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Recent technological advances have greatly diversified the platforms that are available for high-dimensional single-cell immunophenotyping, including mass cytometry, single-cell RNA sequencing, and fluorescent-based flow cytometry. The latter is currently the most commonly used approach, and modern instrumentation allows for the measurement of up to 30 parameters, revealing deep insights into the complexity of the immune system.Here, we provide a practical guidebook for the successful design and execution of complex fluorescence-based immunophenotyping panels. We address common misconceptions and caveats, and also discuss challenges that are associated with the quality control and analysis of these data sets.

Mucosal-associated invariant T cells (MAIT cells) recognize bacterial metabolites as antigen and are found in blood and tissues, where they are poised to contribute to barrier immunity. Recent data demonstrate that MAIT cells located in mucosal barrier tissues are functionally distinct from their blood counterparts, but the relationship and circulation of MAIT cells between blood and different tissue compartments remains poorly understood. Previous studies raised the possibility that MAIT cells do not leave tissue and may either be retained or undergo apoptosis. To directly address if human MAIT cells exit tissues, we collected human donor-matched thoracic duct lymph and blood and analyzed MAIT cell phenotype, transcriptome, and T cell receptor (TCR) diversity by flow cytometry and RNA sequencing. We found that MAIT cells were present in the lymph, despite being largely CCR7- in the blood, thus indicating that MAIT cells in the lymph migrated from tissues and were capable of exiting tissues to recirculate. Importantly, MAIT cells in the lymph and blood had highly overlapping clonotype usage but distinct transcriptome signatures, indicative of differential activation states.

This work describes the first 30-parameter immunophenotyping of the human dendritic cell (DC) compartment using fluorescent-based flow cytometry. The optimized panel allows for simultaneous detection of 21 myeloid-centric markers distinguishing all canonical DC subsets, with parallel enumeration of monocytes, T and B cells as well as NK cells. Thus, this panel will be useful for extensive phenotyping of immune cells from a variety of human samples limited in size., (© 2018 International Society for Advancement of Cytometry.)

Innate lymphoid cells (ILCs) have been classified into "functional subsets" according to their transcription factor and cytokine profiles. Although cytokines, such as IL-12 and IL-23, have been shown to shape plasticity of ILCs, little is known about how the tissue microenvironment influences the plasticity, phenotype, and function of these cells. Here, we show clearly demarcated tissue specifications of Rorc -dependent ILCs across lymphoid and nonlymphoid organs. Although intestinal Rorc fate map-positive ( Rorc fm+ ) ILCs show a clear ILC3 phenotype, lymphoid tissue-derived Rorc fm+ ILCs acquire an natural killer (NK) cell/ILC1-like phenotype. By adoptively transferring Rorc fm+ ILCs into recipient mice, we show that ILCs distribute among various organs and phenotypically adapt to the tissue environment they invade. When investigating their functional properties, we found that only lymphoid-tissue resident Rorc fm+ ILCs can suppress tumor growth, whereas intestinal Rorc fm- ILC1s or NK cells fail to inhibit tumor progression. We thus propose that the tissue microenvironment, combined with ontogeny, provides the specific function, whereas the phenotype is insufficient to predict the functional properties of ILCs., (© 2017 Nussbaum et al.)

Chronic inflammatory diseases are influenced by dysregulation of cytokines. Among them, granulocyte macrophage colony stimulating factor (GM-CSF) is crucial for the pathogenic function of T cells in preclinical models of autoimmunity. To study the impact of dysregulated GM-CSF expression in vivo, we generated a transgenic mouse line allowing the induction of GM-CSF expression in mature, peripheral helper T (Th) cells. Antigen-independent GM-CSF release led to the invasion of inflammatory myeloid cells into the central nervous system (CNS), which was accompanied by the spontaneous development of severe neurological deficits. CNS-invading phagocytes produced reactive oxygen species and exhibited a distinct genetic signature compared to myeloid cells invading other organs. We propose that the CNS is particularly vulnerable to the attack of monocyte-derived phagocytes and that the effector functions of GM-CSF-expanded myeloid cells are in turn guided by the tissue microenvironment., (Copyright © 2017 Elsevier Inc. All rights reserved.)

How and when we use health services or healthcare provision has dominated exploration of and debates around healthcare access. Levels of utilisation are assumed as a proxy for access. Yet, focusing on utilisation conceals an important aspect of the access conundrum: the relationships that patients and potential patients have with the healthcare system and the professionals within those systems. Candidacy has been proposed as an antidote to traditional utilisation models. The Candidacy construct offers the ability to include patient-professional aspects alongside utilisation and thus promotes a deeper understanding of access. Originally applied to healthcare access for vulnerable populations, additional socio-demographic factors, including age and ethnicity, have also been shown to influence the Candidacy process. Here we propose a further extension of the Candidacy construct and illustrate the importance of illness identities when accessing healthcare. Drawing on a secondary data analysis of three data sets of qualitative interviews from colorectal cancer and heart failure patients we found that though similar access issues are apparent pre-diagnosis, diagnosis marks a critical juncture in the experience of access. Cancer patients describe a person-centred responsive healthcare system where their patienthood requires only modest assertion. Cancer speaks for itself. In marked contrast heart failure patients, describe struggling within a seemingly impermeable system to understand their illness, its implications and their own legitimacy as patients. Our work highlights the pressing need for healthcare professionals, systems and policies to promote a person centred approach, which is responsive and timely, regardless of illness category. To achieve this, attitudes regarding the importance or priority afforded to different categories of illness need to be tackled as they directly influence ideas of Candidacy and consequently access and experiences of care., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The CRISPR/Cas9 system can be used to mutate target sequences by introduction of double-strand breaks followed by imprecise repair. To test its use for conditional gene editing we generated mice transgenic for CD4 promoter-driven Cas9 combined with guide RNA targeting CD2. We found that within CD4(+) and CD8(+) lymphocytes from lymph nodes and spleen 1% and 0.6% were not expressing CD2, respectively. T cells lacking CD2 carryied mutations, which confirmed that Cas9 driven by cell-type specific promoters can edit genes in the mouse and may thus allow targeted studies of gene function in vivo.

Ever since its invention half a century ago, flow cytometry has been a major tool for single-cell analysis, fueling advances in our understanding of a variety of complex cellular systems, in particular the immune system. The last decade has witnessed significant technical improvements in available cytometry platforms, such that more than 20 parameters can be analyzed on a single-cell level by fluorescence-based flow cytometry. The advent of mass cytometry has pushed this limit up to, currently, 50 parameters. However, traditional analysis approaches for the resulting high-dimensional datasets, such as gating on bivariate dot plots, have proven to be inefficient. Although a variety of novel computational analysis approaches to interpret these datasets are already available, they have not yet made it into the mainstream and remain largely unknown to many immunologists. Therefore, this review aims at providing a practical overview of novel analysis techniques for high-dimensional cytometry data including SPADE, t-SNE, Wanderlust, Citrus, and PhenoGraph, and how these applications can be used advantageously not only for the most complex datasets, but also for standard 14-parameter cytometry datasets., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

γδ T cells contribute to first line immune defense, particularly through their ability for rapid production of proinflammatory cytokines. The cytokine profile of γδ T cells is hard-wired already during thymic development. Yet, the molecular pathways underlying this phenomenon are incompletely understood. Here we show that signaling via the NFκB-inducing kinase (NIK) is essential for the formation of a fully functional γδ T cell compartment. In the absence of NIK, development of Vγ5(+) dendritic epidermal T cells (DETCs) was halted in the embryonic thymus, and impaired NIK function caused a selective loss of IL-17 expression by γδ T cells. Using a novel conditional mutant of NIK, we could show in vivo that NIK signaling in thymic epithelial cells is essential for the thymic hardwiring of γδ T cell cytokine production.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has emerged as a crucial cytokine produced by auto-reactive T helper (Th) cells that initiate tissue inflammation. Multiple cell types can sense GM-CSF, but the identity of the pathogenic GM-CSF-responsive cells is unclear. By using conditional gene targeting, we systematically deleted the GM-CSF receptor (Csf2rb) in specific subpopulations throughout the myeloid lineages. Experimental autoimmune encephalomyelitis (EAE) progressed normally when either classical dendritic cells (cDCs) or neutrophils lacked GM-CSF responsiveness. The development of tissue-invading monocyte-derived dendritic cells (moDCs) was also unperturbed upon Csf2rb deletion. Instead, deletion of Csf2rb in CCR2(+)Ly6C(hi) monocytes phenocopied the EAE resistance seen in complete Csf2rb-deficient mice. High-dimensional analysis of tissue-infiltrating moDCs revealed that GM-CSF initiates a combination of inflammatory mechanisms. These results indicate that GM-CSF signaling controls a pathogenic expression signature in CCR2(+)Ly6C(hi) monocytes and their progeny, which was essential for tissue damage., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Aims: To summarize the findings and "expert-panel" consensus of the State-of-the-Science Seminar on pelvic floor muscle training (PFMT) adherence held prior to the 41st International Continence Society scientific meeting, Glasgow, 2011., Methods: Summaries of research and theory about PFMT adherence (based on a comprehensive literature search) were presented by subject experts at the 2011 Seminar to generate discussion and guidance for clinical practice and future research. Supplemental research, post-seminar, resulted in, three review papers summarizing: (1) relevant behavioral theories, (2) adherence measurement, determinants and effectiveness of PFMT adherence interventions, and (3) patients' PFMT experiences. A fourth, reported findings from an online survey of health professionals and the public., Results: Few high-quality studies were found. Paper I summarizes 12 behavioral frameworks relevant to theoretical development of PFMT adherence interventions and strategies. Findings in Paper II suggest both PFMT self-efficacy and intention-to-adhere predict PFMT adherence. Paper III identified six potential adherence modifiers worthy of further investigation. Paper IV found patient-related factors were the biggest adherence barrier to PFMT adherence., Conclusion: Given the lack of high-quality studies, the conclusions were informed by expert opinion. Adherence is central to short- and longer-term PFMT effect. More attention and explicit reporting is needed regarding: (1) applying health behavior theory in PFMT program planning; (2) identifying adherence determinants; (3) developing and implementing interventions targeting known adherence determinants; (4) using patient-centred approaches to evaluating adherence barriers and facilitators; (5) measuring adherence, including refining and testing instruments; and (6) testing the association between adherence and PFMT outcome., (© 2015 Wiley Periodicals, Inc.)

Aims: This paper, the first of four emanating from the International Continence Society's 2011 State-of-the-Science Seminar on pelvic-floor-muscle training (PFMT) adherence, aimed to summarize the literature on theoretical models to promote PFMT adherence, as identified in the research, or suggested by the seminar's expert panel, and recommends future directions for clinical practice and research., Methods: Existing literature on theories of health behavior were identified through a conventional subject search of electronic databases, reference-list checking, and input from the expert panel. A core eligibility criterion was that the study included a theoretical model to underpin adherence strategies used in an intervention to promote PFM training/exercise., Results: A brief critique of 12 theoretical models/theories is provided and, were appropriate, their use in PFMT adherence strategies identified or examples of possible uses in future studies outlined., Conclusion: A better theoretical-based understanding of interventions to promote PFMT adherence through changes in health behaviors is required. The results of this scoping review and expert opinions identified several promising models. Future research should explicitly map the theories behind interventions that are thought to improve adherence in various populations (e.g., perinatal women to prevent or lessen urinary incontinence). In addition, identified behavioral theories applied to PFMT require a process whereby their impact can be evaluated., (© 2015 Wiley Periodicals, Inc.)

The thymic epithelium forms specialized niches to enable thymocyte differentiation. While the common epithelial progenitor of medullary and cortical thymic epithelial cells (mTECs and cTECs) is well defined, early stages of mTEC lineage specification have remained elusive. Here, we utilized in vivo targeting of mTECs to resolve their differentiation pathways and to determine whether mTEC progenitors participate in thymocyte education. We found that mTECs descend from a lineage committed, podoplanin (PDPN)-expressing progenitor located at the cortico-medullary junction. PDPN(+) junctional TECs (jTECs) represent a distinct TEC population that builds the thymic medulla, but only partially supports negative selection and thymocyte differentiation. Moreover, conditional gene targeting revealed that abrogation of alternative NF-κB pathway signaling in the jTEC stage completely blocked mTEC development. Taken together, this study identifies jTECs as lineage-committed mTEC progenitors and shows that NF-κB-dependent progression of jTECs to mTECs is critical to secure central tolerance., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: In the UK, the Quality and Outcome Framework (QOF) has specific targets for general practictioners to record body-mass index (BMI) and blood pressure (BP) in major mental illness, diabetes, and chronic kidney disease. Although incentives are given for aspects of major mental illness (schizophrenia, bipolar disorder, and related psychoses), barriers to care can occur. Our aim was to compare recording of specific targets for BP and BMI in individuals with major mental illness relative to diabetes and chronic kidney disease across the UK., Methods: Using 2012 and 2013 QOF data from 9731 general practices across all four countries in the UK, we calculated median payment, population achievement, and exception rates for BP indicators in major mental illness and chronic kidney disease and BMI indicators in major mental illness and diabetes. Differences in unweighted rates between practices in the same UK country were tested with a sign test. Differences in population achievement rate between practices in different countries were compared with those in England by use of a quantile regression analysis., Findings: UK payment and population achievement rates for BMI recording in major mental illness were significantly lower than were those in diabetes (payment 92·7% vs 95·5% and population achievement 84·0% vs 92·5%, p<0·0001) and exception rates were higher (8·1% vs 2·0%, p<0·0001). For BP recording, UK payment and population achievement rates were significantly lower for major mental illness than for chronic kidney disease (94·1% vs 97·8% and 87·0% vs 97·1%, p<0·0001), whereas exception rate was higher (6·5% vs 0·0%, p<0·0001). This difference was observed for all UK countries. Median population achievement rates for BMI and BP recording in major mental illness were significantly lower in Scotland than in England (for BMI -1·5%, 99% CI -2·7 to -0·3, and for BP -1·8%, -2·7 to -0·9; p<0·0001 for both). There were no cross-jurisdiction differences for chronic kidney disease and diabetes., Interpretation: We found lower payment rates, higher exception rates, and lower population achievement rates for BMI and BP recording in major mental illness than in diabetes and chronic kidney disease throughout the UK. We also found variation in these rates between countries. This finding is probably multifactorial, reflecting a combination of patient, clinician, and wider organisational factors; however, it might also suggest inequality in access to certain aspects of health care for people with major mental illness., Funding: None., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Digital technologies are being used as part of international efforts to revolutionize healthcare in order to meet increasing demands such as the rising burden of chronic disease and ageing populations. In Scotland there is a government push towards a national service (Living It Up) as a single point of reference where citizens can access information, products and services to support their health and wellbeing. The aim of the study is to examine implementation issues including the challenges or facilitators which can help to sustain this intervention. We gathered data in three ways: a) participant observation to gain an understanding of LiU (N=16); b) in-depth interviews (N=21) with stakeholders involved in the process; and c) analysis of documentary evidence about the progress of the implementation (N=45). Barriers included the need to "work at risk" due to delays in financing, inadequate infrastructure and skill-set deficiencies, whilst facilitators included trusted relationships, champions and a push towards normalisation. The findings suggest that a Scottish ehealth service is achievable but identifies key considerations for future large scale initiatives.

Patients with chronic conditions or multimorbidity, and often their caregivers, have to adjust their lives and mobilise their capacity (ability) to respond to the workload (demands) imposed by treatments and the care of their conditions. There is a continuous and complex interaction between workload and capacity. When capacity proves insufficient to address the treatment workload, creating a burden, patients may place a lower priority on other aspects of their lives, or reduce engagement with healthcare. Guidelines usually focus on disease-centred outcomes without consideration of limited capacity or demanding workload (burden) from treatment regimens. It seems reasonable to consider that healthcare needs reshaping so that care that pursues goals important to patients as well as those suggested by evidence-based medicine. This can be achieved by using shared decision approaches guided by the expertise of clinicians to deliver optimal care while minimising the burden of treatment on patients, their caregivers, and the healthcare system. What we need is minimally disruptive medicine.

Advances in cell-fate mapping have revealed the complexity in phenotype, ontogeny and tissue distribution of the mammalian myeloid system. To capture this phenotypic diversity, we developed a 38-antibody panel for mass cytometry and used dimensionality reduction with machine learning-aided cluster analysis to build a composite of murine (mouse) myeloid cells in the steady state across lymphoid and nonlymphoid tissues. In addition to identifying all previously described myeloid populations, higher-order analysis allowed objective delineation of otherwise ambiguous subsets, including monocyte-macrophage intermediates and an array of granulocyte variants. Using mice that cannot sense granulocyte macrophage-colony stimulating factor GM-CSF (Csf2rb(-/-)), which have discrete alterations in myeloid development, we confirmed differences in barrier tissue dendritic cells, lung macrophages and eosinophils. The methodology further identified variations in the monocyte and innate lymphoid cell compartment that were unexpected, which confirmed that this approach is a powerful tool for unambiguous and unbiased characterization of the myeloid system.

Background: The Quality and Outcomes Framework (QOF) has specific targets for body mass index (BMI) and blood pressure recording in major mental illness (MMI), diabetes, and chronic kidney disease (CKD). Although aspects of MMI (schizophrenia, bipolar disorder, and related psychoses) are incentivised, barriers to care may occur., Aim: To compare payment, population achievement, and exception rates for blood pressure and BMI recording in MMI relative to diabetes and CKD across the UK., Design and Setting: Analysis of 2012/2013 QOF data from 9731 UK general practices 2 years after the introduction of the mental health, BMI, and blood pressure QOF indicators., Method: Payment, exception, and population achievement rates for the MMI and CKD blood pressure indicators and the MMI and diabetes BMI indicators were calculated and compared., Results: UK payment and population achievement rates for BMI recording for MMI were significantly lower than for diabetes (payment: 92.7% versus 95.5% and population achievement: 84.0% versus 92.5%, P<0.001) and exception rates were higher (8.1% versus 2.0%, P<0.001). For blood pressure recording, UK payment and population achievement rates were significantly lower for MMI than for CKD (94.1% versus 97.8% and 87.0% versus 97.1%, P<0.001), while exception rate was higher (6.5% versus 0.0%, P<0.001). This was observed for all countries. Compared with England, Northern Ireland had higher population achievement rates for both mental health indicators, whereas Scotland and Wales had lower rates. There were no cross-jurisdiction differences for CKD and diabetes., Conclusion: Differences in payment, exception, and population achievement rates for blood pressure and BMI recording for MMI relative to CKD and diabetes were observed across the UK. These findings suggest potential inequalities in the monitoring of physical health in MMI within the UK primary care system., (© British Journal of General Practice 2014.)

Background: Comorbidity in Multiple Sclerosis (MS) is associated with worse health and higher mortality. This study aims to describe clinician recorded comorbidities in people with MS., Methods: 39 comorbidities in 3826 people with MS aged ≥25 years were compared against 1,268,859 controls. Results were analysed by age, gender, and socioeconomic status, with unadjusted and adjusted Odds Ratios (ORs) calculated using logistic regression., Results: People with MS were more likely to have one (OR 2.44; 95% CI 2.26-2.64), two (OR 1.49; 95% CI 1.38-1.62), three (OR 1.86; 95% CI 1.69-2.04), four or more (OR 1.61; 95% CI 1.47-1.77) non-MS chronic conditions than controls, and greater mental health comorbidity (OR 2.94; 95% CI 2.75-3.14), which increased as the number of physical comorbidities rose. Cardiovascular conditions, including atrial fibrillation (OR 0.49; 95% CI 0.36-0.67), chronic kidney disease (OR 0.51; 95% CI 0.40-0.65), heart failure (OR 0.62; 95% CI 0.45-0.85), coronary heart disease (OR 0.64; 95% CI 0.52-0.71), and hypertension (OR 0.65; 95% CI 0.59-0.72) were significantly less common in people with MS., Conclusion: People with MS have excess multiple chronic conditions, with associated increased mental health comorbidity. The low recorded cardiovascular comorbidity warrants further investigation.

Background: This is a time of unprecedented mobility across the globe. Healthcare systems need to adapt to ensure that primary care is culturally and linguistically appropriate for migrants. Evidence-based guidelines and training interventions for cultural competence and the use of professional interpreters are available across European healthcare settings. However, in real-world practice migrants and their healthcare providers 'get by' with a range of informal and inadequate strategies. RESTORE is an EU FP7 funded project, which is designed to address this translational gap., Objectives: The objective of RESTORE is to investigate and support the implementation of guidelines and training initiatives to support communication in cross-cultural consultations in selected European primary care settings., Design: RESTORE is a qualitative, participatory health project running from 2011-2015. It uses a novel combination of normalization process theory and participatory learning and action research to follow and shape the implementation journeys of relevant guidelines and training initiatives. Research teams in Ireland, England, the Netherlands, Austria and Greece are conducting similar parallel qualitative case study fieldwork, with a complementary health policy analysis led by Scotland. In each setting, key stakeholders, including migrants, are involved in participatory data generation and analysis., Expected Results: RESTORE will provide knowledge about the levers and barriers to the implementation of guidelines and training initiatives in European healthcare settings and about successful, transferrable strategies to overcome identified barriers. RESTORE will elucidate the role of policy in shaping these implementation journeys; generate recommendations for European policy driving the development of culturally and linguistically appropriate healthcare systems.

Innate lymphoid cells (ILCs) differ from T and B cells as they do not express genetically rearranged antigen receptors. The most prominent member of this group, NK cells, can be identified by numerous surface receptors such as natural cytotoxicity receptors (NCRs). However, novel groups of ILCs have recently been described and classified based on fate-determining transcription factors and cytokines being produced, similarly to T helper cells. Due to the lack of exclusive markers, ILCs are primarily defined by the paucity of lineage markers. Using RORc-fate-mapping mice, we found that the common lineage exclusion using CD3 yields an ILC population containing a large proportion of T cells with recombined TCR loci and low expression of CD3. Thus, we suggest adding CD5 as a marker for thorough elimination of T cells to avoid erroneous interpretations of ILC function in immunity.

The purpose of this paper is to substantiate the importance of research about barriers and levers to the implementation of supports for cross-cultural communication in primary care settings in Europe. After an overview of migrant health issues, with the focus on communication in cross-cultural consultations in primary care and the importance of language barriers, we highlight the fact that there are serious problems in routine practice that persist over time and across different European settings. Language and cultural barriers hamper communication in consultations between doctors and migrants, with a range of negative effects including poorer compliance and a greater propensity to access emergency services. It is well established that there is a need for skilled interpreters and for professionals who are culturally competent to address this problem. A range of professional guidelines and training initiatives exist that support the communication in cross-cultural consultations in primary care. However, these are commonly not implemented in daily practice. It is as yet unknown why professionals do not accept or implement these guidelines and interventions, or under what circumstances they would do so. A new study involving six European countries, RESTORE (REsearch into implementation STrategies to support patients of different ORigins and language background in a variety of European primary care settings), aims to address these gaps in knowledge. It uses a unique combination of a contemporary social theory, normalisation process theory (NPT) and participatory learning and action (PLA) research. This should enhance understanding of the levers and barriers to implementation, as well as providing stakeholders, with the opportunity to generate creative solutions to problems experienced with the implementation of such interventions.

Background: Multiple sclerosis (MS) is a stressful condition; depression, anxiety, pain and fatigue are all common problems. Mindfulness based interventions (MBIs) mitigate stress and prevent relapse in depression and are increasingly being used in healthcare. However, there are currently no systematic reviews of MBIs in people with MS. This review aims to evaluate the effectiveness of MBIs in people with MS., Methods: Systematic searches were carried out in seven major databases, using both subject headings and key words. Papers were screened, data extracted, quality appraised, and analysed by two reviewers independently, using predefined criteria. Study quality was assessed using the Cochrane Collaboration risk of bias tool. Perceived stress was the primary outcome. Secondary outcomes include mental health, physical health, quality of life, and health service utilisation. Statistical meta-analysis was not possible. Disagreements were adjudicated by a third party reviewer., Results: Three studies (n = 183 participants) were included in the final analysis. The studies were undertaken in Wales (n = 16, randomised controlled trial - (RCT)), Switzerland (n = 150, RCT), and the United States (n = 17, controlled trial). 146 (80%) participants were female; mean age (SD) was 48.6 (9.4) years. Relapsing remitting MS was the main diagnostic category (n = 123, 67%); 43 (26%) had secondary progressive disease; and the remainder were unspecified. MBIs lasted 6-8 weeks; attrition rates were variable (5-43%); all employed pre- post- measures; two had longer follow up; one at 3, and one at 6 months. Socio-economic status of participants was not made explicit; health service utilisation and costs were not reported. No study reported on perceived stress. All studies reported quality of life (QOL), mental health (anxiety and depression), physical (fatigue, standing balance, pain), and psychosocial measures. Statistically significant beneficial effects relating to QOL, mental health, and selected physical health measures were sustained at 3- and 6- month follow up., Conclusion: From the limited data available, MBIs may benefit some MS patients in terms of QOL, mental health, and some physical health measures. Further studies are needed to clarify how MBIs might best serve the MS population.

Within the past years immune cells in general and T cells in particular have been categorized into a vast variety of subsets with different functional properties. One of the key technologies fueling this emerging complexity is intracellular staining for effector cytokines and/or lineage-defining transcription factors. Here we discuss the critical steps for performing successful multicolor immunophenotyping of mouse T cells in combination with analysis of intracellular molecules after ex vivo isolation.

In many areas of immunology it is desirable to be able to track particular cells throughout an assay, whether it be in vivo or in vitro. There are two classes or reagents used for this purpose-general protein labels (reactive compounds that form random covalent bonds with amino groups on cellular proteins) and general membrane labels (lipophilic compounds that partition stably but non-covalently into the plasma membrane). The fluorescein derivative, carboxyfluorescein diacetate succinimidyl ester, CFDA-SE (general protein label), has been found to be particularly well suited for the purpose of cell tracking. Once labeled, cells can be tracked both in vivo and in vitro. Moreover, due to the randomness of the labeling, cells undergoing division maintain half of the staining intensity of the parent cell. This halving of the staining intensity additionally allows for monitoring of cells undergoing division for 6-8 consecutive cycles.

IL-23 is absolutely crucial for the development of T-cell driven autoimmune disease in mice. Even though IL-23 is widely held to be involved in the stabilization of IL-17-secreting T cells, naïve T cells lack the IL-23 receptor. Thus, the primary cellular target of IL-23 in the context of autoimmunity is a subject of some debate. Innate lymphoid cells (ILCs) are a recently discovered family of lymphocytes being involved in early host defense, particularly at mucosal epithelial surfaces. Given the fact that RORγt-dependent ILCs (group 3 ILCs) constitutively express the IL-23-receptor, and that they have been implicated in intestinal autoimmunity, we hypothesized that ILCs could contribute to the early development of autoimmune neuroinflammation. Through systematic analysis, we detected a sizable population of Thy1(+) Sca1(+) ILCs in the inflamed CNS tissue. CNS-infiltrating ILCs were characterized by expression of the IL-7-receptor and production of proinflammatory IL-17 and IFN-γ. Furthermore, genetic fate-mapping revealed their dependence on the transcription factor RORγt. However, upon specific in vivo ablation of this cell population, we found that they do not influence the course of the disease., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objectives: Provision of person-centred generalist care is a core component of quality primary care systems. The World Health Organisation believes that a lack of generalist primary care is contributing to inefficiency, ineffectiveness and inequity in healthcare. In UK primary care, General Practitioners (GPs) are the largest group of practising generalists. Yet GPs fulfil multiple roles and the pressures of delivering these roles along with wider contextual changes create real challenges to generalist practice. Our study aimed to explore GP perceptions of enablers and constraints for expert generalist care, in order to identify what is needed to ensure health systems are designed to support the generalist role., Design: Qualitative study in General Practice., Setting: UK primary care., Main Outcome Measures: A qualitative study - interviews, surveys and focus groups with GPs and GP trainees. Data collection and analysis was informed by Normalisation Process Theory., Design and Setting: Qualitative study in General Practice. We conducted interviews, surveys and focus groups with GPs and GP trainees based mainly, but not exclusively, in the UK. Data collection and analysis were informed by Normalization Process Theory., Participants: UK based GPs (interview and surveys); European GP trainees (focus groups)., Results: Our findings highlight key gaps in current training and service design which may limit development and implementation of expert generalist practice (EGP). These include the lack of a consistent and universal understanding of the distinct expertise of EGP, competing priorities inhibiting the delivery of EGP, lack of the consistent development of skills in interpretive practice and a lack of resources for monitoring EGP., Conclusions: WE DESCRIBE FOUR AREAS FOR CHANGE: Translating EGP, Priority setting for EGP, Trusting EGP and Identifying the impact of EGP. We outline proposals for work needed in each area to help enhance the expert generalist role.

TGF-β is widely held to be critical for the maintenance and function of regulatory T (T(reg)) cells and thus peripheral tolerance. This is highlighted by constitutive ablation of TGF-β receptor (TR) during thymic development in mice, which leads to a lethal autoimmune syndrome. Here we describe that TGF-β-driven peripheral tolerance is not regulated by TGF-β signalling on mature CD4⁺ T cells. Inducible TR2 ablation specifically on CD4⁺ T cells did not result in a lethal autoinflammation. Transfer of these TR2-deficient CD4⁺ T cells to lymphopenic recipients resulted in colitis, but not overt autoimmunity. In contrast, thymic ablation of TR2 in combination with lymphopenia led to lethal multi-organ inflammation. Interestingly, deletion of TR2 on mature CD4⁺ T cells does not result in the collapse of the T(reg) cell population as observed in constitutive models. Instead, a pronounced enlargement of both regulatory and effector memory T cell pools was observed. This expansion is cell-intrinsic and seems to be caused by increased T cell receptor sensitivity independently of common gamma chain-dependent cytokine signals. The expression of Foxp3 and other regulatory T cells markers was not dependent on TGF-β signalling and the TR2-deficient T(reg) cells retained their suppressive function both in vitro and in vivo. In summary, absence of TGF-β signalling on mature CD4⁺ T cells is not responsible for breakdown of peripheral tolerance, but rather controls homeostasis of mature T cells in adult mice., Competing Interests: The authors have declared that no competing interests exist.

The pathology of Alzheimer's disease has an inflammatory component that is characterized by upregulation of proinflammatory cytokines, particularly in response to amyloid-β (Aβ). Using the APPPS1 Alzheimer's disease mouse model, we found increased production of the common interleukin-12 (IL-12) and IL-23 subunit p40 by microglia. Genetic ablation of the IL-12/IL-23 signaling molecules p40, p35 or p19, in which deficiency of p40 or its receptor complex had the strongest effect, resulted in decreased cerebral amyloid load. Although deletion of IL-12/IL-23 signaling from the radiation-resistant glial compartment of the brain was most efficient in mitigating cerebral amyloidosis, peripheral administration of a neutralizing p40-specific antibody likewise resulted in a reduction of cerebral amyloid load in APPPS1 mice. Furthermore, intracerebroventricular delivery of antibodies to p40 significantly reduced the concentration of soluble Aβ species and reversed cognitive deficits in aged APPPS1 mice. The concentration of p40 was also increased in the cerebrospinal fluid of subjects with Alzheimer's disease, which suggests that inhibition of the IL-12/IL-23 pathway may attenuate Alzheimer's disease pathology and cognitive deficits.

Background: The implementation of guidelines and training initiatives to support communication in cross-cultural primary care consultations is ad hoc across a range of international settings with negative consequences particularly for migrants. This situation reflects a well-documented translational gap between evidence and practice and is part of the wider problem of implementing guidelines and the broader range of professional educational and quality interventions in routine practice. In this paper, we describe our use of a contemporary social theory, Normalization Process Theory and participatory research methodology--Participatory Learning and Action--to investigate and support implementation of such guidelines and training initiatives in routine practice., Methods: This is a qualitative case study, using multiple primary care sites across Europe. Purposive and maximum variation sampling approaches will be used to identify and recruit stakeholders-migrant service users, general practitioners, primary care nurses, practice managers and administrative staff, interpreters, cultural mediators, service planners, and policy makers. We are conducting a mapping exercise to identify relevant guidelines and training initiatives. We will then initiate a PLA-brokered dialogue with stakeholders around Normalization Process Theory's four constructs--coherence, cognitive participation, collective action, and reflexive monitoring. Through this, we will enable stakeholders in each setting to select a single guideline or training initiative for implementation in their local setting. We will prospectively investigate and support the implementation journeys for the five selected interventions. Data will be generated using a Participatory Learning and Action approach to interviews and focus groups. Data analysis will follow the principles of thematic analysis, will occur in iterative cycles throughout the project and will involve participatory co-analysis with key stakeholders to enhance the authenticity and veracity of findings., Discussion: This research employs a unique combination of Normalization Process Theory and Participatory Learning and Action, which will provide a novel approach to the analysis of implementation journeys. The findings will advance knowledge in the field of implementation science because we are using and testing theoretical and methodological approaches so that we can critically appraise their scope to mediate barriers and improve the implementation processes.

During the past decade, it has been firmly established that IL-23 is essential for disease development in several models of autoimmune disease, including psoriatic skin inflammation, inflammatory bowel disease (IBD), and experimental autoimmune encephalomyelitis (EAE). The mechanism by which IL-23 exerts its pathogenic role has been mostly scrutinized in the context of Th17 cells, which were thought to mediate autoimmunity by secretion of IL-17 family cytokines. However, the picture emerging now is one of multiple IL-23-responsive cell types, pro-inflammatory cytokine induction, and pathogenic "licensing" following an IL-23-dominated interaction between the T cell and the antigen-presenting cell (APC). This review will focus on our changing view of IL-23-dependent autoimmune pathologies with a particular emphasis on the responder cells and their IL-23-induced factors that ultimately mediate tissue destruction., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Normalization Process Theory (NPT) can be used to explain implementation processes in health care relating to new technologies and complex interventions. This paper describes the processes by which we developed a simplified version of NPT for use by clinicians, managers, and policy makers, and which could be embedded in a web-enabled toolkit and on-line users manual., Methods: Between 2006 and 2010 we undertook four tasks. (i) We presented NPT to potential and actual users in multiple workshops, seminars, and presentations. (ii) Using what we discovered from these meetings, we decided to create a simplified set of statements and explanations expressing core constructs of the theory (iii) We circulated these statements to a criterion sample of 60 researchers, clinicians and others, using SurveyMonkey to collect qualitative textual data about their criticisms of the statements. (iv) We then reconstructed the statements and explanations to meet users' criticisms, embedded them in a web-enabled toolkit, and beta tested this 'in the wild'., Results: On-line data collection was effective: over a four week period 50/60 participants responded using SurveyMonkey (40/60) or direct phone and email contact (10/60). An additional nine responses were received from people who had been sent the SurveyMonkey form by other respondents. Beta testing of the web enabled toolkit produced 13 responses, from 327 visits to http://www.normalizationprocess.org. Qualitative analysis of both sets of responses showed a high level of support for the statements but also showed that some statements poorly expressed their underlying constructs or overlapped with others. These were rewritten to take account of users' criticisms and then embedded in a web-enabled toolkit. As a result we were able translate the core constructs into a simplified set of statements that could be utilized by non-experts., Conclusion: Normalization Process Theory has been developed through transparent procedures at each stage of its life. The theory has been shown to be sufficiently robust to merit formal testing. This project has provided a user friendly version of NPT that can be embedded in a web-enabled toolkit and used as a heuristic device to think through implementation and integration problems.

The canonical NF-κB pathway is a driving force for virtually all aspects of inflammation. Conversely, the role of the noncanonical NF-κB pathway and its central mediator NF-κB-inducing kinase (NIK) remains poorly defined. NIK has been proposed to be involved in the formation of T(H)17 cells, and its absence in T(H) cells renders them incapable of inducing autoimmune responses, suggesting a T cell-intrinsic role for NIK. Upon systematic analysis of NIK function in cell-mediated immunity, we found that NIK signaling is dispensable within CD4(+) T cells but played a pivotal role in dendritic cells (DCs). We discovered that NIK signaling is required in DCs to deliver co-stimulatory signals to CD4(+) T cells and that DC-restricted expression of NIK is sufficient to restore T(H)1 and T(H)17 responses as well as cell-mediated immunity in NIK(-/-) mice. When CD4(+) T cells developed in the absence of NIK-sufficient DCs, they were rendered anergic. Reintroduction of NIK into DCs allowed developing NIK(-/-) CD4(+) T cells to become functional effector populations and restored the development of autoimmune disease. Therefore, our data suggest that a population of thymic DCs requires NIK to shape the formation of most αβ CD4(+) T effector lineages during early development., (© 2011 Hofmann et al.)

Background: The immune modulating properties of statins may benefit smokers with asthma. We tested the hypothesis that short-term treatment with atorvastatin improves lung function or indices of asthma control in smokers with asthma., Methods: Seventy one smokers with mild to moderate asthma were recruited to a randomized double-blind parallel group trial comparing treatment with atorvastatin (40 mg per day) versus placebo for 4 weeks. After 4 weeks treatment inhaled beclometasone (400 μg per day) was added to both treatment arms for a further 4 weeks. The primary outcome was morning peak expiratory flow after 4 weeks treatment. Secondary outcome measures included indices of asthma control and airway inflammation., Results: At 4 weeks, there was no improvement in the atorvastatin group compared to the placebo group in morning peak expiratory flow [-10.67 L/min, 95% CI -38.70 to 17.37, p = 0.449], but there was an improvement with atorvastatin in asthma quality of life score [0.52, 95% CI 0.17 to 0.87 p = 0.005]. There was no significant improvement with atorvastatin and inhaled beclometasone compared to inhaled beclometasone alone in outcome measures at 8 weeks., Conclusions: Short-term treatment with atorvastatin does not alter lung function but may improve asthma quality of life in smokers with mild to moderate asthma., Trial Registration: Clinicaltrials.gov identifier: NCT00463827.

Background: The use of information and communication technologies in healthcare is seen as essential for high quality and cost-effective healthcare. However, implementation of e-health initiatives has often been problematic, with many failing to demonstrate predicted benefits. This study aimed to explore and understand the experiences of implementers -- the senior managers and other staff charged with implementing e-health initiatives and their assessment of factors which promote or inhibit the successful implementation, embedding, and integration of e-health initiatives., Methods: We used a case study methodology, using semi-structured interviews with implementers for data collection. Case studies were selected to provide a range of healthcare contexts (primary, secondary, community care), e-health initiatives, and degrees of normalization. The initiatives studied were Picture Archiving and Communication System (PACS) in secondary care, a Community Nurse Information System (CNIS) in community care, and Choose and Book (C&B) across the primary-secondary care interface. Implementers were selected to provide a range of seniority, including chief executive officers, middle managers, and staff with 'on the ground' experience. Interview data were analyzed using a framework derived from Normalization Process Theory (NPT)., Results: Twenty-three interviews were completed across the three case studies. There were wide differences in experiences of implementation and embedding across these case studies; these differences were well explained by collective action components of NPT. New technology was most likely to 'normalize' where implementers perceived that it had a positive impact on interactions between professionals and patients and between different professional groups, and fit well with the organisational goals and skill sets of existing staff. However, where implementers perceived problems in one or more of these areas, they also perceived a lower level of normalization., Conclusions: Implementers had rich understandings of barriers and facilitators to successful implementation of e-health initiatives, and their views should continue to be sought in future research. NPT can be used to explain observed variations in implementation processes, and may be useful in drawing planners' attention to potential problems with a view to addressing them during implementation planning.

We examined the decisions made about transfer of patients with minor injuries, when telemedicine support was provided to the remote nurses in two different ways: the telemedicine doctor either used a video link, or a telephone call with viewing of digital X-ray images (a low resolution version of Picture Archiving and Communications Systems [PACS]). A quasi-randomized study design was used, with a panel of 20 emergency medicine doctors who independently reviewed previously-stored consultations using the two modalities. In total, 60 case reviews were conducted during five sessions, representing 33 different cases from the routine workload of Minor Treatment Centres in Scotland. More experienced doctors transferred fewer patients than less experienced doctors. The proportion of patients transferred was higher when PACS was used than when video was used in most of the cases. A mixed effects logistic regression model was fitted to the data. The estimated odds for patient transfer were 56% lower when video was used instead of PACS (odds ratio 0.44, 95% confidence interval 0.20, 0.93). Although the cost implications are not yet known, video support for local decision-making should remain the preferred method of telemedicine for minor injuries work.

Background: The past decade has seen considerable interest in the development and evaluation of complex interventions to improve health. Such interventions can only have a significant impact on health and health care if they are shown to be effective when tested, are capable of being widely implemented and can be normalised into routine practice. To date, there is still a problematic gap between research and implementation. The Normalisation Process Theory (NPT) addresses the factors needed for successful implementation and integration of interventions into routine work (normalisation)., Discussion: In this paper, we suggest that the NPT can act as a sensitising tool, enabling researchers to think through issues of implementation while designing a complex intervention and its evaluation. The need to ensure trial procedures that are feasible and compatible with clinical practice is not limited to trials of complex interventions, and NPT may improve trial design by highlighting potential problems with recruitment or data collection, as well as ensuring the intervention has good implementation potential., Summary: The NPT is a new theory which offers trialists a consistent framework that can be used to describe, assess and enhance implementation potential. We encourage trialists to consider using it in their next trial.

Background: The use of Information and Communication Technology (ICT) or e-Health is seen as essential for a modern, cost-effective health service. However, there are well documented problems with implementation of e-Health initiatives, despite the existence of a great deal of research into how best to implement e-Health (an example of the gap between research and practice). This paper reports on the development and formative evaluation of an e-Health Implementation Toolkit (e-HIT) which aims to summarise and synthesise new and existing research on implementation of e-Health initiatives, and present it to senior managers in a user-friendly format., Results: The content of the e-HIT was derived by combining data from a systematic review of reviews of barriers and facilitators to implementation of e-Health initiatives with qualitative data derived from interviews of "implementers", that is people who had been charged with implementing an e-Health initiative. These data were summarised, synthesised and combined with the constructs from the Normalisation Process Model. The software for the toolkit was developed by a commercial company (RocketScience). Formative evaluation was undertaken by obtaining user feedback. There are three components to the toolkit--a section on background and instructions for use aimed at novice users; the toolkit itself; and the report generated by completing the toolkit. It is available to download from http://www.ucl.ac.uk/pcph/research/ehealth/documents/e-HIT.xls., Conclusions: The e-HIT shows potential as a tool for enhancing future e-Health implementations. Further work is needed to make it fully web-enabled, and to determine its predictive potential for future implementations.

New technologies can change healthcare delivery. Cisco HealthPresence, an integrated platform that combines video, audio, and call center technology with medical information to create a virtual clinic experience, was piloted on emergency department patients. The aim was to assess primary care consultations. Patients were supported by an assistant. The doctor was remote from the patient and collected details and a recommended management plan. The same doctor re-examined the patient face-to-face. All patients completed a questionnaire about the experience. Key staff and a small sample of patients were interviewed. One hundred and five (N = 105) patients were included; 42% were given advice, 25% were prescribed analgesia, 26% were prescribed antibiotics, and 15% were x-rayed. There were early problems with the digital stethoscope. Doctors reported that the management plan changed in 7% of cases after seeing the patient. At least 90% of patients reported a positive experience. All patients and staff interviewed were positive. Staff found equipment to be valid and reliable; a concern was the inability to perform "hands on" examination. Telemedicine requires a change in the way of consulting and staff must be interested in using the technology to understand the differences. As one of the doctors said, "HealthPresence was a positive experience." Greater numbers would be required to validate key findings. As judged by clinicians, HealthPresence was successful and potentially safe for triage of unscheduled cases. Different types of staffing models need to be considered to ensure optimum use of health professionals. This study has shown that, despite some limitations, most HealthPresence consultations were found to be safe and appropriate. Further study of this consultation technology is required. HealthPresence has the potential to transform access to services for many patients, and to improve the effectiveness of delivery across a number of services.

The majority of regulatory T cells (T(regs)) are believed to be of thymic origin. It has been hypothesized that this may result from unique intrathymic environmental cues, possibly requiring a dedicated antigen-presenting cell (APC). However, T cell-intrinsic developmental regulation of the susceptibility to T(reg) differentiation remains a mutually non-exclusive scenario. We found that upon exposure of monoclonal T cells of sequential developmental stages to a thymic microenvironment expressing cognate antigen, the efficiency of T(reg) induction inversely correlated with progressive maturation. This inclination of immature thymocytes toward T(reg) differentiation was even seen in an APC-free in vitro system, providing only TCR stimulation and IL-2. In support of quantitative but not qualitative features of external cues being critical, thymic epithelial cells as well as different thymic dendritic cell (DC)-subtypes efficiently induced T(reg) development of immature thymocytes, albeit at strikingly different optimal doses of cognate antigen. We propose that the intrinsically high predisposition of immature thymocytes to T(reg) development may contribute to the predominantly thymic origin of the T(reg) repertoire. The underlying instructive stimulus, however, does not require unique features of a dedicated APC and can be delivered by hematopoietic as well as epithelial thymic stromal cells.

Background: Theories are important tools in the social and natural sciences. The methods by which they are derived are rarely described and discussed. Normalization Process Theory explains how new technologies, ways of acting, and ways of working become routinely embedded in everyday practice, and has applications in the study of implementation processes. This paper describes the process by which it was built., Methods: Between 1998 and 2008, we developed a theory. We derived a set of empirical generalizations from analysis of data collected in qualitative studies of healthcare work and organization. We developed an applied theoretical model through analysis of empirical generalizations. Finally, we built a formal theory through a process of extension and implication analysis of the applied theoretical model., Results: Each phase of theory development showed that the constructs of the theory did not conflict with each other, had explanatory power, and possessed sufficient robustness for formal testing. As the theory developed, its scope expanded from a set of observed regularities in data with procedural explanations, to an applied theoretical model, to a formal middle-range theory., Conclusion: Normalization Process Theory has been developed through procedures that were properly sceptical and critical, and which were opened to review at each stage of development. The theory has been shown to merit formal testing.

Background: Investing in computer-based information systems is notoriously risky, since many systems fail to become routinely used as part of everyday working practices, yet there is clear evidence about the management practices which improve the acceptance and integration of such systems. Our aim in this study was to identify to what extent these generic management practices are evident in e-health projects, and to use that knowledge to develop a theoretical model of e-health implementation. This will support the implementation of appropriate e-health systems., Methods: This study consisted of qualitative semi-structured interviews with managers and health professionals in Scotland, UK. We contacted the Scottish Ethics Committee, who advised that formal application to that body was not necessary for this study. The interview guide aimed to identify the issues which respondents believed had affected the successful implementation of e-health projects. We drew on our research into information systems in other sectors to identify likely themes and questions, which we piloted and revised. Eighteen respondents with experience of e-health projects agreed to be interviewed. These were recorded, transcribed, coded, and then analysed with 'Nvivo' data analysis software., Results: Respondents identified factors in the context of e-health projects which had affected implementation, including clarity of the strategy; supportive structures and cultures; effects on working processes; and how staff perceived the change. The results also identified useful implementation practices such as balancing planning with adaptability; managing participation; and using power effectively., Conclusion: The interviews confirmed that the contextual factors that affect implementation of information systems in general also affect implementation of e-health projects. As expected, these take place in an evolving context of strategies, structures, cultures, working processes and people. Respondents also confirmed that those managing such projects seek to change these contexts through observable implementation processes of planning, adaptation, participation and using power. This study confirms that research to support the delivery of appropriate e-health projects can usefully draw on the experience of information systems in other sectors.