Integrating qualitative research within a clinical trials unit: developing strategies and understanding their implementation in contexts.

BACKGROUND/AIMS: The value of using qualitative methods within clinical trials is widely recognised. How qualitative research is integrated within trials units to achieve this is less clear. This paper describes the process through which qualitative research has been integrated within Cardiff University's Centre for Trials Research (CTR) in Wales, UK. We highlight facilitators of, and challenges to, integration. METHODS: We held group discussions on the work of the Qualitative Research Group (QRG) within CTR. The content of these discussions, materials for a presentation in CTR, and documents relating to the development of the QRG were interpreted at a workshop attended by group members. Normalisation Process Theory (NPT) was used to structure analysis. A writing group prepared a document for input from members of CTR, forming the basis of this paper. RESULTS: Actions to integrate qualitative research comprised: its inclusion in Centre strategies; formation of a QRG with dedicated funding/roles; embedding of qualitative research within operating systems; capacity building/training; monitoring opportunities to include qualitative methods in studies; maximising the quality of qualitative research and developing methodological innovation. Facilitators of these actions included: the influence of the broader methodological landscape within trial/study design and its promotion of the value of qualitative research; and close physical proximity of CTR qualitative staff/students allowing sharing of methodological approaches. Introduction of innovative qualitative methods generated interest among other staff groups. Challenges included: pressure to under-resource qualitative components of research, preference for a statistical stance historically in some research areas and funding structures, and difficulties faced by qualitative researchers carving out individual academic profiles when working across trials/studies. CONCLUSIONS: Given that CTUs are pivotal to the design and conduct of RCTs and related study types across multiple disciplines, integrating qualitative research into trials units is crucial if its contribution is to be fully realised. We have made explicit one trials unit's experience of embedding qualitative research and present this to open dialogue on ways to operationalise and optimise qualitative research in trials. NPT provides a valuable framework with which to theorise these processes, including the importance of sense-making and legitimisation when introducing new practices within organisations.

Using Qualitative Questionnaires in Medical Education Research.

Most students in Health Profession Education courses are new to the world of qualitative research. Faced with the challenge of designing a research project, they are often drawn towards using the questionnaire as a data collection method, commonly assuming that utilising open-ended questions alone constitutes qualitative research design. Designing questionnaires that meet the standards of rigour is challenging, and this common assumption reflects inexperience with and misunderstandings of qualitative ontology, as well as the lack of methodological literature on designing and developing qualitative questionnaires. This paper is written with research supervisors as well as students in mind, as it is aimed to help elucidate the decision-making process and the justification for using a qualitative questionnaire. Drawing upon examples of research conducted by our students, and the wider literature, we demonstrate how qualitative questionnaires can produce rich and meaningful findings when they (1) prioritise qualitative research values, and (2) follow a rigorous design process when the questionnaire is developed. We conclude by offering a simple framework for developing rigorous qualitative questionnaires to those who may consider using this approach.

Rehabilitation experiences following major lower limb amputation due to complications of vascular disease: a UK qualitative study.

PURPOSE: Rehabilitation experiences of lower limb amputees with poorer physical health have not been fully explored. This study aimed to qualitatively explore experiences of rehabilitation amongst patients who had recently undergone amputation due to complications of vascular disease. METHODS: Semi-structured, face-to-face interviews were conducted with 14 patients participating in the PLACEMENT randomised controlled feasibility trial (ISRCTN: 85710690; EudraCT: 2016-003544-37), which investigated the effectiveness of using a perineural catheter for postoperative pain relief following major lower limb amputation. Framework analysis was used to identify key themes and compare participant data. FINDINGS: Three main themes and corresponding sub-themes were identified: (i) other patients as inspiration; (ii) other patients as competition; and (iii) imagined futures. Perceptions relating to other patients played a key role in rehabilitation, providing a source of motivation, support, and competition. Participants' imagined futures were uncertain, and this was compounded by a lack of information and delays in equipment and/or adaptations. CONCLUSIONS: Findings highlight the importance of fellow patients in supporting rehabilitation following lower limb amputation. Enabling contact with other patients should thus be a key consideration when planning rehabilitation. There is a clear unmet need for realistic information relating to post-amputation recovery, tailored to the needs of individual patients.

There is a clear unmet need for patient information on rehabilitation following major lower limb amputation.Information about future mobility - particularly prosthesis use - should be realistic and individually tailored.The key role of fellow patients should be fully considered when planning post-amputation rehabilitation.

Scoping review protocol: exploration of the barriers and facilitators to the uptake of early postnatal contraception.

INTRODUCTION: It is well documented that many women do not desire a short interpregnancy interval. Medical societies, government agencies and leaders in the field recommend that contraception should be part of maternity care. Short spaced and unplanned pregnancies increase the chances of mortality and morbidity in the mother and child. The WHO recommends a 24-month interpregnancy interval; however, short pregnancy intervals remain common. The goal of this scoping review will be to explore barriers and facilitators to the uptake of early postnatal contraception. A review of globally published literature relating to the implementation of a postnatal contraception service provision globally will be carried out which will highlight evidence gaps, strengths and weaknesses of studies associated with uptake and known barriers and facilitators to the uptake of early postnatal contraception. METHODS AND ANALYSIS: This scoping review will be conducted in accordance with the Joanna Briggs Institute methodology for scoping reviews. The search strategy aims to locate both published and unpublished studies. An initial limited search of PubMed and CINAHL was undertaken to identify articles on the provision of postnatal contraception. The search strategy will be adapted for each included database CINAHL, SCOPUS, MEDLINE, PROSPERO and COCHRANE from 1 January 1993 to 1 January 2023 and reviewed by two reviewers. The data will be analysed and presented in tables, diagrams and text. ETHICS AND DISSEMINATION: Ethical approval is not required. This review is a retrospective review of widely and publicly available evidence. The review findings will be disseminated via publication in peer-reviewed journals, as part of a PhD thesis and conference presentation. SCOPING REVIEW QUESTION: What are the barriers and facilitators to early postnatal contraception provision and uptake?

"It's not just about the numbers": Inside the black box of nurses' professional judgement in nurse staffing systems in England and Wales: Insights from a qualitative cross-case comparative study.

BACKGROUND: Whether implicit or explicit, professional judgement is a central component of the many nurse staffing systems implemented in high-income countries to inform workforce planning and staff deployment. Whilst a substantial body of research has evaluated the technical and operational elements of nurse staffing systems, no studies have systematically examined the role of professional judgement and its contribution to decision-making. OBJECTIVE: To explore nurses' use of professional judgement in nurse staffing systems in England and Wales. METHODS: A cross-case comparative design centred on adult in-patient services in three University Health Boards in Wales and three National Health Service Trusts in England. Data generation was undertaken between January 2021 and March 2023 through stakeholder interviews, observations of staffing meetings, and analysis of documents and artefacts. Observations were undertaken in clinical areas but limited to three cases by COVID-19 restrictions. Analysis was informed by translational mobilisation theory. FINDINGS: Two kinds of professional judgement were deployed in the nurse staffing systems: the judgement of clinical nurses and the judgement of senior nurse managers. The research highlighted the reflexive relationship between professional judgement and data, and the circumstances in which organisations placed trust in people and when they placed trust in numbers. Nurses' professional judgement was central to the generation of data, its interpretation and contextualisation. Healthcare organisations relied on the professional judgements of clinical nurses and senior nurse managers in making operational decisions to mitigate risk, where real-world understanding of the status of the organisation was privileged over formal data. Professional judgement had attenuated authority for the purposes of workforce planning, where data was a master actor. Nurses expressed concerns that strategic decision-making prioritised safety and efficiency, and formal measurement systems did not capture important aspects of care quality or staff wellbeing, which made it difficult to articulate their professional judgement. CONCLUSIONS: The implementation of staffing systems is resource intensive. Given limited evidence on which to recommend any specific methodology, the priority for future research is to optimise existing systems. If nurses are to deploy their professional judgement to proactively influence the conditions for care, as well as responding to the challenges of risk mitigation, there is a need for robust systems of nursing measurement aligned with agreed standards of care and a vocabulary through which these judgements can be articulated. TWEETABLE ABSTRACT: Health systems depend on nurses' professional judgement for operational staffing decisions, but data is privileged over professional judgement for workforce planning.

Acceptability and feasibility of a planned preconception weight loss intervention in women with long-acting reversible contraception: the Plan-it mixed-methods study.

BACKGROUND: Women with overweight (a body mass index of ≥ 25 kg/m2) or obesity (a body mass index of ≥ 30 kg/m2) are at greater risk of experiencing complications during pregnancy and labour than women with a healthy weight. Women who remove their long-acting reversible contraception (i.e. coils or implants) are one of the few groups of people who contact services as part of their preparation for conception, creating an opportunity to offer a weight loss intervention. OBJECTIVES: The objectives were to understand if routine NHS data captured the pathway from long-acting reversible contraception removal to pregnancy and included body mass index; to identify the suitable components of a preconception weight loss intervention; and to engage with key stakeholders to determine the acceptability and feasibility of asking women with overweight/obesity to delay the removal of their long-acting reversible contraception in order to take part in a preconception weight loss intervention. DESIGN: This was a preparatory mixed-methods study, assessing the acceptability and feasibility of a potential intervention, using routine NHS data and purposefully collected qualitative data. PARTICIPANTS: The NHS routine data included all women with a long-acting reversible contraception code. There were three groups of participants in the surveys and interviews: health-care practitioners who remove long-acting reversible contraception; weight management consultants; and women of reproductive age with experience of overweight/obesity and of using long-acting reversible contraception. SETTING: UK-based health-care practitioners recruited at professional meetings; and weight management consultants and contraceptive users recruited via social media. DATA SOURCES: Anonymised routine data from UK sexual health clinics and the Clinical Practice Research Datalink, including the Pregnancy Register; and online surveys and qualitative interviews with stakeholders. RESULTS: The records of 2,632,871 women aged 16-48 years showed that 318,040 had at least one long-acting reversible contraception event, with 62% of records including a body mass index. Given the identified limitations of the routine NHS data sets, it would not be feasible to reliably identify women with overweight/obesity who request a long-acting reversible contraception removal with an intention to become pregnant. Online surveys were completed by 100 health-care practitioners, four weight management consultants and 243 contraceptive users. Ten health-care practitioners and 20 long-acting reversible contraception users completed qualitative interviews. A realist-informed approach generated a hypothesised programme theory. The combination of weight discussions and the delay of long-acting reversible contraception removal was unacceptable as an intervention to contraceptive users for ethical and practical reasons. However, a preconception health intervention incorporating weight loss could be acceptable, and one potential programme is outlined. LIMITATIONS: There was very limited engagement with weight management consultants, and the sample of participating stakeholders may not be representative. CONCLUSIONS: An intervention that asks women to delay long-acting reversible contraception removal to participate in a preconception weight loss intervention would be neither feasible nor acceptable. A preconception health programme, including weight management, would be welcomed but requires risk communication training of health-care practitioners. FUTURE WORK: Work to improve routine data sets, increase awareness of the importance of preconception health and overcome health-care practitioner barriers to discussing weight as part of preconception care is a priority. TRIAL REGISTRATION: This trial is registered as ISRCTN14733020. FUNDING: This project was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 27, No. 1.

If a woman has overweight or obesity when she is pregnant, then there is a greater risk of health problems for her and her baby. About half of women of childbearing age have overweight or obesity, so we need to find ways of supporting women to lose weight before they become pregnant (described here as 'preconception'). This can be difficult because women do not usually talk to a health-care practitioner (e.g. general practitioners, sexual health doctors, nurses) about becoming pregnant, but one group of women who do are those who need to have a long-acting reversible contraceptive (e.g. a coil or an implant) removed. This study was designed as preparatory work for a potential future study of a preconception weight loss intervention. We wanted to answer three questions: (1) would women with experience of overweight and of using a long-acting reversible contraceptive think that it would be acceptable to ask women to delay having their long-acting reversible contraceptive removed to take part in a weight loss intervention before pregnancy; (2) what did health-care practitioners think about that idea, and would they be happy to ask women to take part; and (3) can NHS information (routine data) tell us how many women might potentially take part in such an intervention? We looked at NHS routine data and the research on preconception weight loss interventions. A total of 100 health-care practitioners and 243 users of long-acting reversible contraceptives completed surveys, and 10 health-care practitioners and 20 users of long-acting reversible contraceptives took part in interviews. We found that routine data could not be used to identify people reliably. Designing a weight loss intervention that needed women to delay the removal of a long-acting reversible contraceptive was not acceptable to women. A population-based preconception weight loss intervention with a positive focus was acceptable, but, for such a programme to be delivered by the NHS, health-care practitioners need more knowledge, skills and confidence in talking about weight with patients.

Inclusion of sex chromosomes in noninvasive prenatal testing in Asia, Australia, Europe and the USA: A survey study.

OBJECTIVE: To examine the extent to which sex chromosomes are included in current noninvasive prenatal testing (NIPT) and the reporting practices with respect to fetal chromosomal sex and sex chromosome aberrations (SCAs), in addition to an update on the general implementation of NIPT. METHOD: A questionnaire addressing the research objectives was distributed by email to fetal medicine and clinical genetics experts in Asia, Australia, Europe and the USA. RESULTS: Guidelines on NIPT are available in the majority of the included countries. Not all existing guidelines address reporting of fetal chromosomal sex and SCAs. In most settings, NIPT frequently includes sex chromosomes (five Australian states, China, Hong Kong, Israel, Singapore, Thailand, USA and 23 of 31 European countries). This occurs most often by default or when parents wish to know fetal sex. In most settings, a potential SCA is reported by stating the risk hereof as "low" or "high" and/or by naming the SCA. Less than 50% of all pregnant women receive NIPT according to respondents from three Australian states, China, Israel, Singapore, Thailand and 24 of 31 European countries. However, this percentage, the genomic coverage of NIPT and its application as primary or secondary screening vary by setting. CONCLUSION: In most of the studied countries/states, NIPT commonly includes sex chromosomes. The reporting practices concerning fetal chromosomal sex and SCAs are diverse and most commonly not addressed by guidelines. In general, NIPT is variably implemented across countries/states.

The acceptability of asking women to delay removal of a long-acting reversible contraceptive to take part in a preconception weight loss programme: a mixed methods study using qualitative and routine data (Plan-it).

BACKGROUND: Having a body mass index (BMI) which is classified as overweight (BMI ≥ 25) or obese (BMI ≥ 30) increases the risk of complications during pregnancy and labour. Weight-management interventions which target excess gestational weight gain during pregnancy have had limited success. Women who use long-acting reversible contraception (LARC) are in contact with services as part of their preparation for conception, creating a potential opportunity to offer a preconception weight-loss intervention. The aims of this mixed methods study were to assess the acceptability and practicability of a weight-loss intervention which asked people to delay LARC removal in order to lose weight before conceiving. METHODS: Routine UK NHS data were analysed to identify pathways from LARC removal to pregnancy. Qualitative surveys and advisory group discussions with service providers and LARC users with experience of being overweight were conducted and analysed thematically. RESULTS: Three hundred fifteen thousand seven hundred fifty-five UK women aged 16-48 years between 2009-2018 had at least one LARC-related event (e.g. insertion, removal) and 1.7% of those events were recorded as related to planning a pregnancy. BMI was included in 62% of women's records, with 54% of those BMI being classified as overweight or obese. Online surveys were completed by 100 healthcare practitioners and 243 LARC users. Stakeholders identified facilitators and barriers associated with the proposed intervention including sensitivities of discussing weight, service-user past experiences, practitioner skills, the setting and ethical implications of the proposed intervention. CONCLUSIONS: Although women and service providers recognised potential benefits, a preconception weight-loss intervention asking people to delay LARC removal posed many barriers, due mainly to the acceptability of such an intervention to women and healthcare practitioners. Weight-loss interventions that target the general population, together with a focus on improving public knowledge of preconception health, may be more acceptable than interventions which solely focus on LARC users. Many of the barriers identified, including communication, understanding and beliefs about weight and risk, appointment systems and the limitations of routine datasets also have relevance for any preconception weight-loss intervention. Work to improve routine datasets and reducing communication barriers to discussing weight are priorities. TRIAL REGISTRATION: ISRCTN14733020 registered 10.05.2019.

Paediatric early warning systems: not a simple answer to a complex question.

Paediatric early warning systems (PEWS) to reduce in-hospital mortality have been a laudable endeavour. Evaluation of their impact has rarely examined the internal validity of the components of PEWS in achieving desired outcomes. We highlight the assumptions made regarding the mode of action of PEWS and, as PEWS become more commonplace, this paper asks whether we really understand their function, process and outcome.

Development, implementation and evaluation of an evidence-based paediatric early warning system improvement programme: the PUMA mixed methods study.

BACKGROUND: Paediatric mortality rates in the United Kingdom are amongst the highest in Europe. Clinically missed deterioration is a contributory factor. Evidence to support any single intervention to address this problem is limited, but a cumulative body of research highlights the need for a systems approach. METHODS: An evidence-based, theoretically informed, paediatric early warning system improvement programme (PUMA Programme) was developed and implemented in two general hospitals (no onsite Paediatric Intensive Care Unit) and two tertiary hospitals (with onsite Paediatric Intensive Care Unit) in the United Kingdom. Designed to harness local expertise to implement contextually appropriate improvement initiatives, the PUMA Programme includes a propositional model of a paediatric early warning system, system assessment tools, guidance to support improvement initiatives and structured facilitation and support. Each hospital was evaluated using interrupted time series and qualitative case studies. The primary quantitative outcome was a composite metric (adverse events), representing the number of children monthly that experienced one of the following: mortality, cardiac arrest, respiratory arrest, unplanned admission to Paediatric Intensive Care Unit, or unplanned admission to Higher Dependency Unit. System changes were assessed qualitatively through observations of clinical practice and interviews with staff and parents. A qualitative evaluation of implementation processes was undertaken. RESULTS: All sites assessed their paediatric early warning systems and identified areas for improvement. All made contextually appropriate system changes, despite implementation challenges. There was a decline in the adverse event rate trend in three sites; in one site where system wide changes were organisationally supported, the decline was significant (ß = -0.09 (95% CI: - 0.15, - 0.05); p = < 0.001). Changes in trends coincided with implementation of site-specific changes. CONCLUSIONS: System level change to improve paediatric early warning systems can bring about positive impacts on clinical outcomes, but in paediatric practice, where the patient population is smaller and clinical outcomes event rates are low, alternative outcome measures are required to support research and quality improvement beyond large specialist centres, and methodological work on rare events is indicated. With investment in the development of alternative outcome measures and methodologies, programmes like PUMA could improve mortality and morbidity in paediatrics and other patient populations.

Exploring patients' experiences of analgesia after major lower limb amputation: a qualitative study.

OBJECTIVES: To explore patient experiences, understanding and perceptions of analgesia following major lower limb amputation. DESIGN: Qualitative interview study, conducted as part of a randomised controlled feasibility trial. SETTING: Participants were recruited from two general hospitals in South Wales. PARTICIPANTS: Interview participants were patients enrolled in PLACEMENT (Perineural Local Anaesthetic Catheter aftEr Major lowEr limb amputatioN Trial): a randomised controlled feasibility trial comparing the use of perineural catheter (PNC) versus standard care for postoperative pain relief following major lower limb amputation. PLACEMENT participants who completed 5-day postoperative follow-up, were able and willing to participate in a face-to-face interview, and had consented to be contacted, were eligible to take part in the qualitative study. A total of 20 interviews were conducted with 14 participants: 10 male and 4 female. METHODS: Semi-structured, face-to-face interviews were conducted with participants over two time points: (1) up to 1 month and (2) at least 6 months following amputation. Interviews were audio-recorded, transcribed verbatim and analysed using a framework approach. RESULTS: Interviews revealed unanticipated benefits of PNC usage for postoperative pain relief. Participants valued the localised and continuous nature of this mode of analgesia in comparison to opioids. Concerns about opioid dependence and side effects of pain relief medication were raised by participants in both treatment groups, with some reporting trying to limit their intake of analgesics. CONCLUSIONS: Findings suggest routine placement of a PNC following major lower limb amputation could reduce postoperative pain, particularly for patient groups at risk of postoperative delirium. This method of analgesic delivery also has the potential to reduce preoperative anxiety, alleviate the burden of pain management and minimise opioid use. Future research could further examine the comparison between patient-controlled analgesia and continuous analgesia in relation to patient anxiety and satisfaction with pain management. TRIAL REGISTRATION NUMBER: ISRCTN: 85710690; EudraCT: 2016-003544-37.

Current use of noninvasive prenatal testing in Europe, Australia and the USA: A graphical presentation.

INTRODUCTION: Noninvasive prenatal testing (NIPT) using cell-free fetal DNA has increasingly been adopted as a screening tool for fetal aneuploidies. Several studies have discussed benefits and limitations of NIPT compared with both ultrasound and invasive procedures, but in spite of some shortcomings NIPT has become extensively used within the last 5 years. This study aims to describe the current use of NIPT in Europe, Australia and the USA. MATERIAL AND METHODS: We conducted a survey to describe the current use of NIPT. Colleagues filled in a simple email-based questionnaire on NIPT in their own country, providing information on (a) access to NIPT, (b) NIPT's chromosomal coverage, (c) financial coverage of NIPT for the patient and (d) the proportion of women using NIPT in pregnancy. Some data are best clinical estimates, due to a lack of national data. RESULTS: In Europe, 14 countries have adopted NIPT into a national policy/program. Two countries (Belgium and the Netherlands) offer NIPT for all pregnant women, whereas most other European countries have implemented NIPT as an offer for higher risk women after first trimester screening. In Australia, either combined first trimester screening (cFTS) or NIPT is used as a primary prenatal screening test. In the USA, there are no national consensus policies on the use of NIPT; however, NIPT is widely implemented. In most European countries offering NIPT, the proportion of women using NIPT is well below 25%. In the Netherlands, Austria, Italy, Spain and most Australian and American States, 25%-50% of women have NIPT performed and in Belgium testing is above 75%. In most countries, NIPT reports on trisomy 13, 18 and 21, and often also on sex chromosome aneuploidies. Only in Belgium, the Netherlands, Lithuania, Greece, Cyprus and Italy is NIPT offered predominantly as a genome-wide test (including some microdeletions or a whole genome coverage). CONCLUSIONS: Noninvasive prenatal testing has been widely adopted throughout Europe, Australia and the USA, but only a few countries/states have a national policy on the use of NIPT. The variation in NIPT utilization is considerable.

A prospective, mixed-methods, before and after study to identify the evidence base for the core components of an effective Paediatric Early Warning System and the development of an implementation package containing those core recommendations for use in the UK: Paediatric early warning system - utilisation and mortality avoidance- the PUMA study protocol.

BACKGROUND: In hospital, staff need to routinely monitor patients to identify those who are seriously ill, so that they receive timely treatment to improve their condition. A Paediatric Early Warning System is a multi-faceted socio-technical system to detect deterioration in children, which may or may not include a track and trigger tool. It functions to monitor, detect and prompt an urgent response to signs of deterioration, with the aim of preventing morbidity and mortality. The purpose of this study is to develop an evidence-based improvement programme to optimise the effectiveness of Paediatric Early Warning Systems in different inpatient contexts, and to evaluate the feasibility and potential effectiveness of the programme in predicting deterioration and triggering timely interventions. METHODS: This study will be conducted in two district and two specialist children's hospitals. It deploys an Interrupted Time Series (ITS) design in conjunction with ethnographic cases studies with embedded process evaluation. Informed by Translational Mobilisation Theory and Normalisation Process Theory, the study is underpinned by a functions based approach to improvement. Workstream (1) will develop an evidence-based improvement programme to optimise Paediatric Early Warning System based on systematic reviews. Workstream (2) consists of observation and recording outcomes in current practice in the four sites, implementation of the improvement programme and concurrent process evaluation, and evaluation of the impact of the programme. Outcomes will be mortality and critical events, unplanned admission to Paediatric Intensive Care (PICU) or Paediatric High Dependency Unit (PHDU), cardiac arrest, respiratory arrest, medical emergencies requiring immediate assistance, reviews by PICU staff, and critical deterioration, with qualitative evidence of the impact of the intervention on Paediatric Early Warning System and learning from the implementation process. DISCUSSION: This paper presents the background, rationale and design for this mixed methods study. This will be the most comprehensive study of Paediatric Early Warning Systems and the first to deploy a functions-based approach to improvement in the UK with the aim to improve paediatric patient safety and reduce mortality. Our findings will inform recommendations about the safety processes for every hospital treating paediatric in-patients across the NHS. TRIAL REGISTRATION: Sponsor: Cardiff University, 30-36 Newport Road, Cardiff, CF24 0DE Sponsor ref.: SPON1362-14. Funder: National Institute for Health Research, Health Services & Delivery Research Programme (NIHR HS&DR) Funder reference: 12/178/17. Research Ethics Committee reference: 15/SW/0084 [13/04/2015]. PROSPERO reference: CRD42015015326 [23/01/2015]. ISRCTN: 94228292 https://doi.org/10.1186/ISRCTN94228292 [date of application 13/05/2015; date of registration: 18/08/2015]. Prospective registration prior to data collection and participant consent commencing in September 2014.

Effect of disrupted mitochondria as a source of damage-associated molecular patterns on the production of tumor necrosis factor α by splenocytes from dogs.

OBJECTIVE To evaluate the effects of damage-associated molecular patterns (DAMPs) derived from disrupted mitochondria on canine splenocytes and other immune cells. SAMPLES Liver, spleen, and bone marrow samples obtained from 8 cadavers of healthy research Beagles that had been euthanized for other purposes. PROCEDURES Mitochondria were obtained from canine hepatocytes, and mitochondrial DAMPs (containing approx 75% mitochondrial proteins) were prepared. Mitochondrial DAMPs and the nuclear cytokine high-mobility group box protein 1 were applied to splenocytes, bone marrow-differentiated dendritic cells, and a canine myelomonocytic cell (DH82) line for 6 or 24 hours. Cell culture supernatants from splenocytes, dendritic cells, and DH82 cells were assayed for tumor necrosis factor α with an ELISA. Expression of tumor necrosis factor α mRNA in splenocytes was evaluated with a quantitative real-time PCR assay. RESULTS In all cell populations evaluated, production of tumor necrosis factor α was consistently increased by mitochondrial DAMPs at 6 hours (as measured by an ELISA). In contrast, high-mobility group box protein 1 did not have any independent proinflammatory effects in this experimental system. CONCLUSIONS AND CLINICAL RELEVANCE The study revealed an in vitro inflammatory effect of mitochondrial DAMPs (containing approx 75% mitochondrial proteins) in canine cells and validated the use of an in vitro splenocyte model to assess DAMP-induced inflammation in dogs. This experimental system may aid in understanding the contribution of DAMPs to sepsis and the systemic inflammatory response syndrome in humans. Further studies in dogs are needed to validate the biological importance of these findings and to evaluate the in vivo role of mitochondrial DAMPs in triggering and perpetuating systemic inflammatory states.

Tolerance and Cross-Tolerance following Toll-Like Receptor (TLR)-4 and -9 Activation Are Mediated by IRAK-M and Modulated by IL-7 in Murine Splenocytes.

OBJECTIVE: Immune suppression during critical illness predisposes to serious infections. We sought to determine the mechanisms regulating tolerance and cross-tolerance to common pro-inflammatory danger signals in a model that recapitulates the intact in vivo immune response. MATERIALS AND METHODS: Flt3-expanded splenocytes obtained from wild-type or matching IRAK-M knockout (IRAK-M-/-), C57BL/6, male mice (8-10 weeks old) were treated repeatedly or alternately with either LPS or CpGA DNA, agonists of Toll-like receptor (TLR)-4 and -9, respectively, over successive 24-hour periods. Supernatants were collected following each 24-hour period with cytokine release (ELISA) and splenocyte IRAK-M expression (Western blot) determined. Tolerance and cross-tolerance were assessed in the absence or presence of programmed death receptor (PD)-1 blocking antibody or IL-7 pre-treatment. MAIN RESULTS: Splenocytes notably exhibited both tolerance and cross-tolerance to subsequent treatments with either LPS or CpGA DNA. The character of tolerance and cross-tolerance in this model was distinct following initial LPS or CpGA treatment in that TNFα and IFNγ release (not IL-10) were suppressed following LPS; whereas, initial CpGA treatment suppressed TNFα, IFNγ and IL-10 release in response to subsequent stimulation (LPS or CpGA). Tolerance and cross-tolerance were unrelated to IL-10 release or PD-1 but were attenuated in IRAK-M-/- splenocytes. IL-7 significantly suppressed IRAK-M expression and restored TNFα and IFNγ production without influencing IL-10 release. CONCLUSIONS: In summary, acute immune tolerance and cross-tolerance in response to LPS or CpGA were distinct in that LPS selectively suppressed pro-inflammatory cytokine responses; whereas, CpGA suppressed both pro- and anti-inflammatory responses. The induction of tolerance and cross-tolerance in response to common danger signals was mechanistically unrelated to IL-10 or PD-1 but was directly influenced by IRAK-M expression. IL-7 reduced IRAK-M expression and attenuated immune tolerance induced by either LPS or CpGA, and thus may be useful for reversal of immune tolerance in the setting of critical illness.

Continuous retinoic acid induces the differentiation of mature regulatory monocytes but fails to induce regulatory dendritic cells.

BACKGROUND: Myeloid cells (MC) have potent immunoregulatory abilities that can be therapeutically useful to treat inflammatory disease. However, the factors which promote regulatory myeloid cell differentiation remain poorly understood. We have previously shown that estriol (E3) induces mature regulatory dendritic cells in vivo. To determine whether additional steroid hormones could induce mature regulatory myeloid cells, we investigated the effects of retinoic acid (RA) on MCs. Retinoic acid is a steroid hormone important in regulating mucosal immunity in the gut and promoting myeloid differentiation. We hypothesized that the presence of RA during differentiation would promote the formation of mature regulatory myeloid cells (MCregs). METHODS: To determine RA's ability to induce regulatory myeloid cells, we differentiated bone marrow progenitor cells with granulocytic-macrophage colony-stimulating factor (GM-CSF) under the influence of RA. We found that day 7 MCs differentiated in the presence of RA had an increase in the percent positive and relative expression levels of both maturation (CD80, CD86, and MHCII) and inhibitory (PD-L1 and PD-L2) markers compared to control cells. Functionally, these day 7 RA MCs expressed increased intracellular IL-10, induced regulatory T cells in vitro compared to controls and suppressed the proliferation of responder immune cells even after inflammatory challenge with LPS. CONCLUSION: RA induced mature regulatory myeloid cells that were suppressive and had a CD11b+ CD11c-Ly6C low/intermediate monocyte phenotype. Surprisingly, RA CD11c+ dendritic cells were not suppressive and could contribute to enhanced proliferation. These results suggest that continuous RA has unique effects on different myeloid populations during monopoeisis and dendropoiesis and promotes a population of regulatory monocytes.

The fbpABC operon is required for Ton-independent utilization of xenosiderophores by Neisseria gonorrhoeae strain FA19.

Neisseria gonorrhoeae produces no known siderophores but can employ host-derived, iron-binding proteins, including transferrin and lactoferrin, as iron sources. Given the propensity of this pathogen to hijack rather than synthesize iron-sequestering molecules, we hypothesized that the ability to use siderophores produced by other bacteria, or xenosiderophores, may also play a role in the survival of the gonococcus. Among a panel of diverse siderophores, only the catecholate xenosiderophores enterobactin and salmochelin promoted growth of gonococcal strain FA19. Surprisingly, the internalization pathway was independent of TonB or any of the TonB-dependent transporters. Xenosiderophore-mediated growth was similarly independent of the pilin-extruding secretin formed by PilQ and of the hydrophobic-agent efflux system composed of MtrCDE. The fbpABC operon encodes a periplasmic-binding-protein-dependent ABC transport system that enables the gonococcus to transport iron into the cell subsequent to outer membrane translocation. We hypothesized that the FbpABC proteins, required for ferric iron transport from transferrin and lactoferrin, might also contribute to the utilization of xenosiderophores as iron sources. We created mutants that conditionally expressed FbpABC from an IPTG-inducible promoter. We determined that expression of FbpABC was required for growth of gonococcal strain FA19 in the presence of enterobactin and salmochelin. The monomeric component of enterobactin, dihydroxybenzoylserine (DHBS), and the S2 form of salmochelin specifically promoted FbpABC-dependent growth of FA19. This study demonstrated that the gonococcal FbpABC transport system is required for utilization of some xenosiderophores as iron sources and that growth promotion by these ferric siderophores can occur in the absence of TonB or individual TonB-dependent transporters.

Type IV secretion machinery promotes ton-independent intracellular survival of Neisseria gonorrhoeae within cervical epithelial cells.

Survival of Neisseria gonorrhoeae within host epithelial cells is expected to be important in the pathogenesis of gonococcal disease. We previously demonstrated that strain FA1090 derives iron from a host cell in a process that requires the Ton complex and a putative TonB-dependent transporter, TdfF. FA1090, however, lacks the gonococcal genetic island (GGI) that is present in the majority of strains. The GGI in strain MS11 has been partially characterized, and it encodes a type IV secretion system (T4SS) involved in DNA release. In this study we investigated the role of iron acquisition and GGI-encoded gene products in gonococcal survival within cervical epithelial cells. We demonstrated that intracellular survival of MS11 was dependent on acquisition of iron from the host cell, but unlike the findings for FA1090, expression of the Ton complex was not required. Survival was not dependent on a putative TonB-like protein encoded in the GGI but instead was directly linked to T4SS structural components in a manner independent of the ability to release or internalize DNA. These data suggest that expression of selected GGI-encoded open reading frames confers an advantage during cervical cell infection. This study provides the first link between expression of the T4SS apparatus and intracellular survival of gonococci.

Non-medical sex selection: ethical issues.

INTRODUCTION: This paper aims to provide a concise review of the ethical issues that are commonly raised in the UK debate on non-medical sex selection. Background information on sex selection technologies is provided, as is a description of the relevant UK legislation. Arguments for and against non-medical sex selection will be explained and compared and conclusions will be drawn. It is finally suggested that the international debate on non-medical sex selection ought to be regarded as an important area of related interest. SOURCES OF DATA: Data were obtained from a search of existing ethics and policy literature focusing on sex selection. AREAS OF AGREEMENT: There are very few areas of universal agreement in the debate. AREAS OF CONTROVERSY: There is much disagreement between critics over what harms are likely to be caused by sex selection and whether such harms are morally significant. The issue of whether governments can legitimately place limitations upon individual reproductive autonomy, and if so, to what degree, remains controversial.