The Importance of Nitrate Reduction for Oral Health.

Salivary glands concentrate plasma nitrate into saliva, leading to high nitrate concentrations that can reach the millimolar range after a nitrate-rich vegetable meal. Whereas human cells cannot reduce nitrate to nitrite effectively, certain oral bacteria can. This leads to an increase in systemic nitrite that can improve conditions such as hypertension and diabetes through nitric oxide availability. Apart from systemic benefits, it has been proposed that microbial nitrate reduction can also promote oral health. In this review, we discuss evidence associating dietary nitrate with oral health. Oral bacteria can reduce nitrite to nitric oxide, a free radical with antimicrobial properties capable of inhibiting sensitive species such as anaerobes involved in periodontal diseases. Nitrate has also been shown to increase resilience against salivary acidification in vivo and in vitro, thus preventing caries development. One potential mechanism is proton consumption during denitrification and/or bacterial reduction of nitrite to ammonium. Additionally, lactic acid (organic acid involved in oral acidification) and hydrogen sulfide (volatile compound involved in halitosis) can act as electron donors for these processes. The nitrate-reducing bacteria Rothia and Neisseria are consistently found at higher levels in individuals free of oral disease (vs. individuals with caries, periodontitis, and/or halitosis) and increase when nitrate is consumed in clinical studies. Preliminary in vitro and clinical evidence show that bacteria normally associated with disease, such as Veillonella (caries) and Prevotella (periodontal diseases and halitosis), decrease in the presence of nitrate. We propose nitrate as an ecologic factor stimulating eubiosis (i.e., an increase in health-associated species and functions). Finally, we discuss the preventive and therapeutic potential, as well as safety issues, related to the use of nitrate. In vivo evidence is limited; therefore, robust clinical studies are required to confirm the potential benefits of nitrate reduction on oral health.

SMDI: An Index for Measuring Subgingival Microbial Dysbiosis.

An intuitive, clinically relevant index of microbial dysbiosis as a summary statistic of subgingival microbiome profiles is needed. Here, we describe a subgingival microbial dysbiosis index (SMDI) based on machine learning analysis of published periodontitis/health 16S microbiome data. The raw sequencing data, split into training and test sets, were quality filtered, taxonomically assigned to the species level, and centered log-ratio transformed. The training data set was subject to random forest analysis to identify discriminating species (DS) between periodontitis and health. DS lists, compiled by various "Gini" importance score cutoffs, were used to compute the SMDI for samples in the training and test data sets as the mean centered log-ratio abundance of periodontitis-associated species subtracted by that of health-associated ones. Diagnostic accuracy was assessed with receiver operating characteristic analysis. An SMDI based on 49 DS provided the highest accuracy with areas under the curve of 0.96 and 0.92 in the training and test data sets, respectively, and ranged from -6 (most normobiotic) to 5 (most dysbiotic) with a value around zero discriminating most of the periodontitis and healthy samples. The top periodontitis-associated DS were Treponema denticola, Mogibacterium timidum, Fretibacterium spp., and Tannerella forsythia, while Actinomyces naeslundii and Streptococcus sanguinis were the top health-associated DS. The index was highly reproducible by hypervariable region. Applying the index to additional test data sets in which nitrate had been used to modulate the microbiome demonstrated that nitrate has dysbiosis-lowering properties in vitro and in vivo. Finally, 3 genera (Treponema, Fretibacterium, and Actinomyces) were identified that could be used for calculation of a simplified SMDI with comparable accuracy. In conclusion, we have developed a nonbiased, reproducible, and easy-to-interpret index that can be used to identify patients/sites at risk of periodontitis, to assess the microbial response to treatment, and, importantly, as a quantitative tool in microbiome modulation studies.

In Silico Modeling of Hyposalivation and Biofilm Dysbiosis in Root Caries.

Root caries progression is aggravated by hyposalivation, which can accelerate the conversion of a dental biofilm from having a symbiotic microbial relationship with the host (predominance of nonaciduric species) to a dysbiotic one (dominated by aciduric species). Using a mathematical model previously employed to investigate factors associated with biofilm dysbiosis, we systematically explored the deleterious effect of hyposalivation on the composition of the biofilm and the risk of root dentin demineralization. By varying the clearance half-times of sugar (i.e., readily fermented dietary carbohydrates), we simulated hyposalivation and investigated its effect on 1) the time that the biofilm pH spends below the minimum for dentin or enamel demineralization and 2) the conversion of the biofilm from a symbiotic to dysbiotic composition. The effect of increasing sugar clearance half-times on the time that the biofilm pH is below the threshold for demineralization was more pronounced for dentin than for enamel (e.g., increasing the clearance half-time from 2 to 6 min doubled the time that the biofilm pH was below the threshold for dentin demineralization). The effect on biofilm composition assessed at 50 d showed that the conversion from a symbiotic to a dysbiotic biofilm happened around a frequency of 6 sugar intakes per day when the clearance half-time was 2 min but only 3 sugar intakes per day when the clearance half-time was 6 min. Taken together, the results confirm the profound effect that prolonged sugar clearance has on the dynamics of dental biofilm composition and the subsequent risk of root caries. This in silico model should be applied to study how interventions that alter salivary clearance rates or modify biofilm pH can affect clinical conditions such as root caries.

Modeling Normal and Dysbiotic Subgingival Microbiomes: Effect of Nutrients.

Screening for microbiome modulators requires availability of a high-throughput in vitro model that replicates subgingival dysbiosis and normobiosis, with a tool to measure microbial dysbiosis. Here, we tested various formulations to grow health- and periodontitis-associated subgingival microbiomes in parallel, and we describe a new subgingival dysbiosis index. Subgingival plaque samples pooled from 5 healthy subjects and, separately, 5 subjects with periodontitis were used to inoculate a Calgary Biofilm Device containing saliva-conditioned, hydroxyapatite-coated pegs. Microbiomes were grown for 7 d on either nutrient-rich media-including a modification of SHI medium, brain-heart infusion (BHI) supplemented with hemin and vitamin K, and a blend of SHI and BHI, each at 3 sucrose concentrations (0%, 0.05% and 0.1%)-or nutrient-limited media (saliva with 5%, 10%, or 20% inactivated human serum). The microbiomes were assessed for biomass, viability, and 16S rRNA profiles. In addition to richness and diversity, a dysbiosis index was calculated as the ratio of the sum of relative abundances of disease-associated species to that of health-associated species. The supplemented BHI and blend of SHI and BHI resulted in the highest biomass, whereas saliva-serum maximized viability. Distinct groups of bacteria were enriched in the different media. Regardless of medium type, the periodontitis-derived microbiomes showed higher species richness and alpha diversity and clustered with their inoculum separate from the health-derived microbiomes. Microbiomes grown in saliva-serum showed the highest species richness and the highest similarity to the clinical inocula in both health and disease. However, inclusion of serum reduced alpha diversity and increased dysbiosis in healthy microbiomes in a dose-dependent manner, mainly due to overenrichment of Porphyromonas species. The modification of SHI stood second in terms of species richness and diversity but resulted in low biomass and viability and significantly worsened dysbiosis in the periodontitis-derived microbiomes. Overall, saliva with 5% human serum was optimal for replicating subgingival microbiomes from health and disease.

In Sickness and in Health-What Does the Oral Microbiome Mean to Us? An Ecological Perspective.

The oral microbiome is natural and has a symbiotic relationship with the host by delivering important benefits. In oral health, a dynamic balance is reached between the host, the environment, and the microbiome. However, the frequent intake of sugar and/or reductions in saliva flow results in extended periods of low pH in the biofilm, which disrupts this symbiotic relationship. Such conditions inhibit the growth of beneficial species and drive the selection of bacteria with an acid-producing/acid-tolerating phenotype, thereby increasing the risk of caries (dysbiosis). A more detailed understanding of the interdependencies and interactions that exist among the resident microbiota in dental biofilms, and an increased awareness of the relationship between the host and the oral microbiome, is providing new insights and fresh opportunities to promote symbiosis and prevent dysbiosis. These include modifying the oral microbiome (e.g., with prebiotics and probiotics), manipulating the oral environment to selectively favor the growth of beneficial species, and moderating the growth and metabolism of the biofilm to reduce the likelihood of dysbiosis. Evidence is provided to suggest that the regular provision of interventions that deliver small but relevant benefits, consistently over a prolonged period, can support the maintenance of a symbiotic oral microbiome.

Resilience of the Oral Microbiota in Health: Mechanisms That Prevent Dysbiosis.

Dental diseases are now viewed as a consequence of a deleterious shift in the balance of the normally stable resident oral microbiome. It is known that frequent carbohydrate consumption or reduced saliva flow can lead to caries, and excessive plaque accumulation increases the risk of periodontal diseases. However, when these "disease drivers" are present, while some individuals appear to be susceptible, others are more tolerant or resilient to suffering from undesirable changes in their oral microbiome. Health-maintaining mechanisms that limit the effect of disease drivers include the complex set of metabolic and functional interrelationships that develop within dental biofilms and between biofilms and the host. In contrast, "positive feedback loops" can develop within these microbial communities that disrupt resilience and provoke a large and abrupt change in function and structure of the ecosystem (a microbial "regime shift"), which promotes dysbiosis and oral disease. For instance, acidification due to carbohydrate fermentation or inflammation in response to accumulated plaque select for a cariogenic or periopathogenic microbiota, respectively, in a chain of self-reinforcing events. Conversely, in tolerant individuals, health-maintaining mechanisms, including negative feedback to the drivers, can maintain resilience and promote resistance to and recovery from disease drivers. Recently studied health-maintaining mechanisms include ammonia production, limiting a drop in pH that can lead to caries, and denitrification, which could inhibit several stages of disease-associated positive feedback loops. Omics studies comparing the microbiome of, and its interaction with, susceptible and tolerant hosts can detect markers of resilience. The neutralization or inhibition of disease drivers, together with the identification and promotion of health-promoting species and functions, for example, by pre- and probiotics, could enhance microbiome resilience and lead to new strategies to prevent disease.

Novel methodology for determining the effect of adsorbates on human enamel acid dissolution.

OBJECTIVE: The effect of various interventions on enamel demineralisation can be determined by chemically measuring mineral ions dissolved by the attacking acid. Results are usually expressed as mineral loss per surface area of enamel exposed. Acid resistant varnish or adhesive tape are typically used to delineate an area of enamel. However, enamel surface curvature, rugosity and porosity reduce the reliability of simple area measurements made at the macro scale. Our aim was to develop a simple method for investigating the effect of adsorbates on enamel demineralisation that does not rely on knowing the area of enamel exposed. As an exemplar we have used salivary proteins as a model adsorbate. DESIGN: Natural human tooth enamel surfaces were subjected to five sequential acid challenges and then incubated in adsorbate (whole clarified saliva) followed by a further 15 acid challenges. Demineralisation was determined by measuring the phosphate released into the acid during each exposure by a spectrophotometric assay. The initial five challenges established a mean baseline mineral loss for each tooth against which the effect of subsequently adsorbed proteins could be compared. RESULTS: Salivary proteins significantly reduced the acid demineralisation of human enamel by 43% (p<0.01). Loss of proteins during each challenge corresponded to a gradual reduction in the degree of protection afforded. CONCLUSIONS: The methodology provides a simple and flexible means to investigate the effect of any adsorbate on enamel acid dissolution. Knowledge of the area of exposed enamel is irrelevant as each tooth acts as its own negative control.

In silico modelling to differentiate the contribution of sugar frequency versus total amount in driving biofilm dysbiosis in dental caries.

Dental caries is the most prevalent infection globally and a substantial economic burden in developed countries. Dietary sugars are the main risk factor, and drive increased proportions of acid-producing and acid-tolerating (aciduric) bacterial species within dental biofilms. Recent longitudinal studies have suggested that caries is most strongly correlated with total sugar intake, contrasting with the prevailing view that intake frequency is the primary determinant. To explore this possibility, we employed a computational model for supragingival plaque to systematically sample combinations of sugar frequency and total amount, allowing their independent contributions on the ratio of aciduric (i.e. cariogenic) to non-aciduric bacteria to be unambiguously determined. Sugar frequency was found to be irrelevant for either very high or very low daily total amounts as the simulated biofilm was predicted to be always or never cariogenic, respectively. Frequency was a determining factor for intermediate total amounts of sugar, including the estimated average human consumption. An increased risk of caries (i.e. high prevalence of aciduric/non-aciduric species) was predicted for high intake frequencies. Thus, both total amount and frequency of sugar intake may combine to influence plaque cariogenicity. These findings could be employed to support public guidance for dietary change, leading to improved oral healthcare.

An aerosol challenge model of tuberculosis in Mauritian cynomolgus macaques.

BACKGROUND: New interventions for tuberculosis are urgently needed. Non-human primate (NHP) models provide the most relevant pre-clinical models of human disease and play a critical role in vaccine development. Models utilising Asian cynomolgus macaque populations are well established but the restricted genetic diversity of the Mauritian cynomolgus macaques may be of added value. METHODS: Mauritian cynomolgus macaques were exposed to a range of doses of M. tuberculosis delivered by aerosol, and the outcome was assessed using clinical, imaging and pathology-based measures. RESULTS: All macaques developed characteristic clinical signs and disease features of tuberculosis (TB). Disease burden and the ability to control disease were dependent on exposure dose. Mauritian cynomolgus macaques showed less variation in pulmonary disease burden and total gross pathology scores within exposure dose groups than either Indian rhesus macaques or Chinese cynomolgus macaques. CONCLUSIONS: The genetic homogeneity of Mauritian cynomolgus macaques makes them a potentially useful model of human tuberculosis.

Dental biofilm: ecological interactions in health and disease.

BACKGROUND: The oral microbiome is diverse and exists as multispecies microbial communities on oral surfaces in structurally and functionally organized biofilms. AIM: To describe the network of microbial interactions (both synergistic and antagonistic) occurring within these biofilms and assess their role in oral health and dental disease. METHODS: PubMed database was searched for studies on microbial ecological interactions in dental biofilms. The search results did not lend themselves to systematic review and have been summarized in a narrative review instead. RESULTS: Five hundred and forty-seven original research articles and 212 reviews were identified. The majority (86%) of research articles addressed bacterial-bacterial interactions, while inter-kingdom microbial interactions were the least studied. The interactions included physical and nutritional synergistic associations, antagonism, cell-to-cell communication and gene transfer. CONCLUSIONS: Oral microbial communities display emergent properties that cannot be inferred from studies of single species. Individual organisms grow in environments they would not tolerate in pure culture. The networks of multiple synergistic and antagonistic interactions generate microbial inter-dependencies and give biofilms a resilience to minor environmental perturbations, and this contributes to oral health. If key environmental pressures exceed thresholds associated with health, then the competitiveness among oral microorganisms is altered and dysbiosis can occur, increasing the risk of dental disease.

A randomised clinical study to determine the effect of a toothpaste containing enzymes and proteins on plaque oral microbiome ecology.

The numerous species that make up the oral microbiome are now understood to play a key role in establishment and maintenance of oral health. The ability to taxonomically identify community members at the species level is important to elucidating its diversity and association to health and disease. We report the overall ecological effects of using a toothpaste containing enzymes and proteins compared to a control toothpaste on the plaque microbiome. The results reported here demonstrate that a toothpaste containing enzymes and proteins can augment natural salivary defences to promote an overall community shift resulting in an increase in bacteria associated with gum health and a concomitant decrease in those associated with periodontal disease. Statistical analysis shows significant increases in 12 taxa associated with gum health including Neisseria spp. and a significant decrease in 10 taxa associated with periodontal disease including Treponema spp. The results demonstrate that a toothpaste containing enzymes and proteins can significantly shift the ecology of the oral microbiome (at species level) resulting in a community with a stronger association to health.

The oral microbiome - an update for oral healthcare professionals.

For millions of years, our resident microbes have coevolved and coexisted with us in a mostly harmonious symbiotic relationship. We are not distinct entities from our microbiome, but together we form a 'superorganism' or holobiont, with the microbiome playing a significant role in our physiology and health. The mouth houses the second most diverse microbial community in the body, harbouring over 700 species of bacteria that colonise the hard surfaces of teeth and the soft tissues of the oral mucosa. Through recent advances in technology, we have started to unravel the complexities of the oral microbiome and gained new insights into its role during both health and disease. Perturbations of the oral microbiome through modern-day lifestyles can have detrimental consequences for our general and oral health. In dysbiosis, the finely-tuned equilibrium of the oral ecosystem is disrupted, allowing disease-promoting bacteria to manifest and cause conditions such as caries, gingivitis and periodontitis. For practitioners and patients alike, promoting a balanced microbiome is therefore important to effectively maintain or restore oral health. This article aims to give an update on our current knowledge of the oral microbiome in health and disease and to discuss implications for modern-day oral healthcare.

Alternative BCG delivery strategies improve protection against Mycobacterium tuberculosis in non-human primates: Protection associated with mycobacterial antigen-specific CD4 effector memory T-cell populations.

Intradermal (ID) BCG injection provides incomplete protection against TB in humans and experimental models. Alternative BCG vaccination strategies may improve protection in model species, including rhesus macaques. This study compares the immunogenicity and efficacy of BCG administered by ID and intravenous (IV) injection, or as an intratracheal mucosal boost (ID + IT), against aerosol challenge with Mycobacterium tuberculosis Erdman strain. Disease pathology was significantly reduced, and survival improved, by each BCG vaccination strategy, relative to unvaccinated animals. However, IV induced protection surpassed that achieved by all other routes, providing an opportunity to explore protective immunological mechanisms using antigen-specific IFN-γ ELISpot and polychromatic flow cytometry assays. IFN-γ spot forming units and multifunctional CD4 T-cell frequencies increased significantly following each vaccination regimen and were greatest following IV immunisation. Vaccine-induced multifunctional CD4 T-cells producing IFN-γ and TNF-α were associated with reduced disease pathology following subsequent M.tb challenge; however, high frequencies of this population following M.tb infection correlated with increased pathology. Cytokine producing T-cells primarily occupied the CD4 transitional effector memory phenotype, implicating this population as central to the mycobacterial response, potentially contributing to the stringent control observed in IV vaccinated animals. This study demonstrates the protective efficacy of IV BCG vaccination in rhesus macaques, offering a valuable tool for the interrogation of immunological mechanisms and potential correlates of protection.

Evaluation of the Immunogenicity of Mycobacterium bovis BCG Delivered by Aerosol to the Lungs of Macaques.

Nine million cases of tuberculosis (TB) were reported in 2013, with a further 1.5 million deaths attributed to the disease. When delivered as an intradermal (i.d.) injection, the Mycobacterium bovis BCG vaccine provides limited protection, whereas aerosol delivery has been shown to enhance efficacy in experimental models. In this study, we used the rhesus macaque model to characterize the mucosal and systemic immune response induced by aerosol-delivered BCG vaccine. Aerosol delivery of BCG induced both Th1 and Th17 cytokine responses. Polyfunctional CD4 T cells were detected in bronchoalveolar lavage (BAL) fluid and peripheral blood mononuclear cells (PBMCs) 8 weeks following vaccination in a dose-dependent manner. A similar trend was seen in peripheral gamma interferon (IFN-γ) spot-forming units measured by enzyme-linked immunosorbent spot (ELISpot) assay and serum anti-purified protein derivative (PPD) IgG levels. CD8 T cells predominantly expressed cytokines individually, with pronounced tumor necrosis factor alpha (TNF-α) production by BAL fluid cells. T-cell memory phenotype analysis revealed that CD4 and CD8 populations isolated from BAL fluid samples were polarized toward an effector memory phenotype, whereas the frequencies of peripheral central memory T cells increased significantly and remained elevated following aerosol vaccination. Expression patterns of the α4ß1 integrin lung homing markers remained consistently high on CD4 and CD8 T cells isolated from BAL fluid and varied on peripheral T cells. This characterization of aerosol BCG vaccination highlights features of the resulting mycobacterium-specific immune response that may contribute to the enhanced protection previously reported in aerosol BCG vaccination studies and will inform future studies involving vaccines delivered to the mucosal surfaces of the lung.

Evaluation of the safety and immunogenicity of a candidate tuberculosis vaccine, MVA85A, delivered by aerosol to the lungs of macaques.

Tuberculosis (TB) is a reemerging disease. The only available vaccine, Mycobacterium bovis BCG, is delivered intradermally and confers highly variable efficacy against pulmonary disease. There is an urgent need for improved vaccination strategies. Murine studies suggest that immunizations delivered directly to the respiratory mucosa might be a more effective route of vaccination. This study compared the immunogenicity of a leading candidate tuberculosis (TB) vaccine, modified vaccinia virus Ankara expressing antigen 85A (MVA85A), in rhesus macaques, delivered either as an aerosol or as an intradermal boost immunization 12 weeks after an intradermal BCG prime vaccine. Aerosol vaccination was well tolerated. MVA85A delivered by aerosol or by intradermal injection induced antigen-specific immune responses in the periphery and the lung, with a trend toward the highest response when the compartment and route of delivery were matched. The ability of poxvirus-vectored vaccines delivered by the systemic route to induce responses in the mucosal immune compartment in macaques is in contrast to the independent compartmentalization of mucosal and systemic immune systems described in mice. Unlike intradermal vaccination, aerosol vaccination did not induce a detectable serum anti-vector antibody response. The delivery of vaccines to the lungs might provide an immunization strategy that limits the induction of systemic anti-vector immunity, which would be extremely useful in the development of improved vaccine strategies. This is the first study to show a recombinant MVA-vectored vaccine to be highly immunogenic when delivered by the aerosol route to nonhuman primates. These results provide important safety and proof-of-concept data for further evaluation of this route of immunization for use in human clinical trials.

Method for assessing IFN-γ responses in guinea pigs during TB vaccine trials.

AIMS: We sought to develop a new method that enables the assessment of the immune response of guinea pigs during TB vaccine evaluation studies, without the need to cull or anaesthetize animals. METHOD AND RESULTS: Guinea pigs were vaccinated with five different formulations of oral BCG. One week prior to challenge with Mycobacterium bovis, blood (50-200 µl) was taken from the ears of vaccinated subjects. Host RNA was isolated and amplified following antigenic restimulation of PBMCs for 24 h with 30 µg of bovine PPD. The up- or down-regulation of γ-interferon (IFN-γ), a key cytokine involved in protection against tuberculosis, was assessed using real-time PCR. The relative expression of prechallenge IFN-γ mRNA in the vaccinated groups (n=5) correlated (P<0·001) with protection against M. bovis challenge. CONCLUSION: We have demonstrated that it is possible to take blood samples and track IFN-γ responses in guinea pigs that then go on to be exposed to M. bovis, thus providing prechallenge vaccine uptake information. SIGNIFICANCE AND IMPACT OF THE STUDY: This methodology will also be applicable for tracking the immune responses of vaccinated guinea pigs over time that then go on to be challenged with M. tuberculosis during human TB vaccine evaluation studies.

Contemporary perspective on plaque control.

The aim of this review article is to provide a scientific platform that will enable the dental team to develop a rational approach to plaque control based on the latest knowledge of the role of the oral microflora in health and disease. The resident oral microflora is natural and forms spatially-organised, interactive, multi-species biofilms on mucosal and dental surfaces in the mouth. These resident oral microbial communities play a key function in the normal development of the physiology of the host and are important in preventing colonisation by exogenous and often undesirable microbes. A dynamic balance exists between the resident microflora and the host in health, and disease results from a breakdown of this delicate relationship. Patients should be taught effective plaque control techniques that maintain dental biofilms at levels compatible with oral health so as to retain the beneficial properties of the resident microflora while reducing the risk of dental disease from excessive plaque accumulation. Antimicrobial and antiplaque agents in oral care products can augment mechanical plaque control by several direct and indirect mechanisms that not only involve reducing or removing dental biofilms but also include inhibiting bacterial metabolism when the agents are still present at sub-lethal concentrations.

The expression of ferritin, lactoferrin, transferrin receptor and solute carrier family 11A1 in the host response to BCG-vaccination and Mycobacterium tuberculosis challenge.

Iron is an essential cofactor for both mycobacterial growth during infection and for a successful protective immune response by the host. The immune response partly depends on the regulation of iron by the host, including the tight control of expression of the iron-storage protein, ferritin. BCG vaccination can protect against disease following Mycobacterium tuberculosis infection, but the mechanisms of protection remain unclear. To further explore these mechanisms, splenocytes from BCG-vaccinated guinea pigs were stimulated ex vivo with purified protein derivative from M. tuberculosis and a significant down-regulation of ferritin light- and heavy-chain was measured by reverse-transcription quantitative-PCR (P≤0.05 and ≤0.01, respectively). The mechanisms of this down-regulation were shown to involve TNFα and nitric oxide. A more in depth analysis of the mRNA expression profiles, including genes involved in iron metabolism, was performed using a guinea pig specific immunological microarray following ex vivo infection with M. tuberculosis of splenocytes from BCG-vaccinated and naïve guinea pigs. M. tuberculosis infection induced a pro-inflammatory response in splenocytes from both groups, resulting in down-regulation of ferritin (P≤0.05). In addition, lactoferrin (P≤0.002), transferrin receptor (P≤0.05) and solute carrier family 11A1 (P≤0.05), were only significantly down-regulated after infection of the splenocytes from BCG-vaccinated animals. The results show that expression of iron-metabolism genes is tightly regulated as part of the host response to M. tuberculosis infection and that BCG-vaccination enhances the ability of the host to mount an iron-restriction response which may in turn help to combat invasion by mycobacteria.

A quantitative assessment of residual protein levels on dental instruments reprocessed by manual, ultrasonic and automated cleaning methods.

OBJECTIVE: To assess residual protein on dental instruments cleaned in general dental practice by manual, manual plus ultrasonic and automated washer disinfector (AWD) processes. DESIGN AND SETTING: Instruments submitted by 30 dental surgeries in the South West of England. SUBJECTS (MATERIALS) AND METHODS: Instruments analysed were matrix bands, associated retaining clips, diamond and stainless steel burs, extraction forceps and hand scalers. Each instrument was visually assessed under magnification for residual debris. Residual protein was extracted by immersion in detergent and sonication. A collection of used but uncleaned instruments of each type (n = 177) was also analysed for adherent protein using ophthalaldehyde/N-acetylcysteine reagent. MAIN OUTCOME MEASURES: Residual protein levels allowed comparisons to be made on the effectiveness of different cleaning processes. RESULTS: One thousand, three hundred and four instruments were analysed. Observational data demonstrated several shortcomings in cleaning chemistries and operation of the AWD. For uncleaned instruments, median residual protein levels ranged from 0.4 µg (stainless steel burs) to 462 µg (extraction forceps). Following manual washing, median protein levels ranged from 0.3-78 µg; for manual plus ultrasonic washing, levels ranged from 9-39 µg and AWD levels ranged from 0.3-27 µg. Manual washing combined with ultrasonic cleaning was significantly less effective than the other two processes (p <0.008). AWDs reduced the variability in the cleaning process. No correlation was found between visual scoring and residual protein determination. CONCLUSION(S): There was a wide variation in residual protein levels both within and between different methods and instruments and this underlines the complexity of this process.