porphyromonas gingivalis. The regulation of expression of hBD2, IL-8, CXCL2/GRObeta and CCL-20/MIP3alpha by GECs was greatly enhanced by conditioned medium from bacterially activated DCs. This enhancement was primarily mediated via IL-1beta, since induction was largely attenuated by IL-1 receptor antagonist. In addition, the defensins influence DCs by eliciting differential cytokine and chemokine secretion. HBD2 significantly induced IL-6, while hBD3 induced MCP-1 to approximately the same extent as LPS, suggesting a unique role in immune responses. CONCLUSIONS: The results suggest that cytokines, chemokines and beta-defensins are involved in interaction of these two cell types, and the responses are bacteria-specific. Differential and coordinated regulation between GECs and DCs may be important in regulation of innate immune homeostasis and response to pathogens in the oral cavity.

Evaluation of the use of ultrasound within a power toothbrush to dislodge oral bacteria using an in vitro Streptococcus mutans biofilm model.

PURPOSE: To investigate in vitro the use of ultrasound in a power toothbrush to aid in the removal of dental plaque biofilm without bristle contact. METHODS: Dental plaque was modeled using Streptococcus mutans biofilm adherent to hydroxyapatite disks. Treatment arms included positive and negative controls, disks with and without biofilm, respectively. Power toothbrush modes of action tested included a toothbrush with sonic and ultrasonic action (ULT), the same toothbrush with only sonic action (ULN), a sonic toothbrush (SON) and a rotating/oscillating toothbrush (OSC). The active element of the toothbrushes (bristles or point of ultrasound emission) was immersed in toothpaste slurry and held 3 mm away from the disk surface. Treatment included activation of the toothbrush mode of action for 5 seconds. Control disks were exposed to the same fluid environment but not exposed to a power toothbrush. After treatment, biofilm present on the disks was stained using a red dental plaque disclosing solution. Photographs were then taken and the presence of biofilm assessed using digital image analysis. For each disk a normalized pixel volume, related to the presence of biofilm corrected for lighting, was determined. Statistical testing was done with a one-way ANOVA and a Bonferroni post hoc test. RESULTS: Normalized pixel volumes (mean +/- standard deviation) were 0.428 (0.010) for the negative control and 1.022 (0.040) for the positive control. Normalized pixel volumes for the power toothbrush modes of action were 0.641 (0.075) for ULT, 0.972 (0.027) for ULN, 0.921 (0.010) for SON and 0.955 (0.025) for OSC. Statistical analysis showed a significant treatment effect (P<0.001). All power toothbrush modes of action exhibited some biofilm removal without bristle contact in this in vitro assay. Of the modes of action tested, the combined sonic and ultrasonic mode of action (ULT) removed the greatest amount of biofilm from the disk surfaces. The same toothbrush when tested with (ULT) and without (ULN) ultrasound showed a greater amount of biofilm removed when ultrasound was present.

Interplay of protease-activated receptors and NOD pattern recognition receptors in epithelial innate immune responses to bacteria.

Protease-activated receptors (PARs), nucleotide-binding oligomerization domain (NOD) receptors and Toll-like receptors (TLRs) play a role in innate immunity, but little is known about interaction between these receptors. The goal of this study was to investigate how silencing one receptor affects the expression of other receptors and downstream innate immune markers in response to bacteria. Human gingival epithelial cells (GECs) were transfected with siRNA specific for PAR1 or PAR2, then stimulated with periopathogen Porphyromonas gingivalis, bridging organism between pathogens and non-pathogens Fusobacterium nucleatum, or non-pathogen Streptococcus gordonii. PAR1 or PAR2 knock-down resulted in up-regulated NOD1 and NOD2 expression with P. gingivalis or F. nucleatum stimulation (p<0.01), as well as enhanced TLR2 and TLR4 expression when cells were stimulated by bacteria that utilize TLR2 or TLR4, respectively. Involvement of PARs for induction of CC chemokine ligand 20 (CCL20), a cytokine with antimicrobial properties, was observed following stimulation of the three bacterial species. Furthermore, results from multiple cytokine ELISA array showed receptors utilized in the induction of various innate immune markers are tailored to individual bacterium tested. Our data suggest complex interplay of several receptors is required for appropriate innate immune responses to the different types of bacteria present within the oral cavity and that receptor expression itself is altered depending on which organism the cell encounters.

Phospholipase C, p38/MAPK, and NF-kappaB-mediated induction of MIP-3alpha/CCL20 by Porphyromonas gingivalis.

Macrophage inflammatory protein-3alpha/C-C chemokine ligand 20 (MIP-3alpha/CCL20) is an antimicrobial peptide that plays an important role in innate immunity. In addition to direct microbicidal effects, MIP-3alpha/CCL20 also exhibits cytokine-like functions that are critical during dendritic cell activation. The aim of the present study was to investigate further which signaling pathways are involved in the MIP-3alpha/CCL20 mRNA expression in response to whole-cell Porphyromonas gingivalis. Primary gingival epithelial cells (GECs) and the immortalized oral keratinocyte cell-line OKF6/TERT-2 were stimulated with whole-cell P. gingivalis. Prior to stimulation, GECs and OKF6/TERT-2 cells were pretreated with specific inhibitors for nuclear-factor-kappaB (NF-kappaB), mitogen-activated protein kinase (MAPK), phospholipase C (PLC), and phosphatidylinositol-3-kinase (PI3K). In GECs and OKF6/TERT-2 cells, activation of NF-kappaB was examined after exposure to P. gingivalis. The gene expression of MIP-3alpha/CCL20 was significantly induced in response to P. gingivalis (P

Modulation of expression of innate immunity markers CXCL5/ENA-78 and CCL20/MIP3alpha by protease-activated receptors (PARs) in human gingival epithelial cells.

Protease-activated receptors (PARs) are G-protein-coupled receptors with an active role in host defense. The two most highly expressed members of the PAR family in gingival epithelial cells (GECs) are PAR1 and PAR2. The major virulence factors of periodontal pathogen Porphyromonas gingivalis are its proteases which can activate PAR2. However, little is known about the function of PARs in GECs when they are activated by their endogenous agonist enzymes. The purpose of this study was to characterize how the expression of innate immune markers is modulated when PAR1 and PAR2 are activated by their agonist enzymes, thrombin and trypsin, respectively. Here, we report that activation of PAR1 and PAR2 induces cell proliferation at low concentration. Activation of PAR via proteolytic activity of thrombin and trypsin induces expression of CXCL5/ENA-78 and CCL20/MIP3alpha in a concentration-dependent manner. Induction of CXCL5 via PAR1 was inhibited in the presence of PAR1 cleavage blocking antibodies and by PAR1 siRNA. The induction of CXCL5 and CCL20 via PAR2 was inhibited by PAR2 siRNA. These findings indicate an active role in innate immune responses by PAR1 and PAR2 in GECs. Modulation of innate immunity by PARs may contribute to co-ordinated and balanced immunosurveillance in GECs.

Differential effects of periopathogens on host protease inhibitors SLPI, elafin, SCCA1, and SCCA2.

OBJECTIVE: Secretory leukocyte peptidase inhibitors (SLPI), elafin, squamous cell carcinoma antigen 1 and 2 (SCCA1 and SCCA2) are specific endogenous serine protease inhibitors expressed by epithelial cells that prevent tissue damage from excessive proteolytic enzyme activity due to inflammation. To determine the effects of various periopathogens on these protease inhibitors, we utilized human gingival epithelial cells (GECs) challenged with cell-free bacteria supernatants of various periopathogens Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. DESIGN: The gene expression and secretion of SLPI, elafin, SCCA1, and SCCA2 were determined using real-time PCR and ELISA, respectively. The direct effects of periopathogens and P. gingivalis gingipain mutants on these inhibitors were examined in vitro by Western Blot. The effect on the innate immune response of GECs was measured by expression of antimicrobial peptides: human beta-defenisin-2 (hBD2) and chemokine (C-C motif) ligand 20 (CCL20). RESULTS: We found that SLPI, SCCA2, elafin, hBD2, and CCL20 gene expression levels were significantly induced (p<0.001) in response to P. gingivalis, whose virulence factors include cysteine proteases, but not in response to stimulation by other bacteria. P. gingivalis reduced the secretion of SLPI and elafin significantly in GECs, and degraded recombinant SLPI, elafin, SCCA1, and SCCA2. Differential degradation patterns of SLPI, elafin, SCCA1, and SCCA2 were observed with different bacteria as well as P. gingivalis mutants associated with the loss of specific gingipains secreted by P. gingivalis. In addition, pretreatment of GECs with SLPI, SCCA1, or SCCA2 partially blocked hBD2 and CCL20 mRNA expression in response to P. gingivalis, suggesting a protective effect. CONCLUSION: Our results suggest that different periopathogens affect the host protease inhibitors in a different manner, suggesting host susceptibility may differ in the presence of these pathogens. The balance between cellular protease inhibitors and their degradation may be an important factor in susceptibility to periodontal infection.

Association of a genetic polymorphism (-44 C/G SNP) in the human DEFB1 gene with expression and inducibility of multiple beta-defensins in gingival keratinocytes.

BACKGROUND: Human beta-defensins (hBDs) are antimicrobial peptides with a role in innate immune defense. Our laboratory previously showed that a single nucleotide polymorphism (SNP) in the 5' untranslated region of the hBD1 gene (DEFB1), denoted -44 (rs1800972), is correlated with protection from oral Candida. Because this SNP alters the putative mRNA structure, we hypothesized that it alters hBD1 expression. METHODS: Transfection of reporter constructs and evaluation of antimicrobial activity and mRNA expression levels in keratinocytes from multiple donors were used to evaluate the effect of this SNP on constitutive and induced levels of expression. RESULTS: Transfection of CAT reporter constructs containing the 5' untranslated region showed that the -44 G allele yielded a 2-fold increase in CAT protein compared to other common haplotypes suggesting a cis effect on transcription or translation. The constitutive hBD1 mRNA level in human oral keratinocytes was significantly greater in cells from donors with the -44 GG genotype compared to those with the common CC genotype. Surprisingly, the hBD3 mRNA level as well as antimicrobial activity of keratinocyte extracts also correlated with the -44 G allele. Induced levels of hBD1, hBD2, and hBD3 mRNA were evaluated in keratinocytes challenged with Toll-like receptor 2 and 4 ligands, interleukin-1beta, TNFalpha, and interferon-gamma (IFNgamma). In contrast to constitutive expression levels, IFNgamma-induced keratinocyte hBD1 and hBD3 mRNA expression was significantly greater in cells with the common CC genotype, but there was no clear correlation of genotype with hBD2 expression. CONCLUSION: The DEFB1 -44 G allele is associated with an increase in overall constitutive antimicrobial activity and expression of hBD1 and hBD3 in a manner that is consistent with protection from candidiasis, while the more common C allele is associated with IFNgamma inducibility of these beta-defensins and is likely to be more protective in conditions that enhance IFNgamma expression such as chronic periodontitis. These results suggest a complex relationship between genetics and defensin expression that may influence periodontal health and innate immune responses.

Sonic tooth brushing reduces gingival overgrowth in renal transplant recipients.

Cyclosporine (CSA) is a commonly used immunosuppressive medication in pediatric transplantation. Drug-induced gingival overgrowth (DIGO) is a frequent side effect associated with CSA use and can impair the patient's ability to achieve good oral hygiene. This study tested the hypothesis that sonic tooth brushing and oral hygiene instruction can reduce the occurrence or severity of DIGO in CSA-treated pediatric renal transplant recipients. Twenty-three pediatric renal transplant patients with DIGO were randomly allocated to treatment or control groups. The treatment group received oral hygiene instruction and use of a sonic toothbrush, while the control group continued their usual home care with manual brushes. Dental impressions and photographs of all subjects were taken at baseline and every 3 months for a year. The casts and photographs were evaluated by a dental panel to compare the DIGO levels from baseline until the end of the study. After 12 months the control group had significantly more severe DIGO than did the sonic tooth brushing and oral hygiene instruction group (OR=4.5, 95%CI=1.2-16.0, P=0.03). Of the risk factors considered, only male gender was significantly associated with worse outcome (OR=6.1, 95%CI=2.3-16.1, P=0.03). The use of a powered toothbrush, together with oral hygiene instruction, may be an important component of health maintenance for pediatric transplant patients on CSA.

Periodontal disease pathogenesis: genetic risk factors and paradigm shift.

Periodontitis is a widespread disease caused by oral bacteria and their interaction with lifestyle and genetic risk factors. The focus of periodontal research has expanded over the past few years to include significant progress in the understanding of genetic processes provided by the Human Genome Project. This presentation summarizes recent views on periodontal pathogenesis and the genetic risk factors involved in the disease. Additionally, it describes the paradigm shift in periodontal care, which is evolving from a repair-based to a wellness-based model.

Clinical effects of scaling and root planing on untreated teeth.

OBJECTIVE: The aim of this report is to examine whether scaling and root planing (SRP) in one area of the mouth may affect periodontal improvement in untreated areas in the same patient, possibly through systemic effects of treatment. MATERIAL AND METHODS: Twenty patients diagnosed with generalized aggressive periodontitis were randomized into treatment (n=11) and no treatment (n=9) groups. Within the treatment group, three quadrants were treated by SRP at week 0, 3, 12, and 24, while a single experimental quadrant remained untreated throughout the study. The outcome for all teeth was assessed using clinical parameters, subtraction radiography, and pathogenic bacteria levels in the subgingival flora over the 24-week study period. RESULTS: Compared with sites in no treatment patients, the treated sites in the treated patients showed a 1 mm decrease in probing depth (PD) (p<0.01) and a 0.5 mm increase in bone height (p<0.01) by 24 weeks. In untreated sites within treated subjects, however, PDs tended to improve (p=0.09) but at a reduced rate compared with treated sites. The levels of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis (Bacteroides forsythus) remained unchanged in untreated sites while levels of Prevetolla intermedia and Treponema denticola tended to decrease as compared with controls but did not reach significance. CONCLUSIONS: This study indicates that untreated sites in treated periodontitis patients show a trend towards clinical improvement and exhibit reductions in some but not all periodontopathic bacterial species tested.