Hatice Hasturk

Comparisons of Subgingival Microbial Profiles of Refractory Periodontitis, Severe Periodontitis, and Periodontal Health Using the Human Oral Microbe Identification Microarray

BACKGROUND This study compared the subgingival microbiota of subjects with refractory periodontitis (RP) to those in subjects with treatable periodontitis (GRs = good responders) or periodontal health (PH) using the Human Oral Microbe Identification Microarray (HOMIM). METHODS At baseline, subgingival plaque samples were taken from 47 subjects with periodontitis and 20 individuals with PH and analyzed for the presence of 300 species by HOMIM. The subjects with periodontitis were classified as having RP (n = 17) based on mean attachment loss (AL) and/or more than three sites with AL >or=2.5 mm after scaling and root planing, surgery, and systemically administered amoxicillin and metronidazole or as GRs (n = 30) based on mean attachment gain and no sites with AL >or=2.5 mm after treatment. Significant differences in taxa among the groups were sought using the Kruskal-Wallis and chi(2) tests. RESULTS More species were detected in patients with disease (GR or RP) than in those without disease (PH). Subjects with RP were distinguished from GRs or those with PH by a significantly higher frequency of putative periodontal pathogens, such as Parvimonas micra (previously Peptostreptococcus micros or Micromonas micros), Campylobacter gracilis, Eubacterium nodatum, Selenomonas noxia, Tannerella forsythia (previously T. forsythensis), Porphyromonas gingivalis, Prevotella spp., Treponema spp., and Eikenella corrodens, as well as unusual species (Pseudoramibacter alactolyticus, TM7 spp. oral taxon [OT] 346/356, Bacteroidetes sp. OT 272/274, Solobacterium moorei, Desulfobulbus sp. OT 041, Brevundimonas diminuta, Sphaerocytophaga sp. OT 337, Shuttleworthia satelles, Filifactor alocis, Dialister invisus/pneumosintes, Granulicatella adiacens, Mogibacterium timidum, Veillonella atypica, Mycoplasma salivarium, Synergistes sp. cluster II, and Acidaminococcaceae [G-1] sp. OT 132/150/155/148/135) (P <0.05). Species that were more prevalent in subjects with PH than in patients with periodontitis included Actinomyces sp. OT 170, Actinomyces spp. cluster I, Capnocytophaga sputigena, Cardiobacterium hominis, Haemophilus parainfluenzae, Lautropia mirabilis, Propionibacterium propionicum, Rothia dentocariosa/mucilaginosa, and Streptococcus sanguinis (P <0.05). CONCLUSION As determined by HOMIM, patients with RP presented a distinct microbial profile compared to patients in the GR and PH groups.

RvE1 protects from local inflammation and osteoclast- mediated bone destruction in periodontitis

Periodontitis is a well‐appreciated example of leukocyte‐mediated bone loss and inflammation that has pathogenic features similar to those observed in other inflammatory diseases such as arthritis. Resolvins are a new family of bioactive products of omega‐3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammatory signals. Because it is now increasingly apparent that local inflammation plays a critical role in many diseases, including cardiovascular disease, atherosclerosis, and asthma, experiments were undertaken to evaluate the actions of the newly described EPA‐derived Resolvin E1 (RvE1) in regulation of neutrophil tissue destruction and resolution of inflammation. The actions of an aspirin‐triggered lipoxin (LX) analog and RvE1 in a human disease, localized aggressive periodontitis (LAP), were determined. Results indicate that neutrophils from LAP are refractory to anti‐inflammatory molecules of the LX series, whereas LAP neutrophils respond to RvE1. In addition, RvE1 specifically binds to human neutrophils at a site that is functionally distinct from the LX receptor. Consistent with these potent actions, topical application of RvE1 in rabbit periodontitis conferred dramatic protection against inflammation induced tissue and bone loss associated with periodontitis.

Resolvin E1 Regulates Inflammation at the Cellular and Tissue Level and Restores Tissue Homeostasis In Vivo

Resolvin E1 (RvE1) is a potent proresolving mediator of inflammation derived from omega-3 eicosapentaenoic acid that acts locally to stop leukocyte recruitment and promote resolution. RvE1 displays potent counter-regulatory and tissue-protective actions in vitro and in vivo. Periodontal disease is a local inflammatory disease initiated by bacteria characterized by neutrophil-mediated tissue injury followed by development of a chronic immune lesion. In this study, we report the treatment of established periodontitis using RvE1 as a monotherapy in rabbits compared with structurally related lipids PGE2 and leukotriene B4. PGE2 and leukotriene B4 each enhanced development of periodontitis and worsened the severity of disease. Promotion of resolution of inflammation as a therapeutic target with RvE1 resulted in complete restoration of the local lesion, and reduction in the systemic inflammatory markers C-reactive protein and IL-1β. This report is the first to show that resolution of inflammation by a naturally occurring endogenous lipid mediator results in complete regeneration of pathologically lost tissues, including bone.

Elevated Proinflammatory Cytokine Production by a Skewed T Cell Compartment Requires Monocytes and Promotes Inflammation in Type 2 Diabetes

An appropriate balance between proinflammatory (Th17 and Th1) and anti-inflammatory (regulatory T cells [Tregs] and Th2) subsets of T cells is critical to maintain homeostasis and avoid inflammatory disease. Type 2 diabetes (T2D) is a chronic inflammatory disease promoted by changes in immune cell function. Recent work indicates T cells are important mediators of inflammation in a mouse model of T2D. These studies identified an elevation in the Th17 and Th1 subsets with a decrease in the Treg subset, which culminates in inflammation and insulin resistance. Based on these data, we tested the hypothesis that T cells in T2D patients are skewed toward proinflammatory subsets. Our data show that blood from T2D patients has increased circulating Th17 cells and elevated activation of Th17 signature genes. Importantly, T cells required culture with monocytes to maintain Th17 signatures, and fresh ex vivo T cells from T2D patients appeared to be poised for IL-17 production. T cells from T2D patients also have increased production of IFN-γ, but produce healthy levels of IL-4. In contrast, T2D patients had decreased percentages of CD4+ Tregs. These data indicate that T cells in T2D patients are naturally skewed toward proinflammatory subsets that likely promote chronic inflammation in T2D through elevated cytokine production. Potential therapies targeted toward resetting this balance need to be approached with caution due to the reciprocal relationship between Th17 cells and Tregs. Understanding the unique aspects of T2D T cells is essential to predict outcomes of such treatments.

Deep sequencing of the oral microbiome reveals signatures of periodontal disease.

The oral microbiome, the complex ecosystem of microbes inhabiting the human mouth, harbors several thousands of bacterial types. The proliferation of pathogenic bacteria within the mouth gives rise to periodontitis, an inflammatory disease known to also constitute a risk factor for cardiovascular disease. While much is known about individual species associated with pathogenesis, the system-level mechanisms underlying the transition from health to disease are still poorly understood. Through the sequencing of the 16S rRNA gene and of whole community DNA we provide a glimpse at the global genetic, metabolic, and ecological changes associated with periodontitis in 15 subgingival plaque samples, four from each of two periodontitis patients, and the remaining samples from three healthy individuals. We also demonstrate the power of whole-metagenome sequencing approaches in characterizing the genomes of key players in the oral microbiome, including an unculturable TM7 organism. We reveal the disease microbiome to be enriched in virulence factors, and adapted to a parasitic lifestyle that takes advantage of the disrupted host homeostasis. Furthermore, diseased samples share a common structure that was not found in completely healthy samples, suggesting that the disease state may occupy a narrow region within the space of possible configurations of the oral microbiome. Our pilot study demonstrates the power of high-throughput sequencing as a tool for understanding the role of the oral microbiome in periodontal disease. Despite a modest level of sequencing (∼2 lanes Illumina 76 bp PE) and high human DNA contamination (up to ∼90%) we were able to partially reconstruct several oral microbes and to preliminarily characterize some systems-level differences between the healthy and diseased oral microbiomes.

Inflammatory and immune pathways in the pathogenesis of periodontal disease

The pathogenesis of periodontitis involves a complex immune/inflammatory cascade that is initiated by the bacteria of the oral biofilm that forms naturally on the teeth. The susceptibility to periodontitis appears to be determined by the host response; specifically, the magnitude of the inflammatory response and the differential activation of immune pathways. The purpose of this review was to delineate our current knowledge of the host response in periodontitis. The role of innate immunity, the failure of acute inflammation to resolve (thus becoming chronic), the cytokine pathways that regulate the activation of acquired immunity and the cells and products of the immune system are considered. New information relating to regulation of both inflammation and the immune response will be reviewed in the context of susceptibility to, and perhaps control of, periodontitis.

Toll-like receptors regulate B cell cytokine production in patients with diabetes

Aims/hypothesisUnderstanding cellular and molecular events in diabetes mellitus will identify new approaches for therapy. Immune system cells are important modulators of chronic inflammation in diabetes mellitus, but the role of B cells is not adequately studied. The aim of this work was to define the function of B cells in diabetes mellitus patients through focus on B cell responses to pattern recognition receptors.MethodsWe measured expression and function of Toll-like receptors (TLRs) on peripheral blood B cells from diabetes mellitus patients by flow cytometry and multiplexed cytokine analysis. We similarly analysed B cells from non-diabetic donors and periodontal disease patients as comparative cohorts.ResultsB cells from diabetes mellitus patients secrete multiple pro-inflammatory cytokines, and IL-8 production is significantly elevated in B cells from diabetic patients compared with those from non-diabetic individuals. These data, plus modest elevation of TLR surface expression, suggest B cell IL-8 hyperproduction is a cytokine-specific outcome of altered TLR function in B cells from diabetes mellitus patients. Altered TLR function is further evidenced by demonstration of an unexpected, albeit modest ‘anti-inflammatory’ function for TLR4. Importantly, B cells from diabetes mellitus patients fail to secrete IL-10, an anti-inflammatory cytokine implicated in inflammatory disease resolution, under a variety of TLR-stimulating conditions. Comparative analyses of B cells from patients with a second chronic inflammatory disease, periodontal disease, indicated that some alterations in B cell TLR function associate specifically with diabetes mellitus.Conclusions/interpretationAltered TLR function in B cells from diabetes mellitus patients increases inflammation by two mechanisms: elevation of pro-inflammatory IL-8 and lack of anti-inflammatory/protective IL-10 production.

A Molecular Defect in Intracellular Lipid Signaling in Human Neutrophils in Localized Aggressive Periodontal Tissue Damage

Host defense mechanisms are impaired in patients with congenital neutrophil (polymorphonuclear neutrophils (PMN)) defects. Impaired PMN chemotaxis is observed in localized aggressive periodontitis (LAP), a familial disorder characterized by destruction of the supporting structures of dentition. In the present studies, we sought evidence for molecular events underlying this aberrant human PMN phenotype. To this end, PMN transendothelial migration and superoxide anion generation were assessed with LAP patients and asymptomatic family members, as well as patients with other chronic mucosal inflammation. PMN from LAP patients showed decreased transmigration across vascular endothelial monolayers (18 ± 12% of control, n = 4) and increased superoxide anion generation (358 ± 37%, p = 0.003). Gene expression was analyzed using oligonucleotide microarrays and fluorescence-based kinetic PCR. cDNA microarray and kinetic-PCR analysis revealed diminished RNA expression of leukocyte-type diacylglycerol (DAG) kinase α in PMN from LAP patients (4.6 ± 1.7 relative units, n = 6, p = 0.007) compared with asymptomatic individuals (51 ± 27 relative units, n = 7). DAG kinase activity was monitored by DAG phosphorylation and individual DAG molecular species were quantified using liquid chromatography and tandem mass spectrometry-based lipidomics. DAG kinase activity was also significantly decreased (73 ± 2%, p = 0.007) and correlated with increased accumulation of 1,2-diacyl-sn-3-glycerol substrates (p = 0.01). These results implicate defects in both PMN transendothelial migration and PMN DAG kinase α signaling as disordered functions in LAP. Moreover, they identify a potential molecular lesion in PMN signal transduction that may account for their aberrant responses and tissue destruction in this disease.

Adjunctive treatment of chronic periodontitis with daily dietary supplementation with omega-3 Fatty acids and low-dose aspirin.

BACKGROUND Host modulatory therapy has been proposed as a treatment for periodontal diseases. Omega-3 (ω-3) polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), were shown to have therapeutic anti-inflammatory and protective actions in inflammatory diseases including periodontitis. The goal of this study was to test an innovative strategy for periodontal treatment in a clinical experiment. METHODS Eighty healthy subjects (40 in each group) with advanced chronic periodontitis were enrolled in Mansoura, Egypt, in a parallel-design, double-masked clinical study. The control group was treated with scaling and root planing (SRP) and a placebo, whereas the ω-3 group was treated with SRP followed by dietary supplementation of fish oil (900 mg EPA + DHA) and 81 mg aspirin daily. Saliva samples were obtained from all patients at baseline and 3 and 6 months for evaluation of receptor activator of nuclear factor-kappa B ligand (RANKL) and matrix metalloproteinase-8 (MMP-8). Plaque and gingival indices, bleeding on probing, probing depths, and attachment levels were recorded at the same time points. RESULTS Statistical analyses demonstrated a significant reduction in probing depths and a significant attachment gain after 3 and 6 months in the ω-3 group compared to baseline and the control group (P <0.05). Salivary RANKL and MMP-8 levels showed significant reductions in the ω-3 group in response to treatment at 3 and 6 months and compared to the control group at 6 months (P <0.01). Supplementation with ω-3 + aspirin resulted in a significant shift in the frequency of pockets with probing depths <4 mm (P <0.05). CONCLUSION The results of this preliminary clinical study suggest that dietary supplementation with ω-3 PUFAs and 81 mg aspirin may provide a sustainable, low-cost intervention to augment periodontal therapy.

Priming of neutrophil oxidative burst in diabetes requires preassembly of the NADPH oxidase.

Hyperglycemia associated with diabetes mellitus results in the priming of neutrophils leading to oxidative stress that is, in part, responsible for diabetic complications. p47phox, a NADPH oxidase cytosolic subunit, is a key protein in the assembly of the NADPH oxidase leading to superoxide generation. Little is known about the priming mechanism of oxidative pathways in neutrophils of people with diabetes. In this study, the kinetics of p47phox activation was investigated by comparing neutrophils from diabetic and healthy subjects, and the mechanism of hyperglycemia‐induced changes was studied by using neutrophil‐like HL‐60 cells as a model. In resting neutrophils from diabetic subjects, p47phox prematurely translocates to the cell membrane and preassembles with p22phox, a NADPH oxidase membrane subunit. This premature p47phox translocation and preassembly with p22phox were also observed in HL‐60 cells cultured with high glucose (HG; 25 mM) and with the specific ligand for the receptor for advanced glycation end products (RAGE), S100B. Phosphorylation of ERK1/2, but not p38 MAPK, was the primary signaling pathway, as evidenced by PD98059 suppressing the translocation of p47phox in HL‐60 cells incubated with HG and S100B. HL‐60 cells cultured in HG and S100B exhibited a 1.8‐fold increase in fMLP‐induced superoxide generation compared with those cultured in normal glucose (5.5 mM). These data suggest that HG and increased AGE prime neutrophils and increase oxidative stress inducing the translocation of p47phox to the cell membrane and preassembly with p22phox by stimulating a RAGE‐ERK1/2 pathway.