Sterically Enhanced Control of Enzyme-Assisted DNA Assembly.

Traditional methods for the assembly of functionalised DNA structures, involving enzyme restriction and modification, present difficulties when working with small DNA fragments (<100 bp), in part due to a lack of control over enzymatic action during the DNA modification process. This limits the design flexibility and range of accessible DNA structures. Here, we show that these limitations can be overcome by introducing chemical modifications into the DNA that spatially restrict enzymatic activity. This approach, sterically controlled nuclease enhanced (SCoNE) DNA assembly, thereby circumvents the size limitations of conventional Gibson assembly (GA) and allows the preparation of well-defined, functionalised DNA structures with multiple probes for specific analytes, such as IL-6, procalcitonin (PCT), and a biotin reporter group. Notably, when using the same starting materials, conventional GA under typical conditions fails. We demonstrate successful analyte capture based on standard and modified sandwich ELISA and also show how the inclusion of biotin probes provides additional functionality for product isolation.

Pre-conditioning of gingival epithelial cells with sub-apoptotic concentrations of curcumin prevents pro-inflammatory cytokine release.

BACKGROUND AND OBJECTIVE: Plaque-induced gingival inflammation (gingivitis) is ubiquitous in humans. The epithelial barrier reacts to the presence of oral bacteria and induces inflammatory cascades. The objective of this study was to investigate the mechanism by which the small molecule micronutrient curcumin could decrease inflammatory response in vitro to oral bacterium heat-killed Fusobacterium nucleatum as curcumin could be a useful compound for combatting gingivitis already consumed by humans. METHODS: H400 oral epithelial cell line was pre-conditioned with curcumin and the production of cytokines was measured by enzyme-linked immunosorbent assay (ELISA) and translocation of transcription factors was used to monitor inflammatory responses. Haem oxygenase (HO-1) expression and molecules that HO-1 releases were evaluated for their potential to reduce the quantity of cytokine production. Immunofluorescence microscopy and Western blotting were used to evaluate changes in transcription factor and enzyme location. RESULTS: Pre-conditioning of H400 cells with a sub-apoptotic concentration of curcumin (20 µM) attenuated secretion of Granulocyte-Macrophage - Colony-Stimulating Factor (GM-CSF) and reduced NFkB nuclear translocation. This pre-conditioning caused an increase in nuclear Nrf2; an initial drop (at 8 h) followed by an adaptive increase (at 24 h) in glutathione; and an increase in haem oxygenase (HO-1) expression. Inhibition of HO-1 by SnPPIX prevented the curcumin-induced attenuation of GM-CSF production. HO-1 catalyses the breakdown of haem to carbon monoxide, free iron and biliverdin: the HO-1/CO anti-inflammatory pathway. Elevations in carbon monoxide, achieved using carbon monoxide releasing molecule-2 (CORM2) treatment alone abrogated F. nucleatum-induced cytokine production. Biliverdin is converted to bilirubin by biliverdin reductase (BVR). This pleiotropic protein was found to increase in cell membrane expression upon curcumin treatment. CONCLUSION: Curcumin decreased inflammatory cytokine production induced by Fusobacterium nucleatum in H400 oral epithelial cells. The mechanism of action appears to be driven by the increase of haem oxygenase and the production of carbon monoxide.

Periradicular tissue fluid-derived biomarkers for apical periodontitis: An in vitro methodological and in vivo cross-sectional study.

BACKGROUND: Periradicular tissue fluid (PTF) offers a source of diagnostic, prognostic and predictive biomarkers for endodontic disease. AIMS: (1) To optimize basic parameters for PTF paper point sampling in vitro for subsequent in vivo application. (2) To compare proteomes of PTF from teeth with normal apical tissues (NAT) and asymptomatic apical periodontitis (AAP) using high-throughput panels. METHODOLOGY: (1) To assess volume absorbance, paper points (n = 20) of multiple brands, sizes and sampling durations were inserted into PBS/1%BSA at several depths. Wetted lengths (mm) were measured against standard curves to determine volume absorbance (µL). To assess analyte recovery, paper points (n = 6) loaded with 2 µL recombinant IL-1ß (15.6 ng/mL) were eluted into 250 µL: (i) PBS; (ii) PBS/1% BSA; (iii) PBS/0.1% Tween20; (iv) PBS/0.25 M NaCl. These then underwent: (i) vortexing; (ii) vortexing/centrifugation; (iii) centrifugation; (iv) incubation/vortexing/centrifugation. Sandwich-ELISAs determined analyte recovery (%) against positive controls. (2) Using optimized protocols, PTF was retrieved from permanent teeth with NAT or AAP after accessing root canals. Samples, normalized to total fluid volume (TFV), were analysed to determine proteomic profiles (pg/TFV) of NAT and AAP via O-link Target-48 panel. Correlations between AAP and diagnostic accuracy were explored using principal-component analysis (PCA) and area under receive-operating-characteristic curves (AUC [95% CI]), respectively. Statistical comparisons were made using Mann-Whitney U, anova and post hoc Bonferonni tests (α < .01). RESULTS: (1) UnoDent's 'Classic' points facilitated maximum volume absorbance (p < .05), with no significant differences after 60 s (1.6 µL [1.30-1.73]), 1 mm depth and up to 40/0.02 (2.2 µL [1.98-2.20]). For elution, vortexing (89.3%) and PBS/1% BSA (86.9%) yielded the largest IL-1ß recovery (p < .05). (2) 41 (NAT: 13; AAP: 31) PTF samples proceeded to analysis. The panel detected 18 analytes (CCL-2, -3, -4; CSF-1; CXCL-8, -9; HGF; IL-1ß, -6, -17A, -18; MMP-1, -12; OLR-1; OSM; TNFSF-10, -12; VEGF-A) in ≥75% of AAP samples at statistically higher concentrations (p < .01). CXCL-8, IL-1ß, OLR-1, OSM and TNFSF-12 were strongly correlated to AAP. 'Excellent' diagnostic performance was observed for TNFSF-12 (AUC: 0.94 [95% CI: 0.86-1.00]) and the PCA-derived cluster (AUC: 0.96 [95% CI: 0.89-1.00]). CONCLUSIONS: Optimized PTF sampling parameters were identified in this study. When applied clinically, high-throughput proteomic analyses revealed complex interconnected networks of potential biomarkers. TNFSF-12 discriminated periradicular disease from health the greatest; however, clustering analytes further improved diagnostic accuracy. Additional independent investigations are required to validate these findings.

Discovery, validation, and diagnostic ability of multiple protein-based biomarkers in saliva and gingival crevicular fluid to distinguish between health and periodontal diseases.

AIM: To discover and validate differential protein biomarker expression in saliva and gingival crevicular fluid (GCF) to discriminate objectively between periodontal health and plaque-induced periodontal disease states. MATERIALS AND METHODS: One-hundred and ninety participants were recruited from two centres (Birmingham and Newcastle upon Tyne, UK) comprising healthy, gingivitis, periodontitis, and edentulous donors. Samples from the Birmingham cohort were analysed by quantitative mass spectrometry proteomics for biomarker discovery. Shortlisted candidate proteins were then verified by enzyme-linked immunosorbent assay in both cohorts. Leave-one-out cross validation logistic regression analysis was used to identify the best performing biomarker panels. RESULTS: Ninety-five proteins were identified in both GCF and saliva samples, and 15 candidate proteins were selected based upon differences discovered between the donor groups. The best performing panels to distinguish between: health or gingivitis and periodontitis contained matrix metalloproteinase-9 (MMP9), S100A8, alpha-1-acid glycoprotein (A1AGP), pyruvate kinase, and age (area under the curve [AUC] 0.970); health and gingivitis contained MMP9, S100A8, A1AGP, and pyruvate kinase, but not age (AUC 0.768); and mild to moderate and advanced periodontitis contained MMP9, S100A8, A1AGP, pyruvate kinase, and age (AUC 0.789). CONCLUSIONS: Biomarker panels containing four proteins with and without age as a further parameter can distinguish between periodontal health and disease states.

Predicted salivary human protease activity in experimental gingivitis revealed by endoProteo-FASP approach.

Gingivitis is a highly prevalent oral condition that can be studied in humans via the 21-d experimental gingivitis model, which allows for investigations into the induction and resolution of gingivitis. In this study, we used the autolysis of saliva as a source of peptides to predict the activity of human proteases in saliva during induction and resolution of inflammation. Healthy volunteers, with no remarkable oral or systemic conditions, were recruited into the study and stimulated saliva samples were collected at days 0, 21, and 35 of experimental gingivitis. Plaque and gingival indices were recorded to ensure clinical induction and resolution. Saliva was auto-digested at 37°C for 18 h before identification of peptides by mass spectrometry. Protease prediction was carried out using Proteasix in silico with the identified peptides. A comparison of day 0 to days 21 and 35 showed changes in predicted protease activity. Correlation network analysis revealed that at day 21 the proteases became less connected and showed a potential for a dysregulated system; by day 35 the connectivity was returning towards similar conditions at day 0. This study demonstrates that changes in predicted proteases are apparent even in saliva collected from donors experiencing inflammation around three teeth.

The Saliva Proteome of Dogs: Variations Within and Between Breeds and Between Species.

Saliva is a complex multifunctional fluid that bathes the oral cavity to assist in soft and hard tissue maintenance, lubrication, buffering, defense against microbes, and initiating digestion of foods. It has been extensively characterized in humans but its protein composition in dogs remains poorly characterized, yet saliva composition could explain (patho) physiological differences between individuals, breeds and with humans. This pilot discovery study aimed to characterize canine saliva from two breeds, Labrador retrievers and Beagles, and to compare this with human saliva using quantitative mass spectrometry. The analysis demonstrated considerable inter-individual variation and difference between breeds; however these were small in comparison to the differences between species. Functional mapping suggested roles of detected proteins similar to those found in human saliva with the exception of the initiation of digestion as salivary amylase was lacking or at very low abundance in canine saliva samples. Many potential anti-microbial proteins were detected agreeing with the notion that the oral cavity is under continuous microbial challenge.

Longitudinal quantification of the gingival crevicular fluid proteome during progression from gingivitis to periodontitis in a canine model.

AIM: Inflammatory periodontal disease is widespread in dogs. This study evaluated site-specific changes in the canine gingival crevicular fluid (GCF) proteome during longitudinal progression from very mild gingivitis to mild periodontitis. Periodontitis diagnosis in dogs requires general anaesthesia with associated risks and costs; our ultimate aim was to develop a periodontitis diagnostic for application in conscious dogs. The objective of this work was to identify potential biomarkers of periodontal disease progression in dogs. MATERIAL AND METHODS: Gingival crevicular fluid was sampled from a total of 10 teeth in eight dogs at three different stages of health/disease and samples prepared for quantitative mass spectrometry (data available via ProteomeXchange; identifier PXD003337). A univariate mixed model analysis determined significantly altered proteins between health states and six were evaluated by ELISA. RESULTS: Four hundred and six proteins were identified with 84 present in all samples. The prevalence of 40 proteins was found to be significantly changed in periodontitis relative to gingivitis. ELISA measurements confirmed that haptoglobin was significantly increased. CONCLUSIONS: This study demonstrates for the first time that proteins detected by mass spectrometry have potential to identify novel biomarkers for canine periodontal disease. Further work is required to validate additional biomarkers for a periodontitis diagnostic.

Impaired neutrophil directional chemotactic accuracy in chronic periodontitis patients.

AIM: To investigate the chemotactic accuracy of peripheral blood neutrophils from patients with chronic periodontitis compared with matched healthy controls, before and after non-surgical periodontal therapy. MATERIAL & METHODS: Neutrophils were isolated from patients and controls (n = 18) by density centrifugation. Using the Insall chamber and video microscopy, neutrophils were analysed for directional chemotaxis towards N-formyl-methionyl-leucyl-phenylalanine [fMLP (10 nM), or CXCL8 (200 ng/ml)]. Circular statistics were utilized for the analysis of cell movement. RESULTS: Prior to treatment, neutrophils from patients with chronic periodontitis had significantly reduced speed, velocity and chemotactic accuracy compared to healthy controls for both chemoattractants. Following periodontal treatment, patient neutrophils continued to display reduced speed in response to both chemoattractants. However, velocity and accuracy were normalized for the weak chemoattractant CXCL8 while they remained significantly reduced for fMLP. CONCLUSIONS: Chronic periodontitis is associated with reduced neutrophil chemotaxis, and this is only partially restored by successful treatment. Dysfunctional neutrophil chemotaxis may predispose patients with periodontitis to their disease by increasing tissue transit times, thus exacerbating neutrophil-mediated collateral host tissue damage.

Oral Epithelial Cell Culture Model for Studying the Pathogenesis of Chronic Inflammatory Disease.

The interactions between bacteria, epithelium, and neutrophilic polymorphonuclear leukocytes (neutrophils) are the key to the initiation and progression of many chronic inflammatory-immune diseases. In addition, all can be influenced by external factors, such as micronutrients, thereby providing potentially novel approaches to therapy. This chapter will therefore provide detailed methods for core techniques involved in studying cellular and molecular epithelial responses to a bacterial challenge in relation to chronic inflammatory disease pathogenesis and therapy.

Antioxidant Micronutrients and Oxidative Stress Biomarkers.

Chronic inflammatory diseases are the major causes of mortality in humans and recent research has improved our understanding of the major impact of lifestyle factors upon inflammatory diseases and conditions. One of the most influential of these is nutrition, which may drive both pro-inflammatory as well as anti-inflammatory cascades at molecular and cellular levels. There are a variety of model systems that may be employed to investigate the impact of micronutrients and macronutrients upon inflammatory pathways, many of which operate through oxidative stress, either at the level of controlling the redox state of the cell and downstream redox-regulated gene transcription factors, and other acting as free radical generating or scavenging agents. This chapter focuses upon biological sample preparation prior to assay and details methods for analyzing certain antioxidant micronutrients and biomarkers of oxidative stress.

The superantigens SpeC and TSST-1 specifically activate TRBV12-3/12-4+ memory T cells.

Severe bacterial or viral infections can induce a state of immune hyperactivation that can culminate in a potentially lethal cytokine storm. The classic example is toxic shock syndrome, a life-threatening complication of Staphylococcus aureus or Streptococcus pyogenes infection, which is driven by potent toxins known as superantigens (SAgs). SAgs are thought to promote immune evasion via the promiscuous activation of T cells, which subsequently become hyporesponsive, and act by cross-linking major histocompatibility complex class II molecules on antigen-presenting cells to particular ß-chain variable (TRBV) regions of αß T cell receptors (TCRs). Although some of these interactions have been defined previously, our knowledge of SAg-responsive TRBV regions is incomplete. In this study, we found that CD4+ and CD8+ T cells expressing TRBV12-3/12-4+ TCRs were highly responsive to streptococcal pyrogenic exotoxin C (SpeC) and toxic shock syndrome toxin-1 (TSST-1). In particular, SpeC and TSST-1 specifically induced effector cytokine production and the upregulation of multiple coinhibitory receptors among TRBV12-3/12-4+ CD4+ and CD8+ memory T cells, and importantly, these biological responses were dependent on human leukocyte antigen (HLA)-DR. Collectively, these data provided evidence of functionally determinative and therapeutically relevant interactions between SpeC and TSST-1 and CD4+ and CD8+ memory T cells expressing TRBV12-3/12-4+ TCRs, mediated via HLA-DR.

Impact of Gingivitis on Circulating Neutrophil Reactivity and Gingival Crevicular Fluid Inflammatory Proteins.

Gingivitis is an extremely common oral inflammatory condition and can be induced in humans using an acute 21-day experimental gingivitis model. Neutrophils are known to be highly prevalent in the gingival crevice during gingival inflammation; however, the effect of gingivitis and the associated biofilm on peripheral blood neutrophils (PBN) is not well characterised. Thus, the aim of this study was to examine the impact of inflammation induced by experimental gingivitis and its resolution upon the function of PBN. Fifteen systemically healthy volunteers undertook a split-mouth 21-day experimental gingivitis study followed by a resolution phase of 14 days. PBN function, including reactive oxygen species (ROS) production, neutrophil extracellular trap (NET) release, directional chemotactic accuracy and expression of host mediators in gingival crevicular fluid (GCF), were measured at baseline (day 0), on day 21 and on day 35. NET formation and ROS production were significantly elevated at day 21. Chemotactic speed was also elevated in response to bacterial peptide fMLP at day 21. At day 35, ROS production in response to an Fcgamma stimulant, opsonised Staphylococcus aureus, remained elevated. The data presented suggest a lasting biological impact of the experimental gingivitis on PBN function even after clinical symptoms have abated.

Fusobacterium nucleatum Subspecies Differ in Biofilm Forming Ability in vitro.

Development of dysbiosis in complex multispecies bacterial biofilms forming on teeth, known as dental plaque, is one of the factors causing periodontitis. Fusobacterium nucleatum (F. nucleatum) is recognised as a key microorganism in subgingival dental plaque, and is linked to periodontitis as well as colorectal cancer and systemic diseases. Five subspecies of F. nucleatum have been identified: animalis, fusiforme, nucleatum, polymorphum, and vincentii. Differential integration of subspecies into multispecies biofilm models has been reported, however, biofilm forming ability of individual F. nucleatum subspecies is largely unknown. The aim of this study was to determine the single-subspecies biofilm forming abilities of F. nucleatum ATCC type strains. Static single subspecies F. nucleatum biofilms were grown anaerobically for 3 days on untreated or surface-modified (sandblasting, artificial saliva, fibronectin, gelatin, or poly-L-lysine coating) plastic and glass coverslips. Biofilm mass was quantified using crystal violet (CV) staining. Biofilm architecture and thickness were analysed by scanning electron microscopy and confocal laser scanning microscopy. Bioinformatic analysis was performed to identify orthologues of known adhesion proteins in F. nucleatum subspecies. Surface type and treatment significantly influenced single-subspecies biofilm formation. Biofilm formation was overall highest on poly-L-lysine coated surfaces and sandblasted glass surfaces. Biofilm thickness and stability, as well as architecture, varied amongst the subspecies. Interestingly, F. nucleatum ssp. polymorphum did not form a detectable, continuous layer of biofilm on any of the tested substrates. Consistent with limited biofilm forming ability in vitro, F. nucleatum ssp. polymorphum showed the least conservation of the adhesion proteins CmpA and Fap2 in silico. Here, we show that biofilm formation by F. nucleatum in vitro is subspecies- and substrate-specific. Additionally, F. nucleatum ssp. polymorphum does not appear to form stable single-subspecies continuous layers of biofilm in vitro. Understanding the differences in F. nucleatum single-subspecies biofilm formation may shed light on multi-species biofilm formation mechanisms and may reveal new virulence factors as novel therapeutic targets for prevention and treatment of F. nucleatum-mediated infections and diseases.

Free radicals and redox signalling in T-cells during chronic inflammation and ageing.

During chronic inflammation and ageing, the increase in oxidative stress in both intracellular and extracellular compartments is likely to influence local cell functions. Redox changes alter the T-cell proteome in a quantitative and qualitative manner, and post-translational modifications to surface and cytoplasmic proteins by increased reactive species can influence T-cell function. Previously, we have shown that RA (rheumatoid arthritis) T-cells exhibit reduced ROS (reactive oxygen species) production in response to extracellular stimulation compared with age-matched controls, and basal ROS levels [measured as DCF (2',7'-dichlorofluorescein) fluorescence] are lower in RA T-cells. In contrast, exposing T-cells in vitro to different extracellular redox environments modulates intracellular signalling and enhances cytokine secretion. Together, these data suggest that a complex relationship exists between intra- and extra-cellular redox compartments which contribute to the T-cell phenotype.

Pyruvate Kinase, Inflammation and Periodontal Disease.

Pyruvate kinase (PK) is the final and rate-limiting enzyme in glycolysis. It has four isoforms PKM1, PKM2, PKL and PKR. PK can form homo tetramers, dimers or monomers. The tetrameric form has the most catalytic activity; however, the dimeric form has non-canonical functions that contribute to the inflammatory response, wound healing and cellular crosstalk. This brief review explores these functions and speculates on their role in periodontal disease.

Formulation of a reactive oxygen producing calcium sulphate cement as an anti-bacterial hard tissue scaffold.

Prophylactic antibiotic bone cements are extensively used in orthopaedics. However, the development of antimicrobial resistance to antibiotics, demonstrates a need to find alternative treatments. Herein, an antimicrobial honey (SurgihoneyRO-SHRO) has been successfully incorporated into a calcium sulphate (CS) based cement to produce a hard tissue scaffold with the ability to inhibit bacterial growth. Antimicrobial properties elicited from SHRO are predominantly owed to the water-initiated production of reactive oxygen species (ROS). As an alternative to initially loading CS cement with SHRO, in order to prevent premature activation, SHRO was added into the already developing cement matrix, locking available water into the CS crystal structure before SHRO addition. Promisingly, this methodology produced > 2.5 times (715.0 ± 147.3 µM/mL/g) more ROS over 24 h and exhibited a compressive strength (32.2 ± 5.8 MPa) comparable to trabecular bone after 3 weeks of immersion. In-vitro the SHRO loaded CS scaffolds were shown to inhibit growth of clinically relevant organisms, Staphylococcus aureus and Pseudomonas aeruginosa, with comparable potency to equivalent doses of gentamicin. Encouragingly, formulations did not inhibit wound healing or induce an inflammatory response from osteoblasts. Overall this study highlights the prophylactic potential of CS-SHRO cements as an alternative to traditional antibiotics.

Proteomic analysis of a noninvasive human model of acute inflammation and its resolution: the twenty-one day gingivitis model.

The 21-day experimental gingivitis model, an established noninvasive model of inflammation in response to increasing bacterial accumulation in humans, is designed to enable the study of both the induction and resolution of inflammation. Here, we have analyzed gingival crevicular fluid, an oral fluid comprising a serum transudate and tissue exudates, by LC-MS/MS using Fourier transform ion cyclotron resonance mass spectrometry and iTRAQ isobaric mass tags, to establish meta-proteomic profiles of inflammation-induced changes in proteins in healthy young volunteers. Across the course of experimentally induced gingivitis, we identified 16 bacterial and 186 human proteins. Although abundances of the bacterial proteins identified did not vary temporally, Fusobacterium outer membrane proteins were detected. Fusobacterium species have previously been associated with periodontal health or disease. The human proteins identified spanned a wide range of compartments (both extracellular and intracellular) and functions, including serum proteins, proteins displaying antibacterial properties, and proteins with functions associated with cellular transcription, DNA binding, the cytoskeleton, cell adhesion, and cilia. PolySNAP3 clustering software was used in a multilayered analytical approach. Clusters of proteins that associated with changes to the clinical parameters included neuronal and synapse associated proteins.

Crevicular fluid glutathione levels in periodontitis and the effect of non-surgical therapy.

AIM: To quantify reduced and oxidized glutathione (GSH and GSSG) levels in gingival crevicular fluid (GCF) of periodontitis patients pre-therapy (versus periodontally healthy controls) and ascertain whether successful non-surgical therapy alters glutathione levels. MATERIALS AND METHODS: Thirty-second GCF samples (6/subject) were collected on Periopaper() strips from starved, non-smokers (n=20; mean age 43.6 years) with chronic periodontitis, before and 3 months after non-surgical therapy, and periodontally healthy, age- and gender-matched controls (n=20). GSH and GSSG levels were determined using reversed-phase high-performance liquid chromatography with fluorescence detection. RESULTS: Lower concentrations of GSH (p<0.01) and GSSG (p<0.05) were detected in GCF from patients (pre- and post-therapy) than controls and treatment had no significant effect. Amounts per 30-second sample did not differ between patients and controls. However, the amount of GSSG per 30-second sample decreased in patients after therapy (p<0.05). Consequently, therapy increased the GSH:GSSG ratio (p<0.05) in patients compared with the controls (p=0.8). CONCLUSION: These data demonstrate high concentrations of GSH within GCF, which are compromised in chronic periodontitis. While therapy does not appear to fully restore GSH concentrations in GCF, it does restore the redox balance (GSH:GSSG ratio), suggesting that the abnormal redox balance arises secondary to oxidative stress resulting from periodontal inflammation.

Oxygen tension modulates the cytokine response of oral epithelium to periodontal bacteria.

BACKGROUND: There is an inverse relationship between pocket depth and pocket oxygen tension with deep pockets being associated with anaerobic bacteria. However, little is known about how the host tissues respond to bacteria under differing oxygen tensions within the periodontal pocket. AIM: To investigate the effect of different oxygen tensions upon nuclear factor-kappa B (NF-κB) activation and the inflammatory cytokine response of oral epithelial cells when exposed to nine species of oral bacteria. MATERIALS AND METHODS: H400 oral epithelial cells were equilibrated at 2%, 10% or 21% oxygen. Cells were stimulated with heat-killed oral bacteria at multiplicity of infection 10:1, Escherichia coli lipopolysaccharide (15 µg/ml) or vehicle control. Interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-α) levels were measured by enzyme-linked immunosorbent assay and NF-κB activation was measured by reporter vector or by immunohistochemical analysis. RESULTS: Tannerella forsythensis, Porphyromonas gingivalis and Prevotella intermedia elicited the greatest epithelial NF-κB activation and cytokine responses. An oxygen-tension-dependent trend in cytokine production was observed with the highest IL-8 and TNF-α production observed at 2% oxygen and lowest at 21% oxygen. CONCLUSIONS: These data demonstrate a greater pro-inflammatory host response and cell signalling response to bacteria present in more anaerobic conditions, and hypersensitivity of epithelial cells to pro-inflammatory stimuli at 2% oxygen, which may have implications for disease pathogenesis and/or therapy.

Proteomic Investigations of In Vitro and In Vivo Models of Periodontal Disease.

Proteomics has currently been a developing field in periodontal diseases to obtain protein information of certain samples. Periodontal disease is an inflammatory disorder that attacks the teeth, connective tissues, and alveolar bone within the oral cavity. Proteomics information can provide proteins that are differentially expressed in diseased or healthy samples. This review provides insight into approaches researching single species, multi species, bacteria, non-human, and human models of periodontal disease for proteomics information. The approaches that have been taken include gel electrophoresis and qualitative and quantitative mass spectrometry. This review is carried out by extracting information about in vitro and in vivo studies of proteomics in models of periodontal diseases that have been carried out in the past two decades. The research has concentrated on a relatively small but well-known group of microorganisms. A wide range of models has been reviewed and conclusions across the breadth of these studies are presented in this review.