Functional expression of thermo-sensitive TRP channels in human odontoblasts

Introduction: Accumulating evidence supports a role for odontoblasts in initiating tooth pain, however direct ionic mechanisms underlying dentine nociceptive function remain unclear. The transient receptor potential (TRP) ion channels are directly related to cellular mechanisms of nociception and thermo-sensitive function but their expression by human odontoblasts remains to be determined. Objectives: To investigate the expression and functionality of the thermo-sensitive TRP channels TRPV1, TRPV4, TRPM8 and TRPA1 in human odontoblasts. Methods: Human odontoblasts were derived from dental pulp of immature permanent third molars by explant method. Cell lysates of odontoblasts were subject to SDS- polyacrylamide gel electrophoresis and proteins were blotted onto nitrocellulose membranes. Blots were probed with primary antibodies to TRPA1, TRPM8, TRPV4 and TRPV1. Detection of bound primary antibodies was achieved using appropriate anti-species antibody conjugates and chemiluminescent substrates. Functionality of the channels was determined with Ca2+ microfluorimetry, where cells grown in cover slips and incubated with Fura 2AM prior to stimulation with capsaicin (TRPV1 agonist), 4 alpha-phorbol 12,13-didecanoate (4áPDD) (TRPV4 agonist), icilin (TRPA1 agonist) and menthol (TRPM8 agonist). Emitted fluorescence was measured and the fluorescence ratio (R) was calculated as F340/F380 to determine the level of [Ca2+]i. Results: Western blotting confirmed the molecular localisation of thermo-sensitive TRP channels in human odontoblasts. Functionality assays revealed increase in [Ca2+]i in response to capsacin, icillin, methanol and 4áPDD indicating functional expression of TRPV1, TRPA1, TRPM8 and TRPV4 respectively. Conclusions: Functional expression of thermo-sensitive TRP channels in human odontoblasts may indicate a crucial role for odontoblasts in thermally induced dental pain. (Supported by a Research Grant from the Royal College of Surgeons of Edinburgh)

Radioimmunoassay Quantitation of Neuropeptide Y (NPY) in Human Dental Pulps

Neuropeptide Y (NPY) is a 36 amino acid peptide that is abundantly expressed in both the central and peripheral nervous systems. NPY has previously been shown to be present in human dental pulp although its exact role in pulpal health and disease remains to be fully elucidated. In addition to serving a neurotransmitter role, NPY may also have a role in modulating the pulpal response to injury and inflammation. Indeed NPY is known to be a potent vasoconstrictor in a range of tissues. Recent work by our research group has demonstrated changes in sensory neuropeptide levels measured by radioimmunoassay (RIA) in healthy and carious teeth. In addition to elevated levels of sensory neuropeptides, it is also possible that the carious process is associated with increased levels of autonomic neuropeptides such as NPY. Objectives: The aim of the present study was to undertake a comprehensive quantitative RIA analysis of NPY expression in human dental pulps from carious and non-carious teeth. Methods: A total of 22 non-carious and 46 carious teeth were included in the study. NPY was measured in all samples using RIA. Briefly, the RIA system consisted of a total volume of 400 ul, comprising 100 ul anti-NPY antibody (Peninsula Laboratories), 200 ul human NPY synthetic standard or pulp sample, and 100 ul of 125I-labelled NPY as radioactive tracer. Results: The mean concentration of NPY in non-carious teeth was found to be 4.28 ng/g (4.34 SD) compared to 9.57 ng/g (9.39 SD) in carious teeth. Using ANOVA the difference in NPY levels between the non-carious group and the carious group was found to be statistically significant (p= 0.003). Conclusion: The significant increase in the levels of NPY in carious dental pulps reported in this study provides evidence for a role for NPY in the pulpal response to caries.

Northern Ireland

Northern Ireland has been considered a conflict-resolution success story. The 1998 Belfast/Good Friday Agreement provided a framework for managing a long-standing ethnonational conflict, and has ushered in relative political stability. The consociational features of the Northern Ireland Assembly can be seen either as necessary for managing conflict or as institutionalizing sectarianism so that politics along left–right lines cannot emerge. Although there is evidence for the development of a “Northern Irish” identity to counter competing British and Irish identities, Northern Ireland is a long way from transcending the sectarian structures that shape almost all aspects of social and political life. Northern Ireland remains segregated along religious lines and is also prone to tensions around the anniversaries of atrocities and the public use of symbols and rituals. The failure to systematically “deal with the past” through public information recovery and truth-telling mechanisms also seems to have hindered progress toward reconciliation.

Acid-labile protein-adducted heterocyclic aromatic amines in human blood are not viable biomarkers of dietary exposure: A systematic study.

Heterocyclic aromatic amines (HCA) are carcinogenic mutagens formed during cooking of protein-rich foods. HCA residues adducted to blood proteins have been postulated as biomarkers of HCA exposure. However, the viability of quantifying HCAs following hydrolytic release from adducts in vivo and correlation with dietary intake are unproven. To definitively assess the potential of labile HCA-protein adducts as biomarkers, a highly sensitive UPLC-MS/MS method was validated for four major HCAs: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx). Limits of detection were 1e5 pg/ml plasma and recoveries 91e115%. Efficacy of hydrolysis was demonstrated by HCA-protein adducts synthesised in vitro. Plasma and 7-day food diaries were collected from 122 fasting adults consuming their habitual diets. Estimated HCA intakes ranged from 0 to 2.5 mg/day. An extensive range of hydrolysis conditions was examined for release of adducted HCAs in plasma. HCA was detected in only one sample (PhIP, 9.7 pg/ml), demonstrating conclusively for the first time that acid-labile HCA adducts do not reflect dietary HCA intake and are present at such low concentrations that they are not feasible biomarkers of exposure. Identification of biomarkers remains important. The search should concentrate on stabilised HCA peptide markers and use of untargeted proteomic and metabolomic approaches.

Measuring the macrosystem in post-accord Northern Ireland: A social-ecological approach

The macrosystem refers to the overarching patterns that influence behavior at each level of the social ecology (Bronfenbrenner, 1977), making it a necessary component for assessing human development in contexts of political violence. This article proposes a method for systematically measuring the macrosystem in Northern Ireland that allows for a subnational analysis, multiple time units, and indicators of both low-level violence and positive relations. Articles were randomly chosen for each weekday in 2006-2011 from two prominent Northern Irish newspapers and coded according to their reflection of positive relations and political tensions between Catholics and Protestants. The newspaper data were then compared to existing macro-level measurements in Northern Ireland. We found that the newspaper data provided a more nuanced understanding of fluctuations in intergroup relations than the corresponding measures. This has practical implications for peacebuilding and advances our methods for assessing the impact of macro-level processes on individual development.

Justice, Truth and Oral History: Legislating the Past 'From Below' in Northern Ireland

Drawing on the ‘from below’ perspective which has emerged in transitional justice scholarship and practice
over the past two decades, this article critically examines the dealing with the past debate in Northern
Ireland. The paper begins by offering an outline of the from below perspective in the context of post-conflict
or post-authoritarian societies which are struggling to come to terms with past violence and human rights
abuses. Having provided some of the legal and political background to the most recent efforts to deal with
the past in Northern Ireland, it then critically examines the relevant past-related provisions of the Stormont
House Agreement, namely the institutions which are designed to facilitate ‘justice’, truth recovery and the
establishment of an Oral History Archive. Drawing from the political science and social movement
literature on lobbying and the ways in which interests groups may seek to influence policy, the paper then
explores the efforts of the authors and others to contribute to the broader public debate, including through
drafting and circulating a ‘Model Bill’ on dealing with the past (reproduced elsewhere in this issue) as a
counterweight to the legislation which is required from the British government to implement the Stormont
House Agreement. The authors argue that the combination of technical capacity, grass-roots
credibility and ‘international-savvy’ local solutions offers a framework for praxis from below in other
contexts where activists are struggling to extend ownership of transitional justice beyond political elites.
Keywords: transitional justice; from below; dealing with the past; legislation; truth
recovery; prosecutions; oral history

Low-pathogenicity Mycoplasma spp. alter human monocyte and macrophage function and are highly prevalent among patients with ventilator-acquired pneumonia

Background
Ventilator-acquired pneumonia (VAP) remains a significant problem within intensive care units (ICUs). There is a growing recognition of the impact of critical-illness-induced immunoparesis on the pathogenesis of VAP, but the mechanisms remain incompletely understood. We hypothesised that, because of limitations in their routine detection, Mycoplasmataceae are more prevalent among patients with VAP than previously recognised, and that these organisms potentially impair immune cell function.
Methods and setting
159 patients were recruited from 12 UK ICUs. All patients had suspected VAP and underwent bronchoscopy and bronchoalveolar lavage (BAL). VAP was defined as growth of organisms at >104 colony forming units per ml of BAL fluid on conventional culture. Samples were tested for Mycoplasmataceae (Mycoplasma and Ureaplasma spp.) by PCR, and positive samples underwent sequencing for speciation. 36 healthy donors underwent BAL for comparison. Additionally, healthy donor monocytes and macrophages were exposed to Mycoplasma salivarium and their ability to respond to lipopolysaccharide and undertake phagocytosis was assessed.

Results
Mycoplasmataceaewerefoundin49%(95%CI 33% to 65%) of patients with VAP, compared with 14% (95% CI 9% to 25%) of patients without VAP. Patients with sterile BAL fluid had a similar prevalence to healthy donor BAL fluid (10% (95% CI 4% to 20%) vs 8% (95% CI 2% to 22%)). The most common organism identified was M. salivarium. Blood monocytes from healthy volunteers incubated with M. salivarium displayed an impaired TNF-α response to lipopolysaccharide ( p=0.0003), as did monocyte-derived macrophages (MDMs) (p=0.024). MDM exposed to M. salivarium demonstrated impaired phagocytosis ( p=0.005).

Discussion and conclusions
This study demonstrates a high prevalence of Mycoplasmataceae among patients with VAP, with a markedly lower prevalence among patients with suspected VAP in whom subsequent cultures refuted the diagnosis. The most common organism found, M. salivarium, is able to alter the functions of key immune cells. Mycoplasmataceae may contribute to VAP pathogenesis.

Evaluation of quinazolin-4-piperidine sulfamides as inhibitors of human NPP1: relevance in the treatment of pathologic mineralization of valve interstitial cells

Le rétrécissement valvulaire aortique calcifié (RAC) est le trouble valvulaire le plus fréquent chez les personnes âgées des pays développés. La seule option de traitement possible le remplacement de la valve aortique. L’identification du rôle de l’enzyme ecto-nucleotidase NPP1 dans le processus de calcification suggère que cette enzyme pourrait être une cible potentielle pour le développement d’un inhibiteur pharmacologique contre la calcification de la valve aortique. Jusqu’à présent, les composés qui ont été développés en tant qu’inhibiteurs de NPP1 manquent de puissance et de spécificité. Dans la présente étude, nous avons démontré que les dérivés de sulfonamides quinazolin-4-pipéridine sont des inhibiteurs puissants, spécifiques, et non-compétitifs de NPP1. In vitro, dans des cellules isolées de valve aortique nous avons fourni des preuves que l’inhibition de NPP1 par ces dérivés bloque la minéralisation, l’apoptose et la transition ostéogénique des cellules interstitielles de valve aortique.

New approaches for the diagnosis of human papillomavirus infection:relevance for clinical practice and cancer prevention

Tese de doutoramento, Biologia (Microbiologia), Universidade de Lisboa, Faculdade de Ciências, 2014

Neurophysiological origin of human brain asymmetry for speech and language.

The physiological basis of human cerebral asymmetry for language remains mysterious. We have used simultaneous physiological and anatomical measurements to investigate the issue. Concentrating on neural oscillatory activity in speech-specific frequency bands and exploring interactions between gestural (motor) and auditory-evoked activity, we find, in the absence of language-related processing, that left auditory, somatosensory, articulatory motor, and inferior parietal cortices show specific, lateralized, speech-related physiological properties. With the addition of ecologically valid audiovisual stimulation, activity in auditory cortex synchronizes with left-dominant input from the motor cortex at frequencies corresponding to syllabic, but not phonemic, speech rhythms. Our results support theories of language lateralization that posit a major role for intrinsic, hardwired perceptuomotor processing in syllabic parsing and are compatible both with the evolutionary view that speech arose from a combination of syllable-sized vocalizations and meaningful hand gestures and with developmental observations suggesting phonemic analysis is a developmentally acquired process.

Safety of human immunisation with a live-attenuated Mycobacterium tuberculosis vaccine: a randomised, double-blind, controlled phase I trial.

BACKGROUND: Tuberculosis remains one of the world's deadliest transmissible diseases despite widespread use of the BCG vaccine. MTBVAC is a new live tuberculosis vaccine based on genetically attenuated Mycobacterium tuberculosis that expresses most antigens present in human isolates of M tuberculosis. We aimed to compare the safety of MTBVAC with BCG in healthy adult volunteers. METHODS: We did this single-centre, randomised, double-blind, controlled phase 1 study at the Centre Hospitalier Universitaire Vaudois (CHUV; Lausanne, Switzerland). Volunteers were eligible for inclusion if they were aged 18-45 years, clinically healthy, HIV-negative and tuberculosis-negative, and had no history of active tuberculosis, chemoprophylaxis for tuberculosis, or BCG vaccination. Volunteers fulfilling the inclusion criteria were randomly assigned to three cohorts in a dose-escalation manner. Randomisation was done centrally by the CHUV Pharmacy and treatments were masked from the study team and volunteers. As participants were recruited within each cohort, they were randomly assigned 3:1 to receive MTBVAC or BCG. Of the participants allocated MTBVAC, those in the first cohort received 5 × 10(3) colony forming units (CFU) MTBVAC, those in the second cohort received 5 × 10(4) CFU MTBVAC, and those in the third cohort received 5 × 10(5) CFU MTBVAC. In all cohorts, participants assigned to receive BCG were given 5 × 10(5) CFU BCG. Each participant received a single intradermal injection of their assigned vaccine in 0·1 mL sterile water in their non-dominant arm. The primary outcome was safety in all vaccinated participants. Secondary outcomes included whole blood cell-mediated immune response to live MTBVAC and BCG, and interferon γ release assays (IGRA) of peripheral blood mononuclear cells. This trial is registered with ClinicalTrials.gov, number NCT02013245. FINDINGS: Between Jan 23, 2013, and Nov 6, 2013, we enrolled 36 volunteers into three cohorts, each of which consisted of nine participants who received MTBVAC and three who received BCG. 34 volunteers completed the trial. The safety of vaccination with MTBVAC at all doses was similar to that of BCG, and vaccination did not induce any serious adverse events. All individuals were IGRA negative at the end of follow-up (day 210). After whole blood stimulation with live MTBVAC or BCG, MTBVAC was at least as immunogenic as BCG. At the same dose as BCG (5×10(5) CFU), although no statistical significance could be achieved, there were more responders in the MTBVAC group than in the BCG group, with a greater frequency of polyfunctional CD4+ central memory T cells. INTERPRETATION: To our knowledge, MTBVAC is the first live-attenuated M tuberculosis vaccine to reach clinical assessment, showing similar safety to BCG. MTBVAC seemed to be at least as immunogenic as BCG, but the study was not powered to investigate this outcome. Further plans to use more immunogenicity endpoints in a larger number of volunteers (adults and adolescents) are underway, with the aim to thoroughly characterise and potentially distinguish immunogenicity between MTBVAC and BCG in tuberculosis-endemic countries. Combined with an excellent safety profile, these data support advanced clinical development in high-burden tuberculosis endemic countries. FUNDING: Biofabri and Bill & Melinda Gates Foundation through the TuBerculosis Vaccine Initiative (TBVI).