Detection of adrenomedullin and nitric oxide in different forms of periodontal disease.

BACKGROUND AND OBJECTIVE: The multifunctional molecules adrenomedullin (AM) and nitric oxide (NO) are both involved in the host response to microbial challenge during periodontal disease. Whether they coexist in periodontal inflammation and if equally produced in the different forms of periodontal disease has not previously been investigated. The aims of this study were to describe the locations of AM and NO in healthy and inflamed gingival tissues and to determine and compare their levels in the gingival crevicular fluid and saliva of patients with gingivitis, chronic periodontitis and aggressive periodontitis. MATERIAL AND METHODS: AM and inducible nitric oxide synthase (iNOS) were immunolocalized in clinically healthy and inflamed gingival tissue sections. The cells expressing AM and iNOS were characterized using immunocytochemistry with different markers for macrophages [cluster differentiation (CD)68 and CD14)], dendritic cells (CD83), neutrophils [neutrophil gelatinase-associated lipocalin (nGAL)] and natural killer cells (CD56). In an initial study, the levels of AM and NO were also measured in samples of gingival crevicular fluid and saliva obtained from patients with a diagnosis of gingivitis (n = 9), chronic periodontitis (n = 9) and aggressive periodontitis (n = 9) using an ELISA and the nitrate/nitrite (NO metabolites) Griess assay, respectively. RESULTS: Low levels of AM- and iNOS-expressing cells were detected in healthy gingival tissues in comparison with three-fold higher levels of these cells in inflamed tissues. These cells were localized mainly in the epithelial layer but were also present in deeper connective tissue. AM and iNOS were co-localized in particular cells within inflamed tissues, namely CD68(+) (52%) and CD14(+) (36%) macrophages, but also in nGAL(+) neutrophils (16%) and CD83(+) dendritic cells (14%). Interestingly, AM and NO levels in saliva were both found to be higher (p < 0.01) in patients with aggressive periodontitis than in patients with chronic periodontitis or gingivitis. In contrast, in gingival crevicular fluid, the levels of NO showed marked differences among patients with chronic periodontitis, aggressive periodontitis and gingivitis (p < 0.01), and the levels of AM were higher (p < 0.01) in both chronic and aggressive periodontitis compared with gingivitis alone. CONCLUSION: The data presented demonstrate a functional linkage between AM and NO in periodontal disease, with salivary and gingival crevicular fluid levels possibly associated with different forms and severities of periodontal disease. Exacerbated production of both AM and NO in saliva suggests their potential use as salivary markers of aggressive periodontitis.

Anti-biofilm activity of zinc oxide and hydroxyapatite nanoparticles as dental implant coating materials.

OBJECTIVES: Dental implants are prone to failure as a result of bacterial biofilm accumulation. Such biofilms are often resistant to traditional antimicrobials and the use of nanoparticles as implant coatings may offer a means to control infection over a prolonged period. The objective of this study was to determine the antibiofilm activity of nanoparticulate coated titanium (Ti) discs using a film fermenter based system. METHODS: Metal oxide nanoparticles of zinc oxide (nZnO), hydroxyapatite (nHA) and a combination (nZnO+nHA) were coated using electrohydrodynamic deposition onto Ti discs. Using human saliva as an inoculum, biofilms were grown on coated discs for 96 h in a constant depth film fermenter under aerobic conditions with artificial saliva and peri-implant sulcular fluid. Viability assays and biofilm thickness measurements were used to assess antimicrobial activity. RESULTS: Following 96 h, reduced numbers of facultatively anaerobic and Streptococcus spp. on all three nano-coated surfaces were demonstrated. The proportion of non-viable microorganisms was shown to be higher on nZnO and composite (nZnO+nHA) coated surfaces at 96 h compared with nHA coated and uncoated titanium. Biofilm thickness comparison also demonstrated that nZnO and composite coatings to be the most effective. CONCLUSIONS: The findings support the use of coating Ti dental implant surfaces with nZnO to provide an antimicrobial function. CLINICAL SIGNIFICANCE: Current forms of treatment for implant associated infection are often inadequate and may result in chronic infection requiring implant removal and resective/regenerative procedures to restore and reshape supporting tissue. The use of metal oxide nanoparticles to coat implants could provide osteoconductive and antimicrobial functionalities to prevent failure.

Regulation of adrenomedullin and nitric oxide production by periodontal bacteria.

OBJECTIVES: In periodontitis the host response to bacterial challenge includes activity of the multifunctional molecules adrenomedullin (AM) and nitric oxide (NO). The aim of this study was to investigate the role of periodontal bacteria in regulating the production of these molecules from cultured cells. MATERIAL AND METHODS: Regulation of AM and NO production from oral keratinocytes when challenged with culture supernatants from Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Veillonella atypica, Streptococcus salivarius and Candida albicans was examined. AM and NO were measured in cell culture supernatants using an enzyme-linked immunosorbent assay and the nitrate/nitrite (NO metabolites) Griess assay respectively. Cellular production of AM and inducible NO synthase was also analysed in target cells by immunofluorescence and Western blot analysis. The inter-relationship of AM and NO production were further investigated with macrophages. RESULTS: A. actinomycetemcomitans and C. rectus induced maximal levels of both AM and NO after 6 and 48 h respectively from oral keratinocytes. AM production in macrophages was upregulated in response to the NO donor S-nitrosoglutathione and partially blocked by the inducible NO synthase inhibitor, N(ω) -Nitro-l-arginine methyl ester hydrochloride. Likewise, NO production was increased upon exposure to AM, while the AM receptor antagonist AM 22-52 reduced the release of NO. CONCLUSIONS: Pathogens associated with aggressive periodontitis, A. actinomycetemcomitans and C. rectus, were more effective than those associated with chronic periodontitis, P. gingivalis and Prev. intermedia, and commensals, S. salivarius and V. atypica, as regards the upregulation of AM and NO production from oral keratinocytes. Interaction between these molecules was also demonstrated with macrophages. Understanding the coordinated regulation of AM and NO production in response to periodontal bacteria may identify ways to promote their protective effects and minimize destructive potential.

Antimicrobial properties of electrically formed elastomeric polyurethane-copper oxide nanocomposites for medical and dental applications.

With the rapidly advancing field of nanotechnology having an impact in several areas interfacing life and physical sciences, the potential applications of nanoparticles as antimicrobial agents have been realized and offer great opportunities in addressing several viral and bacterial outbreak issues. Polyurethanes (PUs) are a diverse class of polymeric materials which also have applications in several areas of biomedical science ranging from blood contact devices to implantable dental technologies. In this report, copper oxide (CuO) nanoparticles (mean size ∼50 nm) are embedded into a PU matrix via two electrical fabrication processes. To elucidate the antimicrobial activity, a range of different loading compositions of CuO within the PU matrix (0%, 1%, 5%, and 10% w/w) are electrospun to form thin porous films (thickness < 10 µm). After washing, the films are tested for their antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA). Significant reduction of populations was demonstrated with 10% w/w CuO over a 4-h period. This approach demonstrates the potential of generating tailored antimicrobial structures for a host of applications, such as designer filters, patterned coatings, breathable fabrics, adhesive films (as opposed to sutures), and mechanically supporting structures.

Effects of low-dose doxycycline on cytokine secretion in human monocytes stimulated with Aggregatibacter actinomycetemcomitans.

Doxycycline is an antibiotic used in the treatment of a variety of inflammatory conditions, including periodontitis. Apart from its antimicrobial properties, this drug also has independent anti-inflammatory effects at sub-antimicrobial doses. The present study aimed to investigate the effects of low-doses of doxycycline (LDD) on cytokine production by human monocytic cells challenged with the periodontal pathogen Aggregatibacter actinomycetemcomitans, for up to 6 h. The simultaneous regulation of 12 cytokines were measured by a Human Cytokine Array Kit. To validate the array findings, selected cytokines were also measured by enzyme-linked immunosorbant assay (ELISA). A. actinomycetemcomitans stimulated the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1ß, IL-6 and IL-8 by the cells after 6 h of challenge, and doxycycline significantly inhibited this effect. The kinetics of this regulation demonstrated an early (within 2 h) and significant (P<0.05) inhibition of pro-inflammatory cytokines, with a mild (0.5-fold) up-regulation of the anti-inflammatory cytokine IL-10. The results indicate that LDD acts as an anti-inflammatory agent in human monocytic cells stimulated with A. actinomycetemcomitans. This model provides clear evidence that some of the clinically proven benefits of LDD may be related to its ability to regulate inflammatory mediator release by monocytic cells. This property may contribute to the clinically proven benefits of this antibiotic as an adjunctive treatment for periodontitis.

Levels of periodontal pathogens in neonatal gastric aspirates and possible maternal sites of origin.

Maternal periodontal infection has been recognized as a risk factor for preterm and low birthweight infants. It is suspected that pathogens causing periodontal disease may translocate to the amniotic cavity and so contribute to triggering an adverse pregnancy outcome. This study aimed to determine levels and proportions of periodontal bacteria in neonatal gastric aspirates obtained from complicated pregnancies and the respective maternal oral and vaginal samples using a quantitative polymerase chain reaction approach, and also to determine the origin of the neonate's bacteria by sequence comparisons between the three sites. Aggregatibacter actinomycetemcomitans and Tannerella forsythia were not observed in the neonates or in the women's vaginas. Interestingly, Porphyromonas gingivalis was identified in the neonates in two samples (2.98E+02 and 1.75E+02 cells ml(-1)) and in association with Fusobacterium nucleatum, which was observed at high prevalence (10%) and at high levels reaching up to 2.32E+03 cells ml(-1). Although F. nucleatum was also present in the vaginal samples, the results demonstrated that the neonatal strains were more likely to originate from the mother's oral cavity than to be vaginal strains.

Electrohydrodynamic deposition of nanotitanium doped hydroxyapatite coating for medical and dental applications.

Nano-sized titanium containing hydroxyaptite has been prepared, the particle size of nanoTiHA was shown to be 12-20 nm in width and 30-40 nm in length, smaller than that of nanoHA. X-ray diffraction analysis revealed the phase purity of nanoTiHA produced. Antimicrobical assays demonstrated that nanoTiHA has excellent growth inhibitory properties, and is able to inhibit the growth of all bacterial strains tested, both Gram-negative and Gram-positive species, including multi-antibiotic resistant EMRSA 15 and EMRSA 16 'superbugs'. Biocidal activity against all four Staphylococcus spp was also shown at the concentration tested. Nanostuctured TiHA coating was successfully deposited onto Ti surfaces using EHDA spraying under optimized processing conditions with the thickness of the coating being further controlled by the spraying time. All of the nanoTiHA coated Ti surfaces were able to support human osteoblast (HOB) cell attachment and growth. The coating thickness did not significantly influence the proliferation of HOB cells on nanoTiHA coatings, while the ability of nanoTiHA coating to support HOB cell differentiation was demonstrated from the alkaline phosphatase activity. Our study showed that nanoTiHA has excellent anti-bacterial properties and the thin nanoTiHA coating was also able to support the attachment, growth and differentiation of HOB cells. Therefore, nanoTiHA coating could pave the way for the development of the next generation of dental and orthopedic implants by offering anti-infection potential in addition to osteoconductivity.

The use of nanoparticles to control oral biofilm formation.

Nanoparticles are normally considered to be of a size no greater than 100 nm, and the exploitation of their unique attributes to combat infection has increased markedly over the past decade. The potential of nanoparticles to control the formation of biofilms within the oral cavity, as a function of their biocidal, anti-adhesive, and delivery capabilities, is now coming under close scrutiny. Possible uses as constituents of prosthetic device coatings, as topically applied agents, and within dental materials are being explored. The latest insights into the application of nanoparticles in the control of oral infections, including their use in photodynamic therapy, will be discussed in this review. In particular, the use of nanoparticulate silver, copper, zinc, silicon, and their oxides will be considered in relation to their effects on bacterial populations. The recent interest in the applications of nanoparticulate polymers and calcium phosphates will also be assessed. Particular attention will be paid to the toxicity issues surrounding the potential impact of nanoparticles on oral and other tissues.

A review of nanoparticle functionality and toxicity on the central nervous system.

Although nanoparticles have tremendous potential for a host of applications, their adverse effects on living cells have raised serious concerns recently for their use in the healthcare and consumer sectors. As regards the central nervous system (CNS), research data on nanoparticle interaction with neurons has provided evidence of both negative and positive effects. Maximal application dosage of nanoparticles in materials to provide applications such as antibacterial and antiviral functions is approximately 0.1-1.0 wt%. This concentration can be converted into a liquid phase release rate (leaching rate) depending upon the host or base materials used. For example, nanoparticulate silver (Ag) or copper oxide (CuO)-filled epoxy resin demonstrates much reduced release of the metal ions (Ag(+) or Cu(2+)) into their surrounding environment unless they are mechanically removed or aggravated. Subsequent to leaching effects and entry into living systems, nanoparticles can also cross through many other barriers, such as skin and the blood-brain barrier (BBB), and may also reach bodily organs. In such cases, their concentration or dosage in body fluids is considered to be well below the maximum drug toxicity test limit (10(-5) g ml(-1)) as determined in artificial cerebrospinal solution. As this is a rapidly evolving area and the use of such materials will continue to mature, so will their exposure to members of society. Hence, neurologists have equal interests in nanoparticle effects (positive functionality and negative toxicity) on human neuronal cells within the CNS, where the current research in this field will be highlighted and reviewed.

Investigations into the micro-ecology of oral malodour in man and companion animals.

The loads and locations of bacterial types associated with oral malodour on the tongue surface and gingival crevice were investigated in man and the dog respectively. In the human study, samples were taken from 50 subjects with brushes at the dorsal anterior, dorsal middle, dorsal posterior, dorsal posterior to the circumvallate papillae (DPCP), lateral posterior and ventral posterior (VP) surfaces, and cultured appropriately. Malodour was assessed by trained judges. Mean volatile sulfur compound (VSC) producing bacterial counts (colony forming units/brush × 10(5)) were found to be highest (88.94) and lowest (0.33) at the DPCP and VP sites respectively. Anaerobic, gram-negative and VSC counts at DPCP surfaces increased with malodour intensity, whereas aerobic and S. salivarius counts decreased. The prevalence and populations of the VSC producing Porphyromonas and Prevotella species were determined in the dental plaque from 34 dogs. Porphyromonas gulae and Prevotella intermedia were present in 68% and 44% of dogs, and 47% and 23% of plaque samples respectively. P. gulae and Prev. intermedia counts increased with plaque quantity (P < 0.05) and gingivitis (P < 0.1). The close association observed between canine periodontal disease and measurements of oral malodour is supported.

Porphyromonas gingivalis antagonises Campylobacter rectus induced cytokine production by human monocytes.

Porphyromonas gingivalis and Campylobacter rectus are two major bacterial species implicated in the pathogenesis of periodontitis. P. gingivalis can antagonise the inflammatory response to other periodontal pathogens, a property commonly attributed to its lipopolysaccharide (LPS). The aim of this study was to investigate the capacity of P. gingivalis to antagonise C. rectus induced cytokine stimulation from human monocytes, and to investigate the involvement of its LPS. Primary human monocytes and Monomac-6 cells were challenged with culture supernatants from P. gingivalis and C. rectus, and levels of IL-1beta, IL-6 and IL-8 produced were measured by ELISA after 6h incubation. Purified P. gingivalis LPS was also added alone or in combination with C. rectus culture supernatant. Both species significantly stimulated the production of all three cytokines from the two cell lines, but P. gingivalis was considerably weaker inducer. Co-stimulation of the cells with P. gingivalis and C. rectus suppressed the cytokine-stimulatory capacity of the latter. P. gingivalis LPS alone was sufficient to antagonise IL-6 and IL-8, but not IL-1beta stimulation by C. rectus. In conclusion, mixed infections may impair host immune responses by reducing pro-inflammatory cytokine levels, which may be of relevance to the pathogenesis of periodontitis.

Protective effect of salivary nitrate and microbial nitrate reductase activity against caries.

To test the hypothesis that a combination of high salivary nitrate and high nitrate-reducing capacity are protective against dental caries, 209 children attending the Dental Institute, Barts and The London NHS Trust were examined. Salivary nitrate and nitrite levels, counts of Streptococcus mutans and Lactobacillus spp., and caries experience were recorded. Compared with control subjects, a significant reduction in caries experience was found in patients with high salivary nitrate and high nitrate-reducing ability. Production of nitrite from salivary nitrate by commensal nitrate-reducing bacteria may limit the growth of cariogenic bacteria as a result of the production of antimicrobial oxides of nitrogen, including nitric oxide.

Characterization and expression of adjacent proline iminopeptidase and aspartase genes from Eikenella corrodens.

Two adjacent genes involved in nitrogen metabolism from Eikenella corrodens, with a potential role in pathogenesis, were studied. Proline iminopeptidase (Pip) activity, which may be essential for energy production and protection against host immune mechanisms, is exhibited by E. corrodens. Analysis of Pip-expressing clones revealed an ORF of 939 bases with a predicted amino acid sequence identity of 67% to the Pip of Neisseria gonorrhoea. 200 bp downstream from pip, an ORF of 1395 bases, encoding a protein with 87% identity to a putative aspartase from the Neisseria meningitidis genome sequence, was identified. Enzymatic function was confirmed with a complemented Escherichia coli aspartase deficient mutant. The E. corrodens aspartase was found to be 77% identical to the Haemophilus influenzae aspartase sequence, which was originally identified on the basis of its ability to bind plasminogen. However, the E. corrodens aspartase had no such activity. Southern hybridization indicated both genes to be single copy and conserved within the genomes of a diverse panel of E. corrodens isolates from health and disease.

Prevalence of helicobacter pylori at oral and gastrointestinal sites in children: evidence for possible oral-to-oral transmission.

Acquisition of Helicobacter pylori occurs mainly in childhood. However, the mode of transmission remains unclear. To help elucidate this, 100 children attending for upper gastrointestinal endoscopy were investigated for the presence of H. pylori at various sites. H. pylori was detected in antral gastric biopsies by the rapid urease test (13 patients), culture (13 patients), histology (15 patients) and PCR (20 patients). Gastric juice was positive for H. pylori in 3 patients by culture and 11 patients by PCR. The dental plaque from 68% of gastric biopsy-positive patients (as determined by culture or PCR) and 24% of gastric biopsy-negative patients was positive for H. pylori by PCR. The presence of H. pylori in dental plaque was significantly associated with the presence of this organism in the stomach. H. pylori was detected by PCR in the faeces of 25% of gastric biopsy-positive children sampled. H. pylori was not cultured on any occasion from the oral cavity or faeces. The evidence from this study suggests that oral-to-oral transmission may be a possible mode of spread of H. pylori in children.

Adrenomedullin expression in pathogen-challenged oral epithelial cells.

Adrenomedullin, a multifunctional peptide, is expressed by many surface epithelial cells and, previously, we have demonstrated that adrenomedullin has antimicrobial activity. The oral cavity contains an epithelium that is permanently colonized by microflora, yet infections in a host are rare. We exposed oral keratinocytes to whole, live cells from four microorganisms commonly isolated from the oral cavity, Porphyromonas gingivalis, Streptococcus mutans, Candida albicans and Eikenella corrodens. There was upregulation of protein and gene expression in these cells in response to bacterial suspensions, but not with the yeast, Candida albicans. We propose there is a potential role for microbial products in enhancing mucosal defense mechanisms and that adrenomedullin participates in the prevention of local infection, thus contributing to host defense mechanisms.

Antimicrobial effect of acidified nitrite on periodontal bacteria.

The antimicrobial agent nitric oxide (NO) is formed in the mouth and its concentration is directly related to salivary nitrite, which in turn is related to dietary nitrate intake. The aim of this study was to determine whether nitrite under acidic conditions will have an inhibitory effect, possibly occurring through NO production, on the periodontal disease pathogens Fusobacterium nucleatum, Eikenella corrodens and Porphyromonas gingivalis. Whereas the growth of these organisms was inhibited by a more acid pH, the addition of nitrite caused a marked, further dose-dependent reduction in bacterial numbers after exposure. The ability of these bacteria to recover from nitrite exposure was also affected by pH and nitrite concentration. At acidity levels below pH 5.0, low concentrations of nitrite (0.2 mM) caused effective complete killing of the periodontal bacteria. Addition of sodium thiocyanate did not increase the bacteriostatic or bacteriocidal activity of acidified nitrite against any of the 3 bacteria. These results demonstrate the possibility that nitrite in saliva, under appropriate conditions, may have an effect on the growth and survival of the bacteria implicated in periodontal disease.

Morphological analysis of Helicobacter pylori from gastric biopsies and dental plaque by scanning electron microscopy.

Helicobacter pylori is rarely cultured from sites other than the gastric mucosa. The morphology of H. pylori in the stomach and dental plaque of adult dyspeptic patients was investigated to determine whether a difference in morphology at these sites could explain the inability to culture the organism from the oral cavity. Five adult patients attending for an upper gastrointestinal endoscopy were investigated. Dental plaque and gastric antral biopsy samples were analysed by culture and polymerase chain reaction (PCR) both before and after immunomagnetic separation using polyclonal rabbit anti-H. pylori IgG. Bead:bacteria aggregates were then examined by scanning electron microscopy (SEM). Rod and coccoid forms of H. pylori were seen by SEM in all oral and gastric samples which were H. pylori PCR positive. Although rod and coccoid forms have previously been shown to be associated with the gastric mucosa, this is the first time H. pylori cells have been visualized in dental plaque.

Quantitative culture of Helicobacter pylori from gastric juice: the potential for transmission.

The transmission of Helicobacter pylori may occur by spread of organisms from gastric juice which has been introduced into the mouth by gastro-oesophageal reflux. The aim of this study was to quantify the load of H. pylori present in gastric juice available for transmission. Gastric antral biopsy and gastric juice samples were collected from 108 adult dyspeptic patients undergoing routine upper gastroscopy and the presence of H. pylori was determined. In all, 54 (50%) of 108 patients gave positive results in the gastric antral biopsy rapid urease test and for H. pylori histology. The gastric juice of 40 (37%) of patients gave positive results for the urease A gene by PCR assay; 34 (31%) of patients were positive by these three tests and H. pylori was cultured from the gastric juice of 13 (38%) of these patients. The median count of H. pylori in gastric juice was 1.75 x 10(1) cfu/ml. Viable organisms in gastric juice may lead to transmission of H. pylori when refluxed or vomited into the mouth.

An investigation into the antimicrobial effects of adrenomedullin on members of the skin, oral, respiratory tract and gut microflora.

Adrenomedullin, a novel vasoactive peptide, is known to be expressed by many surface epithelial cells and it was postulated that this peptide may have a protective role. The objective of the study was to assess the antimicrobial activity of adrenomedullin against members of the human skin, oral, respiratory tract and gut microflora using disc diffusion and broth microdilution assays. All strains of bacteria screened in an agar diffusion assay were sensitive; gram-positive and gram-negative bacteria were equally susceptible. No activity against the yeast Candida albicans was observed. In a broth microdilution assay, minimum inhibitory and minimum bacteriocidal concentrations ranged from 7.75 x 10(-1) to 12.5 microg ml(-1) and 0.003 to > 25.0 microg ml(-1), respectively. We propose an antimicrobial role for adrenomedullin. participating in the prevention of local infection, thus contributing to host defence systems.