A novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the source of a current pandemic (COVID-19) with devastating consequences in public health and economic stability. Using a peptide array to map the antibody response of plasma from healing patients (12) and heathy patients (6), we identified three immunodominant linear epitopes, two of which correspond to key proteolytic sites on the spike protein (S1/S2 and S2’) known to be critical for cellular entry. We show biochemical evidence that plasma positive for the epitope adjacent to the S1/S2 cleavage site inhibits furin-mediated proteolysis of spike. Th. Thmer pThe S protein of SARS-CoV-2 shares 76% homology with the SARS-CoV-1, , 53 and mer peptide, vide infra) and should thus be broadly neutralizing, since this proteolysis is critical for infection.Analysis of 12 different plasma samples from SARS-CoV-2 infected patients and comparison to 6 samples from uninfected patients clearly highlighted a strong response to specific epitopes . The thrAnother epitope abundantly detected only in healing patients was the 787–822, a peptide segment extending at the periphery of the solvent exposed part of the protein . It has Finally, the epitope 1147–1158 is found at the C terminus of the spike protein. The structural data reported thus far did not suggest a defined structure for this portion of the S protein. This epitope extends from the helix bundle 1140–1147  and had One limitation of epitope mapping with a peptide array is that it is restricted to linear epitopes. Antibodies binding to the RBD have been shown to participate in interactions spanning multiple peptide fragments. Indeed, we did not observe a strong response to linear peptides in the RBD. A control experiment with AI334/CR3022 antibody , 71 showTo validate the results observed on the microarray, a peptide (655–672) was synthesized as a biotin conjugate for pull-down and ELISA experiments. The sequence corresponding to 655-672-biotin and a scrambled version of the biotinylated peptide were individually immobilized on agarose streptavidin beads. Beads were exposed to serum from patients that were either positive or negative for that epitope based on the microarray data and subsequently treated with anti-Human-IgG-FITC. The fluorescence of the beads was quantified by confocal microscopy . As can Next, an alanine scan was performed to assess the contribution of individual amino acids to the interaction with the antibodies of two of the COVID positive patients containing antibodies for this epitope (Sample number 1 and 6) at two different dilutions (1 to 100 and 1 to 400). For this purpose, 17 different peptide-PNA conjugates were synthesized, replacing one amino acid at the time with Ala  and measN-glycosylated by host-derived glycans (22 N-glycosylation sites) with a potential role in camouflaging immunogenic protein epitopes Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: No**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The manuscript entitled “Identification of immunodominant linear epitopes from SARS-CoV-2 patient plasma” describes a full scan of the linear epitopes of the spike of SARS-CoV- 2 with plasma from COVID19 patients collected 3 weeks after the PCR diagnosis. The scanning is done using 12-residue frames overlapping by 6 residues. The design of the experiments is standard and sufficiently reliable. The PNA attachment of the peptides to the microarray chip may produce neoepitopes sometimes but any microarray technique would create unnatural molecular context so this is an inevitable risk. The authors identify three epitopes, two of which overlap with cleavage sites crucial for the conformational change leading to fusion. The further conformation of the binding of patients’ plasma in different molecular context verifies that the actual interaction depends on the peptides themselves. Unfortunately, none of the validation tests addresses the actual binding to the native protein. Although, the hypothetical epitopes are selected on the basis of reactivity correlating with the disease, correlation is not causation. The necessity of native protein binding assay is dictated by the abundant glycosylation of the spike which occludes the greater part of the protein surface ). The fact that p655-672 and p787-822 are adjacent to cleavage sites suggests that those parts of the polypeptide chain are sufficiently exposed. Still the conformational nature of the typical B cell epitopes requires that results of such linear epitope scanning be validated by a binding assay using the native protein. The logic that antibodies targeted away from the RBD and to the region of the cleavage site would prevent viral entry and, thus, ADE is contradicted in the case of the p655-672 by previous studies on SARS -CoV which showed that ADE is promoted by antibodies to the conserved epitope 611LYQDVNCT618 (the numbering is that of SARS-CoV-2) which is adjacent to the epitope p655-672. My humble opinion is that these contradictions need to be addressed in the final version of the paper.Reviewer #2: This relatively straight forward report examines COVID-19 convalescent plasma for reactivity with peptide (12 mer) array spanning the major structural protein of SARS COV-2. The authors identify three linear epitopes reactive within a sample size of 12. The reactivities are validated by agarose bead, ELISA and alanine scanning. A major short coming of the study is that the convalescent plasma is not particularly well characterized for overall reactivity to SARS COV-2 or neutralizing potential. For that reason, the study is only of marginal significance.Comments1. The abstract is insufficiently detailed to be useful to the readers. The authors should refer to journal guidelines and expand on Methods, Results and Conclusions.2. Figure 5E should ideally display individual data points within the bar graph3. The manuscript is missing a single figure at the end that aggregate the results and provides a better sense of frequency of reactivity of the peptides. Statistical analysis should be included as well.4. There is no mention of how glycosylation might affect linear epitope accessibility.5. Finally, while the authors comment on viral neutralization, there the convalescent sera used in the study was not tittered for endpoint (EC50) or neutralizing activity (IC50). Both would have advanced the study considerably.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  30 Jul 2020The response is provide in the cover letter. The text below is copied from the cover letter (without the formatting to distinguish reviewer comment from the response).Dear Prof Mantis, dear editor,Thank you for considering a revised version of the manuscript addressing the point raised by you and the reviewers. The most significant changes in the manuscript is the addition of biochemical evidence that plasma from patient positive for the epitope adjacent to the furin cleavage site does indeed inhibit the furin-mediated proteolysis of spike whereas plasma negative for this epitope does not. The second most important change is a discussion on the glycosylation sites; this discussion is aided by publications that appeared during the review process (new reference: 64 and 72). The frequency of epitope occurrence has been added to the conclusion and a table summarizing all data has been added (Table S1). Following your comment and the comment of reviewer 2, the plasma of two patient positive for epitope 655-672 were tested for neutralization and found to be neutralizing. However, we wish not to include this data because we cannot ascertain which antibody(ies) from the pool contribute(s) to the neutralization. As discussed in the manuscript, we acknowledge that the technique is limited to linear epitopes and might miss neutralizing antibodies directed at non-linear epitopes. One antibody targeting the receptor binding domain was reported to interact with multiple peptide across the RBD surface  and control experiments with this antibody only revealed weak binding, i.e. the presence of such antibody might be missed in the patient analysis. A point-by-point response is provided below. A marked version of the manuscript is submitted to facilitated the analysis of the changes. ____________________________Review Comments to the AuthorReviewer #1: The manuscript entitled “Identification of immunodominant linear epitopes from SARS-CoV-2 patient plasma” describes a full scan of the linear epitopes of the spike of SARS-CoV- 2 with plasma from COVID19 patients collected 3 weeks after the PCR diagnosis. The scanning is done using 12-residue frames overlapping by 6 residues. The design of the experiments is standard and sufficiently reliable. The PNA attachment of the peptides to the microarray chip may produce neoepitopes sometimes but any microarray technique would create unnatural molecular context so this is an inevitable risk. The authors identify three epitopes, two of which overlap with cleavage sites crucial for the conformational change leading to fusion. The further conformation of the binding of patients’ plasma in different molecular context verifies that the actual interaction depends on the peptides themselves. Unfortunately, none of the validation tests addresses the actual binding to the native protein. Although, the hypothetical epitopes are selected on the basis of reactivity correlating with the disease, correlation is not causation. The necessity of native protein binding assay is dictated by the abundant glycosylation of the spike which occludes the greater part of the protein surface ). The fact that p655-672 and p787-822 are adjacent to cleavage sites suggests that those parts of the polypeptide chain are sufficiently exposed. Still the conformational nature of the typical B cell epitopes requires that results of such linear epitope scanning be validated by a binding assay using the native protein. � The point is well taken. Beyond the model proposed by Hyeonuk Woo et al 2020, experimental work characterizing the glycosylation pattern of spike has now been reported  as well as an EM structure showing the orientation of the glycan . The text has now been revised with a discussion including this new information. As detailed in the text, these glycosylation sites are not anticipated to preclude binding to the identified epitopes. Regarding the necessity of native protein binding assay, all tested samples tested positive in a ELISA assay against S1. To test the binding of the specific antibodies would require antibody purification which is not possible from the samples available. We are currently raising antibodies to these epitopes and the raised antibodies will clearly need to be validated for native protein binding, but this work extends beyond the scope of the present manuscript.Text added to the manuscript: It should be noted that the surface of SARS-CoV-2’s spike is heavily N-glycosylated by host-derived glycans (22 N-glycosylation sites) with a potential role in camouflaging immunogenic protein epitopes.[72] Position N657, which is part of the identified epitope (655-672) adjacent to the furin cleavage site is glycosylated. The alanine scan indicated that this position does not contribute significantly to epitope-antibody interaction. A recently reported cryo EM structure with the 657-N-linked GlcNAc (6ZGE[64]) visible shows that the glycan point away from the epitope. Furthermore, a proteomic analysis showed that this position is unoccupied by a glycan in 16% of the monomers (i.e. nearly 50% of trimeric spike have an unoccupied N657) [72]. Glycosylation has also been reported at the edge of the other two prominent epitopes . The fact that antibodies against these epitopes were observed in convalescing patients suggest that the glycans do not effectively shield access to these segments of spike. In the case of N801, the glycan projects away from the identified epitope [64]; in the case of N1158, there is no structural information available to date.The logic that antibodies targeted away from the RBD and to the region of the cleavage site would prevent viral entry and, thus, ADE is contradicted in the case of the p655-672 by previous studies on SARS -CoV which showed that ADE is promoted by antibodies to the conserved epitope 611LYQDVNCT618 (the numbering is that of SARS-CoV-2) which is adjacent to the epitope p655-672. My humble opinion is that these contradictions need to be addressed in the final version of the paper.� We agree that the statement in the original version was confusing and the sentence has now been removed. The point that we wanted to make was that an antibody that would inhibit furin-mediated cleavage of spike might protect against ADE by preventing the required conformational changes to enable membrane fusion, irrespective of a binding-only antibody or RBD-targeted antibody redirecting the virus to FC-binding cells. This point is now strengthened by the recent publication  showing the importance of furin-mediated cleavage to achieve the conformation leading to high ACE2 affinity and membrane fusion. The point is further strengthened by the demonstration that plasma positive for epitope 655-672 inhibits cleavage whereas plasma negative for this epitope does not. Regarding the precedent for ADE mediated by 611LYQDVNCT618 in SARS-CoV-1, this epitope points away from the furin-cleavage site and would most likely result in binding-only antibodies. However, this discussion is immaterial since this epitope was not identified in the present study. Text added to the manuscript regarding the furin-protection assay: Based on the importance of the furin-mediated proteolysis, we next asked if plasma from patient positive for epitope 655-672 could protect the spike protein against proteolysis. To this end, the spike protein was labeled with Dylight 549 for visualization following SDS-PAGE. Treatment of labeled spike with furin for 20, 45 and 60 min afforded a progressive formation of two new lower molecular weight bands consistent with a single proteolytic event  and showed that 45 min was sufficient for nearly quantitative proteolysis under these conditions. Addition of plasma prior to the analysis does slightly alter the migration of the spike protein on the gel due to the increased protein loading however, the fluorescence scan still enabled a selective and unambiguous identification of the spike protein on the gel . Performing the proteolytic experiment with furin in the presence of plasma from a patient positive for epitope 655-672  showed a complete protection against proteolysis while plasma from a patient negative for this epitope (sample 10) did not . The comparison with a second sample from a patient positive for this epitope (sample 1) and the plasma from a healthy individual (sample 14) showed the same result . These experiments support the fact that antibodies binding to epitope 655-672 are protective against furin-proteolysis of spike. This protection could be highly relevant in mitigating ADE by preventing viral entry irrespectively of antibody-mediated cellular interactions.Reviewer #2: This relatively straight forward report examines COVID-19 convalescent plasma for reactivity with peptide (12 mer) array spanning the major structural protein of SARS COV-2. The authors identify three linear epitopes reactive within a sample size of 12. The reactivities are validated by agarose bead, ELISA and alanine scanning. A major short coming of the study is that the convalescent plasma is not particularly well characterized for overall reactivity to SARS COV-2 or neutralizing potential. For that reason, the study is only of marginal significance.Comments1. The abstract is insufficiently detailed to be useful to the readers. The authors should refer to journal guidelines and expand on Methods, Results and Conclusions.� We thank the reviewer for pointing out this shortcoming, the abstract has now been revised. 2. Figure 5E should ideally display individual data points within the bar graph� Figure 5E has been revised to show individual data points.3. The manuscript is missing a single figure at the end that aggregate the results and provides a better sense of frequency of reactivity of the peptides. Statistical analysis should be included as well.� A table summarizing all results as well as the quantification of microarray data has now been included (Table S1)4. There is no mention of how glycosylation might affect linear epitope accessibility.� This is a good point that was also raised by reviewer 1 which was not discussed in the original submission for lack of robust experimental evidence regarding glycosylation pattern. Important new publications addressing this question have been published during the review process. This information is now part of the discussion . 5. Finally, while the authors comment on viral neutralization, there the convalescent sera used in the study was not tittered for endpoint (EC50) or neutralizing activity (IC50). Both would have advanced the study considerably.� The plasma from two patients positive for epitope 655-672 was tested for neutralization and found to be neutralizing (IC50) at 1:80 dilution. However, this information can be misleading and overinterpreted since we cannot be sure that the neutralization does not come from an antibody that is not detected using a linear epitope scan. As discussed in the manuscript, a control experiment with AI334/CR3022, an antibody that binds tightly to the RBD with interactions spanning multiple peptide fragments only gave weak binding using the linear peptide scan. In light of this information, any correlation between the antibody profile and neutralization activity may be misleading.Thank you for your time and efforts during these challenging times, -Nicolas Winssinger 11 Aug 2020Identification of immunodominant linear epitopes from SARS-CoV-2 patient plasmaPONE-D-20-18388R1Dear Dr. Winssinger,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Nicholas J MantisAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: (No Response)**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The additions are relevant and sufficient to address the previous concerns. I have no further comments.Reviewer #2: My concerns have been addressed, although it would have been preferable if neutralizing activity could have been more central to the study.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Yes: Anastas PashovReviewer #1: Reviewer #2: No 18 Aug 2020PONE-D-20-18388R1 Identification of immunodominant linear epitopes from SARS-CoV-2 patient plasma Dear Dr. Winssinger:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. 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Although the group differences were not significant, self-care increased (p < 0.05), and sexual problems decreased (p < 0.05) in the intervention group only. No significant intervention effects were observed for HRQoL, self-care confidence, illness perception and exercise capacity. Overall, results of this proof-of-concept trial suggest that the HeartMan personal health system significantly improved mental and sexual health and self-care behaviour in CHF patients. These observations were in contrast to the lack of intervention effects on HRQoL, illness perception and exercise capacity.This study tested the effectiveness of HeartMan—a mobile personal health system offering decisional support for management of congestive heart failure (CHF)—on health-related quality of life (HRQoL), self-management, exercise capacity, illness perception, mental and sexual health. A randomized controlled proof-of-concept trial (1:2 ratio of control:intervention) was set up with ambulatory CHF patients in stable condition in Belgium and Italy. Data were collected by means of a 6-min walking test and a number of standardized questionnaire instruments. A total of 56 (34 intervention and 22 control group) participants completed the study . All depression and anxiety dimensions decreased in the intervention group ( Despite major improvements in medical treatment, CHF remains a condition with high rates of premature mortality and hospital readmission3. Since there is no cure available, proper disease management in CHF is crucial as it may relieve symptoms, prevent hospitalisation or decrease mortality2. Optimal management of the disease may also affect the patient’s health-related quality of life (HRQoL), considered to be an essential treatment goal4. Despite the availability of evidence-based guidelines for proper disease management, the uptake of these guidelines in clinical practice is generally poor, especially when it comes to meeting the recommendations for physical exercise5. Self-care in CHF management is highly complex since extensive behavioural efforts are required relating to medication adherence, fluid and sodium intake, healthy diet and weight management, smoking and alcohol consumption, and physical exercise6. Patients with CHF—who are typically older persons and frequently suffer from comorbidities and mental health impairment—often struggle to adhere to their complicated treatment scheme and lifestyle advice to manage their disease. Therefore, the use of mHealth applications is promising and holds large potential to optimize self-care and improve clinical outcomes. Results on the effectiveness of mHealth interventions in CHF are however inconclusive10.Congestive heart failure (CHF) is a common cardiovascular disease, with an estimated 26 million adults worldwide living with this condition and its prevalence rising to ≥ 10% among people over 70 years of age11. The core of the system is a mobile application, which is connected to a number of sensing devices—including a custom wristband—and cloud services. A decision support system (DSS) provides recommendations that are shown in the mobile application, which also collects inputs from the patients. It is a multi-disciplinary system combining a number of different intervention modalities to cover both physical health management and psychological support. The expert system for physical health in the DSS includes a comprehensive exercise program with an individualized weekly plan consisting of endurance and resistance exercises. It further comprises personalized systems for nutrition advice, self-monitoring, medication intake and disease education. The expert system for psychological support provides cognitive behavioral interventions and mindfulness exercises implemented according to a weekly plan. Unlike most mHealth interventions targeting hard clinical endpoints like mortality and hospitalisation, the primary outcomes of HeartMan are HRQoL and self-management. The therapeutic goal in CHF is not only prolonging survival, but also improving the quality of life gained. Hard endpoints do not necessarily reflect how a disease, its symptoms and treatment are experienced by the patient. Consequently, an increasing interest occurred in using patient-reported outcomes (PROs) as independent outcome measures in clinical practice over the past years12. Moreover, it has been demonstrated that PROs like self-reported HRQoL and health status have a pathophysiological basis and are predictors of clinical events in patients with CHF14.HeartMan was developed as a comprehensive personal health system to address CHF self-managementThe aim was to evaluate the effectiveness of HeartMan, within a randomized controlled proof-of-concept trial, on HRQoL and self-management as primary endpoints, and on exercise capacity, illness perception, and mental and sexual health as the secondary endpoints.11. The design used a 1:2 randomization ratio of control group receiving standard care vs. intervention group receiving the HeartMan system on top of standard care. The main inclusion criteria were being an adult ambulatory CHF patient (both ischemic or non-ischemic aetiology) in stable condition, having a Functional New York Heart Association (NYHA) class of 2 or 3, and having a reduced left ventricular ejection fraction (LVEF) of ≤ 40%.The HeartMan randomized controlled proof-of-concept trial was implemented in 3 hospitals in Belgium (recruitment from 04/01/2018 until 27/06/2018) and one hospital and a local health authority in Italy (recruitment from 11/07/2018 until 06/03/2019), details have been described elsewhere15. It was anticipated that 90 patients were needed to show an 5.8 beats per minute difference in average daily awake heart rate with 90% power between the two groups. This targeted number was increased to 120 (60 in each participating country) to account for drop-out. After recruitment by the treating cardiologist or general physician, 79 patients signed informed consent, 14 of whom were excluded because they did not fulfil the inclusion criteria or because their health deteriorated shortly after. Of the 65 patients being randomized, 4 patients were excluded due to hospitalisation before the start of the intervention, and 5 were excluded during the intervention period due to withdrawal or clinical event, leaving a sample of 56 participants—i.e. 34 in the intervention and 22 in the control group—to evaluate the intervention effects  was targeted, based on power calculations that considered heart rate changes—a parameter correlating with HRQoL—as derived from the CHIRON study which was undertaken by part of the current investigators groupThis study complies with the Declaration of Helsinki and in both countries, formal approval was obtained from the ethical committees  and from the national institutions for clinical trials . The study was registered at ClinicalTrials.gov on 13/04/2018 (NCT03497871).Electronic health records of the target population were screened for eligibility. At the time of a regular outpatient visit, information about the study was presented to eligible patients by the treating physician or heart failure nurse, after which interested patients signed informed consent. The participants returned to the hospital for a subsequent appointment, during which baseline data were collected by means of clinical and questionnaire assessments. Next, the patients were randomly assigned to either the control group or the intervention group with a 1:2 ratio, using a system with two balanced series (one in each country) of sealed envelopes. All outcome measurements were repeated in both intervention and control group at the end of the trial.Patients in the control group received usual care, i.e. the standard treatment in line with clinical guidelines offered by the cardiologist, general practitioner and CHF nurse. Patients in the intervention group continued to receive usual care, and additionally used the HeartMan personal health system in their home setting. The intervention was initiated during a home visit by a member of the research team, providing all necessary equipment, technical installation and user instructions.The trial equipment included an off-the-shelf blood pressure monitor , weight scale  and pill organizer , and a custom wristband sensor developed by BITTIUM  recording heart rate (variability), galvanic skin response, skin temperature and acceleration. Based on data collected from the sensing devices and patient monitoring methods, the DSS recommends actions that are presented to the patient via a mobile application on a smartphone (Nokia 6 TA_1021) which was provided for the duration of the trial. The main components of the DSS were an expert system to manage physical health  and an expert system providing psychological support , in addition to the availability of overall disease education about CHF and its treatment. Intervention patients used the HeartMan system for a period of 3 up to 6 months, during which a telephone-based helpdesk was operational for addressing technical difficulties and user problems.5. The number of meters walked within 6 min was registered to evaluate exercise capacity. Resting heart rate was measured before the test  and used as additional clinical outcome measure.The outcome measures were collected at baseline (T1) and at the end-of-study visit (T2) in the hospital setting. In line with the recommended standardized protocol of the European Heart Failure Association, a 6-min walking test (6MWT) was conducted by a trained researcher16. The 21-item disease-specific Minnesota Living with Heart Failure Questionnaire (MLHFQ) was used to assess overall HRQoL, with a higher score indicating a more impaired HRQoL, as well as the sub-scales for impaired physical HRQoL and impaired emotional HRQoL, calculated from a subset of 8 and 5 items, respectively17. The brief Illness Perception Questionnaire (IPQ) provided a score calculated from 8 items, with a higher score representing a higher degree to which the illness is perceived as threatening or benign, on a scale from 0 to 1018. The 21-item Beck Depression Inventory II (BDI II) instrument returned a total score and two sub-scores on somatic-affective and cognitive manifestations of depression19. We used the 40-item State Trait Anxiety Inventory Form Y (STAI-Y) to assess state anxiety (Y1: how the subject feels at the time of questionnaire submission) and trait anxiety 20. The Sexual Adjustment Scale (SAS) consists of six items and yields a score varying from 0 to 18, with a higher score indicating more sexual disturbance experienced21. The Needs for Counseling scale in CHF (NSCS-CHF) includes 18 items from which a score from 1  to 4 (very important) is calculated, with a higher score indicating a higher expressed need for counseling about sexual activity in CHF22.The patients filled in a number of standardized questionnaire instruments during the hospital visit. Completion of the self-administered questionnaire package took on average 30 min and was done in the presence of a member from the research team (in case support or clarification was needed). The Self-Care of Heart Failure Index (SCHFI) was used to measure self-care maintenance (10 items) and self-care confidence (6 items), with each subscale being standardized to a score ranging from 0 to 100, higher scores indicating better self-care quality23 and 3C-HF24 scoring systems. If a new assessment of LVEF was available at the end of the intervention study, this was also registered as an additional clinical outcome measure in a sub-sample of participants.Electronic health records were screened to obtain data on clinical characteristics , CHF-related characteristics , and information about comorbidities and medication use. In case the necessary electronic health data were available, the predicted one-year mortality risk was calculated using the MAGGICU test, Chi² or Fisher’s Exact test. The same comparative statistical tests were used for the drop-out analysis and for evaluating potential differences at T1 between Italian and Belgian participants. To evaluate the intervention effects, we assessed the change in the outcome variables from T1 to T2 in the intervention and control group by means of the paired-samples Student’s t-test or Wilcoxon signed-rank test. Group differences in these change scores were furthermore evaluated with independent samples Student’s t-test or Mann–Whitney U test. A p-value at < 0.05 level was considered statistically significant. All analyses were conducted in IBM SPSS Statistics version 24.Continuous variables were checked for normality of distribution before analysis. Descriptive statistics were obtained through proportions/numbers and mean (plus standard deviation (sd)) or median (plus interquartile range (IQR)) values. Descriptive, clinical and outcome variables at T1 were compared between intervention and control group by means of independent samples Student’s t-test, Mann–Whitney The sample of 56 patients had a mean age of 63 years sd 10.5), was predominantly male (77%), and had by design a reduced LVEF of ≤ 40% . An overview of characteristics and outcome variables at T1 is shown in Table 0.5, was p > 0.05; results not shown). Comparison at T1 between both countries showed a more adverse clinical profile in the Belgian (N = 34) vs. Italian (N = 22) sample, with a lower mean LVEF , a higher median predicted 1-year mortality risk according to MAGGIC , and higher mean values for impaired total HRQoL on the MLHFQ  and impaired physical HRQoL on the MLHFQ . On the other hand, Belgian patients scored significantly better on mean self-care maintenance score on the SCHFI at T1 . No significant differences in socio-demographic characteristics, exercise capacity, or mental health at T1 were observed between the Belgian and Italian patient group (results not shown).The drop-out analysis comparing the 56 patients participating throughout the intervention period with the nine patients who were randomized but dropped out before or during the intervention period see Fig.  showed nIntervention effects are reported in Table 25. However, their effectiveness in managing CHF patients remains controversial, and both positive results26 as lacking effects10 on mortality and hospitalisation have been reported. Real-time transmission of patient data enables remote follow-up, but involves substantial interaction with healthcare providers. These systems do not focus on empowering patients to properly manage their disease. There is high need for systems that focus on self-care and enhance individual adherence of patients to their complicated treatment plans and lifestyle advice. Patients who are actively involved in their own care, and who adhere to the medication and lifestyle regimen, are known to have better prognosis6. So in addition to pure tele-monitoring, there is a need for mHealth systems that interpret data generated in home environments directly for the benefit of the patients, but the implementation of DSSs in the context of patient support in home environments is still limited27. HeartMan was designed as an advanced disease management support system for stable CHF patients to use in their home environment11. End users were involved in all development stages using a human-centred design process. Its major strengths are the personalisation to the individual patient’s clinical and psychological profile, and its advanced functionality by integrating several relevant intervention modalities relating to physical exercise, nutrition, medication intake, mental support and disease education.In the past years the use of tele-monitoring systems in cardiac patients has increased tremendously11. The findings suggest that psychological support had an important impact on patients, relieving their somatic-affective and cognitive symptoms of depression (BDI II), as well as their state and trait signs of anxiety (STAI-Y). This is in line with the growing evidence showing the ability of mindfulness to improve psychological well-being in chronic disease overall28, and specifically in CHF29. Although the available evidence from RCT studies is limited and somewhat mixed, several reviews show that CBT strategies can have beneficial effects on mental health and symptom burden in CHF populations32.The results showed that HeartMan was effective in improving mental health in CHF patients. The psychological support module in the HeartMan system was quite advanced, including personalized messages based on cognitive behavioral therapy (CBT) to dissolve patients’ cognitive dissonance, in addition to a weekly plan of mindfulness games and exercises for relaxation and general psychological wellbeing16. In line with documented poor levels of adherence and self-management in this patient group33, the overall level at T1 was below the 70 point cut-off for adequate self-care in both intervention and control group. It approached the threshold of 70 in the intervention group at T2, which is suggested to be the minimal effectiveness cut-point for improving health outcomes16. It should be noted though that no significant group difference was observed for this outcome, so this result should be interpreted with caution.Results of this trial further suggest that using HeartMan improved self-care behaviour. The self-care maintenance score of the SCHFI, indicating overall quality of disease management on a scale from 0 to 100, improved significantly in the intervention group with 6.5 points, which is a little below the threshold of half a standard deviation for a clinically relevant change11. Problems with sexual functioning and activity are very common in CHF, while satisfaction with one’s sexual activity is considered important by most patients35. We observed a reduction in experienced sexual problems in the intervention group in addition to a significant difference in need for sexual counselling which decreased in the control group only. These findings suggest that applying mHealth for sexual counselling—combined with the observed improvement in self-care—might evoke reflection upon the topic and lower the experience of sexual problems. It should be noted though that about 1/5 had incomplete data on these questionnaires, presumably because some felt uncomfortable answering these questions. Therefore, we need to interpret these results with caution since a selection bias is likely, i.e. those who answered the questionnaires are probably those with better outcomes.The mobile application provided access to a depository which users could consult on a voluntary basis to look up educational information about CHF and its treatment, as well as expert knowledge about how the disease impacts on sexual activity and dysfunction10. We used the same instrument to measure HRQoL, i.e. the MLHFQ, but a much smaller sample was involved. The null finding for the emotional dimension of HRQoL is in contrast with the observed intervention effect on depression and anxiety. These conflicting results are against expectations since depressive symptoms have been identified as consistent predictors of HRQoL in CHF patients36. A plausible explanation lies in the different characteristics of the instruments used. The MLHFQ includes five items to cover the emotional dimension of impaired HRQoL, asking rather explicitly about a patient’s overall emotional state17. As such it is less specific and significantly affected by the patient’s momentary perception37, compared to the BDI II and STAI-Y instruments which are more accurate  and sensitive to evaluate psychological predictors of depression or anxiety20.Null findings were observed on the remaining primary and secondary endpoints, i.e. HRQoL, exercise capacity and illness perception. Although most mHealth interventions focus on hard endpoints rather than PROs, the large-scale BEAT-HF trial showed an improvement in HRQoL, while no effect on hospital readmission was observed11. No effect on exercise capacity was observed, neither on distance walked in the 6MWT nor on resting heart rate. Our results suggest that while the HeartMan system empowered patients to improve their self-care behavior and advanced their mental health, the system did not sufficiently succeed in making them adhere to the advanced exercise program. Although there is convincing evidence about the benefits of exercise in CHF, the uptake of exercise is known to be problematic33.The comprehensive physical exercise program in the HeartMan DSS was adapted to each patient's physical capacity and psychological profile, and provided a gradually increasing weekly set of endurance and resistance exercises39. However, in a group of stable CHF patients with large variety in exercise habits at baseline, the initiation of a detailed exercise program through a mobile application, even if it is individualized and gradually progressive, is much more challenging. The effects may thus be less pronounced and remain undetected in this small proof-of-concept trial, especially since 6 patients (3 in each group) with baseline information were not able to perform the 6MWT at T2. Regarding the use of a 6MWT, further research with cardiopulmonary exercise testing as the state-of-the-art method for measuring exercise capacity will be required.Wearables and mHealth applications providing individualized follow-up to patients in their maintenance phase of cardiac rehabilitation have been shown effective for improving cardiorespiratory fitness and exercise capacity40. Again it is possible that the sample was too small or the intervention period too short to induce significant effects on these outcome measures.Contrary to what we hypothesized, the HeartMan intervention did not affect patients’ illness perception (IPQ) nor their level of self-care confidence (SCHFI). The disease education module provided in the depository of the mobile application was meant to improve patients’ comprehension of the disease and to boost their confidence to take actions and self-manage their CHF. Negative illness perceptions are known to be associated with worse clinical outcomes, underlining the importance of changing these representations as a treatment goal11. While a total of 79 patients signed informed consent, only 56 were included in the final effect evaluation. No major drop-out effects were observed when comparing the final sample with those who dropped out after randomisation, although the small and unbalanced numbers in this analysis make reliable conclusions difficult. Strict inclusion and exclusion criteria were adopted, and especially the restriction to patients with reduced LVEF ≤ 40% critically limited the number of eligible patients. Another major bottleneck was the common reluctance of patients to participate in a randomized study in general, or to enter an intervention program requiring intensive use of an mHealth system. Therefore, the number of eligible and consenting patients was much lower than anticipated in both countries, and the restrictions imposed by the funding agency did not allow any extension of the recruitment period. A second limitation is that inevitably a number of technical difficulties or flaws arose in this proof-of-concept trial where the system was evaluated in a real patient setting for the first time. A helpdesk was operational for addressing technical issues and problems with using the system, but nonetheless this likely affected some patients’ adherence to the system. As a result, some of the truly potential intervention effects may have been underestimated. Because of the multi-modal nature of the system only an overall effect evaluation was possible, disregarding the effects of the individual components. Also, there is a risk of multiple testing problem as we targeted multiple endpoints in relation to the multi-disciplinary system. The system was tested in patients from two countries, increasing the generalizability of findings. A number of characteristics differed between the Belgian and Italian samples at T1, but the low number of participants does not allow analysing stratified intervention effects. Overall, the HeartMan patient population was relatively young, including mostly men with mild symptoms.The main limitation of this proof-of-concept trial is the small sample size resulting in preliminary findings. A priori sample size calculations indicated a target number of 90 patients33. In contrast, no effects on HRQoL, exercise capacity and illness perception were detected. In addition to the pre-defined endpoints indicated in the study protocol, additional electronic health record data enabled to verify potential effects of the system on clinical outcomes. Follow-up data on LVEF and predicted one-year mortality risk were available for more than two thirds of the sample and thus provided preliminary findings about the clinical effectiveness. Patients in the intervention group significantly improved their LVEF and the difference with the control group was borderline significant. A new assessment of LVEF at follow-up was not available in 38% of the intervention and 27% of the control group, and although no systematic bias seemed present, caution is required in the interpretation of this finding. Intervention patients significantly reduced their predicted one-year mortality risk according to the MAGGIC23 and 3C-HF24 scoring systems, although similar trends were observed in the control group. These preliminary findings need to be investigated more in depth. Future validation studies are needed to test the overall effectiveness of the HeartMan system in a wider context, i.e. in larger samples of CHF patients using a less restricted participant profile and applying a longer intervention period.In conclusion, the Heartman system significantly improved mental health and a trend for improved self-care behaviour was observed, outcomes which are known to not only reinforce one another but also predict hard clinical endpoints in CHF


Autophagy is a catabolic process, utilized constitutionally by body cells to recycle nutrients and to remove unwanted/damaged intracellular constituents. It is enhanced during periods of stress, such as starvation and hypoxia, aiding in cell survival and it is linked to major cellular processes, such as apoptosis and antigen expression. The process has been extensively studied in vitro models or tumor tissue samples with rare application on human subjects. Plasma samples from 24 advanced solid tumor patients were collected at different time points before and after chemotherapy. Their exosomes were isolate and blotted for microtubule-associated protein-1 light chain-3 (LC-3B) protein as a marker for autophagy. All the subjects received a standard chemotherapy regimen of carboplatin- gemcitabine with chloroquine (CQ)/ hydroxychloroquine (HCQ) in chronic doses throughout their treatment period as an autophagy modulator. CQ/HCQ was given in 50 mg increments as guided by their tolerability to treatment. A total of 267 plasma samples were obtained for the 24 patients and processed. Each sample corresponds to a single time point. The first group included 6 patients, all received 50 mg of CQ with chemotherapy. LC-3B I was detected in their isolated exosomes, while LC3-BII was not detected in their samples. The second cohort of patients included 3 subjects who re-ceived 100mg of HCQ. They demonstrated both LC3-BI and II on day 15 after chemotherapy in one patient, and on third cycle after 24 hours in the second patient. The third cohort included 3 subjects who received 150 mg of HCQ. All cases demonstrated LC3-BI and II on first cycle of treatment after less than 24 hours. The last cohort included 8 subjects, who received a fixed dose of 100 mg of HCQ with treatment. In this cohort, we were able to detect both LC3-B isoforms on advanced time points of second and third cycles. Detection of autophagy protein LC3-B in exosomes serves as a dynamic method to monitor autophagy. It can be utilized to study the effects of anti-neoplastic agents on autophagy and mechanisms of drug resistance, however, to standardize our results a larger specimen of patients should be included. Autophagy is a self-degradative process that occurs in eukaryotic cells to maintain protein he-mostasis and recycling of organelles. It is a dynamic process that occur constitutively and in re-sponse to cellular stressors such as cell starvation and hypoxia . Three forms of autophagy occur in mammalian cells, a) micro-autophagy, describes the simple process of lysosomes directly engulfing small cytosolic components , b) chaperone-mediated autophagy (CMA) which is a more selective process in which cytosolic proteins that will be degraded are marked with KFERQ-like pentapeptide motifs that are specifically recognized by chaperone hsc70, and on the lysosomal surface LAMP-2A acts as a receptor to hsc70 and engulfs these proteins inside the lumen to be degraded, and finally c) macro-autophagy: referred to as autophagy, and the process referred to in our study.Autophagy has been widely studied in vitro to understand its dynamics and interactions with other cellular processes including cancer. It has been pointed at as both a culprit that enables cancers cells to survive  and evade antitumor agents , and a physiological mechanism that helps with protection of normal cell integrity and initiation of apoptosis in occasions of abnormal transformations . Some reports considered autophagy as one way through which anticancer agents can achieve their effect , others went to the con-clusion that it may actually have a direct effect on tumor behavior .Even though, its role in cancer development, progression, and metastases has been widely described with a vast amount of data, most of these data were obtained from in vitro and mice models . The main focus is to establish connection to other process by monitoring effects on their biomarkers. Seldom these data were translated into human subjects to verify its applications. A rather brief description of the process entails its initiation through formation of an isolation membrane, termed phagophore. Phagophore origin is not well defined. Some studies suggested an origin from specialized regions in the endoplasmic reticulum, others suggest that Golgi apparatus and mitochondria. Phagophores start engulfing organelles and macromolecules from the cytoplasm, thus evolving into another structure known as the autophagosome. This is a double membrane vacuole with a size range from 300 to 900nm. It requires maturation through fusion with early and late endosomes , forming a hybride organelle called the amphisome. This the final structure which can fuse with lysosomes for degradation of their contents . However, it was reported that autophagosomes can fuse directly with lysosomes . While this sequence of ongoing events is taking place, exosomes are formed and released by the cell, and their release is a dependent process that is directly linked to autophagy. It was suggested that inhibition of autophagy, increase exosomal release and vice versa .Exosomes are a form of extracellular vesicles with a size ranging between 30-100nm in diame-ter. It is believed that they originate from intraluminal vesicle (ILV) inside the late endosomes or the multi-vesicular bodies (MVB) by inward budding of their membranes. When the MVB fuse with the cell membrane and release its contents in the extracellular environment, which are the exosomes . There was a dominating trend that believed that exosomes are the cell way to get rid of unwanted structures . However, the precisely tailored exosome structure , and the specificities of the mechanisms involved in selective loading of its contents argue against it. Even though it is plausible that this can occur as part of the universal picture regarding the exosomal endocytic origin. It exosomes have been implicated in metastases, abnormal DNA changes and intercellular communication . This is thanks to its content of specialized species of microRNAs (miRNA). Previous reports have suggested the use of exosomes as biomarkers for autophagy in breast cancer cell lines . And the concept is interesting, since exosomes secretion and autophagy are both reflect the dynamicity of the cellular process. Hereby, we tested this hy-pothesis in a population of 24 advanced solid tumor patients . The patients were treated with con-ventional gemcitabine-carboplatin regimen. During their treatment period they received daily dose of chloroquine/hydroxychloroquine, as an autophagy modulator. The effect on the changes on autophagic activity was monitored through exosomes isolated from the patient plasma at different time points. The exosomes LC3-B content was demonstrated through western blotting.Study populationThis study included 24 patients who were recruited in 4 cohorts, cohort 1 (n=6), cohort 2 (n=3), cohort 3 (n=3) and extension cohort (n=8). All patients were histologically diagnosed with ad-vanced solid. All subjects provided a written informed consent before treatment in accordance with the Declaration of Helsinki, and the study protocol was approved by the Institutional Re-view Board of the University of Cincinnati Hospital. The subjects enrolled have failed their previous lines of treatment and the proposed chemotherapy regimen (Carb/Gem) was considered a standard of care Cell CultureWe used four cell lines in this study, human glioblastoma cell line (U251), human pancreatic carcinoma cell line (MiaPaCa2), adenocarcinoma human alveolar basal epithelial cells (A549) and human embryonic kidney cells (HEK293). The variety was intentional to insure matching results regardless of baseline autophagic activity of the cell line. These cells were maintained in DMEM media, supplemented with 10% fetal bovine serum (FBS), antimycotic, antibiotic and L-glutamine. At 40% confluency, the culture dishes were washed twice with PBS and the media replaced with 10% exosome depleted FBS (dFBS) DMEM media  and incubated to settle for 24 hours. After 24 hours, the culture dishes were washed with PBS and new dFBS DMEM media was added and treated with chloroquine diphosphate salt  and incubated. Incubation period was 48 hours for western blotting on HEK293 plates and 72 hours for western blotting on U251. The discrepancy in the incubation time was attributed to the confluency of the plates, CQ concentration used and the number of exosomes expected to be secreted by the cell line. The media was collected and used for exosome extraction directly after the incubation time is over.Exosome extraction1- From Patients PlasmaoC.Patients’ blood samples were collected at the mentioned time points and span down at 1500 g for 15 minutes. The upper phase (plasma) was collected in new tubes and stored at -80 degrees till us. Exosomes extraction was done using an exosome extraction reagent  following manufacturer’s instructions, then, suspended in PBS and stored at -802- From Cell Culture Media Tm by Life TechnologiesTM, ref# 4478359) following manufacturer’s instructions. Extracted exosomes were suspended in PBS and stored at - 80oC. The method for patient extraction is described in full details here .Exosomes were extracted from freshly collected cell culture media using exosome precipitation reagent . CD9 was used as a loading control was blotted using rabbit monoclonal antibody (#3700) from Cell Signaling Technology Inc. All western blots were run on 4-15% gradient gels after estimating and unifying samples protein content by BCA.Flow cytometryHEK293 cells were plated in 10 cm culture dishes in 10% FBS DMEM media. The media was re-placed with dFBS DMEM media once a 40% confluency is reached and incubated for 24 hours. Drugs as single agents, Gem at 20uM concentration (Gem20), CQ at 10uM (CQ10) and 20uM (CQ20) concentrations, and in combinations: Gem+CQ10 and Gem+CQ20 were used to treat the plated HEK293 cells in fresh dFBS DMEM media for 16 hours. Both the cells and the media were collected, spinned down, then washed and finally stained with propedium iodide (PI) and annexin V before running the samples. Cell cycle analysis was performed using fresh cells on a FACS Calibur (Becton Dickinson) after incubation with 25 μg/ml of PI. Cell cycle phases were analyzed with the CellQuest-Pro software program (Becton Dickinson). In vitro experimentsWe started by measuring the basic autophagy activity in the four cell lines that we used in this study through extraction of the exosomes from the cell culture media and blotting them for LC-3B. We used time points that corresponds to the post treatment time points at which the samples were withdrawn from the patients. These time points were: 1h, 2h, 4h, 6h and 24h. Since these cells were not treated with any agents, we calculated the time points from the point when fresh exosome free media was added to the cell culture dishes at 60% confluency. Blotting the cells showed equal expression for LC3-BI and II for all cell lines at all time points, however, the bands were most clear at 24h . The celThen we used CQ treatment on the mentioned cell lines to monitor the effective changes in LC-3B expression in the cells and exosomes compared to baseline. We started with different concentrations 25uM, 50uM, 100uM and 120uM, we found that at the set time point of 24h, the 100uM concentration produces enough inhibition of cellular confluency without cellular necrosis. At a concentration of 100uM of CQ, the exosomes extracted from cell culture media of HEK293 cells showed LC-3BII bands at 24h, however, no bands were detected for LC-3BI , this isSince the treatment regimen given to the patients included an autophagy stimulator , gemcitabine  and CQ which inhibits the final stages of autophagy, we wanted to test their effect on LC-3B expression on released exosomes. We set 4 types of treatments for MiaPaCa2 cells including CQ 20uM, Gem 20uM and a combination of both. These concentrations were decided based on the degree of confluency and necrosis after 24h of treatment. We found out that the combination treatment produced by far a thicker band for LC-3BII isoform compared to the Gem treatment only. Although the band was slightly thin compared to CQ treatment, but the effect of the combination treatment was evident . This exTo further investigate this point, we increased the treatment time to 36h and the effect of inhibition and induction was further augmented in the form of fainter bands with Gem and thicker bands with inhibition and combination treatment. CQ inhibition seems to overcome Gem inducing effect since at the same dose combination treatment resulted in equal effects, however, reducing the dose of CQ for treatment resulted in thinner bands . In vivo experimentsA total of 261 samples were obtained for the 24 participants. The time points for the patients were more extended; day -7 (7 days prior to administration of the first cycle), 48h, 72h, day 8 and day 15 samples were also collected. These time points varied from one patient to another since some patients did not tolerate the treatment or they dropped out from the study. The exact details of the patient data will be reported in a separate report.The first cohort of patients included 6 cases who received Carb/Gem at standard doses. CQ/HCQ was started at 50 mg fixed daily dose 7 days before treatment and continued throughout their course of treatment. Exosomes did not show expression of LC-3B II in the western blotting for most cases, however, exclusion of effect for CQ/HCQ at this dose level cannot be excluded since most of the enrolled cases did not received a full course of Carb/Gem and they were dropped out of the study. One patient complete the full course of treatment and we were able to detect LC-3B II starting from the second cycle. The band was very clear on day 8 of the second cycle and ultimately became the predominant band on third and fourth cycles while LC-3BI faded away and became almost nonexistent . LC-3B II expression at baseline, which was lacking in the second patients and hence the variation in the onset of response. The second cohort included 3 cases who received 100 mg of CQ/HCQ throughout the treatment period and more patients remained longer in the study. One patient showed bands on an earlier time point during his treatment course with LC-3B II evident on day-7 and after 4 hours of the first cycle of the first patient , while aLC-3B II expression at baseline, they expressed LC-3B bands on the first cycle. In Figure 6 LC-3B II was expressed at 24h, another case expressed the protein at 24h and the last patient expressed it on day 15. The third cohort included another 3 patients who received 150 mg of CQ/HCQ during the study period. Although some patients lacked Since, 2 patients were not able to tolerate the 150mg regimen in cohort 3, 8 more cases were enrolled under the 100mg regimen schedule to validate the results. Like the previous cases, the patients expressed LC-3B II starting from the late time points of the second or the third cycle. This validated our conclusion that the effects of CQ/HCQ are not only dose dependent but also have a cumulative effect.Necrosis or apoptosisSince the in vitro experiments have shown more cellular death with combination treatment at 24h of treatment we concluded that the regimen kills tumor cells at a higher rate than each drug alone. To investigate this phenomenon, we ran an annexin V assay to verify if the effect is due to necrosis or apoptosis. HEK295 cells were treated with CQ 10uM, CQ 20uM, Gem 20uM and Gem with either CQ 10uM or 20uM. The cells were stained with annexin V. The results showed that compared to control all treatments had less live cells. Treating the cells with CQ 20uM showed as low as 21.7% live cells compared to 49.7% for control. The combination treatment showed higher percentage of live cells at 40.8% and 44.1 % for Gem+ CQ10 and Gem+CQ20 respectively. Combination therapy showed high percentage of apoptotic and pre-apoptotic cells. The increase in CQ concentration with Gem also played a role in increasing the number of necrotic cell population to 17.4% compared to 6.6% for controls and 2.9% for the Gem+CQ10 .In this study we investigated a new method to monitor autophagy in human plasma. Plasma samples were obtained from 24 advanced solid tumor patients at different time points and planned according to their chemotherapy regimen schedules. The patients received either CQ or HCQ during the full course of their treatment at fixed daily dose and each cohort received increasing increments of 50 mg. The starting dose was 50 mg and the time points were obtained 1 hour before treatment administration and after administration of their scheduled dose at 1h, 2h, 4h, 6h, 24h, 48, 72h and on days 8 and 15. Exosomes were isolated from the patients’ plasma and blotted for autophagy protein LC-3B using increased transformation of LC-BI isoform to LC-3B II expression as an indication of increased autophagic activity. We found that the patients had a different patterns of baseline LC-3B expression before treatment. Patients who expressed LC-3B II before treatment, did not express it in the early time points after receiving chemotherapy. The autophagy modulating effect of CQ/HCQ arguably inhibits the late stage of the process when the late endosome fuses with lysosomes , while Gem effect is increasing the turnover of the process . So, we hypothized from our results that together both agents increased the number of released exosomes facilitating their detection in peripheral blood. However, this theory needs further investigation and exclusion of many other variables including other agent used, which is carboplatin, and the effect of other medications administered for other morbidities. Our in vitro studies had shown a clearer distinction between autophagy induction by Gem and inhibition by CQ. The inhibitory effect was manifested in the form of increased LC-3B II expression and changing the dose affected the band thickness in western blotting rather than its intensity. Meanwhile induction by Gem produced thick but faint bands on western blotting. We assumed that the stimulation of the process resulted in increased conversion of LC-BI to LC-BII isoform, however, the CQ/HCQ resulted in accumulation of the protein rather than its degradation due to inhibition of lysosomal fusion. Annexin V assays showed increase in apoptotic and pre-apoptotic cells compared to control with single and double agent treatments using Gem and/or CQ/HCQ. The percentage of necrosis increased with higher CQ dosage, which might suggest that the increased dosage have a higher toxic effect on cells that is yet to be defined. Many studies have suggested the implication of autophagy as means by which tumors develop resistance to therapeutic effects of anti-neoplastic agents . Also many studies suggested that autophagy is affect by many anti-neoplastic agents . The extent of implication of this pivotal process has been linked to other vital process of the body beyond cancer therapy . And with emergence of immunotherapy such process needs to be further understood since autophagy plays a major role in cellular antigen expression . And it has been implicated in evading immune surveillance . Thus, can be related to various predictive and prognostic markers. However, these studies have always been in vitro experiments or done using murine models. Nowadays with the advanced techniques in obtaining and analyzing blood based biopsies , developing biological markers to monitor such pivotal process in patients becomes necessary to evaluate if our understanding to these concepts applies to the heterogenous nature of our patients and the dynamic changes of the disease. LC-3B conversion from LC-3B I to II has been used as an indicator for autophagy since it measures the dynamicity of the process by reflecting the turnover of autophagosome fusion with lysosomes . However, increased expression of both isoforms is used to measure the activity of both autophagy inducers and inhibitors . Although this method is biologically explained , but there was no explanation on how to differentiate between both actions and unless other quantification methods are used, both actions can be interpreted similarly on western blotting. Our in vitro results had showed different picture for each when LC3-B was measured in exosomes instead of cells. This conclusion can serve as simple and cost-effective method to track stimulus effect on autophagy by interpretation of t western blotting compared to well known controls.All subjects that participated in the study were consented before the study.All necessary approvals were granted by the IRB of the University of Cincinnati.LC3-B: microtubule-associated protein-1 light chain-3 BCA: Bicinchoninic acid assayCarb: CarboplatinCQ: ChloroquineCQ10: Chloroquine 10uMCQ20: Chloroquine 20uMdFBS: Exosome depleted FBSESCRT: Endosomal sorting complexes required for the transport FBS: Fetal bovine serum.Gem: Gemcitabine HCQ: HydroxychloroquineILV: Intraluminal vesicle kDa: Kilo DaltonLC3B: Microtubule-associated protein-1 light chain-3MVB: Multi-vesicular bodiesPI: Propedium iodideWB: Western blottingThe authors whose names are listed certify that they have NO affiliations with or involvement in any organization or entity with any financial interest , or non-financial interest  in the subject matter or materials discussed in this manuscript. The research was funded by an internal grant from the University of Cincinnati.Ola Gaber: Research, data collection, writing.Ihab Eldessouki: research, writing.El-Mustapha Bahassi: supervision of correlative studies.John C. Morris: Clinical management, supervision.Nagla Abdel Karim: Clinical trial rational, supervision.


Although previous phase II and III clinical trials conducted in Japan showed that zonisamide improved parkinsonism in patients with dementia with Lewy bodies (DLB), some differences in efficacy outcomes were observed between the trials.post hoc analysis of pooled data from the previous phase II and III trials.We aimed to further examine the efficacy and safety of zonisamide in DLB patients with parkinsonism in a Both trials featured a 4-week run-in period followed by a 12-week treatment period with a double-blind, placebo-controlled, parallel-group, randomized, multicenter trial design. In our pooled analysis, the primary outcome was the change in Unified Parkinson’s Disease Rating Scale (UPDRS) part III total score. Other outcomes included the changes in Mini-Mental State Examination (MMSE) and Neuropsychiatric Inventory-10 (NPI-10) scores, and the incidence of adverse events.Zonisamide significantly decreased the UPDRS part III total and individual motor symptom scores but did not affect the MMSE or NPI-10 scores at week 12. There was no difference in the incidence of adverse events between the zonisamide and placebo groups except for decreased appetite, which had an increased frequency in the zonisamide 50 mg group compared with placebo.Our findings indicate that zonisamide improved parkinsonism with DLB without deterioration of cognitive function and or worsening behavioral and psychological symptoms of dementia. Dementia with Lewy bodies (DLB) is the second most common type of dementia in older adults after Alzheimer’s disease, accounting for 10% –15% of all dementia cases . A recenThe pathology of parkinsonism in DLB is similar to that in Parkinson’s disease (PD), with both fea-turing the loss of dopaminergic neurons in the nig-rostriatal pathway and presence of Lewy bodies . Major cClinical management of parkinsonism in DLB typically includes levodopa, the standard therapy for PD and commonly administered to patients with DLB. However, patients with DLB appear to be less responsive to levodopa compared with patients with PD, and the risk of exacerbation of psychiatric symptoms remains a dose-limiting factor . CurrentZonisamide, approved globally for the treatment of epilepsy, has been approved for the treatment of PD in Japan, where it is typically prescribed as adj-unctive therapy to levodopa . The propost hoc analysis of pooled data from the phase II and III trials [The clear similarities between PD and DLB  and the I trials  to furthData were pooled from the phase II (JapicCTI-122040) and phase III (JapicCTI-152839) trials of zonisamide in patients with DLB conducted in Japan ; both trPatients with probable DLB, diagnosed according to the Clinical Diagnostic Criteria for DLB  , were ela≥10%, ≥ 20%, or≥30% reduction from baseline in UPDRS part III total score at week 12, in reference to previous reports [The key outcome for the efficacy of zonisamide against parkinsonism in DLB in this analysis was the change from baseline at week 12 in UPDRS part III total score. To reduce inter-/intra-rater variability, training was conducted prior to the trial start. A more detailed analysis of the effects of zonisamide on the following four motor symptoms  was also reports  where mi reports , represe reports , represep-values for the proportion of responders were calculated using logistic regression analysis with Firth’s penalized likelihood approach, with treatment group and trial as fixed effects and baseline value as a covariate. Statistical differences in MMSE and NPI-10 score changes between the zonisamide and placebo groups were evaluated by analysis of covariance with treatment group and trial as the fixed effects and baseline value as a covariate. Interaction p-values were calculated to evaluate the heterogeneity between the trials and were added to each original model. Statistical differences in the incidence of AEs between the zonisamide and placebo groups were analyzed using the Mantel–Haenszel test stratified by trial as a fixed effect. All statistical analyses were carried out at a two-sided significance level of 5%. No multiplicity adjustment for multiple treatment groups, outcomes, or visits was performed. All statistical analyses were performed using SAS version 9.4 .All statistical analyses were performed using one-stage meta-analysis methods with individual patient data. Differences in patient demographics and clinical characteristics at baseline among the placebo and zonisamide 25 mg and 50 mg groups were analyzed using the Cochran–Mantel–Haenszel test stratified with trial for categorical variable and analysis of variance with trial as the fixed effect for continuous variables. Efficacy against parkinsonism was assessed using a mixed-effect model for repeated measures among the placebo and zonisamide 25 mg and 50 mg groups with treatment group, visit, trial, and treatment-by-visit interaction as fixed effects, and baseline value as a covariate. An unstructured covariance matrix was assumed, and the degree of freedom was estimated using Kenward–Roger’s approximation. The efficacy set comprised 498 subjects  with a mean age of 76.6 years, mean DLB duration of 1.4 years, and mean levodopa dose of 262 mg/day . The safZonisamide significantly decreased the UPDRS part III total score compared with placebo, with no significant difference in score change observed between the zonisamide 25 mg and 50 mg groups . Zonisamp < 0.001 versus placebo) for zonisamide 25 mg, 55.0% (p = 0.002) for zonisamide 50 mg, and 37.6% for placebo  at week 12 (LOCF) were 61.2% , with small differences in efficacy on parkinsonism observed between the 25 mg and 50 mg groups. Furthermore, the proportion of responders and changes in score among responders  who received zonisamide were comparable to those reported in a previous study of dopaminergic treatment in patients with DLB who received levodopa . AdditioWith respect to the effect of zonisamide on cognitive function, no significant difference was observed between zonisamide (either dose) and placebo on the change in MMSE score. This finding differed from the outcome observed in the phase III trial, where zonisamide 50 mg significantly decreased the MMSE score compared with placebo . FurtherRegarding the effect of zonisamide on BPSD, no difference in NPI-10 score was observed for either dose of zonisamide versus placebo. Furthermore, in the analysis of the NPI-10 items and the subgroup analysis among patients with severe BPSD, no differences in NPI-10 score change were observed between the zonisamide 25 mg or 50 mg groups and the placebo group . Taken tA low incidence of each AE was observed among patients treated with zonisamide in the present study. With the exception of decreased appetite in patients receiving zonisamide 50 mg, the incidence of AEs did not differ between either zonisamide group and pla-cebo. In addition to the low frequency of AEs related to cognitive function and BPSD discussed above, the incidence of other AEs related to characteristic symptoms of DLB, such as autonomic dysfunction , was alsWhen evaluating the differences between the two doses of zonisamide, our findings show that the cha-nge in UPDRS part III total score was greater in the 50 mg group versus the 25 mg group; however, the difference was not statistically significant, suggesting no difference in efficacy. Regarding safety, although there was no significant difference in the overall incidence rate of adverse events, that in the 50 mg group was slightly higher than that in the 25 mg group, and the incidence rate of decreased appetite was significantly higher in the 50 mg group versus the placebo group. These findings suggest that the recommended dose of zonisamide for DLB patients with parkinsonism is 25 mg.post hoc study design and the inclusion of only Japanese patients, which limits the generalizability of the results. Furthermore, the trials enrolled participants from essentially the same patient population using the same inclusion/exclusion criteria, and the 12-week treatment periods were relatively short. Future studies with longer duration in a broader patient population are therefore required, and real-world study data may also contribute to determining the actual benefits of zonisamide to patients and caregivers.The present study had some limitations, including the post hoc analysis indicate that zonisamide as adjunct therapy to levodopa is efficacious for the treatment of parkinsonism with DLB, particularly the management of bradykinesia, tremor, and rigidity. Importantly, exacerbation of psychiatric symptoms, which is typically associated with levodopa use, was not observed. Zonisamide was shown to be well tolerated in patients with DLB with parkinsonism, with no new safety signals identified in the present analysis. Authors’ disclosures available online .In conclusion, the outcomes of this Click here for additional data file.