Studies on Receptor Activation in Intact and Permeabilized Smooth Muscle

The aim of this thesis was to study agonist-induced contractions in smooth muscle. The majority of the work was undertaken on rat anococcygeus, although some has been performed on guinea pig portal vein longitudinal muscle. The agonists which were examined (noradrenaline, phenylephrine and acetylcholine) all cause contraction in rat anococcygeus. They are thought to have their effect mediated by a G-protein which in turn stimulates the phosphatidylinositol cycle and promotes contraction. Work was carried out therefore, to analyse the effect of receptor activation at two levels: (a) the G-protein; and (b) the phosphatidylinositol cycle. To examine the intracellular effects of receptor activation the muscle was permeabilized. Part of this thesis comprises an investigation into the effectiveness of the three putative permeabilizing techniques. ANALYSIS OF THREE DIFFERENT PERMEABILIZATION TECHNIQUES The three permeabilization techniques examined were: (a) saponin-treatment; (b) EGTA-treatment; and (c) alpha-toxin-treatment. To analyse the effectiveness of each treatment several tests were carried out and the behaviour of the permeabilized smooth muscle after the three different treatments was compared with that of an intact muscle. The muscles were examined to see: (i) if they contracted readily to calcium; (ii) if they produced rigor crossbridges when ATP and CrP were removed from the bathing medium; (iii) how they responded when two factors known to have a direct effect on the contractile proteins, cyclic AMP and inorganic phosphate, were added to the bathing medium; and (iv) how they responded to the application of noradrenaline at a low calcium concentration. It was found that calcium-activated force decayed with time in saponin-and alpha-toxin-treated muscle. Possible mechanisms for the decay were examined. These included: (a) loss of calmodulin; (b) reduced or increased ionic strength; and (c) reduced or increased pH. These were all examined in saponin-treated muscle. The effect of lowering ionic strength was examined in toxin-treated muscle. RECEPTOR ACTIVATION AND CONTRACTION Once a suitable permeabilization technique had been established, further experimentation was undertaken to examine the effect of receptor activation on contraction in intact and permeabilized muscle. The permeabilization technique adopted was alpha-toxin treatment. Originally it had been hoped that this method would allow the analysis of receptor activation by noradrenaline and acetylcholine. However, it was found that alpha-toxin-treated rat anococcygeus muscle was unable to contract in response to acetylcholine. Possible reasons for this is examined in Chapter 4. (a) G-protein Kitazawa et al (1989) had already reported that GTP had to be included in the bathing medium before phenylephrine could produce a contraction in alpha-toxin permeabilized guinea pig portal vein. This led these workers to conclude that a G-protein was involved in receptor activation. The rest of Chapter 4 aims to assess the involvement of G-proteins in receptor activation in alpha-toxin-treated rat anococcygeus and guinea pig portal vein. Receptor activation was examined by looking at its effect on calcium release and calcium-activated force. The involvement of G-proteins was examined by using GTP, GTP-gamma-S (a non-hydrolysable analogue of GTP) and GDP-B-S (a non-hydrolysable analogue of GDP). A striking feature of the noradrenaline-activated contractions in both tissues was that their amplitude decayed upon repeated exposure. Several factors were examined to ascertain the cause of this decay. These included examining the effect of GTP on the decay, the accessibility of the calcium store and the sensitivity of the calcium store to Ins(1,4,5)P3 before and after the decay in noradrenaline-activated force. (b) phosphatidylinositol cycle The fifth chapter attempts to assess the role of the phosphatidylinositol cycle in receptor activation. This was examined by chronically treating rats with lithium chloride which is known to block the phosphatases which normally are responsible for the breakdown of Ins(1,4,5)P3. This blockade eventually causes the rundown of inositol within the cell and, therefore, eventually of PtdIns(4,5)P2, the precursor of Ins(1,4,5)P3 and of diacylglycerol (DG). The effects of chronic lithium treatment of rats on (i) the concentration response curves to different agonists; and (ii) the intracellular component produced by different agonists were examined in intact anococcygeus muscle. In the second set of experiments, chronic lithium treatment was undertaken at the same time as the animals were being given myo-inositol in their drinking water. (Abstract shortened by ProQuest.)

Painful tradeoffs : intimate-partner violence and sexual and reproductive health rights in Kenya

Intimate-partner violence involves multiple violations of sexual and reproductive rights, with devastating impacts on the health and wellbeing of those affected. This paper is the result of an action-research collaboration between a Kenyan gender-based violence rehabilitation NGO and a research programme. Qualitative and descriptive quantitative analysis of seven years of client records were carried out to investigate women’s experiences of intimate-partner violence and their responses to it. The paper departs from the observation that international human rights, while profoundly conceptually relevant to Kenyan women, are frequently practically irrelevant to their lives. Instead, various and often contradictory forms of rights, or legitimate claims, co-exist and interact in personal beliefs, in social relationships and in national legal and judicial systems. We therefore seek to contextualise rights in the lives of women affected by intimate-partner violence, to understand how they are articulated and constrained in each of these dimensions. We find that physical and sexual abuse within relationships often leads to repeated exposure to sexual and reproductive health risks, and abused women lack knowledge about these impacts, experience feelings of hopelessness about their health, and are unable to access the health services they need. Economic factors lead many women to subordinate their sexual and reproductive rights to their material needs and those of their children. There are limitations to the recognition of rights in both social attitudes and in the national legal framework. Social networks and justice institutions sometimes support individuals in exercising their rights and sometimes obstruct them. Legal reform, and strengthened services and referral systems are needed if the barriers to women’s rights are to be overcome. Measures to facilitate access to sexual and reproductive health services and to address forms of vulnerability in ongoing abusive relationships are needed to help those affected to end the violence and mitigate its impacts. Keywords: intimate-partner violence; sexual and reproductive health; rights; service delivery; Kenya

Sensors for foetal hypoxia and metabolic acidosis: a review

This article reviews existing clinical practices and sensor research undertaken to monitor fetal well-being during labour. Current clinical practices that include fetal heart rate monitoring and fetal scalp blood sampling are shown to be either inadequate or time-consuming. Monitoring of lactate in blood is identified as a potential alternative for intrapartum fetal monitoring due to its ability to distinguish between different types of acidosis. A literature review from a medical and technical perspective is presented to identify the current advancements in the field of lactate sensors for this application. It is concluded that a less invasive and a more continuous monitoring device is required to fulfill the clinical needs of intrapartum fetal monitoring. Potential specifications for such a system are also presented in this paper

Interleukin-8 predicts fatigue at 12 months post-injury in children with traumatic brain injury

Despite many children experiencing fatigue after childhood brain injury, little is known about the predictors of this complaint. To date, traditional indices of traumatic brain injury (TBI) severity have not reliably predicted persisting fatigue (up to 3 years post‐injury). This study aimed to establish if persisting fatigue is predicted by serum biomarker concentrations in child TBI. We examined if acute serum biomarker expression would improve prediction models of 12‐month fatigue based on injury severity. Blood samples were collected from 87 children (1 – 17 years at injury) sustaining mild to severe TBI (GCS range 3‐15; mean 12.43; classified as mild TBI (n=50, 57%) vs moderate/severe TBI n=37, 43%), and presenting to the Emergency Departments (ED) and Pediatric Intensive Care Units (PICU) at one of three tertiary pediatric hospitals (Royal Children’s Hospital (RCH); Hospital for Sick Children (HSC), Toronto St Justine Children’s Hospital (SJH), Montreal). Six serum biomarker concentrations were measured within 24 hours of injury [interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), soluble vascular cell adhesion molecule (SVCAM), S100 calcium binding protein B (S100B), neuron specific enolase (NSE), and soluble neural cell adhesion molecule (sNCAM)]. Fatigue at 12 months post‐injury was measured using the Pediatric Quality of Life Inventory Multidimensional Fatigue Scale (parent report), classified as present/absent using previously derived cut‐points. At 12 months post‐injury, 22% of participants experienced fatigue. A model including interleukin‐8 (IL‐8) was the best serum biomarker for estimating the probability of children experiencing fatigue at 12 months post‐injury. IL‐8 also significantly improved predictive models of fatigue based on severity

Implementing a whole-school relationships and sex education intervention to prevent dating and relationship violence: evidence from a pilot trial in English secondary schools

Adolescent dating and relationship violence is associated with health harms and is an important topic for sex education. School-based interventions addressing this have been effective in the USA, but schools in England confront pressures that might hinder implementation. We assessed the feasibility of, and contextual enablers/barriers to implementing Project Respect, a whole-school intervention. We conducted a pilot trial with process evaluation in six English secondary schools. Intervention comprised: training; policy-review; mapping and patrolling ‘hotspots’; parent information; help-seeking app; and a curriculum (including student-led campaigns) targeting dating violence. Process evaluation included assessments of fidelity and interviews with the trainer and school staff. Schools delivered training and lessons partially or completely and made parent and app information available. Two schools conducted policy reviews; none patrolled hotspots or implemented campaigns. Implementation was strengthened where staff saw dating violence as a priority. Delivery was undermined where staff were insufficiently involved, lacked time for planning or struggled to timetable lessons, and where new school challenges undermined engagement. School-based health interventions must work to build staff buy-in and ensure they do not overburden schools. Dating and relationship violence might best be addressed in this context as a broader aspect of sex education

A school intervention for 13- to 15-year-olds to prevent dating and relationship violence: the Project Respect pilot cluster RCT

Background ‘Dating and relationship violence’ is intimate partner violence during adolescence. Among dating adolescents in England, 66–75% of girls and 32–50% of boys report victimisation. Multicomponent school-based interventions might reduce dating and relationship violence. We optimised and piloted Project Respect, a new intervention in secondary schools in England, and study methods, to assess the value of a Phase III randomised controlled trial. Objectives To optimise Project Respect and to then conduct a pilot randomised controlled trial in southern England, addressing whether or not progression to a Phase III trial is justified in terms of prespecified criteria. To assess which of two dating and relationship violence scales is optimal, to assess response rates and to consider any necessary refinements. Design Optimisation activities aimed at intervention development and a pilot randomised controlled trial. Setting Optimisation in four secondary schools across southern England, varying by region and local deprivation. A pilot cluster randomised controlled trial in six other such schools (four intervention schools and two control schools), varying by region, attainment and local deprivation. Participants School students in years 8–10 at baseline and staff. Interventions Schools were randomised to the intervention or control arm in a 2 : 1 ratio; intervention comprised staff training, mapping ‘hotspots’ in school for dating and relationship violence, modifying staff patrols, school policy review, informing parents and carers, an application supporting student help-seeking, and a classroom curriculum for students in years 9 and 10 (including student-led campaigns). Main outcome measures Prespecified criteria for progression to Phase III of the trial, concerning acceptability, feasibility, fidelity and response rates. Primary health outcomes were assessed using the Safe Dates and short Conflicts in Adolescent Dating Relationships Inventory measures collected and analysed by individuals who were masked to allocation. Feasibility of economic analysis was assessed. Data sources Baseline and follow-up student and staff surveys, interviews, observations and logbooks. Results The intervention was optimised and approved by the Study Steering Committee. The student response rates in intervention and control groups were 1057 (84.8%) and 369 (76.6%) at baseline, and 1177 (76.8%) and 352 (83.4%) at follow-up, respectively. Safe Dates and the short Conflicts in Adolescent Dating Relationships Inventory had high levels of completion and reliability. At follow-up, prevalence of past-year dating and relationship violence victimisation was around 35% (Safe Dates scale and short Conflicts in Adolescent Dating Relationships Inventory). Staff response rates were very low. Training occurred in all four schools, with suboptimal fidelity. The curriculum was delivered with optimal fidelity in three schools. Other components were delivered inconsistently. Dating and relationship violence was addressed in control schools via violence prevention and responses, but not systematically. Intervention acceptability among students and staff was mixed. An economic evaluation would be feasible. Limitations One school did not undertake baseline surveys. Staff survey response rates were low and completion of the logbook was patchy. Conclusions Our findings suggest that progression to a Phase III trial of this intervention is not indicated because of limited fidelity and acceptability. Future work High prevalence of dating and relationship violence highlights the ongoing need for effective intervention. Potential intervention refinements would include more external support for schools and enhanced curriculum materials. Any future randomised controlled trials could consider having a longer lead-in from randomisation to intervention commencement, using the short Conflicts in Adolescent Dating Relationships Inventory as the primary outcome and not relying on staff surveys. Trial registration Current Controlled Trials ISRCTN65324176. Funding This project was funded by the National Institute for Health Research (NIHR) Public Health Research programme and will be published in full in Public Health Research; Vol. 8, No. 5. See the NIHR Journals Library website for further project information

The EMBARC European Bronchiectasis Registry:protocol for an international observational study

Bronchiectasis is one of the most neglected diseases in respiratory medicine. There are no approved therapies and few large-scale, representative epidemiological studies. The EMBARC (European Multicentre Bronchiectasis Audit and Research Collaboration) registry is a prospective, pan-European observational study of patients with bronchiectasis. The inclusion criterion is a primary clinical diagnosis of bronchiectasis consisting of: 1) a clinical history consistent with bronchiectasis; and 2) computed tomography demonstrating bronchiectasis. Core exclusion criteria are: 1) bronchiectasis due to known cystic fibrosis; 2) age <18 years; and 3) patients who are unable or unwilling to provide informed consent. The study aims to enrol 1000 patients by April 2016 across at least 20 European countries, and 10 000 patients by March 2020. Patients will undergo a comprehensive baseline assessment and will be followed up annually for up to 5 years with the goal of providing high-quality longitudinal data on outcomes, treatment patterns and quality of life. Data from the registry will be available in the form of annual reports. and will be disseminated in conference presentations and peer-reviewed publications. The European Bronchiectasis Registry aims to make a major contribution to understanding the natural history of the disease, as well as guiding evidence-based decision making and facilitating large randomised controlled trials.info:eu-repo/semantics/publishedVersio

Induction of broad immunity by thermostabilised vaccines incorporated in dissolvable microneedles using novel fabrication methods

Dissolvable microneedle (DMN) patches for immunization have multiple benefits, including vaccine stability and ease-of-use. However, conventional DMN fabrication methods have several drawbacks. Here we describe a novel, microfluidic, drop dispensing-based dissolvable microneedle production method that overcomes these issues. Uniquely, heterogeneous arrays, consisting of microneedles of diverse composition, can be easily produced on the same patch. Robustness of the process was demonstrated by incorporating and stabilizing adenovirus and MVA vaccines. Clinically-available trivalent inactivated influenza vaccine (TIV) in DMN patches is fully stable for greater than 6months at 40°C. Immunization using low dose TIV-loaded DMN patches induced significantly higher antibody responses compared to intramuscular-based immunization in mice. TIV-loaded patches also induced a broader, heterosubtypic neutralizing antibody response. By addressing issues that will be faced in large-scale fill-finish DMN fabrication processes and demonstrating superior thermostable characteristics and immunogenicity, this study progresses the translation of this microneedle platform to eventual clinical deployment