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.

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.

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.

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.

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.

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.

Production of hydrolytic enzymes by oral isolates of Eikenella corrodens.

Eikenella corrodens isolates from periodontally healthy subjects and adult periodontitis patients were compared for their ability to produce a range of potential virulence factors. All were positive for proline aminopeptidase, thiol-dependent haemolysin and esterase activities. Low or negative activities were found against casein, phospholipid, lipid, collagen, aminophosphate, phosphate under acid or alkaline conditions, and eleven other amino acid substrates tested. In oral infections, the haemolytic activity of E. corrodens could be amplified in the reduced environment of the periodontal pocket and damage host cells. Proline aminopeptidase may act against proline residues in collagen, immunoglobulins and complement proteins.

Characterisation of Staphylococcus intermedius isolates from canine pyoderma and from healthy carriers by SDS-PAGE of exoproteins, immunoblotting and restriction endonuclease digest analysis.

Ten Staphylococcus intermedius isolates from cases of canine pyoderma and 10 from healthy carriers were examined by SDS-PAGE of exoproteins, immunoblotting and restriction endonuclease digest analysis. Similarities between banding patterns of the isolates were calculated as Dice coefficients for all three methods. For SDS-PAGE and immunoblotting, no significant differentiation was found between the pyoderma and "healthy" groups. Analysis of DNA digested with BglII indicated that S. intermedius is genetically heterogeneous; Dice coefficients for the pyoderma group were distinct from those for the healthy group (p < 0.001), and cluster analysis confirmed that the pyoderma isolates (9) formed a group separate from the majority (6 of 9) of the normal isolates.

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.

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.

Cytotoxic activity of Staphylococcus hyicus.

Culture supernatants from a number of Staphylococcus hyicus strains caused toxic effects to both murine fibroblast and porcine keratinocyte cells in culture. The extent of cytotoxicity was shown to differ between strains and may provide an indication of strain virulence. Purification of cytotoxic activity produced by S. hyicus (strain P119) using preparative isoelectric-focussing demonstrated it to be cytolytic, haemolytic and non-proteolytic. The cytotoxin demonstrates certain properties in common with the delta haemolysin of Staphylococcus aureus.

Detection of Streptococcus mutans by PCR amplification of the spaP gene in teeth rendered caries free.

OBJECTIVES: To investigate the degree of association between tactile and optical criteria as used to assess the carious status of the enamel-dentine junction (EDJ) during cavity preparation, assessment with a caries detector dye and detection of Streptococcus mutans using culture and polymerase chain reaction (PCR) techniques. METHODS: Twenty-nine teeth, extracted within the previous 30 min, and 15 teeth prepared under rubber dam in vivo, were clinically assessed at the EDJ after the removal of evident carious tissue. Demineralisation was then assessed using a caries detector dye (1% acid red in propylene glycol; Cavex). A rosehead bur was used to remove tissue at the EDJ for culture and PCR analysis. Culture was carried out on a tryptone yeast cystine sucrose bacitracin selective medium, and PCR used to amplify a sequence (192 bp) of the spaP gene, which encodes the surface protein antigen I/II of S. mutans. RESULTS: Demineralised tissue at the EDJ, as shown using the dye, was found in 52% of teeth. Removed tissue was culture and PCR positive for S. mutans in 2 and 47% of teeth, respectively. A highly significant association (77% of cases; P < 0.001) was shown between dye and PCR assessment methods. No association was found between any other combination of assessment methods. CONCLUSIONS: Culture methods may underestimate the presence of S. mutans. Removal of sufficient dye-stained tissue is therefore recommended to prevent further carious assault from residual S. mutans.

Occurrence of Staphylococcus intermedius on the hair and skin of normal dogs.

Aerobic bacterial populations were studied on the distal hair coat and at the skin surface of the shoulder, rump and abdomen of 10 healthy dogs. Coagulase negative staphylococci (CNS) were more frequently isolated from the hair than the skin at the shoulder and rump. There was no difference in the isolation rate of coagulase positive staphylococci (CPS) (Staphylococcus intermedius) between the hair and skin. Total skin counts were greatest on the abdomen whereas CNS counts from the hair were least at this site. There were no differences between CPS counts at the three sites on either hair or skin. The populations on the relatively unfavourable microenvironment of the distal hair may represent contamination rather than colonisation. The low populations of CPS at the skin surface also indicate contamination or transient colonisation rather than true resident status.

Clinical and microbiological effects of topical minocycline in the treatment of elderly patients with periodontitis.

OBJECTIVE: To evaluate a 2% w/w minocycline gel (Dentomycin) in the treatment of older adults with chronic periodontitis. DESIGN: Vehicle-controlled, split-mouth SETTING: Dental Institute, St Bartholomew's and the Royal London School of Medicine and Dentistry. SUBJECTS: 22 adults (mean age 60 years). INTERVENTIONS: At baseline, patients received scaling and root planing. Test and placebo were applied at contralateral disease sites surrounding 22 test and 22 control teeth at baseline, and at weeks 2 and 4. MAIN OUTCOME MEASURE: Probing pocket depth, bleeding on probing and supragingival plaque measurements, and microbiological sampling, at one pocket site per tooth were undertaken at baseline, and at weeks 2, 4, 6 and 16. RESULTS: Microbial counts of Porphyromonas gingivalis, Prevotella intermedia and Eikenella corrodens did not change significantly over 16 weeks. No significant reductions in bleeding on probing and supragingival plaque score occurred with either gel. A larger reduction in mean pocket depth, from baseline to week 16, was found at test (6.50 to 4.95 mm; 24% reduction; P < 0.01) than at control sites (6.41 to 5.53 mm; 14% reduction; P < 0.05). CONCLUSIONS: Minocycline gel administration is a useful adjunct to scaling and root planing in the treatment of older adults with periodontitis.

Staphylococci on the skin of pigs: isolates from two farms with different antibiotic policies.

Staphylococci isolated from pigs on two farms were identified and their antibiotic resistance and plasmid profiles were examined. A highly resistant Staphylococcus hyicus was epidemic on one of the farms which was also that at which antibiotic-containing feedstuffs were used most often. Staphylococci from this farm were more often resistant to two or more antibiotics than were the strains from the other farm. The many plasmids present in these staphylococci prevented the determination of the genetic nature of the antibiotic resistance.

Prevalence of Eikenella corrodens and Actinobacillus actinomycetemcomitans in the dental plaque of dogs.

The prevalence and populations of Eikenella corrodens, Actinobacillus actinomycetemcomitans and black pigmented anaerobic bacilli were determined in three samples of supragingival plaque from each of 30 healthy dogs. E corrodens was present in 62 per cent of the dogs and 43.7 per cent of the individual plaque samples but none of the dogs yielded A actinomycetemcomitans. Black pigmented anaerobic bacilli were present in 46.7 per cent of the dogs and 36.7 per cent of the individual plaque samples, and their numbers were correlated with the age of the dogs (P < 0.05) and the amount of plaque (P < 0.01). It is suggested that the prevalence of E corrodens in wound infections of man from dog bites has been underestimated from lack of appropriate techniques for detecting this fastidious organism.

Prevalence of Porphyromonas and Prevotella species in the dental plaque of dogs.

The prevalence and populations of Porphyromonas and Prevotella species were determined in three samples of dental plaque from each of 34 healthy dogs. Porphyromonas gingivalis was present in 68 per cent of the dogs and 47 per cent of the plaque samples. The counts of P gingivalis increased with age (P < 0.1), the amount of plaque (P < 0.05) and the degree of gingivitis (P < 0.1). Prevotella intermedia was present in 44 per cent of the dogs and 23 per cent of the plaque samples. The counts of P intermedia were correlated with the amount of plaque (P < 0.05) and the degree of gingivitis (P < 0.1). Porphyromonas canoris, P salivosa, P cangingivalis, P cansulci, P crevioricanis and Prevotella denticola were isolated from only 9, 6, 3, 3, 3 and 3 per cent of the dogs, respectively. Porphyromonas gingivicanis was not isolated from any of the animals sampled. There was a highly significant (P < 0.001) correlation between the amount of plaque, the extent of gingivitis and the age of the dog.

Dental plaque flora of the dog with reference to fastidious and anaerobic bacteria associated with bites.

Animal bite wounds are amongst the most common types of traumatic injuries in humans. The organisms isolated from these wounds generally reflect the oral flora of the biting animal, and may be fastidious in nature and difficult to identify. This study was undertaken to determine the prevalence of Eikenella corrodens, Actinobacillus actinomycetemcomitans, Porphyromonas and Prevotella spp. in supragingival dental plaque collected from the right maxillary canine and carnassial teeth and the right mandibular canine tooth of dogs. In part one of the study, 30 dogs were used. E. corrodens was found in 62% of these dogs and 44% of individual plaque samples. A. actinomycetemcomitans was not detected in any of the dogs sampled. In part two, 34 dogs were used to determine the prevalence of the black pigmented anaerobic bacilli (Porphyromonas and Prevotella spp.). Porphyromonas gingivalis was present in 68% of these dogs and 47% of individual plaque samples. Prevotella intermedia was present in 44% of the dogs and 23% of individual plaque samples. The recently described Porphyromonas canoris, Porphyromonas salivosa, Porphyromonas cangingivalis, Porphyromonas cansulci, Porphyromonas crevioricanis and Prevotella denticola species were isolated from only 9%, 6%, 3%, 3%, 3% and 3% of dogs respectively. Porphyromonas gingivicanis was not isolated from any of the animals sampled. In conclusion, black-pigmented anaerobic bacilli were isolated from 91% of the animals sampled and therefore constitute a significant risk with respect to bite wound infections. It is also suggested that the prevalence of E. corrodens in wound infections has been underestimated in previous reports because of use of inappropriate techniques for detecting this organism.

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.