Georgia K. Johnson

Discovery of new human β-defensins using a genomics-based approach

Abstract Epithelial β-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a

Prostaglandin E (PGE) and interleukin-1β (IL-1β) levels in gingival crevicular fluid during human orthodontic tooth movement

The purpose of this study was to examine gingival crevicular fluid (GCF) levels of two potent bone resorbing mediators, prostaglandin E (PGE) and interleukin-1β (IL-1β), during human orthodontic tooth movement. The study included 10 patients, each having one treatment tooth undergoing orthodontic movement and a contralateral control tooth. The GCF was sampled at control sites and treatment (compression) sites before activation and at 1, 24, 48, and 168 hours. Prevention of plaque-induced inflammation allowed this study to focus on the dynamics of mechanically stimulated PGE and IL-1β GCF levels. The PGE and IL-1β levels were determined with radioimmunoassay. At 1 and 24 hours, mean GCF IL-1β levels were significantly elevated at treatment teeth (8.9±2.0 and 19.2±6.0 pg, respectively) compared with control teeth (2.0±1.1 pg, p=0.0049, and 2.9±1.0 pg, p=0.0209, respectively). The GCF levels of PGE for the treatment teeth were significantly higher at 24 and 48 hours (108.9±11.9 and 97.9±7.3 pg) than the control teeth (61.8±7.2 pg, p=0.0071, and 70.8±7.4 pg, p=0.0021, respectively). The GCF levels of PGE and IL-1β remained at baseline levels throughout the study for the control teeth, whereas significant elevations from baseline in GCF IL-1β (24 hours) and PGE levels (24 and 48 hours) were observed over time in the treatment teeth (p≤0.05). These results demonstrate that bone-reorbing PGE and IL-1β produced within the periodontium are detectable in GCF during the early phases of tooth movement and return to baseline within 7 days.

Influence of smoking on gingival crevicular fluid cytokines in severe chronic periodontitis.

AIM The aim of this study was to compare the expression of 22 chemokines and cytokines in gingival crevicular fluid (GCF) from smokers and non-smokers with periodontitis and periodontally healthy control subjects. MATERIALS AND METHODS Forty subjects with generalized severe chronic periodontitis (20 smokers and 20 non-smokers) and 12 periodontally healthy control subjects participated in this study. Four diseased and two healthy sites were selected from each of the periodontitis subjects. GCF samples were collected and cytokines analysed utilizing a multiplexed immunoassay (Luminex(®) ). Statistical analyses employed non-parametric tests including the Mann-Whitney and Wilcoxon matched-pairs signed-rank tests. RESULTS Compared with healthy control subjects, GCF in subjects with chronic periodontitis contained significantly higher amounts of interleukin (IL)-1α, IL-1β, IL-6, IL-12(p40) (pro-inflammatory cytokines); IL-8, macrophage chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, regulated on activation normal T-cell expressed and secreted (RANTES) (chemokines); IL-2, IFN-γ, IL-3, IL-4 (Th1/Th2 cytokines); IL-15 [regulator of T-cells and natural killer (NK) cells]. Smokers displayed decreased amounts of pro-inflammatory cytokines [IL-1α, IL-6, IL-12(p40)], chemokines (IL-8, MCP-1, MIP-1, RANTES), and regulators of T-cells and NK cells (IL-7, IL-15). CONCLUSIONS Periodontitis subjects had significantly elevated cytokine and chemokine profiles. Smokers exhibited a decrease in several pro-inflammatory cytokines and chemokines and certain regulators of T-cells and NK-cells. This reflects the immunosuppressant effects of smoking which may contribute to an enhanced susceptibility to periodontitis.

A multiplex immunoassay demonstrates reductions in gingival crevicular fluid cytokines following initial periodontal therapy

BACKGROUND AND OBJECTIVE Cytokines and chemokines play an important role in the pathogenesis of periodontal diseases. The objective of this study was to quantitatively assess the effect of initial periodontal therapy on gingival crevicular fluid levels of a comprehensive panel of cytokines and chemokines, including several less extensively studied mediators. MATERIAL AND METHODS Clinical examinations were performed and gingival crevicular fluid samples obtained from six subjects with generalized severe chronic periodontitis prior to initial periodontal therapy and at re-evaluation (6-8 weeks). Four diseased and two healthy sites were sampled in each subject. Twenty-two gingival crevicular fluid mediators were examined using a multiplex antibody capture and detection platform. Statistical analyses were performed by fitting mixed effects linear models to log-transformed gingival crevicular fluid values. RESULTS Gingival crevicular fluid interleukin (IL)-1alpha and IL-1beta were the only cytokines to differ in initially diseased vs. initially healthy sites. Following initial therapy, 13 of the 16 detectable cytokines and chemokines decreased significantly in diseased sites, including IL-1alpha, IL-1beta, IL-2, IL-3, IL-6, IL-7, IL-8, IL-12 (p40), CCL5/regulated on activation, normally T cell expressed and secreted (RANTES), eotaxin, macrophage chemotactic protein-1, macrophage inflammatory protein-1alpha and interferon-gamma. At healthy sites, only three of the 16 mediators were significantly altered following therapy. CONCLUSION This is the first study, to our knowledge, to evaluate such an extensive panel of gingival crevicular fluid mediators within the same sample prior to and following initial therapy. The results confirm that periodontal therapy effectively reduces pro-inflammatory cytokines and chemokines, including less well-described mediators that may be important in initiation and progression of periodontitis. The multiplex assay will prove useful for future gingival crevicular fluid studies.

The enhancement of bone regeneration by gene activated matrix encoding for platelet derived growth factor

Gene therapy using non-viral vectors that are safe and efficient in transfecting target cells is an effective approach to overcome the shortcomings of protein delivery of growth factors. The objective of this study was to develop and test a non-viral gene delivery system for bone regeneration utilizing a collagen scaffold to deliver polyethylenimine (PEI)-plasmid DNA (pDNA) [encoding platelet derived growth factor-B (PDGF-B)] complexes. The PEI-pPDGF-B complexes were fabricated at amine (N) to phosphate (P) ratio of 10 and characterized for size, surface charge, and in vitro cytotoxicity and transfection efficacy in human bone marrow stromal cells (BMSCs). The influence of the complex-loaded collagen scaffold on cellular attachment and recruitment was evaluated in vitro using microscopy techniques. The in vivo regenerative capacity of the gene delivery system was assessed in 5 mm diameter critical-sized calvarial defects in Fisher 344 rats. The complexes were ~100 nm in size with a positive surface charge. Complexes prepared at an N/P ratio of 10 displayed low cytotoxicity as assessed by a cell viability assay. Confocal microscopy revealed significant proliferation of BMSCs on complex-loaded collagen scaffolds compared to empty scaffolds. In vivo studies showed significantly higher new bone volume/total volume (BV/TV) % in calvarial defects treated with the complex-activated scaffolds following 4 weeks of implantation (14- and 44-fold higher) when compared to empty defects or empty scaffolds, respectively. Together, these findings suggest that non-viral PDGF-B gene-activated scaffolds are effective for bone regeneration and are an attractive gene delivery system with significant potential for clinical translation.

Human β-defensin 3 binds to hemagglutinin B (rHagB), a non-fimbrial adhesin from Porphyromonas gingivalis, and attenuates a pro-inflammatory cytokine response

Regulatory mechanisms in mucosal secretions and tissues recognize antigens and attenuate pro‐inflammatory cytokine responses. Here, we asked whether human β‐defensin 3 (HBD3) serves as an upstream suppressor of cytokine signaling that binds and attenuates pro‐inflammatory cytokine responses to recombinant hemagglutinin B (rHagB), a non‐fimbrial adhesin from Porphyromonas gingivalis strain 381. We found that HBD3 binds to immobilized rHagB and produces a significantly higher resonance unit signal in surface plasmon resonance spectroscopic analysis, than HBD2 and HBD1 that are used as control defensins. Furthermore, we found that HBD3 significantly attenuates (P<0.05) the interleukin (IL)‐6, IL‐10, granulocyte macrophage colony stimulating factor (GM‐CSF) and tumor‐necrosis factor‐α (TNF‐α) responses induced by rHagB in human myeloid dendritic cell culture supernatants and the extracellular signal‐regulated kinases (ERK 1/2) response in human myeloid dendritic cell lysates. Thus, HBD3 binds rHagB and this interaction may be an important initial step to attenuate a pro‐inflammatory cytokine response and an ERK 1/2 response.

Association Between Periodontal Disease and Osteoporosis in Postmenopausal Women in Jordan

BACKGROUND Some studies suggest that females with osteoporosis are at an increased risk of periodontal attachment loss and tooth loss; however, results have varied. The aim of this study is to determine the relationship between periodontitis and osteoporosis among postmenopausal Jordanian women. METHODS This cross-sectional study includes 400 Jordanian postmenopausal women with a mean age of 62.5 years (SD ± 6.4 years). These subjects were recruited from patients who had received a routine dual-energy x-ray absorptiometry examination in the Radiology Department, King Abdullah Hospital, Jordan University of Science and Technology, between June 2008 and February 2009. The relationship between skeletal bone mineral density (BMD) and radiographic and clinical parameters of periodontal status, including the loss of alveolar crestal height (ACH), clinical attachment level, probing depth, and percentage of sites with bleeding on probing, was evaluated after controlling for known confounders. RESULTS Bivariate analyses revealed no significant differences in the severity and extent of clinical attachment and ACH loss among women with normal BMD, osteopenia, and osteoporosis. However, in the multivariate analysis, women with osteoporosis were more likely to have severe ACH loss (odds ratio [OR]: 4.20; 95% confidence interval [CI]: 1.57 to 11.22) and periodontitis (OR: 2.45; 95% CI: 1.38 to 4.34). CONCLUSION Osteoporosis was significantly associated with severe alveolar crestal bone loss and the prevalence of periodontitis cases in postmenopausal Jordanian women.

Interleukin-1 and interleukin-8 in nicotine- and lipopolysaccharide-exposed gingival keratinocyte cultures

BACKGROUND AND OBJECTIVE Tobacco use is associated with increased periodontal destruction in both cigarette smokers and smokeless tobacco users. Gingival keratinocytes are the first cells in contact with microbial and tobacco components and play a key role in the innate immune response to these agents. The objective of this study was to evaluate the effect of nicotine and bacterial lipopolysaccharide (LPS) alone and in combination on gingival keratinocyte production of interleukin-1 alpha (IL-1 alpha) and interleukin-8 (IL-8). MATERIAL AND METHODS Gingival keratinocyte cultures were established from 10 healthy, non-tobacco-using subjects. The cells were stimulated for 24 h with 1 mum or 1 mm nicotine and/or 10 microg/mL Escherichia coli or Porphyromonas gingivalis LPS. Interleukin-1 alpha and IL-8 proteins were quantified using ELISAs. RESULTS Compared with untreated cultures, 1 mm nicotine stimulated production of IL-1 alpha (p < 0.001); E. coli and P. gingivalis LPS increased IL-8 production (p = 0.0014 and p = 0.0232, respectively). A combination of nicotine and LPS produced the highest cytokine quantities. Amounts of IL-1 alpha and IL-8 following 1 mm nicotine and LPS exposure were significantly greater than in untreated cultures (p < 0.001). Interleukin-8 was also responsive to 0.1 mum nicotine combined with E. coli or P. gingivalis LPS compared with control cultures (p < 0.0001 and p = 0.0029, respectively). Both cytokines tended to be elevated following the combined treatment relative to nicotine or LPS treatment alone. CONCLUSION These results demonstrate that nicotine and LPS differentially regulate IL-1 and IL-8 production by gingival keratinocytes. Combined treatment tended to elevate cytokine production further, which may have implications for the progression of periodontitis in tobacco users.

Quality Assessment of Systematic Reviews on Periodontal Regeneration in Humans

BACKGROUND Systematic reviews represent the highest form of evidence in the current hierarchy of evidence-based dentistry. Critical analysis of published systematic reviews may help to analyze their strengths and weaknesses and to identify areas that need future improvement. The aim of this overview is to determine and compare the quality of systematic reviews published in the field of periodontal regeneration using established checklists, such as the Assessment of Multiple Systematic Reviews (AMSTAR) guidelines. METHODS A systematic search was conducted to retrieve reviews on periodontal regeneration in humans. A total of 14 systematic reviews were selected using a set of inclusion and exclusion criteria. Two independent reviewers appraised the quality of the selected reviews using AMSTAR guidelines. Each article was given an AMSTAR total score, based on the number of AMSTAR criteria that were fulfilled. The quality of included reviews was further assessed using a checklist proposed in 2003. RESULTS Only one of the selected systematic reviews satisfied all the AMSTAR guidelines, whereas two reviews satisfied just two of the 11 items. This study shows that published systematic reviews on periodontal regeneration exhibit significant structural and methodologic variability. Quality assessment using the additional checklist further confirmed the variability in the way systematic reviews were conducted and/or reported. CONCLUSION Consideration of guidelines for quality assessment, such as AMSTAR, when designing and conducting systematic reviews may increase the validity and clinical applicability of future reviews.

Quantitation of SPLUNC1 in saliva with an xMAP particle-based antibody capture and detection immunoassay

The short palate lung and nasal epithelial clone 1 (SPLUNC1) protein may be differentially expressed in oral infections, oral inflammatory disorders, or oral malignancies and may be involved in innate immune responses in the oral cavity. However, the actual concentration of SPLUNC1 in saliva has not previously been determined. In this study, we determined the concentrations of SPLUNC1 in saliva using a particle-based antibody capture and detection immunoassay. A commercial goat anti-rhSPLUNC1 polyclonal antibody (AF1897) was linked to fluorescent polystyrene microspheres and used as the capture antibody. A commercial mouse IgG2b anti-rhSPLUNC1 monoclonal antibody (MAB1897) was biotinylated and used as the detection antibody. Western blot and 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis of immunoprecipitated rhSPLUNC1 and SPLUNC1 from saliva were used to show that the capture AF1897 and detection MAB1897 antibodies both recognized SPLUNC1. Protein concentrations in saliva from 20 subjects ranged from 0.9 to 23.9mg/ml; SPLUNC1 concentrations ranged from 34.7ng/ml to 13.8μg/ml; and SPLUNC concentrations normalized per mg of total salivary protein ranged from 4.7ng/ml to 5.3μg/ml. These results show that SPLUNC1 is detected in saliva in a variety of concentrations. This immunoassay may prove to be useful in determining the concentration of SPLUNC1 in saliva for assessing its role in the pathogenesis of oral infections, oral inflammatory disorders, or oral malignancies.