Ecological momentary assessment (EMA) involves repeated, real-time assessments of phenomena  over a period of time in naturalistic settings. EMA is increasingly used to study both within-person and between-person processes. We will review EMA studies investigating key health behaviours and synthesise: (1) study characteristics , (2) associations between psychological predictors and behaviours and (3) moderators of adherence to EMA protocols.This review will focus on EMA studies conducted across five public health behaviours in adult, non-clinical populations: movement behaviour , dietary behaviour, alcohol consumption, tobacco smoking and preventive sexual health behaviours. Studies need to have assessed at least one psychological or contextual predictor of these behaviours. Studies reporting exclusively on physiological outcomes  or those not conducted under free-living conditions will be excluded. We will search OVID MEDLINE, Embase, PsycINFO and Web of Science using terms relevant to EMA and the selected health behaviours. Reference lists of existing systematic reviews of EMA studies will be hand searched. Identified articles will be screened by two reviewers. This review is expected to provide a comprehensive summary of EMA studies assessing psychological or contextual predictors of five public health behaviours.The results will be disseminated through peer-reviewed publications and presentations. Data from included studies will be made available to other researchers. No ethics are required.CRD42020168314. A protocol for a systematic review is provided for ecological momentary assessment (EMA) studies in adult, non-clinical populations.We will include EMA studies of five key public health behaviours including movement behaviours, dietary behaviours, alcohol consumption, tobacco smoking and preventive sexual health behaviours.We will review characteristics of EMA studies  and associations between psychological predictors and behaviours, examining rates of adherence to EMA protocols across different health behaviours and moderators of adherence .Extracted data will be made available to other researchers, thus allowing for the exploration of additional research questions and potential for setting up a ‘living review’.As included studies are likely to be heterogeneous, this may limit the overarching conclusions that can be drawn, and will likely prevent meta-analysis combining effect sizes from multiple studies and across all behaviours.Ecological momentary assessment (EMA), also known as ambulatory assessment or experience sampling methodology, involves repeated, real-time assessments of phenomena  over a period of time in naturalistic settings.Previous reviews of EMA studies have focused on clinical conditions such as borderline personality disorder,21While systematic reviews of EMA studies focusing on specific health behaviours have been conducted,We will synthesise evidence from EMA studies that report either within-person or between-person predictor-behaviour associations. The review will focus on five key public health behaviours: (1) movement behaviours , (2) dietary behaviours, (3) alcohol consumption, (4) tobacco smoking and (5) preventive sexual health behaviours (including contraceptive use).The review aims are:To summarise adherence to EMAs, total length of data collection of EMAs, prompting frequency of EMAs, and incentives structures across studies.To describe within-person and between-person predictor-behaviour associations across EMA studies .To assess potential moderators of adherence to EMAs .This review is intentionally broad in scope to provide an overview of the field for researchers interested in the application of EMAs to the study of health-related behaviours. We expect this overarching review to help identify patterns and key knowledge gaps.This review will adhere to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist . The rev10.1136/bmjopen-2020-046435.supp1Supplementary dataThis review will focus on five key public health behaviours in healthy adults  aged 18+years, namely:Movement behaviours, including physical activity and sedentary behaviour.Dietary behaviours, including snacking or fruit and vegetable consumption.Alcohol consumption.Tobacco smoking, including cigarette, cigar or pipe smoking.Preventive sexual health behaviours, including contraceptive/condom use.No restrictions on geographical location or publication date will be set. To be included, studies need to incorporate multiple  within-day, daily or weekly assessments of predictors and behaviours, and to have reported either (or both) within-person or between-person predictor-behaviour  associations. The frequency of the EMAs should plausibly match how the target behaviour  theoretically or empirically unfolds over time, for example, daily assessments of steps, weekly assessments of gym class attendance if the class is undertaken only once a week. To be included, studies need to assess one of the aforementioned behaviours and at least one psychological or contextual variable via EMAs.In this review, we defined psychological variables as emergent properties of a distributed network of neurons, including cognition , emotion  and processes operating on these , which are linked to behaviour. We further define contextual variables as any potential environmental  influences on behaviour, including the presence of other people, weather or the availability of unhealthy foods/cigarettes/alcohol. The psychological and contextual variables will be closely assessed by the reviewers as to their suitability for inclusion/exclusion in the review.In addition to self-report measures, included studies can use physiological measures of psychological predictors  or behaviours . Studies reporting associations between behaviours and psychological consequences  will be included providing that they also report psychological or contextual predictor-behaviour associations . We will include individuals with overweight and obesity given that 39% of adults globally fall into this category, with most Western countries averaging above 50%.Laboratory studies will not be included. Studies examining clinical populations, that is, solely recruiting participants on the basis of being diagnosed with a physical or mental health condition such as cancer, cardiovascular disease, depression, binge eating disorder or substance use disorder  will be excluded. Studies focusing only on purchasing behaviours  will not be included. Studies focusing on e-cigarettes will be also excluded. Studies not published in English or where no full text could be obtained will also not be included. Although behaviour–behaviour associations may also be considered relevant, our electronic search is not designed to capture such studies, and behaviour–behaviour associations will hence not be considered further in this review.We will search Ovid MEDLINE, Embase, PsycINFO and Web of Science  OR  OR (ambulatory adj1 assessment*) OR (experience adj1 sampl*) OR (daily adj1 diar*) OR  OR within-person OR within-subject* OR (single adj1 case) OR idiographic OR intraindividualtobacco OR smok* OR cigarette OR alcohol* OR drinking OR addict* OR  OR diet OR weight OR overweight OR obes* OR physical activity OR exercise OR sedentary OR sitting OR leisure OR  OR condom OR contraceptive1 AND 2Electronic and hand searches were conducted in January 2020 and updated in February 2021. We restricted the search to human studies available in English that are published in peer-reviewed journals .Reference lists of existing systematic reviews of EMA studies will be hand searched and expertise within the review team will be used to identify additional articles of interest.Identified articles will be merged using CovidenceA data extraction form will be developed in Microsoft Excel to extract information and to import data into R for analysis. Each study will be allocated a unique study identification number. Data will be extracted on:Study description ;Participant characteristics ; gender ; educational attainment (% university education); population type , ethnicity (% white ethnicity);EMA study type ;EMA delivery mode ;EMA method ;EMA characteristics ; prompting frequency , incentive schedule ;Adherence to EMA ;Health behaviour(s) assessed ; and how the health behaviour(s) were measured ;Psychological and contextual predictors  and how they were measured , whether a single item or multiple items were used);Statistical model used to examine predictor-behaviour association  and whether these associations were analysed at the within-person and/or between-person level;Level of aggregation in data analysis ;Coefficients and effect sizes from statistical models ;Control variables in multivariable models For each study, one reviewer will extract the data. At least 20% of studies stratified by behaviour  will be double-checked for accuracy and completeness by a second reviewer. In case there are any uncertainties related to data extraction , we will double-check additional studies until agreement is achieved. All review authors will be involved in data extraction and double-checking.Included studies may vary in quality, which will be considered through a quality appraisal. The appraisal tool was developed by the review team, based on an existing EMA reporting checklist,All quantitative analyses will be conducted in R V.3.5.1. A narrative (descriptive) synthesis will be conducted. We will summarise the number of EMA studies conducted for each of the five health behaviours, study setting  and sample size . We will then present results in relation to each research question.To address the first aim, we will summarise study and EMA characteristics, for example, study setting, population characteristics, percentage prompting frequency , percentage type of EMA method , percentage type of EMA delivery mode , percentage type of incentive structure , rates of EMA adherence (mean or median), and study duration (mean or median). Where appropriate, moderator analyses will be conducted to examine whether predictor-behaviour associations vary depending on study setting, study characteristics, participant characteristics, or type of incentive schedule used.To address the second aim, we will summarise within-person and between-person predictor-behaviour associations across EMA studies . If there is sufficient homogeneity between studies , within-person or between-person predictor-behaviour associations  will be synthesised with random effects meta-analyses, grouped by behaviour. Analyses will be conducted with the 'metafor' or ‘CTmeta’ packages,To address the third aim, we will assess, with random effects meta-analyses, whether EMA adherence varies depending on study setting, study characteristics, participant characteristics or type of incentive schedule used. We do not have any pre-specified hypotheses.A patient and public involvement representative reviewed a lay summary of the protocol for our systematic review. Positive feedback was received on the review’s aims, the importance of the current research and choice of key behaviours relevant to public health. Once the review is completed, feedback will be sought from additional patient and public involvement representatives about the interpretation of findings and plans for dissemination. We will seek advice on how to best present the study outcomes and use them in order to design studies and interventions that are useful and relevant for the public.This study does not require ethics approval as it will summarise data from previously published studies. A protocol was pre-registered on the international Prospective Register of Systematic Reviews and on the Open Science Framework. The findings of the review will be disseminated through peer-reviewed publications and presentations at relevant conferences. The data set will be made available to other researchers online via the creation of a digital object identifier, thus enabling further research questions to be addressed. We expect this review to be useful for researchers and healthcare practitioners who regularly design and interpret results from EMA studies. We plan to publish overarching review and subsequently five behaviour-specific reviews that will provide a more in-depth synthesis of predictor-behaviour associations.EMA is a frequently used research method; however, an overview of studies using this method across key public health behaviours in healthy adults is lacking. This review will provide a comprehensive overview of associations between a psychological/contextual predictor and a health behaviour in EMA studies focusing on movement behaviours, dietary behaviours, alcohol consumption, tobacco smoking and sexual health behaviours. This review will inform the future design of EMA studies and it will influence practice of assessing individuals in real-life settings and providing interventions that are delivered at the time and place when and where required. This review will set a blueprint for how to conduct EMA studies to improve participants’ adherence and conduct meaningful studies in real-life settings.


Osteosarcoma is the most common bone tumor affecting both adolescents and children. Although localized osteosarcoma has an overall survival of >70% in the clinic, metastatic, refractory, and recurrent osteosarcoma have poorer survival rates. Exosomes are extracellular vesicles released by cells and originally thought to be a way for cells to discard unwanted products. Currently, exosomes have been reported to be involved in intercellular cross-talk and induce changes in cellular behavior by transferring cargoes  between cells. Exosomes regulate osteosarcoma progression, and processes such as tumorigenesis, proliferation, metastasis, angiogenesis, immune evasion, and drug resistance. Increasing evidences shows that exosomes have significant potential in promoting osteosarcoma progression and development. In this review, we describe the current research status of exosomes in osteosarcoma, focusing on the biological functions of osteosarcoma exosomes as well as their application in osteosarcoma as diagnostic biomarkers and therapeutic targets. Bone malignancies are the third leading cause of cancer-related deaths in children and adolescents . OsteosaNanodrug delivery systems have the potential to enhance and maintain the clinical efficacy of chemotherapeutic drugs with less side effects by improving the absorption, penetration, and distribution of chemotherapeutic drugs, and has been widely studied in tumor targeted drug therapy in recent years . The secin vivo and in vitro, including cancer cells and normal cells  containing intraluminal vesicles (ILVs) . Early eWhen exosomes arrive at the surface of receptor cells then may induce intracellular signals by binding to surface receptors . ExosomeRecent evidence has shown that exosomes play a vital role in the tumorigenesis, proliferation, metastasis, anti-apoptosis, immune evasion and chemoresistance of osteosarcoma. Garimella et al. first reported the function of exosomes in osteosarcoma. They detected factors such as MMPs, RANKL, TGFβ, and CD-9 in the exosomes of osteosarcoma cells, as well as changes in intracellular calcium concentration that can affect exosome biogenesis. They suggested a novel function of exosomes in driving bone resorption of osteoclastic by disrupting bone remodeling homeostasis in the osteosarcoma bone microenvironment (BME) . This stExosomes can mediate invasion and metastasis of osteosarcoma to local or distant organs and tissues. Groundbreaking research by Macklin et al. showed that exosomes from highly metastatic osteosarcoma cell subpopulations may increase invasion and chemotaxis of poorly metastatic osteosarcoma cell subpopulations. The authors concluded that highly metastatic clonal variants of osteosarcoma derived exosomes may drive metastatic behavior through cooperation between clones and preferential lung colonization . More rein vitro and in vivo (MSCs are known as the host of the tumor microenvironments (TME) and take part in forming the TME as well as interacting with tumor cells . As nove in vivo . Thus, eAnother important feature of exosomes derived from osteosarcoma is their capacity to induce immune evasion. Troyer et al. demonstrated that exosomes secreted by malignant cells contain a unique cargo that can promote the differentiation of CD4 + cells to the T regulatory phenotype , and eventually leading to immune evasion of the malignant cells . In a suExosomes can also induce osteosarcoma chemotherapy resistance. Torreggiani et al. reported that multidrug-resistant osteosarcoma cells are able to transfer osteosarcoma derived exosomes carrying MDR-1 mRNA and its product P-glycoprotein so as to spread their resistance to doxorubicin treatment on sensitive osteosarcoma cells . Pan et In addition to exosomes, exosomal cargoes can also have direct or indirect effects on the biological function of osteosarcoma cells or other cells. Exosomes-delivered bioactive molecules result in the intercellular exchange of genetic information and recipient cell reprogramming . Exosomein vitro and in vivo have shown that exosomes secreted from miR-195-3p upregulated osteosarcoma cells promote cell proliferation and invasion via exosomal miR-195-3p  and how these specifically affect downstream biological processes. Currently, the best described among these molecules are miRNAs. The existence of exosome derived miRNAs in a series of human osteosarcoma or osteoblastic cell lines with different levels of metastatic potential was identified by next generation miRNA sequencing as reported by Jerez et al. The most notable miRNAs are miR-21-5p, miR-143-3p, miR-148a-3p, and miR-181a-5p. Gene ontology analysis of predicted target genes for these miRNAs suggested that they control proteins that regulate cell apoptosis, angiogenesis, adhesion, and migration . Ye et aR-195-3p . Han et R-195-3p .Osteosarcoma cells-secreted exosomes can also transfer miRNAs to other cells in the TME. Gong et al. observed that invasion and migration of non-malignant osteoblast cells (hFOB 1.19) were increased by exposure to exosomes derived from metastatic osteosarcoma by delivering exogenous miR-675 and downregulating target genes within recipient cells such as CALN1 . Yoshidain vitro , to osteosarcoma cells. Shimbo et al. demonstrated that introducing synthetic miR-143 into MSCs could increase MSCs secreted exosomes and the amount of exosome-formed miR-143 in the conditioned medium, which could easily be transferred into recipient cells and inhibit the migration of the 143B osteosarcoma cell line . Qin et in vitro . The stuin vitro . These cIn addition to miRNAs, exosomes also contain several other series of non-coding RNAs, such as circRNAs and lncRNAs, which also play important roles in osteosarcoma. Groundbreaking research by Zhao et al. revealed that by transporting lncRNA PVT1 to osteosarcoma cells, exosomes derived from BMSC could promote osteosarcoma metastasis and growth . In addiProteins are also one of the main components of exosomes. Exosomes exhibit specific proteins that depend on the type of secretory cell, and also carry specific subsets of cell proteins that are independent of cell type . These pEndo-Munoz et al. demonstrated that metastatic osteosarcoma cells can promote metastasis via locally secreted urokinase plasminogen activator (uPA) and via uPA-containing osteosarcoma derived exosomes at distant sites . Baglio In addition to the direct roles of exosome derived proteins, there are also series of proteins that influence the characteristics of osteosarcoma through the regulation of downstream signaling pathways. Zhang et al. revealed that exosomal Collagen type VI alpha 1(COL6A1) derived from osteosarcoma cells promotes invasion and migration of osteosarcoma cells by secreting pro-inflammatory cytokines including IL-6 and IL-8, transforming normal fibroblasts into CAFs and mediating the TGF-β/COL6A1 signaling pathway . Ge et aTo date, there have been many studies on osteosarcoma exosomes and their exosome derived cargoes, which have specifically described the progression and mechanism of osteosarcoma exosomes and exosomes derived cargoes. On this basis, some exosomes and their cargoes have been synthesized for clinical application in osteosarcoma and are expected to be used formally in clinical trials in the future.DNAs are also one type of exosome derived cargoes and include dsDNA, ssDNA, viral DNA and mtDNA. To date, it is unknown whether specific DNAs will be sorted selectively into exosomes to play physiological roles in cells. Some studies have sequenced exosome-related DNAs and will provide the complete genome sequence of cells that secrete exosomes . The disThe accessibility and stability of exosomes in most body fluids make them a novel candidate for osteosarcoma liquid biopsy. A growing number of studies have shown that exosomes may have potential roles in early diagnosis, prognosis prediction, and efficacy evaluation of osteosarcoma .Recent studies have demonstrated that exosome derived miRNAs may serve as effective biomarkers for osteosarcoma. Xu et al. reported that exosomal miRNAs in serum could be used as a reliable diagnostic biomarker for distinguishing differences in chemotherapy responses in osteosarcoma . A preliExosome derived lncRNAs and circRNAs are also considered important diagnostic markers for osteosarcoma. Recently, Li et al. showed that the release of exosomes containing linc00852, contributed to the metastasis, invasion, and proliferation of osteosarcoma cells with low AXL expression and was promoted by osteosarcoma cells with high AXL expression. A positive feedback loop between exosome derived linc00852 and AXL demonstrated that exosome derived linc00852 may act a novel osteosarcoma biomarker . GrowingIn addition to exosome derived RNAs, exosome derived proteins can also serve as potential biomarkers for predicting prognosis in osteosarcoma patients. Wang et al. demonstrated that in exosomes from the serum of osteosarcoma patients PD-L1 and N-cadherin coexisted. The authors developed biomarkers of exosomal PD-L1 and N-cadherin from serum of osteosarcoma patients to predict pulmonary metastasis .Therefore, exosome derived cargoes have the great potential to become novel diagnostic and prognostic biomarkers for osteosarcoma. We believe that exosomes and exosomes derived cargoes as biological tools will play an important role in the prediction and diagnosis of osteosarcoma soon. Then this application of exosomes could improve the survival of osteosarcoma patients which is important clinically for clinicians.The important functions of exosomes in osteosarcoma prove that it could also be developed as therapeutic targets. The osteosarcoma derived exosomal biomarkers mentioned above could also be utilized as therapeutic targets of osteosarcoma for the same origin of osteosarcoma cells. Baglio et al. reported that the osteosarcoma-secreted exosomes carry functional TGFβ molecules that increase IL-6 expression to promote osteosarcoma growth and metastasis formation, and the combination of IL-6 blocking agents with TGFβ inhibitors could stop osteosarcoma progression while reducing drug resistance . More reRecent studies have also suggested that exosome derived RNAs may serve as therapeutic targets for osteosarcoma. Zhang et al. demonstrated that exosome derived miR-206 derived from BMSC can be transported into osteosarcoma cells and inhibit osteosarcoma progression via targeting TRA2B . A studyIn addition to exosomes derived RNAs, exosomes derived proteins can also act as biological therapeutic targets for the treatment of osteosarcoma patients. Cheng et al. identified osteosarcoma derived exosomes could induce M2 type polarization of macrophages by regulating Tim-3 expression . Zhang eExosomes can also be used to deliver chemotherapy drugs for osteosarcoma therapy, which is a better way to reduce the serious side effects of chemotherapy as well as prolong the half-life of chemotherapy drugs. Recently, Wei et al. reported that the exosome isolated from MSCs could be used as a drug nanocarrier to load chemotherapeutic drug doxorubicin, and Exo-doxorubicin could kill osteosarcoma cells more effectively when compared with free doxorubicin .The application of exosomes as therapeutic targets and drug delivery vectors may provide a novel approach for the treatment of osteosarcoma patients. These targets are relevant and important for clinicians and patients in the clinic as they may contribute to improve survival of osteosarcoma patients.Exosomes could be secreted by different types of cells including osteosarcoma, BMSCs, CAFs, ADSCs, macrophages, and the secreted exosomes may contain a series of cargoes such as miRNAs, lncRNAs, circRNAs, and proteins. The exosomes and exosomal cargoes derived from these cells could exert biological activity and modify the functional activity of recipient cells, including proliferation, invasion, migration, angiogenesis, apoptosis, metastasis and chemoresistance. Moreover, several important branches are being explored to better define the clinical applications for exosomes and exosomal cargoes, including biomarkers, therapeutic targets and drug delivery vehicles .Recent studies on exosomes have changed our understanding of the role of exosomes in osteosarcoma and provided new targets for the diagnosis and treatment of osteosarcoma. The role of exosomes in changing osteosarcoma behavior, remodeling TME and producing osteosarcoma drug resistance has attracted extensive attention. The existing studies of exosomes and their cargoes have partially unraveled the mechanism of action of osteosarcoma-associated exosomes. For example, there is molecular communication between osteosarcoma cells and MSCs that help establish a premetastatic niche in the TME. In addition, exosomes carrying specific cargoes in the circulation have potential diagnostic value, which reflects the progression and metastasis stages of osteosarcoma and predict the prognosis of osteosarcoma. Exosomes-based therapy has shown great promise. Blocking exosome-based cargo and its target or developing exosome-based drug delivery provides a novel strategy for the treatment of osteosarcoma.Circulating exosomes derived cargoes have a great potential for early cancer detection, therapy, and prognosis. Nonetheless, using exosomes as biomarkers in diagnosis and prognosis, may result in false positive and negative findings due to the quality and heterogeneity of exosomes. Although the quality of exosomes has improved recently, and cancer therapy based on exosomes is supported by several early clinical trials. In pancreatic ductal adenocarcinoma (PDAC), engineered MSC derived exosomes carrying a commonly mutated gene KRAS siRNA is under research in early-phase clinical trial to treat metastatic pancreas cancer patients with the KRAS mutation (NCT03608631) . The safAlthough increasing attention has been paid to the application of exosomes in osteosarcoma, there are still several problems to be addressed before they can be used in the clinical treatment of osteosarcoma, including the specific mechanism of action of exosomes in osteosarcoma, isolation techniques, quantification, and the analysis of exosomes in body fluids of patients with osteosarcoma, as well as methods for detecting circulating osteosarcoma-specific exosomal cargoes , and the preparation, storage and drug loading methods of engineered exosomes for treating osteosarcoma. In addition, there are many exosomes and exosomal cargoes awaiting to be discovered, providing more biological support for their role in the progression, treatment, and prognosis of osteosarcoma. Therefore, further efforts are needed to understand the role and mechanism of exosomes in osteosarcoma and to develop exosomes-based diagnosis, prognosis prediction and treatment options for osteosarcoma patients.LY and XH: conceptualization, investigation, methodology, and writing-original draft. ZS: funding acquisition, writing-review and editing. HG, LW, WW, DJ, and WY: software, supervision, and writing-review and editing. All authors have given final approval to this version of the manuscript to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The autoimmune encephalitis (AE) syndromes have been characterised by the detection of autoantibodies in serum and/or cerebrospinal fluid which target the extracellular domains of specific neuroglial antigens. The clinical syndromes have phenotypes which are often highly characteristic of their associated antigen-specific autoantibody. For example, the constellation of psychiatric features and the multi-faceted movement disorder observed in patients with NMDAR antibodies are highly distinctive, as are the faciobrachial dystonic seizures observed in close association with LGI1 antibodies. These typically tight correlations may be conferred by the presence of autoantibodies which can directly access and modulate their antigens in vivo. AE remains an under-recognised clinical syndrome but one where early and accurate detection is critical as prompt initiation of immunotherapy is closely associated with improved outcomes. In this review of a rapidly emerging field, we outline molecular observations with translational value. We focus on contemporary methodologies of autoantibody detection, the evolution and distinctive nature of the clinical phenotypes, generalisable therapeutic paradigms, and finally discuss the likely mechanisms of autoimmunity in these patients which may inform future precision therapies. For over 5 decades, the clinical syndrome characterised by the subacute onset of amnesia, agitation, confusion, hallucinations, seizures and sleep disturbance, often accompanied by medial temporal lobe signal changes on imaging, has been referred to as limbic encephalitis (LE) –3. Some By contrast, in recent years, a group of autoimmune encephalitis (AE) syndromes have been characterised by the detection of autoantibodies in serum and/or cerebrospinal fluid (CSF) which target the extracellular domains of specific neuroglial cell-surface antigens , 3, 7–10All autoantibodies which mediate AE bind conformational antigenic epitopes. The tertiary structure of the target protein is thought to create distinctive three-dimensional domains to which autoantibodies preferentially bind. Conversely, loss of this structure reduces the likelihood of binding and subsequent detection of potentially pathogenic autoantibodies. This principle has underpinned the development of assays used in discovery phase research as well as those optimised for routine diagnostic testing Fig. .Fig. 1NeHEK293 cells provide a robust system for expressing proteins in their native conformational state on a mammalian surface membrane. As a result, they are a key reagent in the successful mammalian expression of the conformationally active extracellular domains of the known target antigens. Indeed, they are commonly employed in cell-based assays (CBAs), and have roles both in research to test for candidate targets, and in diagnostic testing to specifically identify a clinically suspected autoantibody. The patient serum or CSF can be applied to the transfected HEK cells either when they are live and intact (‘live CBA’) or after fixation and permeabilization (‘fixed CBA’). Live CBAs avoid permeabilisation and fixation-induced disruption of autoantigens, so patient autoantibodies are only exposed to the most clinically relevant extracellular epitopes. In our experience, this testing approach contributes to higher sensitivity for detecting immunotherapy-responsive conditions –21. HoweLive cultured rodent neurons or fixed brain slices provide substrates for ‘neuron-based assays’ or ‘tissue-based assays’, respectively. These preparations express the native epitopes from neurons, rather than those expressed by non-neuronal, albeit mammalian, cells such as HEK cells. Therefore, they may further improve the detection of autoantibodies with pathogenic potential. Theoretically, they can be used to screen for binding to any endogenous neuronal antigen, although fixed slices will of course detect antibodies which bind intracellular targets. Additionally, they may yield highly characteristic binding patterns and provide information about subcellular and whole brain localisation of autoantigens. These approaches depend on considerable technical expertise to optimise antigen availability, autoantibody detection, and image interpretation. Consequently, their main role remains in research laboratories for autoantibody discovery, or as an adjunct to confirm CBA-determined autoantibody specificities in more native tissue , 21.clinically irrelevant as they do not currently appear directly translatable to the level of individual patients. In contrast, CSF provides a ‘cleaner’ sample for autoantibody determination and in many cases of AE there is a relative overproduction of the antigen-specific autoantibodies in the intrathecal compartment , as reflected in the high frequencies of antigen-specific B cells in CSF –9795–97]A receptor antibodies plus surface neuronal binding without an identified target [In support of this peripheral-to-central flux of the immune process, a recent clinical observation has shed light on the exposure of brain antigens to the immune system as a potential trigger of AE. Patients with proven herpes simplex virus encephalitis (HSVE) have been noted to develop a clinical worsening a few weeks after initiation of their HSVE. This worsening is associated with the de novo generation of autoantibodies , 98, 99.d target , 42, 100d target . There hd target , 102, adWe have identified naïve pre-germinal centre B cells capable of differentiating to secrete aquaporin-4-specific autoantibodies in patients with another CNS autoantibody-mediated condition, neuromyelitis optica . TherefoStudies in patients with LGI1 and CASPR2 antibodies have also implied a role for germinal centre reactions, via the identification of strong and dichotomous genetic susceptibilities. Over 90% of LGI1-antibody-positive encephalitis patients, of both East Asian and Caucasian extraction, have the HLA-DRB1*07:01allele –70, whilMany of the early clinical observations in AE have been further refined to permit the accurate recognition of distinctive clinical phenotypes. This serves as a starting point not only to guide clinical practice, but also to inform focussed laboratory lines of enquiry. The relative rarity of these conditions on a population level remains a challenge to research, and highlights the notion that international and multicentre collaborations remain essential to move the field forward. AE is now identified as an important and previously under-recognised cause of CNS inflammation—it is highly treatable, and early diagnosis and initiation of immunotherapy are paramount to optimise outcomes. The field is now entering a more mature phase of research with advanced approaches in imaging, molecular immunology, and neuroscience. This will ideally permit a more cohesive understanding of aetiology and pathogenesis, and ultimately advance precision therapy in the future.


There have been few investigations of the role that adolescent cognitive ability might play in predicting physical resilience across the life course, including decreased risk of early mortality. Our limited knowledge of how multiple cognitive ability domains shape trajectories of longevity is due, in part, to a lack of aging cohorts with early life cognitive assessments, and family data that allow for examination of shared family and genetic characteristics that may play a role in cognitive ability-health links. We capitalized on data from the 1960 Project Talent high school cohort  and mortality data  collected as part of two recent follow-ups, the Project Talent Twin & Sibling Study and the Project Talent Aging Study, to examine these potential associations. In 1960, ability was assessed in multiple cognitive domains . Mortality status was ascertained through 2016. Binary logistic generalized estimating equations with race, age, sex, and adolescent family SES covariates, indicated that each 1 standard deviation higher ability in multiple cognitive domains in adolescence predicted lower odds of earlier mortality (ORs of 0.79 - 0.87). Co-sibling control models indicated a similar pattern, suggesting that benefits associated with higher cognitive performance do not simply reflect shared environmental and genetic background, but may represent a direct protective effect. These findings indicate that better performance in multiple cognitive domains in adolescence, above and beyond the influence of genetic and family environmental factors, may be or point to modifiable protective factors against risk of early mortality.


Following a cerebral ischemic event, substantial alterations in both cellular and molecular activities occur due to ischemia-induced cerebral pathology. Mounting evidence indicates that the robust recruitment of immune cells plays a central role in the acute stage of stroke. Infiltrating peripheral immune cells and resident microglia mediate neuronal cell death and blood-brain barrier disruption by releasing inflammation-associated molecules. Nevertheless, profound immunological effects in the context of the subacute and chronic recovery phase of stroke have received little attention. Early attempts to curtail the infiltration of immune cells were effective in mitigating brain injury in experimental stroke studies but failed to exert beneficial effects in clinical trials. Neural tissue damage repair processes include angiogenesis, neurogenesis, and synaptic remodeling, etc. Post-stroke inflammatory cells can adopt divergent phenotypes that influence the aforementioned biological processes in both endothelial and neural stem cells by either alleviating acute inflammatory responses or secreting a variety of growth factors, which are substantially involved in the process of angiogenesis and neurogenesis. To better understand the multiple roles of immune cells in neural tissue repair processes post stroke, we review what is known and unknown regarding the role of immune cells in angiogenesis, neurogenesis, and neuronal remodeling. A comprehensive understanding of these inflammatory mechanisms may help identify potential targets for the development of novel immunoregulatory therapeutic strategies that ameliorate complications and improve functional rehabilitation after stroke. Stroke is one of the leading causes of mortality, with many survivors suffering from long-term disability . ResultsRecruitment of leukocytes is an ongoing process that plays an important role in the pathogenesis of ischemic stroke. In addition to accelerating and expanding tissue damage, the inflammatory cascade may also aggravate or alleviate the ischemic insult to the brain . Unlike Due to the complex influence of the microenvironment and persistent immune cell infiltration, inflammation may exert both beneficial and harmful effects on the pathogenesis and prognosis of ischemic brain injuries. Previous observations suggest that targeting stroke-related neuroinflammation could become an effective adjunct therapy, but this approach requires caution regarding the timing and avoidance of adverse effects , 11. In Due to its apparent inflammatory damage in the acute phase and its possible beneficial effects on neural tissue repair processes, the long-term effects of the immune response in stroke remain controversial. In this review, we summarize the relationship between angiogenesis and neuronal remodeling after stroke. We focus on how immune cells participate in the subacute and chronic phases of stroke. This review aims to clarify the roles of different types of immune cells in stroke recovery and potentially enable better stroke treatment through immune regulation.Neural tissue repair after the acute phase of stroke involves two main processes: angiogenesis and neuronal remodeling, which further includes neurogenesis and synaptic remodeling . After sIschemic stroke is mainly caused by cerebral vascular stenosis or occlusion. The focal blood flow declines sharply, leading to a shortage in the supply of oxygen and nutrients, eventually causing cell death and tissue damage. The main way that ischemic brain tissue compensates for insufficient oxygen supply is by building new vessels. Angiogenesis in the infarcted area determines the recovery of cerebral blood flow, neuronal regeneration, and reconstruction of synaptic connections between nerve cells, which all influence the degree of functional recovery of the patient . The colPost-stroke neuronal remodeling is mainly caused by residual neurons that survive ischemia-reperfusion injury and newborn neurons from neurogenesis. Axonal sprouting from residual neurons does not normally appear until 14 days after ischemia, even in the ischemic penumbra . The occThe number of neurons that survive ischemia-reperfusion injury is limited, and newly formed neural connections are generated by neurogenesis. Neurogenesis is the process of generating new functional neurons from endogenous NSCs, including proliferation, migration, and differentiation into mature neurons . NeurogeThe microvasculature brings oxygen, nutrients, and growth factors to the area of ischemic injury, which creates an appropriate microenvironment for cell migration. Neuroblasts from the SVZ or SGZ expand and migrate to the peri-infarct region, where post-ischemic angiogenesis occurs. Compelling evidence suggests that several growth factors released from ECs are also indispensable for NSC proliferation, such as basic fibroblast growth factor, brain-derived neurotrophic factor, EGF, and VEGF . In addiConsidering that neurogenesis usually begins over 1 week after stroke onset, infiltrated immune cells and cytokines have been found to affect neurogenesis during this period. Cytokines reported to have potential regulatory effects on NSCs are listed in in vivo two-photon microscopy combined with immunohistochemistry, researchers demonstrated that neutrophils rapidly attach to inflammatory brain ECs within a few minutes and peak at 1–3 days, as seen in permanent and transient middle cerebral artery occlusion (tMCAO) models in mice , which act as the “blasting fuse” of reactive oxygen species (ROS). Although ROS plays an important role against pathogens, excess ROS can damage surrounding tissues, such as the BBB, which promotes blood leakage into the brain parenchyma and causes a more severe inflammatory response. Moreover, ROS was shown to mediate the expression of MMP-9 via the phosphatidylinositol-3 kinase-mediated signaling pathway, which further impaired BBB integrity (+) before experimental stroke facilitated the clearance of neutrophils by macrophage, which relief further inflammatory damage and therefore improved long-term recovery -β with enhanced phagocytosis, which gives them more anti-inflammatory properties . In addirophages Figure 3in vitro, which were drastic differences from the microenvironment in pathological state in vivo, such as tumor, infection, aging, physical trauma and stroke.There are also a large number of CNS-resident macrophage-like cells, namely microglia, which have phagocytic abilities during tissue damage. Similar to peripheral macrophages which come in with dozens of varieties, microglia also exhibit tremendous heterogeneity, as evidence by advanced transcriptomic and proteomic profiles. Therefore, it is reluctance to paraphrase the microglia with M1/M2 paradigm since the definition are derived from exposing isolated cells to certain stimuli in vitro , such as neutrophils and monocytes, T lymphocytes (CD3+) are a group of cells with heterogenous subtypes. Both pre-clinical and clinical investigations have revealed that T lymphocyte differentiation is more inclined to pro-inflammatory subtypes in the acute stage of stroke. In patients, circulating T cells were noted from day 1 to at least 124 days after stroke onset, with an expedited increase between days 8 and 20 (+/GITR+) has been reported at 6 h after permanent focal cerebral ischemia  were found to aggravate the ischemic injury, such as compromising BBB integrity and inducing microvascular dysfunction. However, depletion of Tregs results in alleviation of cerebral tissue damage characterized by increased focal cerebral blood flow and reduced aggregate fibrin . The con, and Helios  of stroke d Helios . Treg ced Helios . Depleti5 cells) was also shown to enhance NSC proliferation in the SVZ of normal and ischemic mice. The number of mammalian achaete-scute homolog 1 (MASH1)-expressing type C cells was decreased by the anti-IL-10 antibody, while GFAP+/Nestin+ cells were increased at 4 days after stroke, suggesting that an IL-10-mediated neuroprotective effect may facilitate the proliferation of NSCs in the SVZ. However, the underlying mechanism remains elusive  on astrocytes. In addition, astrocytes facilitate the proliferation of Tregs by releasing IL-33, which in turn downregulates astrocyte activity (+ and CD8+), cytokine production, and astrocytosis in response to traumatic brain damage  through single-cell RNA sequencing. Tregs showed robust accumulation in the injured brain at days 7–35 after ischemic stroke. Interestingly, adoptive cell transfer of WT Tregs increased the number of newborn APC+BrdU+ oligodendrocytes in Rag1-/- lymphopenic mice, but this effect was abolished when microglia were depleted. Further analyses revealed that osteopontin released from Treg cells strengthens the phagocytosis of microglia through activation of integrin receptors, which facilitates oligodendrogenesis and white matter regeneration , or specific depletion of CD4+ T cells, CD8+ T cells, B cells, or IFN-γ, was used to determine the contribution of different lymphocyte subgroups to ischemia reperfusion injury and recovery. Smaller infarction volumes and amelioration of neurological disorders were found in mice lacking lymphocytes (Rag−/−), but no improvements were observed in mice lacking B cells. Furthermore, B cell transfer in Rag−/− mice did not shown any significant improvement, indicating that B cells may not be play a protective role in the chronic stage of stroke (–/– mice (B cell depletion), enlarged areas of infarction, neurological disorders, and higher mortality were observed  were found to invade the ischemic core within the first few hours and gradually increased until 6 days after ischemia , the Science Technology and Innovation Commission of Shenzhen , the International collaboration project of Chinese Academy of Sciences (172644KYSB20200045), and the CAS-Croucher Funding Scheme for Joint Laboratories, and Guangdong Innovation Platform of Translational Research for cerebrovascular diseases.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Anthracycline therapy-related cardiac dysfunction (ATRCD) is the most notorious adverse side-effect of chemotherapy. It has become a significant cardiovascular health concern for long-term cancer survivors. With the emerging concept of subclinical ATRCD and newer diagnostictools (Speckle Tracking Echocardiography (STE) and biomarkers), detecting anthracycline cardiac toxicity at an early stage has become an important step to prevent severe cardiac dysfunction and improve the cardiovascular outcome in cancer survivors. Despite the increasing population at risk in sub-Saharan Africa (SSA), there is no contemporary data in Uganda to address the burden, pathogenesis and risk factors of subclinical ATRCD. This big gap in knowledge has led to a lack of local guidelines for monitoring and management of ATRCD.SATRACD  study is an observational prospective cohort study. Three hundred and fifty-three anthracycline naïve cancer patients will be recruited at baseline. Patients are followed up on completion of anthracycline-based chemotherapy and at 6 months after completion of anthracycline therapy. Data on demographics, cancer profile and clinical presentation will be collected at baseline. Comprehensive cardiac assessment will be performed at each visit, including electrocardiogram, conventional echocardiography, STE, cardiac and oxidative stress markers. We will be able to determine the incidence of subclinical and clinical ATRCD at 6 months after completion of anthracycline therapy, determine whether hypertension is a major risk factor for ATRCD, evaluate the role of conventional echocardiography parameters, and biomarkers for detecting subclinical ATRCD.This SATRACD study will provide contemporary data on Ugandan cancer patients who have subclinical and clinical ATRCD, help in the development of local strategies to prevent and manage ATRCD, and improve cardiovascular outcome for Ugandan cancer survivors. Anthracyclines are potent antineoplastic agents with proven efficacy in the treatment of many pediatric-, adult-hematologic and solid organ cancers. Dose-dependent anthracycline therapy-related cardiac dysfunction (ATRCD) is the most common and well-studied chemotherapy-induced cardiovascular toxicity. It was first described in 1971 in 67 patients treated with Adriamycin for a variety of tumors.11Identification of patients at risk of subclinical or clinical ATRCD is also very important, except when implementing surveillance protocols. Pre-existing cardiac risk factors such as hypertension, diabetes mellitus, smoking, previous left ventricular dysfunction and coronary disease, have shown to increase the risk of anthracycline-related cardiac toxicity.Cancer is emerging as a major public health problem in Sub-Saharan Africa (SSA), due to population ageing and growth.Despite the potential population at risk in SSA, there is a noted paucity of information on the burden of anthracycline-induced cardiotoxicity and related predictors among adult cancer patients receiving anthracycline chemotherapy. Lack of STE modality and expertise is a major obstacle for SSA countries to detect subclinical ATRCD. When STE is not available, conventional ECHO parameters, which measure the longitudinal motion of the left ventricle , peak systolic mitral annular velocity by tissue doppler (S'), may potentially be useful.In Uganda, heart failure caused by ATRCD is not uncommon. Patients usually present with advanced heart failure and carry a poor prognosis. In the Uganda Cancer Institute, 60% of the patients receive anthracycline therapy, but the majority of patients only receive screening ECHO prior to therapy. Follow-up cardiac evaluation is not routinely done. This may leave many anthracycline-treated patients with undetected asymptomatic subclinical cardiac dysfunction at risk of overt heart failure when they encounter another cardiovascular risk later in life.Our knowledge of anthracycline cardiotoxicity derives mainly from data in the developed world.To date, there are no contemporary data in Uganda to address the burden, pathogenesis and risk factors of subclinical ATRCD. This big gap in knowledge has led to a lack of local guidelines for monitoring and management of ATRCD.Detecting ATRCD at the subclinical stage is very important for the long-term outcome of cancer survivors. Therefore, this study aims to fill the gaps in knowledge in Ugandan cancer patients who are treated with anthracycline, and to understand the disease better by studying the burden, risk factors and pathogenesis. The study will also identify other available conventional ECHO parameters and oxidative stress makers that are able to detect or rule out subclinical ATRCD. Therefore, the results of the study will promote the application of available resources in cardio-oncology clinical practice, help to establish national guidelines on cardiac monitoring and management of patients who receive anthracycline therapy.This is a prospective study with two parallel Cohorts (Cohorts 1 and 2). Cohort 1 consecutively recruit 300 adult non-hypertensive cancer patients who receive anthracycline therapy. The time point prior to receiving anthracycline therapy (anthracycline naïve) is defined as baseline. The patient's demographic data, cancer diagnosis, dosage of anthracycline, past medical history, symptoms, physical examinations, ECG, ECHO and laboratory data are collected at baseline. Patient follow-ups are performed at the completion of chemotherapy, and 6 months after completion of chemotherapy. Data on symptoms, physical examinations, ECG, ECHO and blood test are collected at each visit.Cohort 2 recruit 53 adult hypertensive cancer patients. The patient's demographic data, cancer diagnosis, dosage of anthracycline, past medical history, symptoms, physical examinations, ECG, ECHO and laboratory data are collected at baseline. Patient follow- ups are performed at the completion of the chemotherapy, and 6-months after completion of chemotherapy. Data of symptoms, physical examinations, ECG, ECHO and blood test are collected at each visit.To determine the incidence of subclinical and clinical ATRCD in cancer patients who receive anthracycline therapyTo compare the changes of GLS value between hypertensive and non-hypertensive cancer patients at completion of anthracycline therapyTo determine the correlation of conventional ECHO parameters  with GLS, and their ability to diagnose or rule out subclinical ATRCDTo determine the correlation of oxidative stress with cardiac function  in patients who receives anthracycline therapy and their role in diagnosing subclinical ATRCD.Objectives. Our objectives are as follow:We hypothesized that hypertension would be a significant risk factor for developing cardiotoxicity in Ugandan cancer patients treated with anthracycline. Conventional ECHO parameters and oxidative stress markers may play important roles in detecting subclinical ATRCD in Ugandan cancer patients.The patient screening site is at the outpatient clinic of the Uganda Cancer Institute (UCI). Patient recruitment and data collection is at the Uganda Heart Institute (UHI). UCI is a specialized national referral oncology center in Kampala, Uganda. The center receives about 1,000 new cancer patients referred for chemotherapy annually. Currently, it has a bed capacity of about 100 beds with 8 specialist medical oncologists, 2 pharmacists, 10 medical officers and 50 trained oncology nurses. About 60% of patients attending UCI receive anthracycline therapy. Adult patients who receive anthracycline, with expected survival longer than two years, are referred to UHI for re-screening of eligibility. Blood samples collected from patients will be stored and shipped to the Hatter Institute for Cardiovascular Research in Africa in Cape Town, where biomarker analysis will be carried out.Patient demographics: age, gender, tribe, districtCancer profile: Cancer diagnosis and stage, type of anthracycline and accumulated dosage*, other medication*Past medical and social history: hypertension, diabetes mellitus, coronary heart disease, renal disease, HIV, smoking, alcohol intake, any other chronic illnessFamily history of heart disease or cancerCurrent clinical presentation*: symptoms of heart failure, NYHA class, heart failure stagePhysical examination*: weight, height, temperature, blood pressure**, heart rate, SaO2, signs of heart failure The following data will be collected from all enrolled subjects:12-lead resting Electrocardiogram*Comprehensive Echocardiography*: using 2D, M-mode, Doppler, tissue Doppler, Speckle Tracking Echocardiography (STE) image to assess cardiac structure, function and hemodynamics.Blood test*: CBC, LFTs, RFTs, FBS, Fasting lipid profile, Troponin I, N-pro BNP, myeloperoxidase (MOP).The following investigative tests will be performed:*: Will be collected at each visit**: Patient's left arm blood pressure will be taken at sitting position, using “Omron” digital blood pressure machine, after resting for 5 minutes. High blood pressure will be quantitatively defined as systolic blood pressure > 140mmHg or diastolic pressure > 90mmHg.Subclinical ATRCD and clinical ATRCD are diagnosed using criteria recommended by the American Society of Echocardiography and the European Association of Cardiovascular Imaging:9LVEF≥53%andA relative percentage decrease of GLS >15%, compared with baseline, and /orTnI become positive during follow upDecrease in LVEF of >10 percentage points, to a value <53%.Diagnostic criteria for clinical ATRCD:Transthoracic echocardiograms images are acquired using the Vivid E9 , conducted by two formally trained cardiologists (including PI). All echocardiograms are analyzed by a single observer (PI). LVEF is calculated from the apical 4- and 2-chamber views using a modified Simpson biplane method. ATRCD is defined as a reduction in LVEF of >10%, to a value of <53%, as per guidelines.9 Speckle tracking strain analysis is used to measure global and regional longitudinal systolic strain from apical four-, two- and three-chamber views. Regional longitudinal strain is demonstrated on a Bull's eye diagram. MAPSE is measured by m-mode obtained from apical four camber view. Average of septal and lateral MAPSE is calculated. Tissue Doppler-derived indices is measured using the apical four-chamber view. Peak systolic mitral annular velocities (S') is calculated by averaging septal and lateral mitral annular velocities.In all patients, blood samples are obtained from venal puncture. Cardiac troponin I is measured as part of the clinical routine. The blood sample is collected in an EDTA tube and SST tube and immediately centrifuged. Plasma and serum are stored at -80 degree Celsius until assayed. The stored blood samples will be transported on dry ice and shipped to the Hatter Institute for Cardiovascular Research in Africa, where the assay for NT-pro BNP and markers for oxidative stress markers will be carried out. The shipment of the samples and biomarker analyses will only be done on samples of patients who have a complete data set, including all the time points (baseline + two follow up visits.)NT-proBNP plasma levels will be measured by electrochemiluminescence immunoassay . Oxidative stress markers, e.g. serum myeloperoxidase (MPO) will be investigated in our cohort. Serum MPO will be measured using the human MPO Instant ELISA .Appropriate dilution of the plasma or serum samples will be utilized for the assays, as required by the manufacturer of each ELISA kit, and each plasma or serum sample will be assayed in duplicate. The average of the two measurements will be utilized to calculate the levels of each of the biomarkers from the standard curve established with known positive standards supplied with each kit.Study data is entered via specialized online study forms into a central database in REDCap. REDCap is a widely used research information system that facilitates formbased entry of study data with quality assurance tools to check the validity and consistency of results.All continuous variables are expressed as a mean ± SD and categorical variables as a percentage.Student's t-test, paired t-test, and Mann-Whitney U test will be used to compare continuous variables, where appropriate. Correlations will be assessed by Pearson's t-test. Significant relationships will then be examined by multivariate linear regression analysis. Receiver operating characteristic (ROC) curves will be plotted to examine the abilty of MAPSE and S' to detect subclinical ATRCD (objective 3). Data will be analyzed using SPSS version 17 and considered significant if p <0.05.Ethical approvals were obtained from School of Medicine Ethics and Research Committee, College of Health Sciences Makerere University (REC REF 2018-081), Uganda National Council of Science and Technology (HS220ES) and Faculty of Health Sciences Human Research Ethics Committee, University of Cape Town (HREC 054/2020sa)All participants are provided with a written copy of the consent form. Any and all questions that participants have regarding the research will be addressed prior to signing of the consent form. Written informed consent in English or Luganda is collected from each participant.Between November 2018 and February 2020, 353 patients  were enrolled at the baseline. There were 167 patients who were able to complete anthracycline-based chemotherapy and who had returned for the second follow-up visit by April 2020. Of these 167 patients, 36 were able to return for the third (last) follow-up visit by April 2020.We expect to complete patient follow-up by the end of 2020 or early in 2021.Anthracycline therapy-related cardiac dysfunction is a well-known adverse effect resulting from this potent anti-cancer regimen. Recently, the emerging concept of subclinical ATRCD and novel diagnostic tools made close cardiac surveillance highly important. To detect and treat ATRCD at the subclinical stage and improve the cardiovascular outcome for long-term cancer survivors have become one of the major goals of cardio-oncology practices worldwide. With the increased cancer burden and higher exposure to anthracycline in the SSA population, subclinical ATRCD should no longer being ignored. The SATRACD study is the first observational cohort study, using international guidelines, to define the burden and risk factors of subclinical ATRCD in a SSA country (Uganda). It evaluates the role of conventional ECHO parameters and biomarkers in detecting subclinical ATRCD. In this study, for the first time, STE is performed in Ugandan cancer patients. This will not only enable us to monitor patients more effectively, but will also initiate cardio-oncology practices in Uganda, promote capacity-building in cardiovascular medicine and initiate more research with STE at stage A and B heart failure and other heart diseases. Data from the SATRACD study will bridge the gap in knowledge of anthracycline cardiotoxicity in Uganda, and, furthermore, help to develop local guidelines on cardiac monitoring for early diagnosis and treatment of ATRCD. Following the findings of the SATRACD study, a further clinical trial may be designed to study the effectiveness of carvedilol on treating subclinical ATRCD in the future.


Infants born preterm are at a high risk for repeated pain exposure in early life. Despite valid tools to assess pain in non-verbal infants and effective interventions to reduce pain associated with medical procedures required as part of their care, many infants receive little to no pain-relieving interventions. Moreover, parents remain significantly underutilized in provision of pain-relieving interventions, despite the known benefit of their involvement. This narrative review provides an overview of the consequences of early exposure to untreated pain in preterm infants, recommendations for a standardized approach to pain assessment in preterm infants, effectiveness of non-pharmacologic and pharmacologic pain-relieving interventions, and suggestions for greater active engagement of parents in the pain care for their preterm infant. Ubiquitous exposure to acute pain is inevitable for all infants, for most within the initial hour and first day(s) after birth as part of recommended medical care . InfantsFor infants delivered preterm, the majority requiring neonatal intensive care, pain exposure is even higher. Data from Canadian  and EuroPoorly treated and/or prolonged pain exposure in preterm neonates has been linked to lasting consequences during a critical time in brain development. For infants delivered very preterm, controlling for gestational age, severity of illness and morbidity exposure to early pain-related distress is associated with both immediate physiologic instability and pain sensitivity as well as long-lasting deleterious impacts on cognition and behavior ,14 and pThis paper provides a narrative review of the current best evidence regarding the assessment and management of pain for infants born preterm requiring neonatal intensive care.Most national and international guidelines on neonatal pain management declare pain assessment to be essential to achieve optimal pain and stress management ,28,29,30The subjective and complex nature of pain makes pain assessment challenging. Self-evaluation of pain is the first choice for persons who can express themselves, verbally or by indicating the intensity of pain at some linear or pictorial scale, but for preterm infants, this is not an option. Instead, care providers must learn to recognize indicators of pain in infants and determine when they need pain alleviation based on these signs.Signs of neonatal pain are categorized into behavioural, physiological, hormonal, or neurophysiological domains, which can either be observed individually or, preferably, in combination for better accuracy ,33 See . Since tThere are over 40 neonatal pain assessment scales published and even more constructed and used in clinical settings ,43,44,45A good pain assessment tool should detect all painful situations (high sensitivity), discriminate pain from non-painful situations (high specificity), measure a specific type of pain they are supposed to measure , give the same results when used repetitively or by different observers (good reliability), and, establish if a pain-relieving intervention was effective (good responsiveness) . The choThere have been increasing attempts to use artificial intelligence and machine learning to analyze signals from preterm infants to evaluate if they indicate pain. An example is the ABC analyzer, which is an automated computer crying frequency analyzer that has shown correlation with the DAN-score during heel prick . DespiteSeveral studies have examined the effects of implementing neonatal pain assessment, however, even if improved process outcomes have been seen, more research on the outcomes on patient level  is needed . In the n = 1075 infants) and expressed breastmilk feeding  during routine minor acute painful procedures in full-term infants ) .Very few studies have examined the long-term effects of repeated sucrose administration. A systematic review of eight studies examining the efficacy and safety of repeated oral sucrose for procedural pain suggests that evidence is supportive of the safety and efficacy of repeated sucrose administration, however, results were mixed . Pre-cliDespite significant progress that has been made over the past three decades addressing the prevention, assessment, and management of neonatal pain, many questions remain regarding optimal use of pharmacological agents. Lack of efficacy data and uncertainty regarding long-term outcomes are two of the primary research gaps that clinicians face when attempting to manage neonatal pain . MoreoveThe most used non-opioid analgesic to manage pain in children including neonates is paracetamol (acetaminophen). While commonly used in older children and adult populations, non-opioid agents such as NSIADs, gabapentin, and dexmedetomidine are contraindicated or less commonly used in neonatal populations. Local topical and regional agents are also utilized with variable uptake across neonatal units, while the mainstay for post-operative procedures includes opioids such as morphine and fentanyl. Adjuvant agents are also used for the management of pain-related distress and refractory pain. An overview of the use of these agents is included below.Based on current best evidence, paracetamol does not provide effective pain relief for infants undergoing procedural pain, despite its frequent use ,131. In Despite the widespread use of non-steroidal anti-inflammatory drugs (NSAIDs) to treat pain in older pediatric populations, their use for neonatal populations is less common. Given the risk associated with renal injury and gastrointestinal bleeding, most NSAIDs such as ketorolac are not approved for use in infants less than six months of age, with greatest risk reported in infants less than three weeks of age and those delivered preterm .Given the positive benefits of pain relief and opioid sparing in older children and adults, there is increasing interest in the use of dexmedetomidine and gabapentin to treat postoperative pain and pain-related distress in neonates. Their use remains off-label, given the lack of FDA approval, in neonatal populations. To date, only a few small studies have been reported. Dexmedetomidine has been associated with less respiratory depression or gastrointestinal dysmotility when compared to fentanyl , and theThere is increasing interest in the use of spinal and epidural anesthesia for postoperative pain management in neonates. Local agents such as bupivacaine, lidocaine, or ropivacaine can be administered as regional anesthesia in awake patients for minor surgeries such as inguinal hernia repair and circumcision, or for procedures such as chest tube placement ,138. TheConsiderable variation in degree of efficacy regarding the use of topical anaesthetics in neonates has been reported across procedures . There iOpioids are the mainstay for the effective treatment of moderate to severe pain across all ages, including neonates. The most commonly used agents for neonatal populations include morphine and fentanyl. Despite the benefits, safe and effective dosing of systemic drugs  for pain relief in neonates is challenging, as their neurodevelopmental stage makes them highly sensitive to drug effects  and can It is somewhat reassuring to note that studies examining the longer-term impact of morphine exposure in ex-preterm infants at age five , eight, n = 83), while there were no group differences in oxygen saturations levels, infants receiving intermittent boluses demonstrated greater pain scores indicating distress  compared to infants receiving continuous morphine infusions of 20 ng/mL. In a randomized trial comparing the pharmacokinetics of fentanyl across 100 neonates, serum fentanyl concentrations were steady in the infants receiving a continuous infusion (1 mcg/kg loading dose followed by 1 mcg/kg/hour infusion) in contrast to wide fluctuations in serum concentration with high-peak concentrations in infants receiving bolus administration (1 mcg/kg/dose administered every four hours) [n = 28) [n = 450) or no treatment (n = 472). Infants in the treated group had improved survival without any difference in moderate or severe sensorimotor impairments at age two years [One significant question which remains unanswered relates to the optimal administration of opioids or adjuvant therapies, either as a continuous infusion or intermittent bolus, to achieve effective pain reduction and reduce cumulative opioid exposure and risk for adverse outcome. In the few studies addressing this question, results are mixed. In post-operative infants (r hours) . Fentany[n = 28) . Similarwo years . More reModern neonatal care is family-centered and supports parental presence in the neonatal unit and parental involvement in care procedures, including pain management, with proper counselling and information ,173,174 There is increasing recognition that parents are aware of their infant’s pain. Although this awareness is stressful for parents ,179, theDespite an increased awareness that newborn infants experience pain, which can lead to short- and long-term negative health outcomes, management of this pain remains varied with mixed results on pain alleviation. Validated and appropriate pain assessment tools should be implemented as a standardized approach for healthcare providers treating neonates to guide pain-relieving treatments, including pharmacological and non-pharmacological interventions. Finally, it is important to engage families in the pain management of newborns, as parents are a valuable resource who want to be involved in alleviating the pain of their infant.


Clinical practice guidelines differ in their recommendations on first-line antihypertensive drug classes. No adequately powered randomised controlled trial have assessed all major drug classes against each other, and previous meta-analyses have mainly relied on pairwise meta-analyses for treatment comparisons.A systematic review and network meta-analysis will be carried out to assess the efficacy and acceptability of all major antihypertensive drug classes. PubMed and CENTRAL were searched on 21 February 2020 to identify randomised controlled trials with at least 1000 person-years of follow-up, assessing any antihypertensive agent against other agents or placebo. All trials fulfilling the inclusion criteria will be assessed for risk of bias using the second version of Cochrane’s risk of bias assessment tool. The study selection process, risk of bias assessment and data extraction are done by two authors in duplicate. Relative risks from individual trials will be combined in pairwise meta-analyses; in the absence of important intransitivity, random-effects network meta-analysis will be performed. The primary outcome for efficacy will be major adverse cardiovascular events, whereas the primary acceptability outcome will be treatment discontinuation for any reason. Additional outcomes include all-cause mortality, cardiovascular mortality, stroke, myocardial infarction, heart failure and acute renal failure. The impact of differences within drug classes will be explored through alternative networks, including analysing thiazide-like and thiazide-type diuretics separately.This review will only process aggregated study level data and does not require ethical approval. The findings will be published in a peer-reviewed medical journal.CRD42020205482. This systematic review and network meta-analysis will be the first contemporary review to assess the efficacy and acceptability of all major antihypertensive drug classes, incorporating direct and indirect evidence.Broad inclusion criteria in terms of patient characteristics ensure wide applicability of the study findings; potential differences between patient groups will be explored through subgroup and sensitivity analyses.Alternative networks, with different node definitions, will be used to explore differences within drug classes, specifically differences between thiazide-like and thiazide-type diuretics.The main limitation of this review is the use of study-level data, with no possibility to account for individual patient characteristics in the analyses.Hypertension (blood pressure ≥140/90 mm Hg) is estimated to affect more than one billion people globally.2 3Five major drug classes have been shown to reduce the risk of CVD compared with placebo or no treatment; ACE inhibitors (ACEi), angiotensin II receptor blockers (ARB), calcium channel blockers (CCB), diuretics and beta blockers (BB).9Even though many antihypertensive agents have been compared with one another or placebo separately, no adequately powered trial has compared all major drug classes head-to-head, assessing CVD outcomes. Several systematic reviews and pairwise meta-analyses aiming to assess this have been published, but all have limited their analyses to comparative trials assessing two or more agents against each other, with the exclusion of placebo-controlled trials.14All major systematic reviews have used the conventional classification of antihypertensive agents described above.19In summary, clinical practice guidelines differ in their recommendations on first-line antihypertensive treatment. This reflects the absence of head-to-head comparisons between major drug classes, and hence uncertainty regarding the comparative efficacy between classes. The aim and purpose of this study is to compare the efficacy and acceptability of different antihypertensive drug classes for CVD prevention through a systematic review and network meta-analysis, incorporating evidence from comparative as well as placebo-controlled trials. Further, we will explore the potential differences among different subclasses of diuretics, RAAS inhibitors and CCB.We will perform a systematic review guided by the recommendations from the Cochrane Collaboration.23Randomised controlled trials comparing any single antihypertensive agent, or combination of antihypertensive agents, against another agent, combination of agents, placebo or no treatment will be included. Trials using a prospective randomised open-label blinded endpoint (PROBE) design will be accepted, whereas cluster-randomised trials, crossover trials and trials of combined or complex interventions will be excluded. Trials assessing different treatment strategies, such as more intensive versus less intensive treatment, will also be excluded, because they convey no information on drug-specific or drug class-specific effects. We will restrict our analyses to trials with at least 1000 person-years of follow-up to avoid small trials not primarily assessing the effect of blood pressure lowering agents on cardiovascular events, but rather the effect of different agents on blood pressure levels. Hundreds of such trials exist; cardiovascular events are uncommon, and when present recorded as adverse events and therefore not properly adjudicated.2). Because the main drug classes listed below (see the Types of intervention section) may be combined , and prescribed in any order without important interactions on cardiovascular outcome level, we will include trials of treatment naïve patients and previously treated patients in the network meta-analysis. Studies including more than 50% of participants with heart failure, left ventricular dysfunction, acute myocardial infarction, macroalbuminuria, end-stage renal disease or chronic kidney disease stage 3 or higher (eGFR <60 mL/min/1.73 m2) at baseline will be excluded. The reason for these exclusions is that one or several of the included drug classes have specific effects in these patient groups, that possibly goes beyond blood pressure lowering.Trials will be included if participants are either primary preventive, have stable coronary artery disease, previous cerebrovascular disease, diabetes mellitus or mild chronic kidney disease (estimated glomerular filtration rate (eGFR) 60–89 mL/min/1.73 mThe main interventions of interest are antihypertensive drug classes which have previously been shown to reduce the risk of cardiovascular events; ACE, ARB, BB, CCB and diuretics. These are the main treatment options in clinical practice for the types of participants listed above (see the Types of participants section), and will therefore constitute the decision set of interventions in this review. Other drug classes, such as potassium-sparing diuretics, alpha-blockers, peripheral vasodilators and central-acting agents, as well as drug combinations including any of the above-mentioned drug classes, placebo and no treatment, will be included in the network as supplementary interventions to maximise the amount of evidence through indirect comparisons .The primary outcome for efficacy will be the composite outcome, major adverse cardiovascular events (MACE). Definitions of MACE vary to some extent across trials, but commonly include cardiovascular mortality, stroke and myocardial infarction. Inclusion of heart failure, revascularisation and peripheral artery disease differ across trials. If available, we prefer the strict definition using cardiovascular mortality, myocardial infarction and stroke. If other definitions of MACE have been applied, they will generally be accepted but appropriateness for inclusion will be assessed on a case-by-case basis. All deviations from the preferred definition will be denoted, and the impact of MACE definition will be explored in sensitivity analyses, excluding trials not presenting results according to the preferred definition.The primary outcome for acceptability will be permanent discontinuation of study medication. We will include discontinuations for any reason because this reflects safety as well as efficacy, and because the reporting of reasons for discontinuation differs to a large extent between trials.Secondary outcomes are all-cause mortality, cardiovascular mortality, myocardial infarction, stroke, heart failure and acute kidney injury. Mortality outcomes, as well as myocardial infarction and stroke, have generally accepted universal definitions, although for myocardial infarction the definition has changed slightly over the years. For these outcomes, the definitions applied in individual trials will generally be accepted. For myocardial infarction and stroke we primarily aim to include fatal and non-fatal events, combined. If only fatal or non-fatal events are reported separately, however, we will accept and include the reported numbers. We will also accept ‘acute coronary syndrome’ as equivalent to myocardial infarction, but we will not include composite coronary events including revascularisations. For heart failure, we will accept definitions including new diagnosis of heart failure, heart failure hospitalisations and heart failure mortality. For acute kidney injury, we will apply the Kidney Disease: Improving Global Outcomes (KDIGO) definition, including at least 50% increase in serum creatinine, where appropriate. However, we will also include discontinuation of treatment due to kidney injury, as many trials were designed before the KDIGO definition of acute kidney injury was published.Preliminary searches in PubMed and CENTRAL were performed on 21 February 2020. We used search terms for blood pressure lowering treatment combined with several CVD terms in both databases. We restricted our search to randomised controlled trials, but applied no date or language restrictions.Full search strategy in PubMed was  AND  AND ).Full search strategy for CENTRAL was (antihypertensive near agents OR “blood pressure-lowering” OR “blood pressure lowering” OR “blood-pressure lowering”) AND  in All Text. Major systematic reviews and meta-analyses were further scrutinised for individual trials possibly missed by the database searches.3 5 10 11 14 29–33Records from PubMed and CENTRAL were combined using EndNote reference software, removing duplicate records. Titles were screened by one reviewer (HJ) to remove apparently irrelevant publications. Abstracts and full-text articles were assessed against eligibility criteria by two reviewers independently (HJ and BC). Discrepancies on study inclusion were resolved by discussion or third-party involvement (MB). All studies fulfilling the above eligibility criteria will be reported in the systematic review, regardless of outcome reporting.Data will be extracted from original publications into Excel spreadsheets by two authors independently. Extracted data will include descriptive study characteristics as well as data for analytical purposes. A preliminary list of variables to extract is presented in Differences in extracted data will be assessed going back to original publications and, if uncertainty prevails, resolved by discussion or involvement of a third investigator.Mean systolic and diastolic blood pressure level at baseline and during follow-up will be collected for each treatment arm in each trial. If baseline blood pressure levels are not presented for each treatment arm separately, values for the total study population will be extracted and assumed to be similar for randomised groups. For follow-up blood pressure values, we will aim to include mean levels during the follow-up period. If this is not reported, we will calculate mean levels for the full follow-up manually when several values are presented, or include values at a single time point . Blood pressure differences between groups will be recorded as reported, or calculated manually from follow-up values when not reported separately.We will extract the number of participants and events for each treatment arm in each study, whenever possible. If the number of events is not presented, we will estimate the number of events from the measure reported, for example, calculating backwards from incidence rates, or manually counting number of events from Kaplan-Meier curves.The number of participants lost to follow-up will be recorded for cardiovascular events and mortality outcomes, for each treatment arm, separately. It is common in cardiovascular trials to follow participants for mortality through national or regional registers even if participants drop out from clinical follow-up during the intervention. Loss to follow-up will be reported descriptively, and the impact of attrition on primary outcomes will be estimated in sensitivity analyses using different assumptions for the outcome of the participants lost to follow-up allowing for some uncertainty in these assumptions.35 36All eligible trials will be assessed for risk of bias using the Cochrane Collaboration’s risk of bias assessment tool, second version (RoB2).The first two domains will be assessed on study level, as the same judgement apply for all outcomes. Trials judged to be at high risk of bias in the randomisation process will be excluded from further analyses. In the second domain, PROBE trials will be considered to be at low risk of bias as long as no specific concerns are noted. The third domain will be assessed for mortality outcomes and non-mortality outcomes separately, as clinical events are usually not available for participants lost to follow-up, whereas vital status may still be available through population registers. If the number of participants with missing outcome data are of the same magnitude as the number of events, this will raise some concerns, whereas high risk of bias will generally require asymmetric missingness of such magnitude it could plausibly impact effect estimates in a clinically important way. For the fourth domain, a blinded endpoint committee will be regarded as the appropriate way of assessing outcomes. Outcome data reported as adverse events have generally been assessed in an open fashion to allow safety monitoring. This gives rise to some concerns, but will not be judged as high risk of bias because cardiovascular outcomes are fairly objective, and the value of blinding in such circumstances is probably limited.The risk of bias assessment will be incorporated in the overall assessment of the quality of evidence, within the Confidence In the results from Network Meta-Analysis (CINeMA) framework, as described under the subheading ‘Confidence in network evidence’.In the presence of two or more studies comparing the same pair of interventions, we will synthesise results using random effects pairwise meta-analysis. The clinical heterogeneity  of the studies within the same treatment comparison will be evaluated prior to synthesis. We will estimate the between-study variance , for each outcome separately in the two main networks, and for the primary efficacy and acceptability outcomes in the sensitivity analyses with combined RAAS inhibitors and subdivided CCB. SUCRAs for the primary efficacy and acceptability outcomes will be presented in two-dimensional graphs.In case several studies evaluating drug class combinations are identified, we will perform a component-level analysis assuming each drug class forming an intervention component.39 40https://cran.r-project.org/web/packages/netmeta/netmeta.pdf).Analyses will be performed in R using the netmeta package , potential differences in treatment effect based on the presence or absence of diabetes, coronary artery disease and kidney disease, will be explored in subgroup or meta-regression analyses. Although meta-regression is more powerful in detecting associations between trial characterises and treatment effect compared with subgroup analyses, our experience is that many trials do not report the exact percentage of participants with certain diseases. It may still be clear from the methods, or from other numbers presented, that it is well below certain thresholds. Thus, meta-regression will be used if permitted by the available data, otherwise trials will be dichotomised based on if ≥50% of the included participants had each characteristic at baseline. The impact of systolic blood pressure at baseline, as well as mean age at baseline, will be assessed through meta-regression analysis.For some trials comparing different active treatments, the blood pressure level during follow-up will differ between treatment arms. From an intention-to-treat perspective, this is not a problem, since any class that reduces blood pressure more effectively would be expected to reduce the number of events to a larger extent. However, to be able to answer the question if any drug class has blood pressure-independent effects on any outcome, such differences need to be taken into account. This will be explored in meta-regression analysis, adjusting for mean blood pressure difference between comparisons within trials.Comparison-adjusted funnel plots will be used to assess small-study effects for each outcome separately. If asymmetry is detected, we will employ network meta-regression models to test whether asymmetry is statistically significant and whether small studies tend to systematically favour specific drug classes.The overall confidence in the results will be assessed using the CINeMA framework.This review does not require ethical approval. The findings will be published in a peer-reviewed medical journal and presented at medical conferences.