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1.  [No title available] 
PMCID: PMC3732561  PMID: 23488730
2.  [No title available] 
PMCID: PMC3758777  PMID: 23662917
3.  Establishment and characterization of a telomerase immortalized human gingival epithelial cell line 
Journal of periodontal research  2013;48(6):10.1111/jre.12059.
Background and Objective
Gingival keratinocytes are used in model systems to investigate the interaction between periodontal bacteria and the epithelium in the initial stages of the periodontal disease process. Primary gingival epithelial cells (GECs) have a finite lifespan in culture before they enter senescence and cease to replicate, while epithelial cells immortalized with viral proteins can exhibit chromosomal rearrangements. The aim of this study was to generate a telomerase-immortalized human gingival epithelial cell line and compare its in vitro behavior to that of human GECs.
Material and Methods
Human primary gingival epithelial cells were immortalized with a bmi1/hTERT combination to prevent cell cycle triggers of senescence and telomere shortening. The resultant cell-line, Telomerase Immortalized Gingival Keratinocytes (TIGKs), were compared to GECs for cell morphology, karyotype, growth and cytokeratin expression, and further characterized for replicative lifespan, expression of toll-like receptors (TLRs) and invasion by P. gingivalis.
TIGKs showed morphologies, karyotype, proliferation rates and expression of characteristic cytokeratin proteins comparable to GECs. TIGKs underwent 36 passages without signs of senescence and expressed transcripts for TLRs 1-6, 8 and 9. A subpopulation of cells underwent stratification after extended time in culture. The cytokeratin profiles of TIGK monolayers were consistent with basal cells. When allowed to stratify, cytokeratin profiles of TIGKs were consistent with suprabasal cells of the junctional epithelium. Further, TIGKs were comparable to GECs in previously reported levels and kinetics of invasion by wild type Porphyromonas gingivalis and an invasion defective ΔserB mutant.
Results confirm bmi1/hTERT-immortalization of primary gingival epithelial cells generated a robust cell-line with similar characteristics to the parental cell type. TIGKs represent a valuable model system for the study of oral bacteria interactions with host gingival cells.
PMCID: PMC3709015  PMID: 23441958
Telomerase; Immortalization; hTERT; bmi1; Gingival Epithelial Cells; Porphyromonas gingivalis
4.  Neutrophils in chronic and aggressive periodontitis in interaction with Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans 
Journal of periodontal research  2009;44(3):368-377.
The aim of this in vitro study was to study phagocytosis and killing of Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 by peripheral blood PMNs taken from aggressive and chronic periodontitis patients. Also, the release of reactive oxygen species (ROS) and human neutrophil elastase (HNE) upon the interaction of PMNs with bacteria was measured.
Peripheral blood PMNs obtained from 12 chronic periodontitis patients, 6 aggressive periodontitis patients and 12 healthy controls were exposed to Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans following opsonisation of the bacteria using the patient’s own serum. Phagocytosis and killing of the bacteria as well as the extracellular HNE activity were quantified for up to 2 hours. The total amount and the extracellular release of ROS were measured by luminol and isoluminol dependent chemiluminescence.
PMNs from chronic (62.16 ± 19.39 %) and aggressive periodontitis (43.26 ± 26.63 %) patients phagocytosed more P. gingivalis than the healthy controls (24.43 ± 19.87 %) at 30 mins after exposure to the bacteria (p < 0.05). PMNs from subjects with chronic and aggressive periodontitis released significantly more ROS and demonstrated greater HNE activity in the absence of any stimulus than PMNs from healthy controls (p < 0.05). The total release of ROS increased in chronic periodontitis PMNs and in the control group PMNs after interaction with P. gingivalis. The interaction with A. actinomycetemcomitans resulted in greater total ROS release in chronic periodontitis PMNs and in the control group PMNs than P. gingivalis.
PMNs in aggressive and chronic periodontitis are hyperactive even without any particular stimulus. The extracellular release of ROS and neutrophil elastase by PMNs may not only affect bacterial virulence and/or viability, but also contribute to damage of the surrounding periodontal tissues.
PMCID: PMC4180098  PMID: 19210340
PMN; phagocytosis; ROS; aggressive periodontitis; chronic periodontitis; Aggregatibacter actinomycetemcomitans; Porphyromonas gingivalis
5.  Cleavage of IgG1 in GCF is associated with presence of Porphyromonas gingivalis 
Journal of periodontal research  2012;48(4):458-465.
Background and Objectives
Immunoglobulin (Ig) G1 plays an important role in the adaptive immune response. Kgp, a lysine-specific cysteine protease from Porphyromonas gingivalis, specifically hydrolyses IgG1 heavy chains. The purpose of this study was to examine whether cleavage of IgG1 occurs in gingival crevicular fluid (GCF) in vivo, and whether there is any association with the presence of P. gingivalis and other periodontopathogens.
Material and methods
GCF was obtained from nine patients with aggressive periodontitis, nine with chronic periodontitis, and five periodontally-healthy individuals. The bacterial loads of P. gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Prevotella intermedia, and Tannerella forsythia were analysed by real-time PCR, and the presence and cleavage of IgG1 and IgG2 were determined using Western blotting. Kgp levels were measured by ELISA.
Cleaved IgG1 was identified in the GCF from 67% of patients with aggressive periodontitis and in 44% of patients with chronic periodontitis. By contrast, no cleaved IgG1 was detectable in the healthy controls. No degradation of IgG2 was detected in any of the samples, regardless of health status. P. gingivalis was found in high numbers in all samples in which cleavage of IgG1 was detected (p < 0.001 compared with samples with no IgG cleavage). Furthermore, high numbers of T. forsythia and P. intermedia were also present in these samples. The level of Kgp in the GCF correlated with the load of P. gingivalis (r = 0.425, p < 0.01). The presence of Kgp (range 0.07–10.98 ng/ml) was associated with proteolytic fragments of IgG1 (p < 0.001). However, cleaved IgG1 was also detected in samples with no detectable Kgp.
In patients with periodontitis cleavage of IgG1 occurs in vivo and may suppress antibody-dependent antibacterial activity in subgingival biofilms especially those colonized by P. gingivalis.
PMCID: PMC3566341  PMID: 23116446
IgG; GCF; periodontitis Porphyromonas gingivalis; gingipains
6.  Systemic endotoxin levels in chronic indolent periodontal infections 
Background and Objective:
Periodontal disease has been linked with an increased risk of various systemic diseases. A plausible biologic explanation for this link includes the opportunity for oral pathogens to translocate to the circulation as a result of breakdown in integrity of the oral epithelium. This study refined a methodology used to detect endotoxin activity in the serum of subjects with indolent periodontal infections.
Material and Methods:
The QCL® Kinetic Chromogenic Assay (Cambrex) is a kinetic measure of endotoxin activity. Sera from 211 pregnant women with periodontitis enrolled in the Obstetrics and Periodontal Therapy Trial were used to develop the assay further and to evaluate the detection of endotoxin activity that might accompany a low-level bacteremia in chronic periodontitis.
We optimized the system to increase the sensitivity and reproducibility of the assay. The refined system was able to detect endotoxin activity in serum at > 0.0125 EU/mL. At baseline (13–16 wk of gestation), 35.5% of the women were positive for endotoxin activity (1.62 ± 2.21; range: 0.38–15 EU/mL).
This report describes a sensitive measure of endotoxin activity in serum. The procedure allowed us to document levels of this microbial virulence factor in serum of individuals with indolent infections such as periodontal disease.
PMCID: PMC4102883  PMID: 20465752
periodontitis; endotoxin; serum; pregnancy; indolent infection
7.  Periodontitis-specific molecular signatures in gingival crevicular fluid 
Journal of periodontal research  2010;45(3):345-352.
Background and Objective
Periodontitis is currently diagnosed almost entirely on gross clinical manifestations that have been in situ for more than 50 years without significant improvement. The general objective of this study was, therefore, to evaluate whether mid-infrared spectroscopy can be used to identify disease-specific molecular alterations to the overall biochemical profile of tissues and body fluids.
Material and Methods
A total of 190 gingival crevicular fluid samples were obtained from periodontitis (n = 64), gingivitis (n = 61) and normal sites (n = 65). Corresponding infrared absorption spectra of gingival crevicular fluid samples were acquired and processed, and the relative contributions of key functional groups in the infrared spectra were analysed. The qualitative assessment of clinical relevance of these gingival crevicular fluid spectra was interpreted with the multivariate statistical analysis-linear discriminant analysis.
Using infrared spectroscopy, we have been able to identify four molecular signatures (representing vibrations in amide I, amide II/tyrosine rings and symmetric and asymmetric stretching vibrations of phosphodiester groups in DNA) in the gingival crevicular fluid of subjects with periodontitis or gingivitis and healthy control subjects that clearly demarcate healthy and diseased periodontal tissues. Furthermore, the diagnostic accuracy for distinction between periodontally healthy and periodontitis sites revealed by multivariate classification of gingival crevicular fluid spectra was 98.4% for a training set of samples and 93.1% for a validation set.
We have established that mid-infrared spectroscopy can be used to identify periodontitis-specific molecular signatures in gingival crevicular fluid and to confirm clinical diagnoses. Future longitudinal studies will assess whether mid-infrared spectroscopy represents a potential prognostic tool, recognized as key to advancement of periodontics.
PMCID: PMC4078927  PMID: 20337898
gingival crevicular fluid; inflammation; infrared spectroscopy; periodontitis
8.  The Role of Physiological Markers of Health in the Association between Demographic Factors and Periodontal Disease 
Journal of periodontal research  2012;48(3):367-372.
This study examines whether the association between periodontal disease and demographic factors is mediated by physiological measures of health.
Age is highly related to oral health status. The higher prevalence of oral disease within sub-groups of the population may reflect a tendency towards “early aging” and dysregulation of multiple physiological systems.
Logistic regression was used to examine whether biomarkers and demographic factors, such as SES and race/ethnicity, were associated with periodontal disease, and then whether the strength of these relationships could be attributed to associations between demographic variables and physiological measures of systemic health.
Periodontal disease was associated with measures of SES and race/ethnicity. Furthermore, one unit increases in CMV optical density, CRP, and HbA1c were associated with a 25% (OR=1.25; 95% CI: 1.14–1.36), 13% (OR: 1.13; 95% CI: 1.03–1.24), and 19% (OR: 1.19; 95% CI: 1.12–1.27) increased likelihood of periodontal disease, respectively. However, when biomarkers and sociodemographic variables were both included in the model, their associations with periodontal disease were significantly reduced or eliminated.
The risk of periodontal disease is higher among blacks and/or low income individuals; however, these associations appear to be partly due to the greater probability of elevated levels of CRP, CMV, or HbA1c among these groups.
PMCID: PMC3609891  PMID: 23231345
Periodontal disease; Cytomegalovirus; Inflammation
9.  Association of CD14-260 Polymorphisms, Red-complex Periodontopathogens and Gingival Crevicular Fluid Cytokine Levels with Cyclosporine A-induced Gingival Overgrowth in Renal Transplant Patients 
Journal of periodontal research  2012;48(2):203-212.
Backgroud and Objective
Genetic factors may influence the colonization of pathogenic bacteria, therefore increasing the risk for the initiation and development of periodontal disease. The present study was carried out to investigate the association of CD14-260 polymorphisms, subgingival microbiota, and gingival crevicular fluid (GCF) cytokine levels with cyclosporine A (CsA)-induced gingival overgrowth (GO) in renal transplant patients.
Material and Methods
204 patients were dichotomized into two groups: 124 with GO and 80 without GO. The CD14-260 polymorphisms were measured using an allele-specific PCR method. The levels of periodontal pathogens were determined by real-time PCR of subgingival samples. GCF levels of IL-1β and sCD14 were detected by ELISA.
The frequency of CD14-260 genotype CT + TT was found to be similar in both groups. Patients with GO presented increased prevalence of Pg, Td, and Tf (red complex) and significantly higher levels of IL-1β than those without GO. GO patients carrying CT+TT genotypes were found to have higher frequencies of Pg, Td, and Tf than those carrying CC genotype. Furthermore, in the presence of red complex, CT+TT genotypes were associated with higher IL-1β levels and severe GO. Multiple logistic regression analysis demonstrated that the severity of GO is not dependent on age, gender and pharmacological variables, being only associated with CD14-260 genotype and red complex periodontopathogens.
No association between CD14-260 polymorphisms and the prevalence of GO was revealed in renal transplant patients administered CsA. However, CD14-260 CT+TT genotypes are associated with the prevalence of red complex periodontopathogens in GO patients, and may thus play some role in the development of severe CsA-induced GO.
PMCID: PMC3514605  PMID: 22934794
cyclosporine A; gingival overgrowth; polymorphism; CD14; cytokines; periodontal pathogens
Journal of periodontal research  2012;48(2):221-227.
Background and Objective
Dentin sialophosphoprotein (DSPP) and its cleaved products, dentin phosphoprotein (DPP) and dentin sialoprotein (DSP), play important roles in biomineralization. Recently, we observed that DSPP is highly expressed in the alveolar bone and cementum, indicating that this molecule may play an important role in the formation and maintenance of a healthy periodontium, and its deletion may cause increased susceptibility to periodontal diseases. The objective of this investigation was to study the effects of Dspp ablation on periodontal tissues by analyzing Dspp null mice.
Newborn to 6-month-old Dspp null mice were examined, and the 3-month and 6-month-old Dspp null mice were characterized in detail using X-ray radiography, histology and scanning electron microscopy (backscattered as well as resin-infiltrating). Wild-type mice of the same age groups served as the normal controls.
The Dspp null mice showed a significant loss of alveolar bone and cementum, particularly in the furcation and the interproximal regions of the molars. The alveolar bone appeared more porous while the quantity of cementum was reduced in the apical region. The canalicular systems and osteocytes in the alveolar bone were abnormal, with reduced numbers of canaliculi and an altered osteocyte morphology. The loss of alveolar bone and cementum along with the detachment of the periodontal ligaments (PDL) led to the apical migration of the epithelial attachment and the formation of periodontal pockets.
Inactivation of DSPP leads to the loss of the alveolar bone and cementum and increased susceptibility to bacterial infections in the PDL of Dspp null mice. The fact that the loss of DSPP results in periodontal diseases indicates that this molecule plays a vital role in maintaining the health of the periodontium.
PMCID: PMC3514631  PMID: 22934831
11.  Antibacterial Effects of Blackberry Extract Target Periodontopathogens 
Background and Objective
Antimicrobial agents provide valuable adjunctive therapy for prevention and control of oral diseases. Limitations in their prolonged use have stimulated the search for new natural occurring agents with more specific activity and fewer adverse effects. Here we sought to determine the anti-bacterial properties of blackberry extract (BBE) in vitro against oral bacterial commensals and periodontopathogens.
Material and Methods
Effects of whole and fractionated BBE on the metabolism of 10 different oral bacteria were evaluated by colorimetric water-soluble tetrazolium-1 (WST-1) assay. Bactericidal effects of whole BBE against F. nucleatum were determined by quantitating colony forming units (CFUs). Cytotoxicity was determined in oral epithelial (OKF6) cells.
BBE at 350-1,400 μg/mL reduced the metabolic activity of P. gingivalis, F. nucleatum and S. mutans. The reduced metabolic activity observed for F. nucleatum corresponded to a reduction in CFUs following exposure to BBE for as little as 1 hour, indicative of its bactericidal properties. An anthocyanin-enriched fraction of BBE reduced the metabolic activity of F. nucleatum but not P. gingivalis or S. mutans, suggesting the contribution of species specific agents in the whole BBE. Oral epithelial cell viability was not reduced following ≤ 6 h exposures to whole BBE (2.24-1400 μg/mL).
BBE alters the metabolic activity of oral periodontopathogens while demonstrating minimal effect on commensals. The specific antibacterial properties of BBE shown in this study along with its anti-inflammatory and antiviral properties previously demonstrated make this natural extract a promising target as an adjunct for prevention and/or complementary therapy of periodontal infections.
PMCID: PMC3540108  PMID: 22812456
Blackberry; oral bacteria; antibacterial effect; periodontitis; periodontopathogens; Fusobacterium nucleatum; topical antimicrobial
12.  Wnt/β-catenin pathway regulates Bmp2-mediated differentiation of dental follicle cells 
Journal of periodontal research  2011;47(3):10.1111/j.1600-0765.2011.01433.x.
Background and Objectives
Bmp2-induced osteogenic differentiation has been shown to occur through the canonical Wnt/β-catenin pathway, whereas factors promoting canonical Wnt signaling in cementoblasts inhibited cell differentiation and promoted cell proliferation in vitro. The aim of this study was to investigate whether putative precursor cells of cementoblasts, dental follicle cells (murine SVF4 cells), when stimulated with Bmp2, would exhibit changes in genes/proteins associated with the Wnt/β-catenin pathway.
Materials and Methods
SVF4 cells were stimulated with Bmp2, and the following assays were carried out: 1) Wnt/β-catenin pathway activation assessed by western blot, β-catenin/TCF reporter assay, and gene expression of lymphoid enhancer-binding factor-1 (Lef1), transcription factor 7 (Tcf7), Wnt inhibitor factor 1 (Wif1) and Axin2, and 2) cementoblast/osteoblast differentiation assessed by mineralization in vitro, and mRNA levels of runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), osteocalcin (Ocn) and bone sialoprotein (Bsp) by qPCR after Wnt3a treatment and knockdown of β-catenin.
Wnt3a induced β-catenin nuclear translocation and upregulated the transcriptional activity of a canonical Wnt-responsive reporter, suggesting the Wnt/β-catenin pathway functions in SVF4 cells. Activation of Wnt signaling with Wnt3a suppressed Bmp2-mediated induction of cementoblast/osteoblast maturation of SVF4 cells. However, β-catenin knockdown showed that Bmp2-induced expression of cementoblast/osteoblast differentiation markers requires endogenous β-catenin. Wnt3a down-regulated transcripts for Runx2, Alp and Ocn in SVF4 cells compared to untreated cells. In contrast, Bmp2 induction of Bsp transcripts occurred independent of Wnt/β-catenin signaling.
These data suggest that stabilization of β-catenin by Wnt-3a treatment inhibits Bmp2-mediated induction of cementoblast/osteoblast differentiation in SVF4 cells, although Bmp2 requires endogenous Wnt/β-catenin signaling to promote cell maturation.
PMCID: PMC3865600  PMID: 22150562
dental follicle cells; Wnt; cementoblast; maturation; BMP
13.  Levels of Selenomonas species in generalized aggressive periodontitis 
Journal of periodontal research  2012;47(6):711-718.
To compare the levels of Selenomonas sputigena and uncultivated/unrecognized Selenomonas species in subgingival biofilms from generalized aggressive periodontitis (GAgP) and periodontaly healthy (PH) subjects.
Material and Methods
GAgP (n=15) and PH (n=15) subjects were recruited and their clinical periodontal parameters were evaluated. Subgingival plaque samples were collected (9 samples/subject) and analyzed for the levels of 10 bacterial taxa, including cultivated and uncultivated/unrecognized microorganisms using the RNA-oligonucleotide quantification technique (ROQT). Differences in the levels of the test taxa between groups were sought using the Mann-Whitney test.
GAgP subjects showed significantly higher mean counts of Porphyromonas gingivalis, Selenomonas sputigena and Selenomonas oral clone CS002 (Human Oral Microbial Database (HOMD) Oral Taxon 131), while Actinomyces gerencseriae and Streptococcus sanguinis were found in higher mean counts in PH subjects (p<0.01). Selenomonas EW084 (HOMD OT 146) was only detected in the GAgP group. In the GAgP group, levels of P. gingivalis and S. sputigena were higher in sites with probing depth (PD) ≥5mm than in shallow sites (PD ≤3mm) (p<0.01). Furthermore, sites with PD≤3mm in GAgP subjects harbored higher levels of these two species than sites in PH subjects. There were positive correlations between PD and levels of P. gingivalis (r=0.77; p<0.01), S. sputigena (r=0.60; p<0.01) and Selenomonas sp. EW076 (OT 139) (r=042, p<0.05).
S. sputigena, Selenomonas sp. oral CS002 (OT 131) and Selenomonas sp. oral clone EW084 (OT 146) may be associated with the pathogenesis of GAgP, and their role in the onset and progression of this infection should be further investigated.
PMCID: PMC3678906  PMID: 22612405
Selenomonas sputigena; molecular biology; 16S rRNA; generalized aggressive periodontitis; not-yet-cultivated species
14.  Subgingival Microbiota in Adult Down Syndrome Periodontitis 
Journal of Periodontal Research  2012;47(4):500-507.
The subgingival microbiota in Down syndrome and non-Down syndrome (non-DS) adults receiving periodic dental care was examined for 40 bacterial species with checkerboard DNA-DNA hybridization, and related to clinical periodontal attachment loss (AL).
A total of 44 Down syndrome, 66 non-DS mentally retarded, and 83 mentally normal adults were clinically evaluated, with subgingival specimens removed and pooled per subject from three interproximal sites on different teeth, and assessed for the presence and levels of 40 bacterial species using species-specific whole genomic DNA probes and checkerboard DNA-DNA hybridization. Significant group differences in species proportions averaged across subjects were evaluated using the Kruskal-Wallis test, and associations between subgingival species and mean subject AL within Down syndrome and non-DS subject groups were quantified with Pearson correlation and multiple linear regression analysis.
Down syndrome subjects exhibited greater AL than non-DS subjects (P = 0.05). Down syndrome subjects yielded most microbial species at levels similar to non-DS subjects, except for higher proportions of Selenomonas noxia, Propionibacterium acnes, Streptococcus gordonii, Streptococcus mitis, and Streptococcus oralis as compared to non-DS study subjects, higher Treponema socranskii than non-DS mentally retarded subjects, and higher Streptococcus constellatus relative to mentally normal subjects. Down syndrome adults classified with periodontitis revealed higher subgingival levels of T. socranskii than non-periodontitis Down syndrome subjects (P = 0.02). Higher subgingival proportions of S. constellatus, Fusobacterium nucleatum subsp. nucleatum, S. noxia and Prevotella nigrescens showed significant positive correlations (r = 0.35 to 0.42), and Actinomyces naeslundii II and Actinomyces odontolyticus negative correlations (r = −0.36 to −0.40), with increasing mean subject AL in Down syndrome adults.
Individuals with DS show higher levels of some subgingival bacterial species and specific associations between certain subgingival bacterial species and loss of periodontal attachment. These findings are consistent with the notion that certain subgingival bacteria may contribute to the increased level of periodontal disease seen in DS individuals, and raise the question as to the reason for increased colonization in DS.
PMCID: PMC3323756  PMID: 22221039
subgingival microbiota; Down syndrome; periodontitis; periodontal
15.  Bone Remodeling Associated Salivary Biomarker MIP-1α Distinguishes Periodontal Disease from Health 
Journal of Periodontal Research  2011;47(3):389-395.
Background and objective
The field of salivary diagnostics lacks an accepted and validated biomarker of alveolar bone remodeling. To address this we examined levels of salivary biomolecules specifically associated with biological aspects of bone remodeling in subjects with chronic periodontitis in a case-control study.
Levels of macrophage inflammatory protein (MIP)-1α, osteoprotegerin (OPG), C-telopeptide pyridinoline cross-links of type I collagen (ICTP), and β-C-terminal type I collagen telopeptide (β-CTX) in unstimulated whole saliva of 80 subjects (40 subjects with moderate to severe chronic periodontitis and 40 gender- and age-matched healthy control subjects) were measured using enzyme immunosorbent assays. Saliva was collected before clinical examination that included probing depth (PD), clinical attachment loss (CAL), and bleeding on probing (BOP).
The mean level of MIP-1α in periodontitis subjects was 18-fold higher than in healthy subjects (p < 0.0001). Clinical periodontal indices significantly correlated with MIP-1α levels (p < 0.0001). MIP-1α, of the biomolecules examined, demonstrated the highest ability to discriminate between periodontal disease and health as determined by area under the curve (AUC = 0.94) and classification and regression tree analysis (sensitivity 94%, specificity 92.7%). OPG levels were elevated 1.6-fold (P = 0.055), whereas ICTP and β-CTX levels were below the level of detection in the majority of subjects.
These findings suggest that the chemokine MIP-1α may aid in identifying periodontitis. Future longitudinal studies are warranted to determine whether this biomarker can help to ascertain progression of bone loss in subjects with periodontal disease.
PMCID: PMC3292624  PMID: 22126530
periodontal health, periodontal disease; saliva; biomarkers; bone remodeling; macrophage inflammatory protein (MIP)-1α; osteoprotegerin (OPG); C-telopeptide pyridinoline cross-links of type I collagen (ICTP), β-C-terminal type I collagen telopeptide (β-CTX)
16.  Protease Inhibitor Levels in Periodontal Health and Disease 
Journal of Periodontal Research  2011;47(2):228-235.
Background and Objectives
Our previous study showed protease inhibitors were attenuated by periopathogen Porphyromonas gingivalis in cultured gingival epithelial cells. We hypothesize fewer protease inhibitors would be present in more advanced periodontal sites where the level of P. gingivalis may be high. The goal of this study was to investigate the relationship between the protease inhibitor (SLPI, ELAFIN, SCCA) levels in gingival crevicular fluid (GCF) and the number of P. gingivalis in subgingival plaque.
Materials and Methods
Plaque samples from subjects without (n=18) and with moderate to advanced periodontitis (n=41) were used to quantify P. gingivalis using real-time PCR. Protease inhibitor levels in the GCF of all the subjects were determined by ELISA.
P. gingivalis was detected in 68.3% of subjects with periodontitis, while 16.7% of patients without periodontitis had detectable level of P. gingivalis. Subjects with periodontitis and P. gingivalis in their plaque exhibited lower SLPI and ELAFIN levels (p<0.001) compare to control subjects without periodontitis. SLPI was also reduced (p<0.05) in GCF of periodontal patients without detectable level of P. gingivalis. Periodontal patients with high vs. low levels of P. gingivalis exhibited reciprocal mean levels of SLPI and ELAFIN concentrations.
The reduced concentrations of SLPI and ELAFIN may contribute to the loss of host protective capacity and increase susceptibility to breakdown from chronic infection. The work of this investigation may aid in finding diagnostic and prognostic markers in periodontal health and disease and may further help in finding pharmacological targets directed against periodontal inflammation.
PMCID: PMC3270203  PMID: 22029638
protease inhibitors; periodontitis; Porphyromonas gingivalis; gingival crevicular fluid
17.  Proteome Dataset of Human Gingival Crevicular Fluid from Healthy Periodontium Sites by Multi-Dimensional Protein Separation and Mass Spectrometry 
Journal of Periodontal Research  2011;47(2):248-262.
Background and Objective
Gingival crevicular fluid (GCF) has been of major interest for many decades as valuable body fluid that may serve as a source of biomarkers for both periodontal and systemic diseases. Because of its very small sample size, sub-μl level, identification of its protein composition by classical biochemical methods has been limited. The advent of highly sensitive mass spectrometric technology has permitted large-scale identification of protein components of many biological samples. This technology has been employed to identify protein composition of GCF from inflamed and periodontal sites. In this report we present a proteome dataset of GCF from healthy periodontium sites.
A combination of periopaper collection method with application of multidimensional protein separation and mass spectrometric (MS) technology led to a large-scale documentation of the proteome of GCF from healthy periodontium sites.
The approaches utilized have culminated in identification of 199 proteins in GCF of periodontally healthy sites. The current GCF proteome from healthy sites was compared and contrasted with those proteomes of GCF from inflamed and periodontal sites as well as serum. The cross-correlation of the GCF and plasma proteomes permitted dissociation of the 199 identified GCF proteins into, 105 proteins (57%) that can be identified in plasma and 94 proteins (43%) which are distinct and unique to GCF microenvironment. Such analysis also revealed distinctions in protein functional categories between serum proteins and those specific to GCF microenvironment.
Firstly, the data presented herein provide the proteome of GCF from periodontally healthy sites through establishment of innovative analytical approaches for effective analysis of GCF from periopapers both at the level of complete elusion and removal of abundant albumin which restricts identification of low abundant proteins. Secondly, it adds significantly to the knowledge of GCF composition and highlights new groups of proteins specific to GCF microenvironment.
PMCID: PMC3272151  PMID: 22029670
Gingival crevicular fluid; mass spectrometry; proteomics; saliva; oral; biomarkers; diagnostics
18.  Early microbial succession in re-developing dental biofilms in periodontal health and disease 
Journal of Periodontal Research  2011;47(1):95-104.
To determine the order of bacterial species succession in re-developing supra and subgingival biofilms.
Supra and subgingival plaque samples were taken separately from 28 teeth in 38 healthy and 17 periodontitis subjects immediately after professional cleaning. Samples were taken again from 7 teeth in randomly selected quadrants after 1, 2, 4 and 7 days of no oral hygiene and analyzed using checkerboard DNA-DNA hybridization. % DNA probe counts were averaged within subjects at each time point. Ecological succession was determined using a modified moving window analysis.
Succession in supragingival biofilms from periodontitis and health was similar. At 1 day, Streptococcus mitis and Neisseria mucosa showed increased proportions, followed by Capnocytophaga gingivalis, Eikenella corrodens, Veillonella parvula and Streptococcus oralis at 1–4 days. At 4–7 days, Campylobacter rectus, Campylobacter showae, Prevotella melaninogenica and Prevotella nigrescens became elevated. Subgingival plaque redevelopment was slower and very different from supragingival. Increased proportions were first observed for S. mitis, followed by V. parvula and C. gingivalis and, at 7 days by Capnocytophaga sputigena and P. nigrescens. No significant increase in proportions of periodontal pathogens was observed in any of the clinical groups or locations.
There is a defined order in bacterial species succession in early supra and subgingival biofilm re-development after professional cleaning.
PMCID: PMC3253172  PMID: 21895662
ecology; succession; oral bacteria; periodontal; periodontitis; biofilms; supragingival; subgingival
19.  Immune response to cytolethal distending toxin of Aggregatibacter actinomycetemcomitans in periodontitis patients 
Journal of periodontal research  2010;45(4):471-480.
Background and Objective
Cytolethal distending toxin (CDT) is a genotoxin produced by Aggregatibacter actinomycetemcomitans. In spite of its association with pathogenesis, little is known about the humoral immune response against the CDT. This study aimed to test whether subgingival colonization and humoral response to A. actinomycetemcomitans would lead to a response against CDT.
Material and Methods
Sera from periodontally healthy, localized and generalized aggressive periodontitis and chronic periodontitis subjects (n = 80) were assessed for immunoglobulin G titers to A. actinomycetemcomitans serotypes a/b/c and to each CDT subunit (CdtA, CdtB and CdtC) by ELISA. A. actinomycetemcomitans subgingival levels and neutralization of CDT activity were also analyzed.
Sera from 75.0% localized and 81.8% generalized aggressive periodontitis patients reacted to A. actinomycetemcomitans. A response to serotype b was detected in localized (66.7%) and generalized aggressive periodontitis (54.5%). Reactivity to A. actinomycetemcomitans correlated with subgingival colonization (R = 0.75, p < 0.05). There was no correlation between A. actinomycetemcomitans colonization or response to serotypes and the immunoglobulin G response to CDT subunits. Titers of immunoglobulin G to CdtA and CdtB did not differ among groups; however, sera of all generalized aggressive periodontitis patients reacted to CdtC. Neutralization of CDT was not correlated with levels of antibodies to CDT subunits.
Response to CdtA and CdtB did not correlate with the periodontal status of the subject in the context of an A. actinomycetemcomitans infection. However, a response to CdtC was found in sera of generalized but not of localized aggressive periodontitis subjects. Differences in response to CdtC between generalized and localized aggressive periodontitis subjects indicate that CDT could be expressed differently by the infecting strains. Alternatively, the antibody response to CdtC could require the colonization of multiple sites.
PMCID: PMC3534802  PMID: 20337882
Aggregatibacter actinomycetemcomitans; cytolethal distending toxin; enzyme-linked immunosorbent assay (ELISA); immunoglobulin G; serotype
20.  Comparison of randomly cloned and whole genomic DNA probes for the detection of Porphyromonas gingivalis and Bacteroides forsythus 
Whole genomic and randomly-cloned DNA probes for two fastidious periodontal pathogens, Porphyromonas gingivalis and Bacteroides forsythus were labeled with digoxigenin and detected by a colorimetric method. The specificity and sensitivity of the whole genomic and cloned probes were compared. The cloned probes were highly specific compared to the whole genomic probes. A significant degree of cross-reactivity with Bacteroides species. Capnocytophaga sp. and Prevotella sp. was observed with the whole genomic probes. The cloned probes were less sensitive than the whole genomic probes and required at least 106 target cells or a minimum of 10 ng of target DNA to be detected during hybridization. Although a ten-fold increase in sensitivity was obtained with the whole genomic probes, cross-hybridization to closely related species limits their reliability in identifying target bacteria in subgingival plaque samples.
PMCID: PMC3516878  PMID: 8636873
Porphyromonas gingivalis; Bacteroides forsythus; DNA probes; non-isotopic labeling
21.  Reduced functional loads alter the physical characteristics of the bone-PDL-cementum complex 
Journal of periodontal research  2011;46(6):730-741.
Adaptive properties of the bone-PDL-tooth complex have been identified by changing the magnitude of functional loads using small-scale animal models such as rodents. Reported adaptive responses as a result of lower loads due to softer diet include decreased muscle development, change in structure-function relationship of the cranium, narrowed PDL-space, changes in mineral level of the cortical bone and alveolar jaw bone, and glycosaminoglycans of the alveolar bone. However, the adaptive role of the dynamic bone-PDL-cementum complex due to prolonged reduced loads has not been fully explained to date, especially with regards to concurrent adaptations of bone, PDL and cementum. Hence, the temporal effect of reduced functional loads on physical characteristics such as morphology and mechanical properties, and mineral profiles of the bone-periodontal ligament (PDL)-cementum complex using a rat model was investigated.
Materials and Methods
Two groups of six-week-old male Sprague-Dawley rats were fed nutritionally identical food with a stiffness range of 127–158N/mm for hard pellet or 0.32–0.47N/mm for soft powder forms. Spatio-temporal adaptation of the bone-PDL-cementum complex was identified by mapping changes in: 1) PDL-collagen orientation and birefringence using polarized light microscopy, bone and cementum adaptation using histochemistry, and bone and cementum morphology using micro X-ray computed tomography, 2) mineral profiles of the PDL-cementum and PDL-bone interfaces by X-ray attenuation, and 3) microhardness of bone and cementum by microindentation of specimens at ages six, eight, twelve, and fifteen weeks.
Reduced functional loads over prolonged time resulted in 1) altered PDL orientation and decreased PDL collagen birefringence indicating decreased PDL turnover rate and decreased apical cementum resorption; 2) a gradual increase in X-ray attenuation, owing to mineral differences, at the PDL-bone and PDL-cementum interfaces without significant differences in the gradients for either group; 3) significantly (p<0.05) lower microhardness of alveolar bone (0.93±0.16 GPa) and secondary cementum (0.803±0.13 GPa) compared to the higher load group (1.10±0.17 GPa and 0.940±0.15 GPa respectively) at fifteen weeks indicating a temporal effect of loads on local mineralization of bone and cementum.
Based on the results from this study, the effect of reduced functional loads for a prolonged time could differentially affect morphology and mechanical properties, and mineral variations and of the local load-bearing sites in a bone-PDL-cementum complex. These observed local changes in turn could help explain the overall biomechanical function and adaptations of the tooth-bone joint. From a clinical translation perspective, our study provides an insight into modulation of load on the complex for improved tooth function during periodontal disease, and/or orthodontic and prosthodontic treatments.
PMCID: PMC3200425  PMID: 21848615
functional loads; tissue interfaces; cementum; bone-tooth biomechanics; alveolar bone; periodontal ligament
22.  Type 1 diabetes predisposes to enhanced gingival leukocyte margination and macromolecule extravasation in vivo 
Journal of periodontal research  2010;45(6):748-756.
Background and Objective
Diabetes predisposes to periodontal disease. However, the cellular and molecular mechanisms linking the two conditions are not clear. The impact of chronic hyperglycemia on leukocyte margination and macromolecule extravasation was determined in gingival vessels in vivo.
Materials and Methods
Gingival intravital microscopy was employed to measure extravasation of fluorescein isothiocyanate (FITC)–dextran in diabetic Akita and healthy wild-type (WT) mice. Rhodamine 6G and FITC–LY6G were injected for nonspecific and polymorphonuclear-specific leukocyte labeling, respectively. Surface expression of leukocyte adhesion molecules was determined with flow cytometry and western blotting.
Vascular permeability was significantly increased in Akita gingival vessels compared with WT [permeability index (PI): WT, 0.75 ± 0.05; Akita, 1.1 ± 0.03: p < 0.05). Wild-type gingival vessels reached comparable permeability 2 h after intragingival injection of tumor necrosis factor α (TNFα), used here as positive control (PI, 1.17 ± 0.16). The number of rolling leukocytes was significantly elevated in diabetic gingiva (WT, 25 ± 3.7 cells/min; Akita, 42 ± 8.5 cells/min; p < 0.03). Similar rolling cell counts were obtained in WT after intragingival injection of TNFα (10 ng TNFα, 47 ± 1.3 cells/min; 100 ng TNFα, 57.5 ± 5.85 cells/min). The number of leukocytes firmly attached to the endothelium was similar in WT and Akita mice. Leukocyte cell-surface expression of P-selectin glycoprotein ligand-1 and CD11a was increased in Akita mice, while L-selectin remained unchanged when compared with WT. Moreover, P-selectin expression in Akita gingival tissues was elevated compared with that of WT.
Chronic hyperglycemia induces a proinflammatory state in the gingival microcirculation characterized by increased vascular permeability, and leukocyte and endothelial cell activation. Leukocyte-induced microvascular damage, in turn, may contribute to periodontal tissue damage in diabetes.
PMCID: PMC3501126  PMID: 20682016
diabetes; leukocyte; intravital microscopy; selectin; gingiva; microcirculation
23.  Potent anti-inflammatory effects of systemically-administered curcumin modulates periodontal disease in vivo 
Journal of periodontal research  2011;46(2):269-279.
Curcumin is a plant-derived dietary spice with various biological activities, including anti-tumoral and anti-inflammatory. Its therapeutic applications have been studied in a variety of conditions, including rheumatoid arthritis, colon cancer and depression; but no studies evaluated the effects of curcumin on periodontal disease in vivo.
Experimental periodontal disease was induced in rats by placing cotton ligatures around both lower first molars. Curcumin was given to the rats intragastrically daily in two doses (30 and 100 mg/Kg) during 15 days. Control animals received ligatures but only the corn oil vehicle by gavage and no treatment negative control animals were included. Bone resorption was assessed by microcomputer tomography and the inflammatory status was evaluated by stereometric analysis. RT-qPCR and ELISA were used to determine the expression of interleukin (IL)-6, tumor necrosis factor (TNF)-alpha and prostaglandin E2 (PGE2) synthase on the gingival tissues. Modulation of p38 mitogen-activated protein kinase (MAPK) and NK-kB activation was assessed by western blot.
Bone resorption was effectively induced in the experimental period, but it was not affected by either dose of curcumin. Curcumin effectively inhibited cytokine gene expression at mRNA and protein levels and dose-dependently inhibited activation of NF-kB in the gingival tissues. p38 MAPK activation was not inhibited by curcumin. Curcumin-treated animals also presented a marked reduction on the inflammatory cell infiltrate and increased collagen content and fibroblastic cell numbers.
Curcumin did not prevent alveolar bone resorption, but its potent anti-inflammatory effect suggests it may have a therapeutic potential in periodontal diseases.
PMCID: PMC3086370  PMID: 21306385
Curcumin; inflammation; periodontal disease; NF-kB; p38 MAPK
24.  Periodontal pathogens interfere with quorum-sensing-dependent virulence properties in Streptococcus mutans 
Journal of periodontal research  2010;46(1):105-110.
Background and objective
The mechanism by which periodontal pathogens dominate at disease sites is not yet understood. One possibility is that these late colonizers antagonize quorum-sensing systems of early colonizers and render those early colonizers less resistant to environmental factors. In this study, we utilized Streptococcus mutans, a well-documented oral Streptococcus with many quorum-sensing dependent properties, as an example of targeted earlier colonizers that are antagonized by periodontal pathogens.
Material and Methods
S. mutans NG8, LT11, and BM71 were used in this study for assessment of transformation and bacteriocin production, respectively. The effects of Porphyromonas gingivalis and Treponema denticola on these competence-stimulating peptide (CSP)-dependent properties were evaluated in mixed broth assays.
Both P. gingivalis (either live bacteria or membrane vesicles) and T. denticola antagonized transformation in S. mutans NG8 and LT11. S. mutans BM71 bacteriocin production was also inhibited by P. gingivalis and T. denticola. Boiling of these late colonizers before mixing the broth cultures abolished their ability to inhibit S. mutans transformation and bacteriocin production. P. gingivalis and T. denticola inactivated S. mutans exogenous CSP, whereas the boiled bacteria did not.
This study demonstrated that periodontal pathogens antagonized S. mutans quorum-sensing properties. This may render S. mutans less virulent and less resistant to environmental antibacterial factors.
PMCID: PMC3058863  PMID: 21108642
periodontal pathogen; Porphyromonas gingivalis; Treponema denticola; Streptococcus mutans; quorum sensing
25.  Expression of periodontal interleukin-6 protein is increased across patients with neither periodontal disease nor diabetes, patients with periodontal disease alone and patients with both diseases 
Journal of periodontal research  2010;45(5):688-694.
Background and objectives
Epidemiological studies have established that patients with diabetes have an increased prevalence and severity of periodontal disease. Interleukin (IL)-6, a multifunctional cytokine, plays a role in the tissue inflammation that characterizes periodontal disease. Our recent study has shown a trend of increase in periodontal IL-6 expression at the mRNA level across patients with neither periodontal disease nor diabetes, patients with periodontal disease alone and patients with both diseases. However, the periodontal IL-6 expression at the protein level in these patients has not been investigated.
Material and Methods
Periodontal tissue specimens were collected from eight patients without periodontal disease and diabetes (group 1), from 17 patients with periodontal disease alone (group 2) and from 10 patients with both periodontal disease and diabetes (group 3). The frozen sections were prepared from these tissue specimens and IL-6 protein expression was detected and quantified.
The nonparametric Kruskal-Wallis test showed that differences in IL-6 protein levels among the three groups were statistically significant (p = 0.035). Nonparametric analysis using Jonckheere-Terpstra test showed a tendency of increase in periodontal IL-6 protein levels across group 1 to group 2 to group 3 (p = 0.006). Parametric analysis of variance (ANOVA) on IL-6 protein levels showed that neither age nor gender significantly affected the difference of IL-6 levels among the groups.
Periodontal IL-6 expression at the protein level is increased across patients with neither periodontal disease nor diabetes, patients with periodontal disease alone, and patients with both diseases.
PMCID: PMC2949977  PMID: 20682019
Periodontal diseases; Diabetes mellitus; IL-6; Gene expression

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