Animals and plants have NLRs (nucleotide-binding leucine-rich repeat receptors) that recognize the presence of pathogens and initiate innate immune responses. In plants, there are three types of NLRs distinguished by their N-terminal domain: the CC (coiled-coil) domain NLRs, the TIR (Toll/interleukin-1 receptor) domain NLRs and the RPW8 (resistance to powdery mildew 8)-like coiled-coil domain NLRs. CC-NLRs (CNLs) and TIR-NLRs (TNLs) generally act as sensors of effectors secreted by pathogens, while RPW8-NLRs (RNLs) signal downstream of many sensor NLRs and are called helper NLRs. Recent studies have revealed three dimensional structures of a CNL (ZAR1) including its inactive, intermediate and active oligomeric state, as well as TNLs (RPP1 and ROQ1) in their active oligomeric states. Furthermore, accumulating evidence suggests that members of the family of lipase-like EDS1 (enhanced disease susceptibility 1) proteins, which are uniquely found in seed plants, play a key role in providing a link between sensor NLRs and helper NLRs during innate immune responses. Here, we summarize the implications of the plant NLR structures that provide insights into distinct mechanisms of action by the different sensor NLRs and discuss plant NLR-mediated innate immune signalling pathways involving the EDS1 family proteins and RNLs. Plant pathogenic microbes pose a major threat to global crop production. To induce efficient defence responses and confer disease resistance, plants rely on two distinct types of innate immune receptors. Cell-surface pattern recognition receptors, including receptor-like kinases (RLKs) and receptor-like proteins (RLPs), perceive conserved microbial molecular structures such as pathogen-associated molecular patterns (PAMPs) and elicit defence responses known as PAMP-triggered immunity (PTI), leading to resistance to a broad range of non-adapted pathogens  proteins and Caenorhabditis elegansCED-4 (van der Biezen and Jones NAIP (NLR family apoptosis inhibitory protein), CIITA (MHC class II transcription activator), HET-E (incompatibility locus protein from Podospora anserina) and TP1 (telomerase-associated protein)  and a C-terminal leucine-rich repeat (LRR) domain  through ASCCARD-pro-caspase-1CARD interaction , containing a CARD (caspase activation and recruitment domains); NLRPs, containing the PYD (pyrin domain); NLRBs, containing multiple BIR (baculovirus inhibitor of apoptosis protein repeat) domains; and NLRAs, containing an AD (acidic transactivating domain) , there are 445 NLRs consisting solely of non-TNL classes, as TNLs have been lost in grasses -NLRs (TNLs) and non-TNLs. The latter includes members with a coiled-coil (CC) motif, termed CC-NLRs (CNLs), and a resistance to powdery mildew 8 (RPW8) domain, termed RPW8-NLRs (RNLs)  and cell death  and ROQ1 (recognition of XopQ 1) from Arabidopsis and Nicotiana benthamiana, respectively  hydrolase (NADase) activity   structures of a plant NLR come from the CNL, Arabidopsis ZAR1 (HopZ-activated resistance), including its ADP-bound inactive state, a ligand-free transition state and an ATP-bound active oligomeric state referred to as a resistosome . The uridylylated PBL2UMP exclusively binds RKS1 of the preformed complex to form ZAR1:RKS1:PBL2UMP, which triggers ZAR1 to exchange ADP for ATP, followed by oligomerization of activated ZAR1 into a pentameric wheel-like structure  characterized by the expanded LRR motifs  of the NB-ARC domain through the other lateral side of LRRs and packs against WHD and the helical domain . This LRR positioning of ZAR1 is different from that of animal NLRs, such as NLRC4 and APAF-1 (WD motif instead of LRRs), which rather contact the NBD  , also known as the Ma resistance gene, encodes a TNL containing five C-JID motifs  as well as the tobacco TNL N failed to trigger cell death in N. benthamiana , potato RxCC (residues 1–112) and Arabidopsis NRG1.1CCR , a solution 3D structure of wheat Sr33CC (residues 6–120) and cryo-EM structures of full-length ZAR1, including inactive and active states  protein which functions as a cation channel , which possibly contributes to pore formation upon activation in a similar way as the ZAR1 α1 helix. While the deletion (Δ16) allows NRG1.1 oligomerization, it results in loss of Ca2+ influx  movement of WHD away from NBD, (2) ADP release and subsequent ATP binding, (3) rearrangement of the N-terminal α1 helix, which protrudes out towards the surface, and (4) oligomerization of ZAR1 through NBD. The resulting pentameric ZAR1 resistosome displays an α-helical barrel structure with pore formed through the CC domains  activity, able to cleave NAD+ into ADPR and nicotinamide, and, in the case of plant and bacterial TIRs, a yet unknown variant of cyclic ADPR (v-cADPR) , triggering auto-immune responses   domains identified in fungal and animal membrane pore-forming proteins  family, consisting of EDS1, PAD4  and SAG101 (senescence-associated gene 101)  and TIR-NBD (TN), have been described in the Arabidopsis Columbia-0 ecotype  are likely capable of autonomously triggering cell death without the need of downstream helper NLRs (Saile et al. Analysis of the N-terminal CC domains of Arabidopsis CNLs classified CNLs into four groups (Wroblewski et al. + cleavage by TNLs are potential candidates that serve as signalling molecules for bringing an RNL into the EDS1 scaffold (Fig. The structures of CNL and TNL resistosomes significantly expand our understanding of the activation mechanisms of plant NLRs. However, there are still important questions to be answered about the nature of NLR activation and the ETI pathways. So far, ZAR1, ROQ1 and RPP1 structures demonstrate that activated monomers, each bound to the activating ligand, form a resistosome, which is similar to how human APAF-1 monomers, bound to cytochrome c, form a wheel-like structure (Dorstyn et al. old Fig. . Whetherold Fig. . How the


In this article, the operational characteristics of coverage with evidence development (CED) programs in Asia-Pacific regions, focusing on two countries―Japan and South Korea―are reviewed. Both countries recommended the introduction of CED to overcome the barrier of lack of robust clinical evidence in the early stages of the introduction of a medical technology. However, each country has a unique approach to CED implementation that reflects the differences in establishment and healthcare and policy environments. Japan adopted a “Challenge Application (CA)” program in 2018, and South Korea introduced the “Conditional Selective Benefit (CSB)” program in 2014. Despite the positive effects of CED programs, their governance and implementation should be improved to benefit patients in both countries from the improved access to new and innovative medical technologies. To this end, CED practices in the United States (the USA) can provide insights on how to improve CED operations in both countries. In an effort to provide patient access to new innovative medical technologies while managing their budgetary impact and use, a range of alternative payment models, known as Managed Entry Agreements (MEAs), has been proposed (2). MEAs are not new; indeed, the pharmaceutical sector have actively pursued these arrangements with payers for decades to provide access to medicines where uncertainties exist (4). In general, MEAs are largely composed of finance-based agreements, such as price-volume agreements, and performance-based agreements (6), which includes risk-sharing and payment upon achieving certain outcomes. Coverage with evidence development (CED) programs are an example of one population-level performance-based MEA(7).While new innovative medical technologies have substantially contributed in saving lives and improving the health outcomes of patients, governments and payers around the world continue to face the challenge of providing access to these technologies while ensuring healthcare expenditure and budgets remain sustainable. Indeed, even in countries where the evaluations of the health benefits of a new technology are undertaken to justify its additional costs, such as cost-effectiveness analyses or value-for-money assessments, decisions on access and funding remain contentious, particularly where uncertainties about clinical effectiveness and value exist (9). CED, which is defined as a conditional coverage and payment program whereby temporary or interim funding and access to new medical technologies is granted under the condition that evidence is collected in parallel to answer the uncertainties surrounding their clinical and/or economic value, has been introduced to overcome these limitations (9).In the early stages of medical technology commercialization, a lack of sufficient and robust evidence results in uncertainties around the clinical and cost-effectiveness of the technology. As a result of such uncertainties, coverage determination procedures may be challenged, limiting access to patients in need. CED initiatives have been introduced to help answer uncertainties around costs and outcomes by generating evidence on the impact and utilization of new medical technologies in vitro diagnostics, and even wearable devices, are fundamentally different from pharmaceuticals in ways that have been well documented (12). Some of these differences include but are not limited to the speed and incremental nature of device innovation, device-operator interactions, and the learning curve effect and licensing or regulatory requirements. Of course, many of these key differences translate into differences in the amount and type of evidence that can be produced to demonstrate the safety and effectiveness of nondrug technologies, particularly the ability to conduct double-blinded randomized studies due to recruitment issues and ethical challenges (13). This lack of evidence leads to challenges for those undertaking health technology assessment (HTA) or making coverage decision. In Korea, if no clinical study evidence pertaining to a medical technology exists, it cannot even be the subject of HTA review.Nondrug medical technologies, such as implantable or disposable medical devices, surgical equipment, (14). Since then, the CMS CED program has evolved with the recent successful inclusion of transcatheter aortic valve replacement (TAVR) technologies and leadless pacemakers in this CED program (15). Other countries such as France (17) and Sweden (19) are also actively undertaking CED programs in various forms, demonstrating the usefulness of CED as an important tool to secure access to new drugs and medical technologies (20).As a mechanism to address the challenges confronting medical technologies, decision-makers and manufacturers have used CED programs to fill the gap of required evidence such as safety and effectiveness in the early market entry stage of medical technologies. In the United States (the USA), the concept of CED emerged over a decade ago when the Centers for Medicare and Medicaid Services (CMS) introduced the CED program in 2006In this study, the authors examined the operational characteristics of CED programs in Asia-Pacific regions, focusing on two countries ―Japan and South Korea (hereafter Korea)―both of which have similar universal healthcare systems and mechanisms to reimburse new medical technologies.Both Japan and Korea have universal social health insurance systems and numerous similarities in the way medical technologies are paid for, such as fee-for-service, limited operation of prospective payment systems known as diagnosis-related groups, and combined fee schedules for hospital and physician fees. Both countries have a functional category payment system for managing medical technologies, including a stand-alone payment for implantable or high-cost disposable devices, low-cost disposable devices and instruments paid under procedure fees, and a premium pricing rule for innovative new medical technologies by creating new functional categories. However, some differences exist between the two countries, for instance, how premium prices are determined .(21).Both countries adopted CED programs suitable for their own healthcare environment. Japan adopted the “Challenge Application (CA)” program, and Korea introduced the “Conditional Selective Benefit (CSB)” program. In April 2018, the Ministry of Health, Labour and Welfare (MHLW) of Japan released revised notifications outlining reimbursement schemes for medical technologies of which the CA program was newly established as one of the major revisions. The program was developed to address the difficulties of verifying the final assessment of coverage determinations for long-term implantable technologies or highly innovative medical technologies before they are listed for coverage and reimbursement. Under the CA program, medical technologies that lack appropriate clinical evidence at the initial listing stage are eligible for reevaluation after evidence has been generated. It provides an opportunity of earlier patient access and premium prices by proving clinical value with additional evidence In Korea, the Ministry of Health and Welfare (MoHW) introduced the CSB program in 2014 to provide preliminary benefits for services and items that required additional evidence to demonstrate its safety, effectiveness, or cost-effectiveness but has potential benefit to patient care. Before the introduction of the program, the services and items under the CSB program were generally determined as noncovered services or items resulting in out-of-pocket costs and often a large financial burden to patients.(22). The expert panel reports to the CSIMC, and it assesses and deliberates the reimbursement application of STMs, including the CA program (23). Manufacturers applying for new STMs can request to be included in the CA program if their application falls into the following group: B1, B2, B3, C1, and C2. In the case of C2, STMs are only subject to CA program, and the details of each category are described in The CA program evaluates clinical evidence generated during the post-market period. It was initiated to provide an opportunity for medical technology manufacturers to apply for the reevaluation of their technologies based on evidence that will be generated after the initial listing of the eligible technologies for coverage and reimbursement. The program was created as a response to a request from the medical technology industry. The development of the CA program involved detailed discussions in an expert panel of Special Treatment Materials . Findings and recommendations from the expert panel are then followed by an approval from the “Central Social Insurance Medical Council .” The CSIMC is an advisory body of MHLW, which deliberate over revisions of the health insurance system and medical fees Not all categories were subject to CA program in the beginning. Initially, the target of CA was limited to B3, C1, and C2 categories. After 2 years, the target was extended to B1 and B2 categories. Products listed before the introduction of the CA program were given the opportunity to apply for the program for certain period. B3, C1, and C2 categories listed before the initiation of the CA program were allowed to submit a CA program application by March 31, 2020. Medical technologies designated and listed as a B1 or B2 category between April 1, 2018, and March 31, 2020, can submit a CA program application until March 31, 2022. The process for reevaluation after granting CA eligibility is similar to that of C1 category application regardless of initial application categories.Applications are permitted for the manufacturers or distributors of medical technologies only. Manufacturers who want to apply for the CA program must submit a plan for clinical data collection and evaluation. There are three formal requirements for the CA program, which are outlined in (24).In Korea, there has been increasing demand on the coverage expansion of the national health insurance (NHI) system due to increased healthcare burden and high ratio of noncovered services and items. Since 2014, the government has implemented a CED system known as the “Selective Benefit (SB)” program to expand the health insurance coverage for four major diseases areas . SB mandates a high percentage of patient copayment rates ranging 30%, 50%, 80%, or 90% against normal patient copayment rate . The scope of coverage has been even further expanded with the introduction of the “Preliminary Benefit (PB)” program by the new government, which came into power in 2017. The new PB program is similar to the SB in terms of operation; however, the scope of items and services covered has been significantly expanded to noncovered services and items, with the exception of cosmetic and plastic surgery(26). The MoHW decides the requirements for the eligible services and items for CSB in advance by official announcement. In addition, only healthcare providers that meet the specific requirements, including prerequisite clinical experience with the technology and possessing physician and facility qualification, are permitted to use the CSB services and items for their patients. In addition, they have an obligation to generate and submit the clinical data of their patients treated with the technologies (27).The determination of eligible services and items for the SB program is made by considering clinical effectiveness, cost-effectiveness, and social demands on reimbursement coverage . TechnolUnder the CSB program, healthcare providers have an obligation to collect and report the clinical data to the Health Insurance Review and Assessment Service (HIRA). The HIRA, which has similar roles and responsibilities to the CMS in the USA, is accountable for operating the CSB program. Hospitals must obtain the approval from the MoHW and HIRA to participate in the CSB program and renew once a year to maintain its accreditation.(28). The manufacturer submitted a C1 category application for the pacemaker in 2007 and obtained a new functional category with 5% utility premium for managed ventricular pacing mode. However, the rATP algorithm was not adequately assessed at the time because of a lack of appropriate clinical evidence. Five years later, a next generation of pacemaker, which was equipped with improved functionality of conditional magnetic resonance imaging (MRI) scan compatibility and rATP algorithm, was introduced. A C1 category application for the new version of pacemaker was submitted and obtained a new functional category with 5% improvement premium based on the MRI compatibility in 2012. While the conditional MRI scan compatibility was evaluated as it was considered a major improvement, the rATP algorithm was not taken into consideration again.A pacemaker with enhanced functionality, namely, the reactive anti-tachycardia pacing (rATP) algorithm, was submitted for the CA program in June 2019. The rATP algorithm delivers atrial anti-tachycardia pacing (ATP) to terminate an ongoing atrial fibrillation (AF) episode after a programmed interval or when the rhythm organizes and/or slows (29). In addition, additional clinical evidence indicated that AF progression increases the risks of stroke and heart failure (32). After the introduction of the CA program, newly developed evidence was used to request a reevaluation of the rATP algorithm in 2019. The CA program was processed similar to that of a C1 category application, and the manufacturer showed the value of the enhanced functionality (rATP) that contributes to a reduction in the number of strokes and episodes of heart failure due to its suppression of AF progression. The MHLW acknowledged that the high probability of improvement in patient outcomes was due to the enhanced functionality of the pacemaker. The MHLW also created a new functional category and the provision of a 3% premium price in 2019. This was the first case of successful CA program implementation.The new clinical evidence to indicate the feature of rATP algorithm was published in an international journal in 2014 (33). Two years after the submission, in June 2015, a positive CSB determination on TAVR was made after a thorough review of the clinical and economic benefit of TAVR to patients with AS (34).As of August 2020, three new medical technologies have been selected for the CSB program in Korea. These technologies include TAVR for the treatment of aortic stenosis (AS), percutaneous left atrial appendage occlusion for AF, and the next-generation sequencing technology-based genetic panel test. The TAVR, a minimally invasive technology treating symptomatic AS, was the first CSB item selected by the MoHW/HIRA. Healthcare providers submitted an application for a new procedure creation for TAVR; simultaneously, the manufacturers of TAVR technologies submitted a reimbursement application for the technology coverage and payment in 2013 after the new HTA approval was made in 2012 by the National Evidence-based Healthcare Collaborating Agency (HTA agency) (35). In the case of TAVR, a national registry has been established to collect outcomes data and is currently in operation since 2017. An advisory group was established for the development and implementation of the TAVR registry protocol. The advisory group consisted of three clinical experts recommended by the Korean Society of Cardiology and Korean Society for Thoracic & Cardiovascular Surgery, and two experts in research methodology and statistics were added to form a total of eight members of the TAVR clinical and methodology advisory groups. Unlike foreign CED programs, key stakeholders, such as manufacturers and regulatory authorities , are not involved in the governance of data collection.Implementation method, records and management, and clinical data submission for CSB are roughly regulated by the law After the initial decision on CED implementation, a reassessment is made every five year to determine whether the CSB needs to be maintained or transformed into formal reimbursement coverage benefit without any conditions for evidence collection. However, if conducting an earlier reassessment by considering the content, characteristics, and effects of the CSB services and items is deemed necessary, the reassessment cycle may change. To date, the reassessment for TAVR remains in progress.The CSB program is limited to healthcare providers who have obtained approval from the MoHW/HIRA to participate by fulfilling the requirements specified in the program. As of December 2020, a total of 45 medical institutions have obtained approval for TAVR CSB implementation.While the demand for the revaluation of safety, effectiveness, and cost-effectiveness of medical technology is increasing at the post-market stage , CED is (36). This example provides insight and learning opportunities for countries that aim to introduce or improve CED programs in terms of transparency and collaborative commitment to generate evidences. In particular, when new medical technologies are introduced in the USA, local Medicare authorities determine whether to pay through local coverage determinations (LCDs) for a certain period of time; then, national coverage determinations (NCDs) are made. The CED program improves patient access to new medical technologies by directly incorporating them into NCDs without going through lengthy and cumbersome LCD process. In Japan and Korea, CED programs are similar in that they are applied nationwide; however, in fact, there is a big difference. In Korea, CSB is used under the restricted condition as divergence of SB program. As the patient copayment rate is quite high (80% for TAVR), it negatively impacts patient access to new medical technologies. In Japan, CED ensures patient access to new medical technologies, but lack of price premiums does not provide an incentive to companies to introduce new medical technologies. A premium price could be sought through post-market reevaluation after the development of robust local or international clinical evidence. Although the regulation for premium price determination currently exists, the ability to obtain a premium price for new and innovative medical technologies is currently very low , and this percentage has been decreasing since 2008 (37). The CED programs in Japan and Korea substantially differ from those in the USA, particularly in how they recognize the value of new medical technologies and also the presence of a patient copayment burden that unfavorably impacts patient access to new medical technologies.It would be insightful for Japan and Korea to look for improvement areas by referring to the USA where CED programs have been effectively operating for several years. The operation and governance of CED programs in the USA is not exclusionary, with the active participation of multiple key stakeholders at specified timepoints in the CED pathway. An example of a CED program in the USA is the case of TAVR, which was established in May 2012 (38). Multi-stakeholder discussions with government authorities meant that the registry was quickly established and incorporated variables that were to be used for the future evaluation of CED. In addition, an important characteristic of medical technologies, namely, the short product life cycle, was considered in the US CED program development (38). On the other hand, for CSB in Korea, the HIRA formed the advisory group to establish a systematic protocol for a TAVR registry in June 2017 (26), which was two years after the CSB program determination for TAVR was made. Unlike the case in the USA, the CSB program in Korea has been slow in communication because of lack of experience and preparedness. For the CA program in Japan, developing a registry is one option for applicants who want to generate evidence for reevaluation. If a registry is officially required, the evidence generation and collection plan is the responsibility of the applicant, and future reassessment by the MHLW is based on evidence collected by applicant. However, there have been no requirements for the establishment of a registry for CA to date.Another important insight Japan and Korea can gain from the US CED programs is the speed and collaboration by which these programs are operated. Indeed, discussions on the development of a Transcatheter Valve Therapy (TVT) Registry began in July 2011, before the medical technologies used for TAVR were even approved by the US Food and Drug Administration (FDA) (38). In Korea, an advisory group for the TAVR registry was formed by inviting six clinical experts from the relevant specialty societies, and two methodological and statistical experts were consulted to establish the registry protocol (26). Unfortunately, representatives from medical technology manufacturers who have extensive knowledge and expertise in their technologies, patient populations, and expected outcomes were not included in the group. In Japan, CA is based on a protocol developed by the manufacturer, and the target of the CA program is only for STMs, not for procedures. Therefore, the leading role and participation of the manufacturers, which are familiar with medical technology related with STMs, are appropriate in CA initiation and process. While the medical technology industry requested the MHLW to extend the CA program for procedures in the C2 category, this is still under consideration. As the CA program has only recently been established, more discussions are needed to identify the advantages and disadvantages of this program. In addition, because of a lack of transparency on the internal decision-making processes of these programs, applicants have been unable to gain insight into what factors enable success or, more importantly, what could be rectified in the case of negative submissions. In the case of Japan, successful CA cases are available through the public notification of the MHLW, and failure cases are not publicized from the health authority. In the case of Korea, so far, a total of three technologies have been designated as CSB, and there are no results of success or failure yet.Another key reason why the TAVR case in the USA is a successful example is the collaboration among multiple stakeholders. The Society of Thoracic Surgeons, American College of Cardiology, US FDA, and CMS participated in the creation of the TVT registry in partnership. Furthermore, they established the Stakeholder Advisory Committee to reflect multiple perspectives. The advisory committee includes a wide range of interest groups, including government, the medical technology industry, patient organizations, professional societies, etc(40).Both countries have opportunities to include more active collaboration and participation in developing and governing CED programs with multiple stakeholders to ensure that the future success and value of these programs are realized. In the meantime, we need to be aware of the challenges of CED implementation, including the difficulties of withdrawal of the technologies because of safety or ethical issues. In particular, safety issue is very critical because the safety and effectiveness of the technologies subject to CED has not been established yet at the initial stage of CED implementation. As an example, the CMS provided coverage of lung volume reduction surgery for Medicare patients enrolled in the National Emphysema Treatment Trial over 7 years. However, it turned out that the surgery was associated with an increase in mortality, and the number of surgeries has significantly decreased Both Japan and Korea have been implementing CED programs since 2018 and 2014, respectively. Each country has established and operated its own unique approach to CED implementation, reflecting the contextual differences in their healthcare and policy environments. It is encouraging that both countries have introduced CED as a method to overcome the barrier of lack of robust clinical evidence in the early stages of medical technology introduction. The medical technology industry is open to CED because it provides an alternative mechanism for patient access to promising technologies that do not yet meet current evidence requirements. Despite the positive impact of CED programs in both countries, improvements in their governance and implementation should be made. The CED case in the USA provides insight on how to improve the CED operation in both countries. In particular, stakeholder engagement during the early stages of development and collaboration and partnership among the relevant stakeholders, including treating clinicians, governments/payers, patients/consumers, and medical technology industry, are required. The improvement of CED programs in Japan and Korea will enable patients in both countries to benefit by improved access to new and innovative medical technologies.All authors are the employees of Company Medtronic and stockholder.Sang-Soo Lee and Jae-Eun Myung made substantial contributions to the conception and design of the work and drafted and critically revised it for important intellectual content. Yuji Tanaka, Hyunsook Choi, and Liesl Strachan revised the part of the manuscript regarding practical CED implementation in each country. Tomohiro Watanuki, Ji-Hyun Lee, and Hyojung Hwang helped edit and revised the manuscript. All of the authors approved the final manuscript.Not applicable


Emotional Literacy (EL) is a well-designed, field-tested curriculum that enhances social, emotional, and academic learning. A total of 565 students, 53% female, from 17 elementary schools, participated in the study. Approximately half of the students participated in the eight-week-long EL program, while the other half was in control conditions. Both the experimental and control group fulfilled the same set of trait and ability emotional intelligence measures in three timepoints: pre-, immediately after, and six months after the program. The effect of the EL program was different for girls and boys at different measurement points. Boys placed in experimental group improved their scores at both post-treatment measurements, meaning that they rated themselves and felt more emotionally competent after being a part of the EL program. Emotional understanding improved consistently with time, measured with both the Vocabulary of Emotions Test (VET) and Test of Emotional Understanding (TEU), regardless of the participation in the EL program. The effect of maturation was slightly more visible in girls, and girls had consistently better scores on the VET and TEU tests than boys. The feedback from school psychologists working with children was positive; they agreed that the children responded well to the activities and willingly participated. Under the umbrella term of emotional intelligence, introduced to the scientific community in the present form by Salovey and Mayer at the beginning of the 1990s , and popEmotional literacy refers to the ability to understand others’ emotions or the way others feel, and is a fundamental and important aspect of EI. Lately, this term has been replaced by the term social–emotional learning or SEL, with the word “learning” deliberately used to emphasize the fact that the acquisition of skills and attitudes is a process . Elias eThe Emotional Literacy (EL) program that was implemented in this study is a predecessor of the RULER program. The RULER was designed based on the Mayer and Salovey  four-braResearch into the benefits of SEL programs found that social and emotional learning in school-aged children (both the ones implemented in schools and extracurricular programs) is improving social and emotional skills, attitudes about self and others, connection to school, positive social behavior and academic performance; they also reduced student’s conduct problems and emotional distress ,16. ProgMost of the theories that explain behavioral changes share a central concept of self-efficacy ,19. BandThe effectiveness of the SEL program, like Emotional Literacy, has not been extensively studied in Croatia. Although several SEL programs were implemented in Croatian schools, there are only a few published studies ,24. The In total, 565 students from 17 elementary schools situated in different regions of Croatia participated in all three measurement sessions. Children attended the 5th and 6th grades. Approximately half of the students participated in the eight-week-long Emotional Literacy program, while the other half was in the control condition, meaning that they were given the same instruments as the experimental group but had no extra activities . Every sBoth the experimental and control group filled out the same set of measures: The Emotional Skills and Competence Questionnaire—Children’s form (ESCQ-C) ,26; The The Emotional Skills and Competence Questionnaire—Children’s form [never to 5—always). In the original version, ESCQ [n’s form ,26 is a on, ESCQ  has 45 ion, ESCQ . The scaon, ESCQ . The relThe Self-Efficacy Questionnaire for Children (SEQ-C) [social self-efficacy (perceived abilities in peer relationships and assertiveness); academic self-efficacy (perceived ability to manage one’s own learning behavior and master academic subjects); and emotional self-efficacy (the perceived ability to cope with negative emotions). It has 24 items, and each item is scored on a 5-point scale . The questionnaire was translated into Croatian by Vulić-Prtorić et al. [ (SEQ-C) ,28 measuć et al. .Vocabulary of Emotions Test (VET) [st (VET)  measuresst (VET)  hierarchst (VET) . The tesst (VET) .Test of Emotional Understanding (TEU) [ng (TEU)  is an abng (TEU) . It is ang (TEU)  structurng (TEU) . The TEUng (TEU) . A shortEmotional Literacy program, school psychologists were contacted with a request to participate in the validation study. Psychologist from 17 schools answered positively, and a booklet with detailed information regarding the validation procedures was sent to their e-mail addresses. Although authors [Limitation and Future directions.After receiving a short education about the The EL program validated in the study consisted of eight words/emotions . There are six steps for every word, which create a complete and holistic understanding of words. During sessions with children, school psychologists went through all eight feeling-words in the same order and same six steps, so that the program was implemented in the same way in all schools.Upon receiving the necessary permissions, a set of questionnaires was given to all participants (1st measurement point). Then, half of the children participated in the EL program, and others had only regular school subjects. Immediately after the last session of the program, all children filled out questionnaires (regardless of their participation in the program) and completed them again after 6 months. Therefore, we had three measurement points, before the implementation of the EL program (at the beginning of the school year), immediately after the last session of the program, and at the end of school year (approximately 5 months after the sessions).Permission for this study was obtained from Ethics Committee for Scientific Research from the Faculty of Humanities and Social Studies, University of Rijeka, Croatia.This study used a pre-test–post-test design, extended across nine months (from September to June), during the 2017/2018 academic year. Pre-test data were collected in September 2017 in a 1-h session before the beginning of the program. Post-test data were collected on two occasions, in January 2018 (immediately after the end of the program) and in June 2018 (approximately 5 months after the program). At all three timepoints, all participants  completed the surveys.To check if participation in the Emotional Literacy program had an effect on the measured variables, we conducted three-way mixed ANOVA with one within-group (time) and two between-group factors . We summarized all the findings in First, we checked the homogeneity of variance for all used measures, with Levene’s test. We also checked the sphericity with Mauchly’s sphericity test and applied the appropriate Huynh–Feldt correction of degrees of freedom. The results are presented in For the Emotional Skills and Competence Questionnaire (ESCQ-C) a statistically significant three-way interaction between group, gender and time was obtained, which means that simple group–gender interactions are different at different timepoints . As can Regarding the results for the Vocabulary of Emotions Test (VET), no statistically significant three-way interaction between group, gender, and time was obtained. A statistically significant two-way interaction between time and gender was obtained, as can be seen in Other two-way interactions were not significant. The statistical significance of a simple main effect was accepted at a Bonferroni-adjusted alpha level of 0.017. There was a statistically significant simple main effect of gender at all three testing timepoints, meaning that girls had better results on the Vocabulary of Emotions Test on all three measurement times, regardless of their placement in the experimental/control group.When results of the Test of Emotional Understanding (TEU) and the Self-efficacy questionnaire (SEQ-C) were compared, no statistically significant three-way interaction of group, gender, and time was obtained. There were no significant two-way interactions.To conclude, the effects of maturation  were obtained for Vocabulary of Emotions Test (VET) and Test of Emotional Understanding (TEU). Emotional understanding as measured with both tests improved consistently with time. There were no changes over time seen on the Emotional Skills and Competence Questionnaire (ESCQ-C) and the Self-Efficacy Questionnaire (SEQ-C).Emotional competence measured with ESCQ-C showed different group x gender patterns at different timepoints. Although two-way interactions were not statistically significant, boys placed in the treatment group showed an improvement in post-test measurements and this effect was not seen for girls. There were no effects of gender or treatment on self-efficacy. A significant interaction between gender and VET was found at different timepoints, suggesting that the effect of maturation was slightly more visible in girls. Additionally, girls had consistently better scores on the VET test than boys. Test of Emotional Understanding also showed that girls had better emotional understanding.touching, spiritless (only 9% of students knew the correct answer), monotonous, lonely, intolerant, pessimistic, tolerant, and shocked. The easiest word was abandoned, with 61% of answers being correct. After the program (Time 2 measurement), correct answers were under 30% for only four words, and over 50% of answers were correct for ten words. Similar results were obtained for Time 3 (5 months after the program).Since this program is expected to develop emotional literacy, answers on the Vocabulary of Emotions Test were analyzed in more detail. Although participation in the program did not have an effect on the student’s emotional vocabulary, our results suggest that it is very important to keep educating students on feeling-words. As can be seen in x group x gender) in the Emotional Skills and Competence Questionnaire, which showed that the effect of the Emotional Literacy program was different for girls and boys at different measurement points. Boys placed in the experimental group improved their scores at both post-treatment measurements, meaning that they rated themselves and felt more emotionally competent after being a part of the EL program—an effect that was not found for girls. Although the 2-way group of x gender interactions at all measurement points were not significant, it seems that there is an indication that the program may be more efficient for boys.The aim of our study was to explore the effects and possible benefits of the Emotional Literacy program on students’ emotional and social competences, emotional understanding and vocabulary, and perceived emotional, social, and academic self-efficacy. We found a significant three-way interaction  and Test of Emotional Understanding (TEU). Surprisingly, we found no gender differences in terms of perceived emotional competence and self-efficacy, although different studies show that girls usually perceive themselves better than boys ,38. FernBesides gender differences, maturation effects were also found for both tests measuring ability , suggesting that students had better emotional understanding (TEU), and emotion vocabulary (VET) at the end of the school year, compared to their results at the beginning of the school year. It is important to emphasize that the observed changes were small, and one school year is not a sufficient period for significant variations in EI to occur among adolescents. Esnaola et al.  analyzedAlthough we found no other significant three-way interaction, we believe that the necessity of the program is visible in some other respects. For example, students, on average, have a poor emotional vocabulary and know the meaning of from 11 to 13 words (out of 35). More than 80% of the answers to some words were wrong, which is very indicative and shows that greater attention should be paid to teaching students emotion-related or feeling words. The ability to correctly understand and use emotion words is an essential part of communication and vital part of emotional and social competence, both of which are particularly important in the period of early adolescence, when forming social relationships becomes of special value. Ekman  states tIn our study we found no significant effects of the Emotional Literacy program on either perceived or objectively measured emotional competence. There are several possible explanations for those findings and the most probable is that the program was too short for any significant change to occur. Due to the tough schedule in Croatian schools, it was possible to arrange only eight sessions, on average (covering eight emotions), which were held after regular school classes. The authors of the program strongly suggest that the program should be implemented as a part of regular school activities, and that it should last for at least the whole school year.Another major limitation is that participants were not assigned randomly to a condition; a school psychologist decided which class would participate in the program, and which class would only participate in the research part and fill in the questionnaires. Several reasons could have influenced this decision—the school psychologist could have chosen the class which they thought would benefit the most from the program  or a class they knew it would be easy to work with. They also may have had to choose a class which was not engaged in other school programs and had room for some extra activities.Moreover, several people were engaged in facilitating the sessions, and although they all had received the same detailed plan on how to work with students, we must assume that there were differences in their approach. An experimental design in which a classes are chosen based on similar characteristics would improve the power of the findings. Several other factors should also be controlled, such as the size of a school, or urban–rural location of a school. Although there were eight schools situated in cities and nine schools from smaller towns or rural areas, from different parts of the country, we did not control for the regional development or socioeconomic status of students and their parents, which could have had an effect on the results.Nevertheless, the feedback received from the school psychologists working with children was positive: they agreed that the children responded well to the activities and willingly participated. Additionally, especially important information for on-going curriculum reform in Croatia was obtained: the social and emotional learning courses must be planned for at least two academic years and should be part of the various regular subjects .


After a cardiac event, a large majority of patients with cardiac conditions do not achieve recommended behavior change targets for secondary prevention. Mental health issues can also impact the ability to engage in health behavior change. There is a need for innovative, flexible, and theory-driven eHealth programs, which include evidence-based strategies to assist patients with cardiac conditions with their recovery, especially in behavioral and emotional self-management.Back on Track web-based self-management program. In addition, this study will test whether there is enhanced benefit of providing one-on-one telephone support from a trained lifestyle counselor, over and above benefit obtained through completing the web-based program alone.The aim of this study is to determine the short- and longer-term behavioral and emotional well-being outcomes of the Back on Track program (without assistance) or “supported” completion of the Back on Track program . All participants will have access to the web-based Back on Track program for 2 months. Telephone sessions with the supported arm participants will occur at approximately 2 and 6 weeks post enrollment. Measures will be assessed at baseline, and then 2 and 6 months later. Outcome measures assessed at all 3 timepoints include dietary intake, physical activity and sitting time, smoking status, anxiety and depression, stage of change, and self-efficacy in relation to behavioral and emotional self-management, quality of life, and self-rated health and well-being. A demographic questionnaire will be included at baseline only and program acceptability at 2 months only.People who have experienced a cardiac event in the previous 12 months and have access to the internet will be eligible for this study (N=120). Participants will be randomly assigned to one of the two study conditions: either “self-directed” completion of the Recruitment began in May 2020 and concluded in August 2021. Data collection for the 6-month follow-up will be completed by February 2022, and data analysis and publication of results will be completed by June 2022. A total of 122 participants were enrolled in this study.Back on Track trial will enable us to quantify the behavioral and emotional improvements obtained and maintained for patients with cardiac conditions and, in particular, to compare two modes of delivery: (1) fully self-directed delivery and (2) supported by a lifestyle counselor. We anticipate that the web-based Back on Track program will assist patients in their recovery and self-management after an acute event, and represents an effective, flexible, and easily accessible adjunct to center-based rehabilitation programs.The Australian New Zealand Clinical Trials Registry ACTRN12620000102976; http://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=378920&isReview=trueDERR1-10.2196/34534 Cardiovascular disease is the primary cause of death and disability, both globally  and in ASecondary prevention, including behavior modification and mood management, is essential for people who have experienced a cardiac event . Up to 9Unfortunately, despite the known benefits of behavior change, a large majority of cardiac event survivors do not achieve recommended targets for secondary prevention, with high rates of unhealthy diets, physical inactivity, and resumption of smoking in the postevent year ,13,14. EWhile CR is recommended for all cardiac event survivors after an acute event  and has Up to 50% of cardiac event survivors have expressed the desire for flexible alternatives to center-based programs, many preferring home-based options, including eHealth . SystemaTo our knowledge, there are no programs that aim to equip people with CHD with cognitive and behavioral skills to maintain psychosocial and behavioral health in the long term. People need evidence-based strategies and tools to develop skills to self-manage their health in the longer term . ResearcBeating Heart Problems, which was underpinned by a framework of self-regulation theory and patient-centered care. Based on the principles and strategies of CBT and MI, the program was designed to provide cardiac event survivors with the cognitive and behavioral skills to self-manage their mood and health behaviors in the long term. A randomized controlled trial (RCT) of the Beating Heart Problems program, involving 275 cardiac event survivors, demonstrated that the intervention group showed a superior reduction in 2-year risk of a recurrent event, greater improvements in functional capacity and behavioral health, and greater reductions in depression incidence and severity, compared to the control group [Back on Track, is underpinned by the same frameworks and theories as the face-to-face program and includes modules that focus on both behavioral and emotional aspects of recovery. In a pilot study, the web-based Back on Track program was shown to be acceptable and useful in terms of self-management support following a cardiac event [With the aim of addressing gaps in secondary preventive care for people with CHD, the Australian Centre for Heart Health (ACHH) developed a face-to-face, group-based self-management program, ol group ,48. To oac event .Back on Track self-management program, and to test whether participants obtain enhanced benefit through the provision of one-on-one telephone support from a trained lifestyle counselor (supported arm), over and above benefit obtained through completing the web-based program alone (self-directed arm). By including 2 follow-up assessments, one on postprogram completion (2 months) and one at 4 months post program (6 months postenrollment), we aim to assess both immediate and sustained benefits.The aim of this study is to assess the benefits, in terms of behavioral and emotional well-being, of the web-based We hypothesize that participants randomly allocated to each arm of the trial will show significant improvement in their behavioral and emotional well-being, self-efficacy, and self-rated health from the preprogram to postprogram assessments, with benefits sustained 4 months after program completion. We also hypothesize that participants allocated to the supported program will demonstrate superior improvements in behavioral and emotional well-being, self-efficacy, and self-rated health compared to those allocated to the self-directed arm, at both the postprogram and follow-up assessments.Ethical approval was granted by the Deakin University Human Research Ethics Committee (2019-438). This project has been funded by the HCF Research Foundation. This study will be conducted and reported in accordance with the CONSORT-EHEALTH guidelines .Back on Track program or “supported” completion of the Back on Track program. The self-directed arm involves participants completing the web-based Back on Track program at their own pace without assistance. The supported arm involves the addition of 2 telephone sessions with a trained lifestyle counselor. The telephone sessions are protocol driven, with a written manual developed by a health psychologist.This project uses a 2-armed RCT design, with participants who register to be involved in the trial being randomly allocated to one of two arms: either “self-directed” completion of the Measures are assessed at three timepoints across the study: a baseline questionnaire is completed prior to participants receiving access to the web-based program, a postprogram questionnaire at the conclusion of the web-based program access (2 months after baseline), and a follow-up questionnaire 4 months later (6 months after baseline). Back on Track program, either self-directed or supported. By comparing outcomes for participants in the 2 groups, we will be able to determine whether the supported approach provides benefits over and above the self-directed use of the web-based Back on Track program. The result will then inform the optimal form of delivery of the Back on Track program for future rollout to people with CHD across Australia.An RCT design was chosen for this trial to accurately compare outcomes for the 2 modes of delivery of the web-based Back on Track program, which is currently available in English only. Participants are also required to have access to the internet and a computer or tablet.A total of 120 participants will be recruited into the trial, with 60 randomly allocated to each of the 2 arms of the trial. Participants will be adults (over 18 years of age) who have had an acute cardiac event, such as AMI, coronary artery bypass graft surgery (CABGS), or percutaneous coronary intervention (PCI), in the past 12 months. Participants are required to be fluent in English to comprehend the Participants are being recruited using both direct engagement via a nationwide social media recruitment drive, and indirect engagement via cardiac health professionals and community-based cardiac support organizations, namely HeartBeat Victoria and Heart Support Australia. A promotional flyer is used for all promotion and recruitment. The nationwide social media recruitment drive involves announcements about the trial on internet-based social media platforms such as Facebook, Twitter, and LinkedIn. HeartBeat Victoria and Heart Support Australia also promote the study to their members, who are all people living with heart disease. Details of eligibility criteria are provided on the relevant websites and organization promotional material. Likewise, the trial is being promoted among cardiac health professionals, based on a list of health professionals who are involved in cardiac rehabilitation services across Australia or have attended past ACCH training programs.In all cases of recruitment, both direct and indirect, potential participants are directed to the ACHH website for detailed information about the study and for instructions on how to register on the internet. Screening for eligibility is undertaken at the time of participant registration via the ACHH website using a set of brief screening questions; specifically, participants are asked to provide their name and contact details, type, and date of cardiac event and to confirm that they are able to read and understand English and that they have access to the internet and a computer or tablet device. All participants are asked to read, sign, and return the participant information and consent form (PICF) after registration and prior to randomization and being given program access. The PICF is emailed to participants via the secure web-based data management and survey program, Research Electronic Data Capture (REDCap), which they sign and return electronically to ACHH.Back on Track program is granted to participants for a 2-month period after they complete the baseline questionnaire.Once registered, participants are randomly allocated to either the self-directed or the supported arm of the trial using an independent and automatic random allocation numbering system. The person undertaking the random allocation is blind to group and participant details. All participants are then asked to complete the web-based baseline questionnaire, also disseminated using REDCap. The link to questionnaires is emailed to participants at each timepoint. Access to the Participants are asked to recomplete the main outcome measures again post program  and at 4-month follow-up (6 months after program access). Again, the REDCap survey link is emailed to participants.Our sample size is based on a systematic review of telehealth-based cardiac rehabilitation  on lifesBack on Track program begins with a goal-setting module, followed by 4 self-selected modules relating to (1) healthy eating, (2) physical activity and reduced sitting, (3) smoking cessation, and (4) cardiac blues and depression. Participants in both the self-directed and supported arms are asked to complete modules relevant to them, in their own time and in any order over the course of 2 months. Within each module, participants undertake exercises that enable them to review situations in their lives, and using concepts from CBT, to identify, challenge, and change the unhelpful thoughts and beliefs associated with risk factors and negative emotions. Participants are prompted to develop action plans and coping plans for implementing practical health behaviors, including goal-setting, and identifying motivators for change, resources, barriers, rewards, and relapse prevention strategies. Other activities include using strategies to increase self-efficacy, including stories of role models, and exploring ratings of importance of and confidence to undertake behavior change.The Back on Track program, it is important to present, up-to-date, relevant information on these guidelines and recommendations. This can help participants with aspects of their self-management, including goal-setting. Back on Track modules.The behavioral recommendations included in the modules  are all based on the current National Heart Foundation of Australia (NHFA) guidelines . AlthougSupported group participants are offered 2 telephone sessions with a trained lifestyle counselor to assist them in goal-setting and overall self-management while undertaking program modules. The session duration is 45-60 minutes for session 1 and up to 30 minutes for session 2. The first session occurs around 1-2 weeks postenrollment. The counselor begins the session with rapport development, then screens for anxiety and depression using the Patient Health Questionnaire-4 (PHQ-4) . Self-efMotivational interviewing strategies are used to support participants to articulate clear and achievable goals and to identify areas they wish to address in behavioral and emotional self-management. Action and coping planning interventions are used to support translation of goals into actions. At completion of the first session, the second telephone session is scheduled for a mutually agreed time (approximately 4 weeks after the first session). The second telephone session includes a reflection on anxiety or depression symptoms, self-efficacy, coping and action plans, relapse prevention, and maintenance of behaviors. In addition, if the counselor considers that the individual requires more intensive mental health support, referral options for counseling are discussed.An SMS reminder is sent 24 hours prior to each telephone session appointment to facilitate engagement. To ensure consistency and quality of program delivery, lifestyle counselors receive fortnightly supervision with an experienced registered health psychologist.Sociodemographic information is collected at baseline only. All participants complete the outcome measures of dietary intake, physical activity and sitting time, smoking status, anxiety and depression, stage of change, and self-efficacy in relation to behavioral and emotional self-management, quality of life, self-rated health, and well-being at baseline, post program , and at 4-month follow-up (6 months after program access). Program acceptability is assessed post program only using a 6-item scale developed specifically for this study.Depression is assessed using the PHQ-9 . The PHQPhysical activity and sitting time are measured using the Active Australia Survey (AAS)  with sitDiet quality is assessed using the Diet Quality Tool (DQT) , a 13-itSmoking is assessed on the basis of self-reports by asking participants “Do you smoke?” Current smokers are asked how many cigarettes they smoke per day. Former smokers are asked when they quit smoking.Anxiety is assessed using the 7-item Generalized Anxiety Disorder instrument (GAD-7) . The GADWell-being is assessed using the World Health Organization–Five Well-Being Index (WHO-5) . This isQuality of life is measured using the Short Form Health Survey (SF-12) . The SF-Self-efficacy is assessed using an 8-item scale, which has been designed specifically for this study. Participants indicate on a 5-point Likert scale how confident they are that they can make or sustain relevant behavioral and emotional changes.Participants are asked to indicate their readiness to change relevant behaviors on a 5-point Likert scale from not thinking about making changes to have maintained changes for more than 6 months. This scale has been developed specifically for this study using wording from the Stages of Change model.Basic sociodemographic , medical , and event-related information  is collected via a self-report questionnaire, using standard questions used in previous ACHH studies .P<.05. In the case that ITT produces a null finding, it is possible that this is due to participants in the supported group not participating in the additional telephone sessions . To test that hypothesis, we will then undertake treatment-received analysis  whereby only those who received the treatment in accordance with the protocol are included. This will enable us to determine whether any differences were obtained between those who received the self-directed treatment and those who received the supported treatment. P values less than .05 will be considered significant for the primary outcome and those less than .01 will be considered significant for secondary outcomes. The effect of the intervention on the primary and secondary outcomes at 6 months will be assessed using analysis of covariance (ANCOVA) models that include the baseline value of the outcome as a covariate and the group assignment (self-directed versus supported) as a categorical variable. The treatment effect, its effect size (Hedge g), and 95% CIs for the treatment effect and within-group changes from baseline to 6 months will be calculated from the ANCOVA models. The sensitivity of the results to missing data will be evaluated using a data-based multiple imputation procedure.In evaluating outcomes, there will be two groups of participants based on the mode of delivery: “self-directed” and “supported.” All statistical analyses will be performed on an intention-to-treat (ITT) basis with As the main outcomes, we will examine differences between the 2 groups in the proportion of participants who are:nondepressed at baseline, post program, and follow-up (based on a PHQ-9 score of <6) [classified as e of <6) ,56,achieving physical activity guidelines of ≥150 minutes of physical activity per week  at baselachieving healthy dietary guidelines of DQT scores >60%  at baselAmong smokers, the proportion of people who have quit smoking will be compared for the 2 groups.Change over time in scores on the GAD-7, WHO-5, and SF-12 and in self-efficacy and readiness to change behaviors and emotions will be compared between the self-directed and supported groups using repeated-measures analysis of variance. Multiple regression analysis, with groups entered as potential predictors, will be used to predict participants with greatest improvements in GAD-7, WHO-5, SF-12, and self-efficacy scores, and readiness to change behaviors and emotions.Proportions will be calculated for all items on the program acceptability scale.Recruitment began in May 2020 and concluded in August 2021. A total of 122 participants were enrolled in this study. Data collection, including 2- and 6-month follow-up is expected to be completed by February 2022, and data analysis and publication of results will be completed by June 2022.Back on Track program will assist people in their recovery after an acute event and will represent a flexible, easily accessible, user-friendly, and effective adjunct to center-based programs. Our findings will enable us to quantify the behavioral and emotional improvements obtained and sustained for people with CHD who participate in the Back on Track program, while also comparing two methods of delivery: one fully self-directed and the other supported by a lifestyle counselor.With cardiac event survivors at a heightened risk of a recurrent events and premature death, and with suboptimal attendance at center-based rehabilitation and secondary prevention programs, there is a need for innovative eHealth programs to support people in their behavioral and emotional self-management after an acute cardiac event. We anticipate that the


Borrelia burgdorferi sensu lato (sl) and to analyze their association with nonspecific symptoms. The study group comprised individuals with persistent IgM antibodies in the absence of IgG. The relation between ELISA values and time elapsed since past erythema migrans (EM) was analyzed. Previous antibiotic treatments were assessed. The association between persistent IgM and nonspecific symptoms was evaluated statistically. Specificity of IgM antibodies for outer surface protein C (OspC) of B. burgdorferi sl was examined by immunoblotting. Further, we investigated the cross-reactivity with Borrelia-unrelated proteins. Fifty-nine patients  were included in the study group. The mean IgM-ELISA values did not change significantly during follow-up (median 6.2 months). The mean ELISA value in the study group was dependent on time elapsed since past EM. Nonspecific symptoms improved significantly more often in patients with lower IgM ELISA results. Persistent IgM antibodies were specific for the C-terminal PKKP motif of OspC. Cross-reacting C-terminal PKKP antigens from both human and prokaryotic origins were identified. We demonstrate that the C-terminal PKKP motif plays a main role for the reactivity of persistent Borrelia IgM toward OspC. However, cross-reactivity to other eukaryotic and/or prokaryotic antigens may hamper the specificity of OspC in the serological diagnosis of Lyme borreliosis. Lack of improvement of nonspecific symptoms was associated with higher IgM ELISA values.The aim of the study was to investigate the etiology of persistent IgM antibodies against IMPORTANCE The reactivity of human IgM with the outer surface protein C (OspC) of Borrelia burgdorferi sensu lato is frequently used to detect Borrelia specific IgM in commercial immunoassays, and such antibodies usually occur in the early phase of the infection. We identified a group of individuals with persistent Borrelia IgM without symptoms of Lyme borreliosis. We used their sera to demonstrate that the C-terminal epitope of OspC binds the IgM. Strikingly, we found that the same epitope occurs also in certain proteins of human and environmental origin; the latter include other bacteria and food plants. Our experimental data show that these Borrelia-unrelated proteins cross-react with the OpsC-specific IgM. This knowledge is important for the development of serologic assays for Lyme borreliosis and provides a cross-reactive explanation for the persistence of Borrelia-IgM. Borrelia burgdorferi sensu lato (sl) is a two-step process comprising a screening test and a supplemental test  is based on clinical manifestation and supportive laboratory test results in the event of disseminated and late manifestations , 2. Detetal test . B. burgresponse . HoweverBorrelia commonly appears in Europe and North America  to study cross-reactions to Borrelia-unrelated antigens; (iii) to examine whether the number and the improvement of reported symptoms are associated with enzyme-linked immunosorbent assay (ELISA) values; and (iv) to determine the frequency of antibiotic prescriptions in patients with persistent anti-Borrelia IgM.The aims of the study were (i) to identify the antigens which are reactive in patients with persistent anti-Epstein Barr Virus (EBV), Herpes simplex virus 1 and 2 (HSV), and cytomegalovirus (CMV) were detected in four (7%), in two (4%), and in one participants (2%), respectively. All sera were negative for Parvovirus B19 (P19V) IgM. The samples were also tested for IgG antibodies against viruses, with 41 (69%) being positive for HSV1, 35 (59%) for CMV, 31 (53%) for P19V, and 13 (22%) for HSV2. IgG to EBV was detected in all samples.IgM antibodies to B. afzelii strain Pko and a version lacking the 20 C-terminal amino acids (variant 0), we ascertained by IB that all tested sera reacted with the full-length OspC but not with the deletion mutant . This finding allowed us to investigate the possibility of cross-reactions.The sera from the first 20 participants were used to test the specificity of the IgM antibodies toward a previously identified epitope of OspC comprising the final 10 C-terminal amino acids . Using fn mutant . To furtn mutant . Becausen mutant  caused nn mutant . The sten mutant , Fig. 3.Borrelia. However, when we applied the same search algorithm for the C-terminal “PKKP” tetramer, the number of non-Borrelia proteins increased to 2638 . From these hits, we selected two proteins, one of human origin (TPSAB1) and another of bacterial origin (bchI), for subsequent binding assays. We recombinantly expressed these two proteins in E. coli either as a full-length or a PKKP-deleted form and tested them for reactivity with three individual sera. Similar to our findings with OspC, the sera from patients with persistent anti-Borrelia IgM reacted to these antigens but lost reactivity when the C-terminal “PKKP” motif was deleted  showed that this sequence is specific for Lyme Thirty-two of the 59 patients reported a history of physician-determined EM and four of them had had two such episodes. The nonlinear relation between IgM ELISA values at the first visit and the time since last EM is shown in P = 0.0406).The differences in IgM ELISA values between patients with and without a history of EM were analyzed as shown in F test, P = 0.76).In total, 45 study participants reported nonspecific symptoms and the mean number of symptoms was 2.4 (range 0–7). Thirty-one participants reported joint pain, 5 joint swellings, 4 back pain, 31 muscle pain, 22 fatigue, 8 forgetfulness, 16 headache, 3 visual problems, and 11 had vertigo. Fourteen study participants reported no complaints. P = 0.0136)  .In total, 27 (46%) study participants had been treated with antibiotics because of persistent IgM before study inclusion. The treatment had been repeated in some participants because the IgM antibody levels had not decreased. Six participants had been treated twice and three had been prescribed a total of three treatment courses.Borrelia species that cause Lyme borreliosis (LB) in Europe differ from those in the United States, and this affects serological diagnosis  and synaptojanin-2 isoform X5. As surrogates of a potential cross-reactive autoantigen and a cross-reactive pathogen, we recombinantly expressed human TPSAB1 and P. aeruginosa Mg-chelatase with and without the C-terminal “PKKP,” respectively. Strikingly, the sera of patients with persistent IgM recognized both wild-type proteins but not when PKKP was deleted. Thus, it is tempting to speculate that IgM persistence in LB potentially originates from a previous infection with Lyme borreliae but is kept up in some individuals by continuous stimulation with cross-reactive autoantigens or antigens from other microorganisms or environmental factors. In respect to the latter, components of our diet could also contribute to the phenomenon of IgM persistence. For example, glutathioine transferase of important crops, such as common wheat (Triticum aestivum) and barley (Hordeum vulgare) also feature a C-terminal “PKKP”. Finally, polyclonal stimulation of B cells by nonrelated agents could also elicit IgM antibodies against Borrelia, as described for Parvovirus B19  has been suggested . The participants gave their written consent.Borrelia-specific IgM antibody reactivity. The main inclusion criterion for participation in the study was a positive Borrelia IgM and a negative Borrelia IgG tested by ELISA and IB at three independent time points. Participants were enrolled between January 2012 and December 2014. None of the participants had symptoms of LB according to published clinical case definitions (Borrelia IgM were used as a control group. Blood was drawn before antibiotic treatment. Four (28%) of these participants were male and 10 (72%) female, with a mean age of 46 years (range 30–71 years).The participants were enrolled at the outpatient clinic of the Institute for Hygiene and Applied Immunology of the Medical University of Vienna. They were referred by physicians or self-referred mainly because of unspecific complaints and/or All study participants were seen by the first author of the study. At the initial visit, the participants were asked about the history and exact date of physician-diagnosed EM. The nonspecific complaints were documented with a questionnaire and included joint pain, joint swelling, back pain, muscle pain or muscle cramp, fatigue, forgetfulness, headache, visual disorders, vertigo, and sensory disorders. The participants were also asked about previous antibiotic treatment for IgM persistence. Follow-up visits were scheduled 4 weeks and 6 months after the initial visit. At the 6-month follow-up visit, the participants were asked whether their symptoms had improved or deteriorated compared to their physical condition at study inclusion.Borrelia-specific VlsE peptide and outer surface protein (Osp) C antigen. The persistence of IgM was defined by a repeated positive test result at the 4-week follow-up visit and the 6-month follow-up visit. All results identified as >200 AU/ml were presented as 200 AU/ml. For inclusion in the study, a negative IgG ELISA was required at all three time points. Each patient was also tested by IB . IgM reactions to the following antigens were measured by automated, photometric reading of the visualized nitrocellulose membranes: VlsE, p25 (OspC), p39 (Borrelia membrane protein A), and p41. Presence of OspC, VlsE, or p39 band, either alone or in combination, was considered as a positive IB result.The Borrelia ELISA manufactured by medac  is based on The presence of cross-reactive viral antibodies was tested by IB  according to the manufacturer’s instructions. The investigation focused on HSV, CMV, EBV, and P19V.Borrelia burgdorferi sl. Their IgM-positive sera (n = 14) were used as controls for the study group with persistent IgM antibodies. 10 samples were also IgG positive.Patients with current EM were tested by ELISA and IB for IgG and IgM against E. coli host strains TOP10F´  or NEB 5-alpha  were used and cultivated in lysogeny broth medium at 37°C. The primer sequences used for cloning are listed in Table S1 in the supplemental material. Recombinant full-length OspC and mutant variants  giving rise to vectors pRSET-C-bapko-OspC or pRSET-C-bapko-variant 0. Correct insertion of the respective amplicons was confirmed by sequencing . The resulting vector pRSET-C-bapko-OspC was used as a template for site-directed mutagenesis (sdm) using the Q5 site-directed mutagenesis kit  as instructed by the manufacturer. The primers for sdm were designed with NEBase Changer v.1.2.6.  and are listed in Table S2. The clones were screened for the correct mutation by sequencing . For expression cloning of recombinant human tryptase 1 (TPSAB1), the myeloid cell line THP-1 was cultivated as previously described , in-house strain P. aeruginosa 26071 was used. After the initial isolation, this strain was cultivated in brain heart infusion medium at 37°C. Before cloning of bchI, the presence of the C-terminal “PKKP” was confirmed by sequencing using primers P_aeruginosa_mg_seq_fwd and P_aeruginosa_mg_seq_rev. Subsequently, bchI was cloned into pRSET-A as described above, using primers Mg-Chelatase_fwd and Mg-Chelatase_rev. Deletion of PKKP was achieved using primers MgCh_ΔPKKP_fwd and MgCh_ΔPKKP_rev in combination with the Q5 site-directed mutagenesis kit  as above.For cloning of E. coli Bl21(DE3)pLysS  or Rosetta 2 . After expression, the six histidine (His-) tagged proteins were purified by gravity flow chromatography using Ni-NTA agarose .For protein expression from the cloned genes, the inserts were sub-cloned into Purified proteins were separated by SDS-PAGE and blotted on a nitrocellulose membrane, which was blocked with Tris-buffered saline-Tween 20 solution  containing 5% wt/vol milk powder. Visualization of successful expression of the recombinant proteins was confirmed using a His-probe antibody . The membrane was developed using Pierce ECL Western Blotting Substrate . For testing patient sera, the respective serum was diluted 1:100 in TBS-T and the membranes were incubated for 1 h at room temperature. After thorough washing, the membrane was incubated for 1 h with an anti-human-IgM antibody conjugated with alkaline phosphatase . The bands were visualized with 1-Step NBT/BCIP Substrate Solution .P values were adjusted according to a resampling-based max-T procedure.Differences in IB band intensities between the study group and EM patients were assessed by two-sample t-tests. To correct for multiple testing, the To analyze the association of EM and IgM development in the study group, a linear mixed model was calculated with ELISA as the dependent variable and history (yes or no), time during follow-up, and the interaction between the latter two as independent variables.An exponential regression model was estimated to describe the nonlinear association between ELISA and the time since last EM (in years).The relation between the ELISA value at the first visit and the number of nonspecific symptoms was calculated using one-way ANOVA.A linear mixed model was estimated modeling ELISA as a function of the independent variables improvement of symptoms (yes or no), time (in months), and the interaction between time and improvement. A random intercept term for patients was included in both mixed models to account for the correlation of repeated measurements from the same patient.The significance level for all tests was set to 5%. The software package R 3.3.0 was used for all analyses . The mul


BackgroundLymphovascular invasion (LVI) is included in the criteria of high-risk stage II colon cancer. However, there are limitations to detecting LVI by routine hematoxylin and eosin (H&E) staining. Alternatively, immunohistochemistry (IHC) for the lymphatic endothelial marker D2-40 may help detect LVI, but its prognostic significance remains unknown. This study aimed to evaluate the prognostic significance of LVI, detected by IHC for D2-40, in low-risk stage II colon cancer. Materials and MethodsA total of 69 patients with low-risk stage II colon cancer were tested for D2-40 to assess LVI. Then, the relationships between IHC-detected LVI and clinical outcomes, including disease-free survival (DFS) and overall survival (OS), were analyzed using both univariate and multivariate analyses. ResultsIHC for D2-40 revealed that 24 out of the 69 cases (34.78%) had LVI-positive tumors. IHC-detected LVI was significantly associated with adverse clinical outcomes on univariate analysis, i.e., both reduced DFS (P = 0.002) and OS (P = 0.0163). In multivariate analysis, controlling for age, IHC-detected LVI remained a significant predictor of reduced DFS with a hazard ratio (HR) of 3.37 and a 95% confidence interval (CI) of 1.39-8.15 (P = 0.007) and OS .ConclusionsOur results demonstrated that IHC analysis for D2-40 enhanced LVI detection in patients with low-risk stage II colon cancer and that cases with a missed diagnosis of LVI by routine H&E staining had adverse clinical outcomes, that is, reduced DFS and OS.  Colon cancer is the third most commonly diagnosed cancer  in both sexes combined worldwide, accounting for 1,096,601 new cases and 551,269 deaths in 2018 . Strateg Lymphovascular invasion (LVI) represents one of the criteria for a high-risk condition. LVI is an independent risk factor for unfavorable outcomes -11. CurrStudy populationThis study was performed at the Department of Pathology, Faculty of Medicine, King Chulalongkorn Memorial Hospital . We used the hospital database to retrospectively review patients’ electronic medical records, surgical reports, and pathological slides. The Institutional Review Board at the Faculty of Medicine, Chulalongkorn University, approved the study protocol for using tissue and clinical data . A total of 69 patients diagnosed with low-risk stage II colon cancer between 2007 and 2014 were enrolled in the study. All of them had been treated with colectomy and lymph node resection and had available paraffin blocks as well as official pathology reports. The study excluded cases with fewer than five years of follow-up. Data regarding age at initial diagnosis, gender, histological grade or subtype, date of first recurrence, and date of death were collected from electronic medical records and official surgical pathology reports.IHC procedureAll original H&E slides were examined by two pathologists and all of the faded slides were recut to ensure the absence of LVI and the other high-risk features. To select two paraffin blocks from each patient for IHC staining, we targeted the advancing front of the tumor and the area with tumor budding. IHC analysis was performed using the commercially available lymphatic endothelial marker podoplanin  detected with EnVision FLEX Visualization Systems  . Briefly, after deparaffinization and rehydration, the sections were pretreated with tris aminomethane-ethylenediaminetetraacetic acid (Tris-EDTA) buffer (pH 9.0) at 95°C for 20 minutes. After the sections were cooled down and immersed in Tris-EDTA buffer, they were preincubated with peroxidase blocking reagent (FLEX) for five minutes. The sections were then incubated with the ready-to-use anti-D2-40 antibody for 20 minutes, followed by incubation with horseradish peroxidase-labeled secondary antibody (FLEX). The complex was visualized with 3,3’-diaminobenzidine tetrahydrochloride solution (FLEX DAB+ chromogen), and hematoxylin was used as the nuclear counterstain. Each run included appropriate positive and negative controls.LVI assessmentAll D2-40-stained slides were evaluated for LVI by an experienced gastrointestinal pathologist blinded to clinical data. LVI was defined as the presence of at least one tumor cell within D2-40-stained lymphatic spaces, as shown in Figure Statistical analysisPatient characteristics, including demographic data and clinicopathological parameters, were analyzed by descriptive statistics. The two-sample t-test was used to compare mean values of numerical variables  between groups . Categorical variables, for example, frequency distributions between LVI-positive and LVI-negative cases, were evaluated using the chi-square or Fisher exact test, as appropriate.Survival analysis was conducted to evaluate the clinical outcome of patients with low-risk stage II colon cancer. Disease-free survival (DFS) was defined as the length of time patients lived without any evidence of the disease and was calculated from the date of colon cancer diagnosis to the date of first recurrence or death. Overall survival (OS) was defined as the length of time from the date of colon cancer diagnosis until death. DFS and OS were estimated by Kaplan-Meier survival analysis, and the log-rank test was used to assess statistical significance between LVI-positive and LVI-negative cases. To evaluate the independent effect of IHC-detected LVI, we performed univariate and multivariate Cox proportional hazard regression analyses. Hazard ratios (HRs) were calculated, and 95% confidence intervals (CIs) were also presented. Proportional hazard assumptions were verified using Schoenfeld residuals and log-log plots. All P values were two-sided, with statistical significance evaluated at the 0.05 α level. All analyses were performed using Stata Statistical Software: Release 15 .Patient characteristicsThe study enrolled 69 patients with low-risk stage II colon cancer, including 37 men (53.62%). The overall median age at initial diagnosis was 66 (range 39-90) years, and nearly two-thirds of the patients  had well-differentiated tumors histologically.Using IHC for D2-40, we identified 24 (34.78%) participants with LVI. There was no statistically significant relationship between the presence of LVI (as detected by D2-40 immunostaining) and clinicopathological variables, including age at initial diagnosis, sex, and histological grade or subtype. Table LVI detected using D2-40 is associated with reduced DFS and OSAt the end of the study period, 62 (89.86%) patients were alive (48 without colon cancer and 14 with colon cancer), and there were seven (10.14%) deaths (five without colon cancer and two with colon cancer). Kaplan-Meier regression analysis and log-rank test revealed a statistically significant association between the presence of IHC-detected LVI and DFS (P = 0.002) Figure . The ratIn our univariate Cox proportional hazard model, age at initial diagnosis was shown to be a statistically significant factor impacting both DFS  and OS . However, in multivariate Cox regression analysis, IHC-detected LVI continued to be a significant predictor of reduced DFS  and OS . Table The main objective of this study was to determine whether additional LVI detected by IHC staining for D2-40 significantly correlated with worse outcomes in a cohort of patients diagnosed with low-risk stage II colon cancer.LVI detectionOur study supports the results of several previous studies reporting that IHC analysis, compared with conventional H&E staining, significantly increased the frequency of LVI detection in colon cancer. Wada et al. found that D2-40 immunostaining identified LVI in 20 out of 120 patients with T1 rectal cancer 16.67%), while H&E staining detected LVI in only seven cases .67%, whi. Ervine Association between IHC-detected LVI and clinical outcomeBased on several studies, LVI positivity in colorectal cancer is widely accepted as an independent indicator of unfavorable prognosis -11. AccoThe major limitations of our study were its single-institution, retrospective design, and a quite small number of participants. Therefore, the risk of false-positive results, or type I errors, may be concerned. Nonetheless, our results are in agreement with several related studies. In summary, IHC staining for the lymphatic endothelial marker D2-40 significantly enhanced LVI detection in patients with low-risk stage II colon cancer, and LVI-positive cases failing to be detected by routine histology had significantly worse clinical outcomes . Thus we propose the use of D2-40 immunostaining for the evaluation of LVI in every LVI-negative case by focusing on the advancing front or the tumor budding.