Assessing the use of minimally invasive self-sampling at home for long-term monitoring of the microbiota within UK families.

Monitoring the presence of commensal and pathogenic respiratory microorganisms is of critical global importance. However, community-based surveillance is difficult because nasopharyngeal swabs are uncomfortable and painful for a wide age range of participants. We designed a methodology for minimally invasive self-sampling at home and assessed its use for longitudinal monitoring of the oral, nasal and hand microbiota of adults and children within families. Healthy families with two adults and up to three children, living in and near Liverpool, United Kingdom, self-collected saliva, nasal lining fluid using synthetic absorptive matrices and hand swabs at home every two weeks for six months. Questionnaires were used to collect demographic and epidemiological data and assess feasibility and acceptability. Participants were invited to take part in an exit interview. Thirty-three families completed the study. Sampling using our approach was acceptable to 25/33 (76%) families, as sampling was fast (76%), easy (76%) and painless (60%). Saliva and hand sampling was acceptable to all participants of any age, whereas nasal sampling was accepted mostly by adults and children older than 5 years. Multi-niche self-sampling at home can be used by adults and children for longitudinal surveillance of respiratory microorganisms, providing key data for design of future studies.

Natural history of subaortic stenosis in 166 dogs (1999-2011).

INTRODUCTION: Subaortic stenosis (SAS) is one of the most common congenital cardiac diseases in dogs. The objective of this study was to provide survival times on a large population of dogs with SAS and to propose a redefined pressure gradient (PG) scale to include a mild, moderate, severe and very severe disease group. ANIMALS, MATERIALS AND METHODS: Dogs were divided into four groups based on the Doppler-derived PG across the stenosis. Disease severity was defined as follows: mild = PG < 50 mmHg; moderate = PG range 50-80 mmHg; severe = PG range 80-130 mmHg; and very severe = PG > 130 mmHg. Over the study period (1999-2011), 166 client-owned dogs were diagnosed with SAS of which 129 had follow-up information available. RESULTS: Unadjusted median survival time for severity groups were as follows: mild 10.6 years; moderate 9.9 years; severe 7.3 years; and very severe 3.0 years. Univariable analysis examining the effect of the PG, age at diagnosis and sex found only the PG and age at diagnosis had a significant effect on survival. Adjusted survival curves showed that the survival time in the very severe group was decreased compared with all other groups. CONCLUSION: Based on the results of this study, a revised SAS classification system with four PG groups is appropriate. Dogs with a PG > 130 mmHg were identified as those with the lowest median survival time.

Author Correction: Phase variation in pneumococcal populations during carriage in the human nasopharynx.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Neutrophil function is impaired in paediatric patients with malignancy and may be a useful clinical marker.

PURPOSE: Patients treated with cytotoxic chemotherapy are at risk of neutropenia, neutropenic fever and neutropenic sepsis. We hypothesised that pre-existing neutrophil function dysfunction may increase susceptibility to neutropenic fever in paediatric patients receiving cytotoxic chemotherapy. METHODS: Prospective cohort study recruited patients at Alder Hey Children's NHS Foundation Trust, United Kingdom. We measured neutrophil phagocytic function using a validated flow cytometric whole blood phagocytosis assay in paediatric patients (n = 16) with oncological disease before and after chemotherapy in a prospective cohort study. We recruited healthy children as a control comparator (n = 10). RESULTS: We found significantly decreased phagocytic function in oncology patients compared to healthy participants. In five patients who developed neutropenic fever, we observed increased pre-dose neutrophil respiratory burst. CONCLUSION: With further validation, measurement of neutrophil function could potentially be used to personalise appropriate prophylactic antimicrobial administration for patients receiving cytotoxic chemotherapy.

The whole blood phagocytosis assay: a clinically relevant test of neutrophil function and dysfunction in community-acquired pneumonia.

OBJECTIVE: To refine and validate a neutrophil function assay with clinical relevance for patients with community-acquired pneumonia (CAP). DESIGN: Two phase cross-sectional study to standardise and refine the assay in blood from healthy volunteers and test neutrophil phagocytic function in hospital patients with CAP. PARTICIPANTS: Phase one: Healthy adult volunteers (n = 30). Phase two: Critical care patients with severe CAP (n = 16), ward-level patients with moderate CAP (n = 15) and respiratory outpatients (no acute disease, n = 15). RESULTS: Our full standard operating procedure for the assay is provided. Patients with severe CAP had significantly decreased neutrophil function compared to moderate severity disease (median phagocytic index 2.8 vs. 18.0, p = 0.014). Moderate severity pneumonia neutrophil function was significantly higher than control samples (median 18.0 vs. 1.6, p = 0.015). There was no significant difference between critical care and control neutrophil function (median 2.8 vs. 1.6, p = 0.752). CONCLUSIONS: Our whole blood neutrophil assay is simple, reproducible and clinically relevant. Changes in neutrophil function measured in this pneumonia cohort is in agreement with previous studies. The assay has potential to be used to identify individuals for clinical trials of immunomodulatory therapies, to risk-stratify patients with pneumonia, and to refine our understanding of 'normal' neutrophil function in infection.

Phase variation in pneumococcal populations during carriage in the human nasopharynx.

Streptococcus pneumoniae is one of the world's leading bacterial pathogens, responsible for pneumonia, septicaemia and meningitis. Asymptomatic colonisation of the nasopharynx is considered to be a prerequisite for these severe infections, however little is understood about the biological changes that permit the pneumococcus to switch from asymptomatic coloniser to invasive pathogen. A phase variable type I restriction-modification (R-M) system (SpnIII) has been linked to a change in capsule expression and to the ability to successfully colonise the murine nasopharynx. Using our laboratory data, we have developed a Markov change model that allows prediction of the expected level of phase variation within a population, and as a result measures when populations deviate from those expected at random. Using this model, we have analysed samples from the Experimental Human Pneumococcal Carriage (EHPC) project. Here we show, through mathematical modelling, that the patterns of dominant SpnIII alleles expressed in the human nasopharynx are significantly different than those predicted by stochastic switching alone. Our inter-disciplinary work demonstrates that the expression of alternative methylation patterns should be an important consideration in studies of pneumococcal colonisation.

Dietary nitrate attenuates high-fat diet-induced obesity via mechanisms involving higher adipocyte respiration and alterations in inflammatory status.

Emerging evidence indicates that dietary nitrate can reverse several features of the metabolic syndrome, but the underlying molecular mechanisms still remain elusive. The aim of the present study was to explore mechanisms involved in the effects of dietary nitrate on the metabolic dysfunctions induced by high-fat diet (HFD) in mice. Four weeks old C57BL/6 male mice, exposed to HFD for ten weeks, were characterised by increased body weight, fat content, increased fasting glucose and impaired glucose clearance. All these metabolic abnormalities were significantly attenuated by dietary nitrate. Mechanistically, subcutaneous primary mouse adipocytes exposed to palmitate (PA) and treated with nitrite exhibited higher mitochondrial respiration, increased protein expression of total mitochondrial complexes and elevated gene expression of the thermogenesis gene UCP-1, as well as of the creatine transporter SLC6A8. Finally, dietary nitrate increased the expression of anti-inflammatory markers in visceral fat, plasma and bone marrow-derived macrophages (Arginase-1, Egr-2, IL-10), which was associated with reduction of NADPH oxidase-derived superoxide production in macrophages. In conclusion, dietary nitrate may have therapeutic utility against obesity and associated metabolic complications possibly by increasing adipocyte mitochondrial respiration and by dampening inflammation and oxidative stress.

Protective effect of PCV vaccine against experimental pneumococcal challenge in adults is primarily mediated by controlling colonisation density.

Widespread use of Pneumococcal Conjugate Vaccines (PCV) has reduced vaccine-type nasopharyngeal colonisation and invasive pneumococcal disease. In a double-blind, randomised controlled trial using the Experimental Human Pneumococcal Challenge (EHPC) model, PCV-13 (Prevenar-13) conferred 78% protection against colonisation acquisition and reduced bacterial intensity (AUC) as measured by classical culture. We used a multiplex qPCR assay targeting lytA and pneumococcal serotype 6A/B cpsA genes to re-assess the colonisation status of the same volunteers. Increase in detection of low-density colonisation resulted in reduced PCV efficacy against colonisation acquisition (29%), compared to classical culture (83%). For experimentally colonised volunteers, PCV had a pronounced effect on decreasing colonisation density. These results obtained in adults suggest that the success of PCV vaccination could primarily be mediated by the control of colonisation density. Studies assessing the impact of pneumococcal vaccines should allow for density measurements in their design.

Nonspecific effects of oral vaccination with live-attenuated Salmonella Typhi strain Ty21a.

Epidemiological and immunological evidence suggests that some vaccines can reduce all-cause mortality through nonspecific changes made to innate immune cells. Here, we present the first data to describe the nonspecific immunological impact of oral vaccination with live-attenuated Salmonella Typhi strain Ty21a. We vaccinated healthy adults with Ty21a and assessed aspects of innate and adaptive immunity over the course of 6 months. Changes to monocyte phenotype/function were observed for at least 3 months. Changes to innate and adaptive immune cell cytokine production in response to stimulation with vaccine and unrelated nonvaccine antigens were observed over the 6-month study period. The changes that we have observed could influence susceptibility to infection through altered immune responses mounted to subsequently encountered pathogens. These changes could influence all-cause mortality.

Reproducibility of quantitative sensory testing applied to musculoskeletal orofacial region: Site and sex differences.

BACKGROUND: This study estimated the inter-rater reliability and agreement of the somatosensory assessment performed at masseter and temporomandibular joint (TMJ) region in a group of healthy female and male participants. METHODS: Forty healthy participants (20 men and 20 women) were evaluated in two sessions by two different examiners. Cold detection threshold (CDT), warm detection threshold (WDT), thermal sensory limen (TSL), cold pain threshold (CPT), heat pain threshold (HPT), mechanical detection threshold (MDT), mechanical pain threshold (MPT), wind-up ratio (WUR) and pressure pain threshold (PPT) were assessed on the skin overlying TMJ and masseter body. Mixed ANOVA, intraclass correlation coefficients (ICC) and standard error of measurement (SEM) were applied to the data (α = 5%). Nonoverlapping 95% confidence intervals (95% CI) of ICCs were considered significantly different. RESULTS: The ICCs of 77% of all quantitative sensory testing (QST) measurements were considered fair to excellent (ICCs: 0.47-0.97), and WUR presented the lowest values. The reliability of WDT, TSL and HPT of masseter was significantly higher than TMJ, whereas the MDT reliability of TMJ was higher than masseter. In addition, the following combination of test/sites presented significantly lower ICCs for women: HPT, MDT of TMJ and MPT of both TMJ and masseter. Finally, the highest SEM values were presented for CPT and MPT. CONCLUSION: The overall somatosensory assessment of the masticatory structures performed by two examiners can be considered sufficiently reliable to discriminate participants, except WUR. Possible site and sex influences on the reproducibility parameters should be taken into account for an appropriate interpretation and clinical application of QST. SIGNIFICANCE: The test site and participant's sex can significantly influence the relative reliability and agreement of quantitative sensory testing applied to musculoskeletal orofacial region, which affect the capacity to discriminate participants and to evaluate changes over time.

The weight of nutrients: kynurenine metabolites in obesity and exercise.

Obesity ultimately results from an imbalance between energy intake and expenditure. However, in addition to their bioenergetic value, nutrients and their metabolites can function as important signalling molecules in energy homeostasis. Indeed, macronutrients and their metabolites can be direct regulators of metabolism through their actions on different organs. In turn, target organs can decide to use, store or transform the incoming nutrients depending on their physiological context and in coordination with other cell types. Tryptophan-kynurenine metabolites are an example of a family of compounds that can serve as systemic integrators of energy metabolism by signalling to different cell types. These include adipocytes, immune cells and muscle fibres, in addition to the well-known effects of kynurenine metabolites on the central nervous system. In the context of energy metabolism, several of the effects elicited by kynurenic acid are mediated by the G-protein-coupled receptor, GPR35. As GPR35 is expressed in tissues such as the adipose tissue, immune cells and the gastrointestinal tract, this receptor could be a potential therapeutic target for the treatment of obesity, diabetes and other metabolic diseases. In addition, metabolic disorders often coincide with states of chronic inflammation, which further highlights GPR35 as an integration node in conditions where inflammation skews metabolism. Defining the molecular interplay between different tissues in the regulation of energy homeostasis can help us understand interindividual variability in the response to nutrient intake and develop safe and efficient therapies to fight obesity and metabolic disease.

Soft tissue assessment before and after mandibular advancement or setback surgery using three-dimensional images: systematic review and meta-analysis.

The aim of this systematic review was to evaluate the effects of mandibular advancement or setback surgery on the facial soft tissue assessed using three-dimensional images. An electronic search was performed in the MEDLINE, Cochrane Library, Scopus, Virtual Health Library (VHL), Web of Science, and OpenGrey databases. Inclusion criteria comprised studies that evaluated the soft tissues of adult patients before and after mandibular advancement or setback surgery using computed tomography or magnetic resonance imaging. The risk of bias was analyzed. A total of 3501 studies were retrieved. The titles and abstracts of 2071 studies were read and 2040 of them were excluded. Thirty-one articles were read in full and six fulfilled the eligibility criteria and were included. A meta-analysis was performed using three of these articles. The correlation was significant and strong in the chin area (r=0.876 and r=0.868) and moderate for the lower lip/lower incisor (r=0.690). The ratio for lower lip/infradentale was 78% and for Pog'/Pog was 98%. Due to the limited number of articles included, there is weak evidence to infer that changes in the facial soft tissue due to mandibular advancement or setback are significant, but changes in the lower lip tend to be smaller than changes in the chin area.

Muscle hardness and masticatory myofascial pain: Assessment and clinical relevance.

The impression of increased muscle hardness in painful muscles is commonly reported in the clinical practice but may be difficult to assess. Therefore, the aim of this review was to present and discuss relevant aspects regarding the assessment of muscle hardness and its association with myofascial temporomandibular disorder (TMD) pain. A non-systematic search for studies of muscle hardness assessment in patients with pain-related TMDs was carried out in PubMed, Cochrane Library, Embase and Google Scholar. Mechanical devices and ultrasound imaging (strain and shear wave elastography) have been consistently used to measure masticatory muscle hardness, although an undisputable reference standard is yet to be determined. Strain elastography has identified greater masseter hardness of the symptomatic side in patients with unilateral myofascial TMD pain when compared to the contralateral side and healthy controls (HC). Likewise, shear wave elastography has shown greater masseter elasticity modulus in patients with myofascial TMD pain when compared to HC, which may be an indication of muscle hardness. Although assessment bias could partly explain these preliminary findings, future randomised controlled trials are encouraged to investigate this relationship. This qualitative review indicates that the muscle hardness of masticatory muscles is still a rather unexplored field of investigation with a good potential to improve the assessment and potentially also the management of myofascial TMD pain. Nonetheless, the current evidence in favour of increased hardness in masticatory muscles in patients with myofascial TMD pain is weak, and the pathophysiological importance and clinical usefulness of such information remain unclear.

Small molecule PGC-1α1 protein stabilizers induce adipocyte Ucp1 expression and uncoupled mitochondrial respiration.

OBJECTIVE: The peroxisome proliferator-activated receptor-γ coactivator-1α1 (PGC-1α1) regulates genes involved in energy metabolism. Increasing adipose tissue energy expenditure through PGC-1α1 activation is potentially beneficial for systemic metabolism. Pharmacological PGC-1α1 activators could be valuable tools in the fight against obesity and metabolic disease. Finding such compounds has been challenging partly because PGC-1α1 is a transcriptional coactivator with no known ligand-binding properties. While, PGC-1α1 activation is regulated by several mechanisms, protein stabilization is a crucial limiting step due to its short half-life under unstimulated conditions. METHODS: We designed a cell-based high-throughput screening system to identify PGC-1α1 protein stabilizers. Positive hits were tested for their ability to induce endogenous PGC-1α1 protein accumulation and activate target gene expression in brown adipocytes. Select compounds were analyzed for their effects on global gene expression and cellular respiration in adipocytes. RESULTS: Among 7,040 compounds screened, we highlight four small molecules with high activity as measured by: PGC-1α1 protein accumulation, target gene expression, and uncoupled mitochondrial respiration in brown adipocytes. CONCLUSIONS: We identify compounds that induce PGC-1α1 protein accumulation and show that this increases uncoupled respiration in brown adipocytes. This screening platform establishes the foundation for a new class of therapeutics with potential use in obesity and associated disorders.

Assessing the ideal microwave duration for disinfection of sinus irrigation bottles-A quantitative study.

OBJECTIVES: Saline irrigation of the nasal cavity and paranasal sinuses has a recognised role in the management of chronic rhinosinusitis. However, bacterial recontamination of irrigation bottles through backflow from the sinonasal cavity is a concern in recurrent sinus cavity infections. While patients are encouraged to clean the irrigation bottles regularly, there remains significant concern that the use of contaminated bottles may perpetuate chronic rhinosinusitis. This study assesses the optimal microwave duration to achieve decontamination for each irrigation bottle component part (reservoir, tube and nozzle) using a standard, commercially available microwave. In addition, the irrigation fluid was also tested for contamination after each microwave cycle. STUDY DESIGN: Laboratory-based experimental study. PARTICIPANTS: No patients were involved in this study. MAIN OUTCOME MEASURES: The percentage in vitro decontamination of the bottles' components was determined following 30, 60, 90, 120, 150 seconds of microwave cycles. RESULTS: Complete decontamination of the bottles was not achieved at any of the tested microwave cycles. Levels of decontamination differed for the different bottle components, and the greatest degree of decontamination for all bottle components occurred at 90 seconds. Although higher levels of decontamination were observed at microwave durations exceeding 90 seconds, this was at the expense of thermal degradation and deformation of the reservoir plastic component of the irrigation bottle. Similarly, lowest contamination of irrigation fluid was observed at 120 seconds. CONCLUSIONS: This study highlights the importance of establishing precise decontamination procedures and recommends a microwave cycle of 90 seconds for optimal decontamination.

Effect of Lactobacillus on body weight and body fat in overweight subjects: a systematic review of randomized controlled clinical trials.

Gut microbiota is important for maintaining body weight. Modulation of gut microbiota by probiotics may result in weight loss and thus help in obesity treatment. The aim of this systematic review was to evaluate the effects of Lactobacillus on weight loss and/or fat mass in overweight adults. A search was performed on the Medline (PubMed) and Scopus electronic databases using the search terms: 'probiotics', 'Lactobacillus, 'obesity', 'body weight changes', 'weight loss', 'overweight', 'abdominal obesity', 'body composition', 'body weight', 'body fat' and 'fat mass'. In the total were found 1567 articles, but only 14 were included in this systematic review. Of these nine showed decreased body weight and/or body fat, three did not find effect and two showed weight gain. Results suggest that the beneficial effects are strain dependent. It can highlight that Lactobacillus plantarum and Lactobacillus rhamnosus when combined with a hypocaloric diet, L. plantarum with Lactobacillus curvatus, Lactobacillus gasseri, Lactobacillus amylovorus, Lactobacillus acidophilus and Lactobacillus casei with phenolic compounds, and multiple species of Lactobacillus.

Agglutination by anti-capsular polysaccharide antibody is associated with protection against experimental human pneumococcal carriage.

The ability of pneumococcal conjugate vaccine (PCV) to decrease transmission by blocking the acquisition of colonization has been attributed to herd immunity. We describe the role of mucosal immunoglobulin G (IgG) to capsular polysaccharide (CPS) in mediating protection from carriage, translating our findings from a murine model to humans. We used a flow cytometric assay to quantify antibody-mediated agglutination demonstrating that hyperimmune sera generated against an unencapsulated mutant was poorly agglutinating. Passive immunization with this antiserum was ineffective to block acquisition of colonization compared to agglutinating antisera raised against the encapsulated parent strain. In the human challenge model, samples were collected from PCV and control-vaccinated adults. In PCV-vaccinated subjects, IgG levels to CPS were increased in serum and nasal wash (NW). IgG to the inoculated strain CPS dropped in NW samples after inoculation suggesting its sequestration by colonizing pneumococci. In post-vaccination NW samples pneumococci were heavily agglutinated compared with pre-vaccination samples in subjects protected against carriage. Our results indicate that pneumococcal agglutination mediated by CPS-specific antibodies is a key mechanism of protection against acquisition of carriage. Capsule may be the only vaccine target that can elicit strong agglutinating antibody responses, leading to protection against carriage acquisition and generation of herd immunity.

Modulation of nasopharyngeal innate defenses by viral coinfection predisposes individuals to experimental pneumococcal carriage.

Increased nasopharyngeal colonization density has been associated with pneumonia. We used experimental human pneumococcal carriage to investigate whether upper respiratory tract viral infection predisposes individuals to carriage. A total of 101 healthy subjects were screened for respiratory virus before pneumococcal intranasal challenge. Virus was associated with increased odds of colonization (75% virus positive became colonized vs. 46% virus-negative subjects; P=0.02). Nasal Factor H (FH) levels were increased in virus-positive subjects and were associated with increased colonization density. Using an in vitro epithelial model we explored the impact of increased mucosal FH in the context of coinfection. Epithelial inflammation and FH binding resulted in increased pneumococcal adherence to the epithelium. Binding was partially blocked by antibodies targeting the FH-binding protein Pneumococcal surface protein C (PspC). PspC epitope mapping revealed individuals lacked antibodies against the FH binding region. We propose that FH binding to PspC in vivo masks this binding site, enabling FH to facilitate pneumococcal/epithelial attachment during viral infection despite the presence of anti-PspC antibodies. We propose that a PspC-based vaccine lacking binding to FH could reduce pneumococcal colonization, and may have enhanced protection in those with underlying viral infection.

Long-term prognosis of tooth autotransplantation: a systematic review and meta-analysis.

The aim of this study was to systematically review the prognosis of autotransplanted teeth followed up for a period of 6 years or more. A literature search was conducted in five databases and the eligibility criteria were established. The outcomes evaluated were the survival rate, percentage of abnormal mobility, pulpal conditions, and percentage of root resorption. The searches identified 1848 articles, and after evaluation against the eligibility criteria, six were included. Data related to outcome measures were extracted from the studies and a meta-analysis was performed. Survival rates ranged from 75.3% to 91% and the meta-analysis showed an effect size of 81% (P<0.0001). The percentage ankylosis ranged from 4.2% to 18.2% and the effect size was 4.8% (P<0.0001). Root resorption percentages ranged from 3% to 10% and the effect size was equal to 4% (P<0.0001). It was not possible to perform a meta-analysis of data on pulpal conditions and percentage of teeth with abnormal mobility. The results of this study showed the survival rate to be excellent, considering the observation period. The rates of ankylosis and root resorption, despite their low values, influence the prognosis of transplanted teeth.

Upper airway dimensions in patients undergoing orthognathic surgery: a systematic review and meta-analysis.

The objective of this systematic review was to evaluate the effect of different types of orthognathic surgery on the dimensions of the upper airways assessed using three-dimensional images. An electronic search was performed in Cochrane Library, Medline, Scopus, VHL, Web of Science, and the System for Information on Grey Literature in Europe, ending January 2015. Inclusion criteria encompassed clinical studies in humans, patient age >15 years, patients submitted to maxillary or mandibular advancement or setback surgery, isolated or in combination, and presentation of airway measures, specifically volume and/or minimum cross-sectional area (CSA), obtained from computed tomography or magnetic resonance imaging. Additional searches were conducted on the references of included articles and in the NLM catalogue. An assessment of the risk of bias was performed. A total of 1180 studies were retrieved, of which 28 met the eligibility criteria; one was later excluded as it presented a high risk of bias. A meta-analysis was performed. There is moderate evidence to conclude that the upper airway minimum CSA increases significantly (124.13 mm(2)) after maxillomandibular advancement (MMA); the total volume increases significantly after MMA (7416.10mm(3)) and decreases significantly after maxillary advancement+mandibular setback (-1552.90 mm(3)) and isolated mandibular setback (-1894.65 mm(3)).