A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes

AbstractA newly-described first-line immune defence mechanism of neutrophils is the release of neutrophil extracellular traps (NETs). Immune complexes (ICxs) induce low level NET release. As such, the in vitro quantification of NETs is challenging with current methodologies. In order to investigate the role of NET release in ICx-mediated autoimmune diseases, we developed a highly sensitive and automated method for quantification of NETs. After labelling human neutrophils with PKH26 and extracellular DNA with Sytox green, cells are fixed and automatically imaged with 3-dimensional confocal laser scanning microscopy (3D-CLSM). NET release is then quantified with digital image analysis whereby the NET amount (Sytox green area) is corrected for the number of imaged neutrophils (PKH26 area). A high sensitivity of the assay is achieved by a) significantly augmenting the area of the well imaged (11%) as compared to conventional assays (0.5%) and b) using a 3D imaging technique for optimal capture of NETs, which are topologically superimposed on neutrophils. In this assay, we confirmed low levels of NET release upon human ICx stimulation which were positive for citrullinated histones and neutrophil elastase. In contrast to PMA-induced NET release, ICx-induced NET release was unchanged when co-incubated with diphenyleneiodonium (DPI). We were able to quantify NET release upon stimulation with serum from RA and SLE patients, which was not observed with normal human serum. To our knowledge, this is the first semi-automated assay capable of sensitive detection and quantification of NET release at a low threshold by using 3D CLSM. The assay is applicable in a high-throughput manner and allows the in vitro analysis of NET release in ICx-mediated autoimmune diseases

Using an artificial intelligence tool incorporating natural language processing to identify patients with a diagnosis of ANCA-associated vasculitis in electronic health records

Background: Because anti-neutrophil cytoplasmatic antibody (ANCA)-associated vasculitis (AAV) is a rare, lifethreatening, auto-immune disease, conducting research is difficult but essential. A long-lasting challenge is to identify rare AAV patients within the electronic-health-record (EHR)-system to facilitate real-world research. Artificial intelligence (AI)-search tools using natural language processing (NLP) for text-mining are increasingly postulated as a solution.Methods: We employed an AI-tool that combined text-mining with NLP-based exclusion, to accurately identify rare AAV patients within large EHR-systems (>2.000.000 records). We developed an identification method in an academic center with an established AAV-training set (n = 203) and validated the method in a non-academic center with an AAV-validation set (n = 84). To assess accuracy anonymized patient records were manually reviewed.Results: Based on an iterative process, a text-mining search was developed on disease description, laboratory measurements, medication and specialisms. In the training center, 608 patients were identified with a sensitivity of 97.0 % (95%CI [93.7, 98.9]) and positive predictive value (PPV) of 56.9 % (95%CI [52.9, 60.1]). NLP-based exclusion resulted in 444 patients increasing PPV to 77.9 % (95%CI [73.7, 81.7]) while sensitivity remained 96.3 % (95%CI [93.8, 98.0]). In the validation center, text-mining identified 333 patients (sensitivity 97.6 % (95%CI [91.6, 99.7]), PPV 58.2 % (95%CI [52.8, 63.6])) and NLP-based exclusion resulted in 223 patients, increasing PPV to 86.1 % (95%CI [80.9, 90.4]) with 98.0 % (95%CI [94.9, 99.4]) sensitivity. Our identification method outperformed ICD-10-coding predominantly in identifying MPO+ and organ-limited AAV patients.Conclusions: Our study highlights the advantages of implementing AI, notably NLP, to accurately identify rare AAV patients within large EHR-systems and demonstrates the applicability and transportability. Therefore, this method can reduce efforts to identify AAV patients and accelerate real-world research, while avoiding bias by ICD-10-coding.Nephrolog

BEING ME: Project report on best practices in learning and education to support LGBT ageing care and wellbeing

This report is based on our experiences of using a collaborative approach to identifying best practices for those involved in professional, vocational and community-based education and learning, in order to facilitate improved support for Lesbian, Gay, Bisexual and Transgender (LGBT) older people in health and social care. The best practices discussed here on learning and teaching, emerged from cross national collaboration and intercultural dialogue with a variety of stakeholders, including older LGBT people, educators, practitioners and learners using the World Café method. As one of the workstreams within the BEING ME European Project funded by EU Erasmus Plus (https://www.beingme.eu/), we aimed to promote and support the social inclusion of LGBT older people through positive interaction with educational institutions that prepare future professionals to work with older people. The best practices described here include a) identifying pedagogic approaches (the method and practice of teaching) b) generating examples of tailored educational resources c) recommendations on how to improve the knowledge and capabilities of future care professionals in the area of LGBT affirmative practices. Through a process of learning and exchange during two World Cafés, these areas were able to be more clearly articulated and should be read in conjunction with the BEING ME ‘Best Practice principles’ (https://beingme.eu/public/application/downloads/resources/being-me-best-practice-principles-20190212.pdf) which underpin good practice in the area of LGBT ageing care. Giving specific attention to identify ways of enhancing the skills, knowledge and capabilities of practitioners through education, should place them in a better position to develop a culture of support, openness and respect for LGBT identities which in turn are essential to LGBT older people's inclusiveness in care environments. The Best Practices Report provides the foundation for the development of the BEING ME educators online ‘toolkit’ aimed at both formal and informal learning settings and to a range of disciplines professions and vocations in health and social care

CD34+ cells home, proliferate, and participate in capillary formation, and in combination with

Objective - Emerging evidence suggests that human blood contains bone marrow (BM)-derived endothelial progenitor cells that contribute to postnatal neovascularization. Clinical trials demonstrated that administration of BM-cells can enhance neovascularization. Most studies, however, used crude cell populations. Identifying the role of different cell populations is important for developing improved cellular therapies. Methods and Results - Effects of the hematopoietic stem cell-containing CD34+ cell population on migration, proliferation, differentiation, stimulation of, and participation in capillary-like tubule formation were assessed in an in vitro 3-dimensional matrix model using human microvascular endothelial cells. During movement over the endothelial monolayer, CD34+ cells remained stuck at sites of capillary tube formation and time- and dose-dependently formed cell clusters. Immunohistochemistry confirmed homing and proliferation of CD34+ cells in and around capillary sprouts. CD34+ cells were transduced with the LNGFR marker gene to allow tracing. LNGFR gene-transduced CD34 + cells integrated in the tubular structures and stained positive for CD31 and UEA-1. CD34+ cells alone stimulated neovascularization by 17%. Coculture with CD34- cells led to 68% enhancement of neovascularization, whereas CD34- cells displayed a variable response by themselves. Cell-cell contact between CD34+ and CD34- cells facilitated endothelial differentiation of CD34+ cells. Conclusions - Our data suggest that administration of CD34+-enriched cell populations may significantly improve neovascularization and point at an important supportive role for (endogenous or exogenous) CD34- cells. © 2005 American Heart Association, Inc. Chemicals / CAS: nitric oxide, 10102-43-9; Antigens, CD34; Biological Marker

Pedagogical principles and methods underpinning education of health and social care practitioners on experiences and needs of older LGBT+ people: findings from a systematic review

There is a growing awareness of the need for LGBT + competency training to ensure that the health and social care services offered to older LGBT + people is affirmative and gender sensitive. To conduct a synthesis of the literature that describes the pedagogical principles, curriculum content and methods (teaching and assessment) used to educate health and social care practitioners on the experiences and needs of older LGBT + people. Systematic thematic review of literature. MEDLINE, CINAHL, PsycINFO, EMBASE, Web of Science, Social Sciences Index, ERIC. In accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statement, this review examined peer-reviewed papers published in English, prior to April 2018 that addressed pedagogical and curriculum issues on the inclusion of needs and experiences of older LGBT + people. The combined searches yielded 2214 papers of which 17 papers were eligible for inclusion, 10 discussion papers and 7 evaluation studies. Analysis identified the following themes: i) Acknowledging the wider historical context of older LGBT + people's lives; ii) Recognising that older LGBT + people are not a homogenous group; iii) Incorporating a multitude of theories and models from different perspectives; iv) Alerting practitioners to the health issues and disparities facing older LGBT + people; v) Including content that supports inclusive care for older LGBT + people; vi) Addressing barriers to older LGBT + people accessing health care; vii) Interactive activities are the preferred pedagogical strategy; viii) Involving older LGBT + people in curriculum development is a core principle; and ix) Mandatory education is not always the solution. As the field matures there is a need for more exploration of curriculum principles, assessment strategies and strategies to overcome barriers to the inclusion of issues experienced by older LGBT + people within curricula. [Abstract copyright: Copyright © 2019 Elsevier Ltd. All rights reserved.

Learning to deliver LGBT+ aged care: exploring and documenting best practices in professional and vocational education through the World Café method

Substantial evidence on the adverse impact of ageing on lesbian, gay, bisexual and transgender (LGBT+) populations through the lack of inclusive care services has highlighted the need for education and training of the health and social care workforce to enhance their skills, knowledge and capabilities in this area. We describe a cross-national collaboration across four European Union countries called BEING ME. This collaboration examined the current pedagogic environment within professional, vocational and community-based education to identify what is most valuable for addressing these needs. The World Café method enabled a process of structured learning and knowledge exchange between stakeholders resulting in: (a) identification of best practices in pedagogies, (b) generation of tailored co-produced educational resources, and (c) recommendations on how to improve the knowledge and capabilities of future care professionals in the area of LGBT+ affirmative practices. Combined with themes from the post-Café evaluation, our findings suggest that underpinning professional and vocational education with a person-in-environment perspective facilitates going some way to acknowledging the historical context of older LGBT+ people's lives. Addressing the unique needs of sub-populations within LGBT+ communities and setting these in the context of holistic and person-centred care may better enable the meeting of their unique diverse needs for ageing. Recommendations are made for learning and teaching strategies to support improved LGBT+ aged care

Degenerated cones in cultured human retinas can successfully be optogenetically reactivated

Biblical references aside, restoring vision to the blind has proven to be a major technical challenge. In recent years, considerable advances have been made towards this end, especially when retinal degeneration underlies the vision loss such as occurs with retinitis pigmentosa. Under these conditions, optogenetic therapies are a particularly promising line of inquiry where remaining retinal cells are made into "artificial photoreceptors". However, this strategy is not without its challenges and a model system using human retinal explants would aid its continued development and refinement. Here, we cultured post-mortem human retinas and show that explants remain viable for around 7 days. Within this period, the cones lose their outer segments and thus their light sensitivity but remain electrophysiologically intact, displaying all the major ionic conductances one would expect for a vertebrate cone. We optogenetically restored light responses to these quiescent cones using a lentivirus vector constructed to express enhanced halorhodopsin under the control of the human arrestin promotor. In these 'reactivated' retinas, we show a light-induced horizontal cell to cone feedback signal in cones, indicating that transduced cones were able to transmit their light response across the synapse to horizontal cells, which generated a large enough response to send a signal back to the cones. Furthermore, we show ganglion cell light responses, suggesting the cultured explant's condition is still good enough to support transmission of the transduced cone signal over the intermediate retinal layers to the final retinal output level. Together, these results show that cultured human retinas are an appropriate model system to test optogenetic vision restoration approaches and that cones which have lost their outer segment, a condition occurring during the early stages of retinitis pigmentosa, are appropriate targets for optogenetic vision restoration therapies.Therapeutic cell differentiatio