Severe dengue can be lethal caused by manifestations such as severe bleeding, fluid accumulation and organ impairment. This study aimed to investigate the role of dengue non-structural 1 (NS1) protein and host factors contributing to severe dengue. Electrical cell-substrate impedance sensing system was used to investigate the changes in barrier function of microvascular endothelial cells treated NS1 protein and serum samples from patients with different disease severity. Cytokines and metabolites profiles were assessed using a multiplex cytokine assay and liquid chromatography mass spectrometry respectively. The findings showed that NS1 was able to induce the loss of barrier function in microvascular endothelium in a dose dependent manner, however, the level of NS1 in serum samples did not correlate with the extent of vascular leakage induced. Further assessment of host factors revealed that cytokines such as CCL2, CCL5, CCL20 and CXCL1, as well as adhesion molecule ICAM-1, that are involved in leukocytes infiltration were expressed higher in dengue patients in comparison to healthy individuals. In addition, metabolomics study revealed the presence of deregulated metabolites involved in the phospholipid metabolism pathway in patients with severe manifestations. In conclusion, disease severity in dengue virus infection did not correlate directly with NS1 level, but instead with host factors that are involved in the regulation of junctional integrity and phospholipid metabolism. However, as the studied population was relatively small in this study, these exploratory findings should be confirmed by expanding the sample size using an independent cohort to further establish the significance of this study. Aedes aegypti or Aedes albopictus . Thein vitro models for dengue virus infection. The cells were maintained at 37°C with 5% CO2 supplemented with endothelial cell growth medium (ECM) (Sciencell) for HDMEC and HPMEC, and CSC complete medium (CSC) for HRtMEC.Three microvascular endothelial cell lines including primary human dermal microvascular endothelial cells (HDMEC) , primary human pulmonary microvascular endothelial cells (HPMEC) (Sciencell) and primary human retinal microvascular endothelial cells (HRtMEC)  were cultured as The three MEC lines were grown to a confluent monolayer in an eight-well electrode array (8W10E+)  in triplicates prior to the treatment with NS1 antigen or serum samples for ECIS measurement. The NS1 antigen (Bio-rad) used is a recombinant dengue virus serotype 1 NS1 protein of sequence strain Nauru/Western Pacific/1974 expressed in 293 human cells. It is produced with a C-terminal 6x His-tag and at a purity of >95% by SDS PAGE analysis diluted in buffer solution Dulbecco’s phosphate buffered saline, which was further diluted during the experiment using the respective media of each cell line used. High (1ng/mL), medium (10pg/mL) and low (0.1pg/mL) levels of recombinant NS1 antigen were selected based on the values obtained in NS1 ELISA test that corresponds to the level of NS1 detected in the selected serum samples. The cells were treated with NS1 antigens at the three selected concentrations, five serum samples that had varying levels of NS1 antigen from each category  and five healthy individuals (HC). The real time changes in impedance (resistance and capacitance) induced in the MECs after treatment was detected using multiple alternative current (AC) frequencies in an ECIS Zθ 16-well array station (Applied Biophysics) for 72 hours. The raw data was first normalized to cell-free wells to eliminate background signals, followed by normalization to 0 hours post infection (hpi) in the untreated cells. The standard error of mean between the triplicates was determined and presented at regular intervals.The level of 14 commonly studied cytokines, chemokines, adhesion molecules and growth factors  in the 40 selected serum samples were assessed using the Human Magnetic Luminex multiplex screening assay based on flow cytometric analysis of magnetic antibody-coated microbeads targeting the analytes of interest . These 14 cytokines have been shown to have a role during dengue virus infection in terms of endothelial immune activation , 37, 38 The metabolic profiling analysis of the 40 selected serum samples was conducted using quadrupole time of flight liquid chromatography mass spectrometry (QTOF-LCMS) technique. Protein precipitation and metabolite extraction from serum samples was performed by adding 3 volumes of cold (-20°C) mixture of methanol and ethanol (volume ratio of 1:1) to 1 volume of serum. Samples were vortex-mixed and stored at -20°C for 5 minutes, followed by centrifugation at 16,000 × g at 4°C for 10 minutes. The pellet was removed and the supernatant was filtered through a 0.22μm nylon filter and subjected to LCMS procedure.10μL of extracted serum sample was applied to a reversed-phase column   in QTOF-LCMS system (Agilent 6550 iFunnel) operated at a flow rate of 0.6mL/min in positive electrospray ionization (ESI) mode. The solvents used were solvent A, water with 0.1% formic acid and solvent B, acetonitrile with 0.1% formic acid. The gradient started from 25% B to 95% B in the first 35 minutes, returned to 25% B within the next 1 minute, and maintained at 25% B for the remaining 9 minutes. Samples were analysed in one randomized run with a capillary voltage of 3000V and nebulizer gas flow rate of 10.5L/min. During the analysis, two reference masses: 121.0509 m/z (C5H4N4) and 922.0098 m/z (C18H18O6N3P3F24) were continuously measured to allow constant mass correction. Data were required in positive ion mode with a full scan from 50 to 1000 m/z at a rate of 1.02 scans per second.The raw data files obtained from QTOF-LCMS were extracted using an untargeted batch-processing feature extraction software, MassHunter Profinder (Agilent) to generate processed .cef file to be input into a chemometrics software, Mass Profiler Professional (Agilent) for data analysis described below . UnsuperThe clinical data and diagnostic results of twenty serum samples selected for ECIS measurement is shown in The NS1 concentration of the fifteen serum samples selected for ECIS measurement were categorized into high (Panbio Unit 60–80), medium (Panbio Unit 40–60) and low (Panbio Unit 20–40) levels. The NS1 ELISA results for 1ng/mL, 10pg/mL and 0.1pg/mL of NS1 antigen are 68.7, 51.2 and 32.1 Panbio Unit respectively, representing high, medium and low level of NS1 corresponding to the level of NS1 detected in the serum samples.The changes in the impedance of the MECs treated with three levels of NS1 were measured using ECIS system for 72 hpi and the results are presented in The five serum samples randomly selected from each category for the ECIS measurement consisted of samples with high (Panbio Unit 60–80), medium (Panbio Unit 40–60) and low levels (Panbio Unit 20–40) of NS1. Dermal, pulmonary and retinal MECs were incubated with all serum samples for 72 hours and the changes in impedance triggered by NS1 protein contained in the serum samples were assessed using ECIS system to investigate the modulation to MECs barrier function.The changes in impedance of retinal MECs incubated with five serum samples from each category are presented in Cytokine profiling was performed on 40 serum samples from four categories, HC, DWOWS, DWWS and SD . Most ofCytokine/chemokine HMGB-1 and adhesion molecule ICAM-1 were expressed significantly higher in patients with SD and the comparison is shown in QTOF-LCMS was performed to compare the pattern of differential metabolic expression among DWOWS, DWWS and SD categories. Two dimensional PCA analysis revealed no outliers and the samples in the same category were tightly clustered. A total of 35,042 compounds were detected from 40 samples, where 1377 compounds were known metabolites identified based on their molecular weight and retention time crosschecked with Agilent database. Fold change (FC) analysis and one-way ANOVA was performed and a total of 500 metabolites met the standard of FC > 2.0 and p < 0.05 subjected to hierarchical clustering by complete linkage. The expression and regulation of metabolites present in the serum from each category is summarized in supplementary Filtering by volcano plot  revealedThe up-regulated and down-regulated metabolites were further analysed into Venn diagrams , where mTiliacora racemosa, Camptotheca acuminata and citrus, where some have been categorized as medicinal plant to have antibiotic and antioxidant properties. Further analysis of the metabolites in lipid metabolism pathway revealed that most of the metabolites (50%) in DWOWS involved fatty acid metabolism, while 63% and 70% of the metabolites in DWWS and SD respectively involved phospholipid metabolism.Pathway analysis was performed on metabolites that were expressed specifically in each category of DWOWS, DWWS and SD and the most regulated pathways were arranged according to the percentages of metabolites involved in each pathway . The finThe metabolites, namely L-isoleucine, L-phenylalanine, 2-amino-3-methyl-1-butanol, C16 sphinganine, indoleacrylic acid and neoabietic acid, were detected in most of the healthy individuals (70–100%), but had a lower prevalence in dengue patients . Among tTwo metabolites, Etn-1-P-cer and Palmitic amide  were notGlycocholic acid and glyco-ursodeoxycholic acid was detected only in dengue patients but the prevalence was low ranging from 30–40% in each category . The meaOur study showed that NS1 alone can induce loss of barrier function in microvascular endothelium in a dose dependent manner. Based on ECIS measurement, the magnitude of vascular leakage observed was positively correlated with the concentration of NS1 protein, especially pronounced in pulmonary MECs. However, serum NS1 level in dengue patients did not directly correlate to the extent of vascular leakage observed in MECs, indicating that there might be other factors overshadowing the direct effect of circulating NS1 to the endothelium, but still leading to the vascular leakage observed in patients with SD. The association of NS1 antigen with disease severity in dengue virus infection have been commonly studied, however the results are contradictory and inconclusive. Several studies reported that the presence of circulating NS1 antigen was associated with a higher risk of developing severe manifestations and the serum NS1 levels were higher in patients with SD than those with mild dengue fever , 17, 43.The loss of barrier function observed in the NS1-treated MECs has also been reported in other studies where NS1 was shown to degrade endothelial glycocalyx components , 44, incTo address the presence of factors other than NS1 protein in determining disease severity in clinical cases, 40 serum samples were subjected to cytokines and metabolomics profiling. Among all the studied cytokines, HMGB-1 and ICAM-1 were found to be correlated with disease severity, where the expression was significantly higher in patients with SD. HMGB-1 (high mobility group box 1) is a DNA binding protein that regulates innate immunity and partially the adaptive immunity . This prMetabolomics study using QTOF-LCMS revealed that metabolites specifically regulated in DWOWS, DWWS and SD mostly interact with lipid metabolism pathway. DENV has been shown to mediate lipid synthesis and metabolism in their replication cycles  by takinOur metabolomics findings have also identified amino acids such as L-isoleucine, L-phenylalanine and 2-amino-methyl-1-butanol, which are essential in the biosynthesis of proteins were lacking in the sera of dengue patients, with the lowest prevalence in patients with SD. Changes in the plasma free amino acid pattern, indicating infection-related alterations of amino acid metabolism, has been reported in dengue virus infection . The disContrarily, some metabolites were detected at a higher prevalence in dengue patients, where healthy individuals expressed such metabolites at a lower occurrence or none at all. Etn-1-P-cer and palmitic amide, the membrane fatty acids in the family of sphingolipids, were detected higher in dengue patients, especially in patients with SD. Studies have shown that sphingolipids and other bioactive signalling molecules were up-regulated in DENV infected mosquitoes cells  and enhachlamydia trachomatis 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: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know**********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: No**********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: 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: This study by Soe and collegues presents a thorough characterization of the effect of patients’ sera with a broad range of disease severity, or NS1 antigen itself, on the integrity of microvascular endothelial cells. In the attempt of clarifying the ongoing debate on the role of NS1 in disrupting functional endothelial barrier, and its causal relation with Dengue disease progression, the authors further profiled the level of several cytokines and multiple metabolites using a multiplex flow cytometric assay and untargeted metabolomics, respectively. Overall the study is clearly written, results presented in an intelligible fashion and discussed in the context of the vast body of literature on the topic. Although no major breakthrough is presented, and most of the results are actually supportive of a lack of correlation between Dengue disease severity and any of the analysed variables , the presented results are still important and worth publishing as they consolidate the notion that no specific/individual factor present in the sera  can be clearly and unequivocally used as prognostic marker for disease severity. Furthermore, the study present a metabolic-based survey of patients sera, which have been well-characterized/normalized (i.e. based on NS1 levels and IgG), and therefore could be of relevance for the community.Major points:My only experimental concern relates to the ECIS experiment, with respect to the control used and the degree of details provided in the methods section.More specifically :- The purified NS1 protein used in Fig.1 was of which origin , to which extent was purified, and in which diluent was diluted? This information is quite critical as it sets the ground for interpretation of the assay in the following figures. Furthermore, in order to exclude additional factors, the purified NS1 protein used here should have been diluted in healthy individual sera .- What are the positive ctrls used in this assay? - What is the dynamic range of this assay? Without the ctrls described above, is difficult to assess whether the amplitude of the changes described in Fig. 1-3 is significant, and whether the variance across and within groups is acceptable.Minor points:Figures 7-10 could be easily grouped in one figure without losing clarity.Furthermore it would be important for the reader, to include in the volcano plots and in the Venn diagrams  full names of at least some of the top metabolites identified as significantly regulated by one or more classes. These figures as such are currently not conveying any meaningful information.Figures-7-13: The raw data and related statistical analysis of MS results should be presented in full in supplementary tables, to provide the reader with a mining-friendly resource. This is one of novelty points of the study, but it is virtually hidden as only selected candidates are presented in details in Fig.11-13.**********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,  28 Jan 2020Response to Reviewers: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: YesResponse: Thank you. 2. Has the statistical analysis been performed appropriately and rigorously?Reviewer #1: I Don't KnowResponse: For cytokine profiling, the precision and reproducibility of the assays were assured by calculating the coefficient of variance of the replicates. Then we proceeded to access the significant differences of the cytokine expression profile between the categories using student’s T test. For untargeted metabolomics, all the statistical analysis was performed using the chemometrics software Mass Profiler Professional designed for the such purposes. 3. Have the authors made all data underlying the findings in their manuscript fully available?The 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.Reviewer #1: NoResponse: The raw data of untargeted metabolomics have been prepared in full in supplementary S1 table (at end of manuscript). 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: YesResponse: Thank you. 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: This study by Soe and collegues presents a thorough characterization of the effect of patients’ sera with a broad range of disease severity, or NS1 antigen itself, on the integrity of microvascular endothelial cells. In the attempt of clarifying the ongoing debate on the role of NS1 in disrupting functional endothelial barrier, and its causal relation with Dengue disease progression, the authors further profiled the level of several cytokines and multiple metabolites using a multiplex flow cytometric assay and untargeted metabolomics, respectively. Overall the study is clearly written, results presented in an intelligible fashion and discussed in the context of the vast body of literature on the topic. Although no major breakthrough is presented, and most of the results are actually supportive of a lack of correlation between Dengue disease severity and any of the analysed variables , the presented results are still important and worth publishing as they consolidate the notion that no specific/individual factor present in the sera  can be clearly and unequivocally used as prognostic marker for disease severity. Furthermore, the study present a metabolic-based survey of patients sera, which have been well-characterized/normalized (i.e. based on NS1 levels and IgG), and therefore could be of relevance for the community.Major points:My only experimental concern relates to the ECIS experiment, with respect to the control used and the degree of details provided in the methods section.More specifically :- The purified NS1 protein used in Fig.1 was of which origin , to which extent was purified, and in which diluent was diluted? This information is quite critical as it sets the ground for interpretation of the assay in the following figures. Response: The purified NS1 protein is a recombinant dengue virus serotype 1 NS1 protein of sequence strain Nauru/Western Pacific/1974 expressed in 293 human cells. It is a purified recombinant protein with a C-terminal 6x His-tag and purity of >95% by SDS PAGE analysis diluted in buffer solution Dulbecco’s phosphate buffered saline, which was further diluted during the experiment using the respective media of each cell line used. The above information has been added to the methodology. Furthermore, in order to exclude additional factors, the purified NS1 protein used here should have been diluted in healthy individual sera .Response: Healthy individual served as negative control for the experiment and hence it contains no NS1 antigen. It was included to compare against the patients from the three categories of dengue. On the other hand, the purified NS1 protein was included not only to investigate the effect of viral factor (NS1) on endothelium, but also as a comparison for the different NS1 level present in the serum samples. The characteristics of all samples were included in table 1. - What are the positive ctrls used in this assay? Response: A positive control was not included in the experiment as a standard disruptor of the endothelial barrier is yet to be established. We therefore included the purified NS1 protein to compare the modulation of endothelial barrier against patient sera that contain NS1 antigen at varying level with the presence of other host factors. - What is the dynamic range of this assay? Without the ctrls described above, is difficult to assess whether the amplitude of the changes described in Fig. 1-3 is significant, and whether the variance across and within groups is acceptable.Response: The use of ECIS system allow us to monitor real-time cell barrier function, cell to cell adhesion and cell to matrix interactions, which is an incredibly sensitive (described below) method for evaluating barrier function of cells in vitro. It was used to compare the relative pattern of modulation between treated and untreated cells, rather than an absolute reading on the impedance changes. The results presented in this manuscript have been normalized and standardized to eliminate the effect of any gross influences during the experiment. The values were detected at multiple frequencies for three biological replicates with 10 detection points each, and the standard error of the mean was plotted as error bar in Fig. 1-3. The statistical measures included the normalization to a cell-free electrode, standardization of the fluctuations due to cell mobility such as micromotion, and a mathematical model that was implied to determine the changes in the endothelial barrier function. The readings were also normalized to the untreated endothelial cells and therefore, only values with significant difference as compared to the untreated cells were plotted in the graphs to represent the real-time changes of the barrier function of the treated cells. The following studies provide information on the sensitivity of ECIS system with regards to detecting low viral effects:1. Mouse brain MEC infected with West Nile virus at MOI of 0.01 increased TEER up to 6 hours [1]2. Reduced TEER was observed in Mouse Hepatitis Virus type 3-infected brain MEC at a MOI of 0.1 up to 72 hours [2]3. Human Bocavirus-1 at the MOI of 0.001, 0.01, 0.1, 1, 10 and 100 reduced the TEER of human airway epithelial cells [3]The studies above on the infection kinetics of different type of viruses on endothelial / epithelial cells provide information that the ECIS system is sensitive enough to detect small modulation of epithelial/endothelial cells infected with viruses at a relatively low MOIs. Limitation of the ECIS has also been included in the last paragraph of the discussion.References quoted:[1] Lazear et al. (2015) Interferon-λ restricts West Nile virus neuroinvasion by tightening the blood-brain barrier, Sci Transl Med, 7(284): 284ra59.[2] Bleau et al. (2015) Brain Invasion by Mouse Hepatitis Virus Depends on Impairment of Tight Junctions and Beta Interferon Production in Brain Microvascular Endothelial Cells, J Virol, 89(19): 9896-9908. [3] Deng et al (2013) In Vitro Modeling of Human Bocavirus 1 Infection of Polarized Primary Human Airway Epithelia, J Virol, 87(7): 4097– 4102. Minor points:Figures 7-10 could be easily grouped in one figure without losing clarity.Response: Figures 7-9 have been combined into Figure 7 and be divided into parts i), ii) and iii). To better discuss the results for pathway analysis as a separated outcome, Figure 10 was kept as a single figure and has been changed to Figure 8. Furthermore it would be important for the reader, to include in the volcano plots and in the Venn diagrams  full names of at least some of the top metabolites identified as significantly regulated by one or more classes. These figures as such are currently not conveying any meaningful information.Response: We agree with the reviewer’s comment, as such, the top regulated metabolites of each category have been added into the figures. Figures-7-13: The raw data and related statistical analysis of MS results should be presented in full in supplementary tables, to provide the reader with a mining-friendly resource. This is one of novelty points of the study, but it is virtually hidden as only selected candidates are presented in details in Fig.11-13.Responses: The raw data of metabolites present in serum samples from each category of DWOWS, DWWS and SD has been presented in supplementary S1 table (at end of manuscript).AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 5 Jun 2020PONE-D-19-24309R1Correlation of viral NS1 protein, host inflammatory cytokines and immune-related metabolites with disease severity of dengue virus infectionPLOS ONEDear Dr. Sekaran,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Specifically try to answer the two reviewers comments about the manuscript organisation and the number of sample tested. As noted you results are in accordance with other studies that NS1 is not a potential marker for early diagnosis of dengue. your title might be: Correlation of host inflammatory cytokines and immune-relatedmetabolites, but not viral NS1 protein, with disease severity of dengue virus infectionplosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Jul 20 2020 11:59PM. 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Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsIf applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Pierre Roques, Ph.D.Academic EditorPLOS ONE[Note: HTML markup is below. Please do not edit.]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 #2: (No Response)Reviewer #3: 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 #2: YesReviewer #3: No**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: YesReviewer #3: 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 #2: NoReviewer #3: No**********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 #2: YesReviewer #3: Yes**********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 #2: In manuscript titled “Correlation of viral NS1 protein, host inflammatory cytokines and immune-related metabolites with disease severity of dengue virus infection”, the authors tested sera from 40 dengue patients, and intended to evaluate the relation between the presence of NS1/host cytokines/host metabolites and the severity of dengue diseases. The overall conclusion is that no significant correlation has been found between the two. Therefore their original intention to develop a potential marker for early diagnosis of dengue failed. The data collected in this manuscript can be valuable to understanding the dengue diseases and to the broader virology field if some of the essential controls are included in the experiments.1) Introduction lacks a clear elaboration of the purpose of the study. It did not establish a clear rationale that supports the logic of testing NS1. For example, in lines 48-50, the authors said “Hence, this study aims to investigate the role of the viral non-structural 1 (NS1) protein, the inflammatory cytokines and the immune-related metabolites in contributing to the degree of severity of dengue virus infection”. However, the sentence before this conclusion is broad statement “The pathogenesis of severe dengue is theorized to be due to the intricate interactions between viral factors, host genetics and host immune activation” with no facts or details to explain why NS1 needs to be tested. More information should be included to elaborate the significance of why these experiments are done.2) Some information introduced in the discussion, such as the first paragraph of discussion explaining ECIS, should be moved to introduction. It is strange to explain a method in discussion after the results have already been presented.3) Figure 1 showed purified NS1 increases vascular permeability in pulmonary MEC. Figures 2-4 did not show substantial differences when they used different groups of patient sera on different cells. To examine whether NS1 has the potential to become a diagnostic marker for dengue, an experiment that dilute the purified NS1 in uninfected human sera would give a clear answer. It is quite possible that certain sera composition blocks the NS1 effect.4) The authors did metabolites analysis with the patient sera. However, without a control of healthy human sera, these data have no meaning for future analysis. Another possible control is to use human sera from another viral infection to show whether conclusion drawn from Figure 7-10 is dengue specific or not.Reviewer #3: The manuscript Correlation of viral NS1 protein, host inflammatory cytokines and immune-related metabolites with disease severity of dengue virus infection presented by Sekaran SD et al, is an interesting study regarding an important cause of disease, dengue.It is a laboratory study that analyze the effect of the addition of Dengue sera to Electrical cell-substrate impedance senting (ECIS). Also, the citokynes and metabolic profile were measured.I have several question.1. The primaty and secondary dengue have differences in several geographic areas. The authors did no give any commentary about that.2. The ECIS was performed with only 5 serum of dengue cases and none the original NS1 Ag was tested.3. The cytokine profile were determine in only 40 serum . The authors need to include more patients.4. The same observations are done regarding the metabolic expression in dengue patients.**********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  digital diagnostic tool,  16 Jun 2020Response to Reviewers:Reviewer #2: In manuscript titled “Correlation of viral NS1 protein, host inflammatory cytokines and immune-related metabolites with disease severity of dengue virus infection”, the authors tested sera from 40 dengue patients, and intended to evaluate the relation between the presence of NS1/host cytokines/host metabolites and the severity of dengue diseases. The overall conclusion is that no significant correlation has been found between the two. Therefore, their original intention to develop a potential marker for early diagnosis of dengue failed. The data collected in this manuscript can be valuable to understanding the dengue diseases and to the broader virology field if some of the essential controls are included in the experiments.1) Introduction lacks a clear elaboration of the purpose of the study. It did not establish a clear rationale that supports the logic of testing NS1. For example, in lines 48-50, the authors said “Hence, this study aims to investigate the role of the viral non-structural 1 (NS1) protein, the inflammatory cytokines and the immune-related metabolites in contributing to the degree of severity of dengue virus infection”. However, the sentence before this conclusion is broad statement “The pathogenesis of severe dengue is theorized to be due to the intricate interactions between viral factors, host genetics and host immune activation” with no facts or details to explain why NS1 needs to be tested. More information should be included to elaborate the significance of why these experiments are done.Response: The role of NS1 in inducing vascular leakage have been described in the second paragraph. Nevertheless, the interactions between viral factors and host factors have been elaborated in the first paragraph to justify the rationale of investigating the correlation of viral factors and host factors with vascular leakage observed in patients with severe dengue. 2) Some information introduced in the discussion, such as the first paragraph of discussion explaining ECIS, should be moved to introduction. It is strange to explain a method in discussion after the results have already been presented.Response: The paragraph discussing the principle of ECIS has been moved to introduction. 3) Figure 1 showed purified NS1 increases vascular permeability in pulmonary MEC. Figures 2-4 did not show substantial differences when they used different groups of patient sera on different cells. To examine whether NS1 has the potential to become a diagnostic marker for dengue, an experiment that dilute the purified NS1 in uninfected human sera would give a clear answer. It is quite possible that certain sera composition blocks the NS1 effect.Response: The main objective of this manuscript is to assess the role of viral factor (NS1) and host factors (cytokines and metabolites) in contributing to the degree of vascular leakage in the microvascular endothelium during DENV infection, therefore, we focus on the sera of DENV infected patients with varying disease severity, which also contain NS1 protein at varying level. Since the findings showed that the serum NS1 level in dengue patients did not directly correlate to the extent of vascular leakage observed in MECs, we then proceeded to investigate the composition of the sera, in term of cytokines and metabolites, that might had overshadowed the effect of NS1. We have successfully identified some of the host proteins that were highly expressed in dengue patients and they might have played a bigger role as compared to NS1 in inducing vascular leakage. However, we would also like to highlight that no single factor alone should be concluded to be responsible for the progression of severe dengue, as the pathogenesis of dengue involves complex interactions between multiple factors. Nevertheless, the last paragraph of the introduction has been rephrased to better describe the main objectives of this study. 4) The authors did metabolites analysis with the patient sera. However, without a control of healthy human sera, these data have no meaning for future analysis. Another possible control is to use human sera from another viral infection to show whether conclusion drawn from Figure 7-10 is dengue specific or not.Response: Control of healthy human sera was included for all the experiments of ECIS, cytokines and metabolomics profiling. The expression of cytokines and metabolites in dengue patients was compared against the expression profile of healthy individuals. The methodology of sample collection and selection, as well as the results of samples characteristics have been revised to better explain the samples used in this study.  Reviewer #3: The manuscript Correlation of viral NS1 protein, host inflammatory cytokines and immune-related metabolites with disease severity of dengue virus infection presented by Sekaran SD et al, is an interesting study regarding an important cause of disease, dengue.It is a laboratory study that analyze the effect of the addition of Dengue sera to Electrical cell-substrate impedance sensing (ECIS). Also, the cytokines and metabolic profile were measured.I have several questions.1. The primary and secondary dengue have differences in several geographic areas. The authors did not give any commentary about that.Response: The introduction has been revised to include the differences of primary and secondary infections in different geographic areas. 2. The ECIS was performed with only 5 serum of dengue cases and none the original NS1 Ag was tested.Response: For ECIS, we investigated the effect of direct stimulation of NS1 antigen on microvascular endothelium, as well as the effect of NS1 in the presence of other host factors in the patient sera towards microvascular endothelial barrier function. A total of 20 samples were used, 5 from each of the categories of healthy individual, dengue without warning signs, dengue with warning signs and severe dengue to provide an insight to the pattern of the changes in the barrier function of four microvascular endothelial cell lines in response to NS1/sera stimulation. 3. The cytokine profile were determined in only 40 serum . The authors need to include more patients. The same observations are done regarding the metabolic expression in dengue patients.Response: One of the limitations for this study was the small sample size of only 40 sera due to the difficulties in obtaining more samples from patients with severe dengue. Although the study has less statistical power, we were able to identify a few cytokines and metabolites that would provide insight on the role of host factors in the pathogenesis of dengue virus infection and possibly determine or predict disease severity that should be assessed with other approaches using a larger sample size in the future. This limitation has been discussed in the discussion.AttachmentResponse to Reviewers 2.docxSubmitted filename: Click here for additional data file. 7 Jul 2020PONE-D-19-24309R2Correlation of host inflammatory cytokines and immune-related metabolites, but not viral NS1 protein, with disease severity of dengue virus infectionPLOS ONEDear Dr. Sekaran,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.The fact that at the end the studied population is very small taking in account all the variables as noted by the reviewers have to be indicated in the abstract as a limitation of the study. Some results confirmed previous studies from independant groups but new exploration done here remained exploratory and deserved to be confirmed or extended using independant cohort. All this limitation deserved to be highligted in the discussion but also in the conclusion of the abstract.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Aug 21 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). 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For instructions see: We look forward to receiving your revised manuscript.Kind regards,Pierre Roques, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments (if provided):Because the very low number of patients studied at the end the abstract should include this information. Ie the work is interesting but exploratory and deserved to be confirmed on independant cohort concerning the ECIS assay. This is mandatory[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. 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Reviewer #2: YesReviewer #3: 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 #2: YesReviewer #3: 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 #2: YesReviewer #3: Yes**********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 #2: 1) The authors addressed my questions. However, the newly added Figure 12 cannot be found in the paper.2) Now that it is clearly described in the materials and methods that the 40 samples include 30 from patients and 10 from healthy people, the sample size is really small.Reviewer #3: The manuscript Correlation of viral NS1 protein, host inflammatory cytokines and immune-related metabolites with disease severity of dengue virus infection presented by Sekaran SD et al, is an interesting study regarding an importantcause of disease, dengue.It is a laboratory study that analyze the effect of the addition of Dengue sera to Electrical cell-substrate impedance sensing (ECIS). Also, the cytokines and metabolic profile were measured.I have several questions.Altough the authors say that they have include the differences of primary and secondary infections in different geographic areas, in the results the differences are scarse.Moreover, the cytokibes were determined in only 40 serum samples from four categories, HC, DWOWS, DWWS and SD. This explained that only eight serum were analized by categorie.The metabolic expresion of dengue patients have the same difficulty. Only 40 sera were studied, correspondind to foue categories.**********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  digital diagnostic tool,  18 Jul 2020Response to ReviewersReviewer #2: 1) The authors addressed my questions. However, the newly added Figure 12 cannot be found in the paper.Response: Figure 12 was named after the graphical abstract; it is now being renamed as Graphical abstract and was uploaded separately. 2) Now that it is clearly described in the materials and methods that the 40 samples include 30 from patients and 10 from healthy people, the sample size is really small.Response: This limitation is now highlighted in the abstract to state that the findings deserved to be confirmed or extended using independent cohort with a bigger sample size. Reviewer #3: The manuscript Correlation of viral NS1 protein, host inflammatory cytokines and immune-related metabolites with disease severity of dengue virus infection presented by Sekaran SD et al, is an interesting study regarding an important cause of disease, dengue.It is a laboratory study that analyzes the effect of the addition of Dengue sera to Electrical cell-substrate impedance sensing (ECIS). Also, the cytokines and metabolic profile were measured.I have several questions.1) Although the authors say that they have include the differences of primary and secondary infections in different geographic areas, in the results the differences are scarce.Response: The differences of primary and secondary infections in different geographic areas were included in the introduction as supplementary information to the topic. However, we did not include the differences between primary and secondary infections in the experiment planning as we would like to focus on the categories of disease severity. 2) Moreover, the cytokines were determined in only 40 serum samples from four categories, HC, DWOWS, DWWS and SD. This explained that only eight serum were analyzed by categories. The metabolic expressions of dengue patients have the same difficulty. Only 40 sera were studied, corresponding to four categories.Response: The limitation of small sample size has been included in the discussion, as we feel that the exploratory findings merits further investigation, this limitation is now highlighted in the abstract to state that the findings deserved to be confirmed or extended using independent cohort with a bigger sample size.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 22 Jul 2020Correlation of host inflammatory cytokines and immune-related metabolites, but not viral NS1 protein, with disease severity of dengue virus infectionPONE-D-19-24309R3Dear Dr. Sekaran,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. 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For more information, please contact Kind regards,Pierre Roques, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:


We would argue that collaboration should involve specialties outside of surgery, for example, infectious diseases, virology, and public health.(2)1 refer to “very well-documented cases of widespread infection of operating-room personnel” yet without reference to such cases. Please note that similar claims have been rejected in other studies.2The authors(3)3 that showed 20.6% of asymptomatic patients who unwittingly had elective surgery during the incubation period of COVID-19 died. This cohort, however, may not be comparable with those undergoing elective facial plastic surgery (elFPS) as simpler surgery had more favorable outcomes.3 Although an ongoing global collaboration (CovidSurg) is informing decision making with 12,000 patients in 600 hospitals in 70 countries, this may not be authentic to the needs elFPS.There is an omission in the recommendations regarding consent. This may form the basis of a legal challenge similar to the UK Supreme Court, Montgomery v Lanarkshire Health Board case. Concern that surgery may accelerate and exacerbate disease progression of COVID-19 is evident in a small but important study(4)Telemedicine is not uniformly feasible and prone to failures as evident with the Attend Anywhere platform within the U.K.'s NHS. The use of local anesthetic and office-based procedures is controversial and may enhance risk of infection transmission by bioaerosol.(5)4 It may, in fact, augment the olfactory effects of COVID-19. Furthermore, tranexamic acid as a potential repurposed drug has not been approved in this role by any regulatory authority.There is a clear need to mitigate for false negative COVID-19 patients and care workers in a cold COVID-19 site. Yet the use of povidone iodine (PVP-I) disinfectant is problematic and concerns as to its use are expressed in the literature of neurotoxicity.(6)Quality of surgery is important. The challenge will be to reduce exposure risk while not diminishing surgical technique. We foresee the increased use of plume extractors or novel extraoral suction devices such as DuraMax.(7)Multidisciplinary collaboration and accurate data dissemination are vital at the prerecommendation stage. Proper consensus and accountability protect against legal challenge and unintended consequence.(8)elFPS will coexist with COVID-19 for the foreseeable future providing increased evidential opportunities to understand actual outcomes. Global multidisciplinary collaboration is paramount together with opportunities to scrutinize and comment transparently on these data.We read your article with interest and wish to note the following:


Breast milk is a complex biofluid that nourishes infants, supports their growth and protects them from diseases. However, at the same time, breastfeeding is a transmission route for human cytomegalovirus (HCMV), with preterm infants being at a great risk of congenital disease. The discrepancy between high HCMV transmission rates and the few reported cases of infants with severe clinical illness is likely due to the protective effect of breast milk. The aim of this study was to investigate the anti-HCMV activity of human preterm colostrum and clarify the role of colostrum-derived extracellular vesicles (EVs). Preterm colostrum samples were collected and the EVs were purified and characterized. The in vitro anti-HCMV activity of both colostrum and EVs was tested against HCMV, and the viral replication step inhibited by colostrum-purified EVs was examined. We investigated the putative role EV surface proteins play in impairing HCMV infection using shaving experiments and proteomic analysis. The obtained results confirmed the antiviral action of colostrum against HCMV and demonstrated a remarkable antiviral activity of colostrum-derived EVs. Furthermore, we demonstrated that EVs impair the attachment of HCMV to cells, with EV surface proteins playing a role in mediating this action. These findings contribute to clarifying the mechanisms that underlie the protective role of human colostrum against HCMV infection. Human milk (HM) is considered the most important biofluid for the nutrition of infants and for the development of a child’s immune system by the World Health Organization . HM provExtracellular vesicles (EVs) from human milk have recently become a subject of increasing interest for their protective role for infants . EVs areColostrum samples  were obtained from 13 healthy mothers admitted to the Sant’Anna Hospital  for preterm delivery between February 2017 and January 2020. An ethical review process was not required for this study since it was not a clinical trial. Each milk donor involved in this research signed a written consent form in which the data protection of the mother and infant was assured. Moreover, the donors were informed that only milk samples stored in excess would be used for research purposes, and the study design was explained. The donors cleaned their hands and breasts according to the Italian Human Milk Bank (HMB) guidelines ,2613 par50 value) ranging from 38.71 µg protein/mL to 213.9 μg protein/mL. The observed variability in the anti-HCMV activity of human colostrum was likely related to the well-known interpersonal variability of HM composition and to the HCMV serostatus of the milk donors. Furthermore, we demonstrated a remarkable antiviral activity in all the EV preparations, with EC50s ranging from 3.98 to 75.93 µg protein/mL.After the isolation and characterization of the EVs, colostrum samples and the corresponding isolated EVs were tested in vitro on HFF-1 cells against HCMV Towne strain expressing the GFP protein; the GFP marker facilitated the identification of infected cells. As reported in 50 value of 41.56 µg protein/mL) was observed, even when a 10-fold greater viral inoculum was used (sample 3). Moreover, we excluded the possibility that the antiviral activity of the EVs was due to cytotoxicity, since the EVs did not affect the cellular viability when the cells were treated under the same conditions as the antiviral assays and processed by means of an MTS assay . To the best of our knowledge, this is the first study to have reported the intrinsic antiviral activity of human colostrum-derived EVs against HCMV infection. Näslund et al. (2014) demonstrated a similar antiviral activity of breast milk exosomes and showed that milk exosomes, but not plasma exosomes, significantly reduced the productive HIV-1 infection of monocyte-derived dendritic cells by approximately 50%, compared with untreated cells, and blocked HIV-1 transfer from monocyte-derived dendritic cells to CD4+ T cells . Only a 90) for 2 h at 37 °C and then added to cells at dilutions at which the vesicles were no longer active. In order to identify the major mechanism of action, three EV preparations  were selected and subjected to further studies. First, a virus inactivation assay was performed in which the virus was incubated with an effective high concentration of EVs  in the comparison between the number of HCMV foci in the untreated wells and those in the treated wells, with no active EV dilutions in the attachment assays, thus suggesting that the viral titers did not change. On the other hand, when EVs were added immediately after virus attachment to assess their ability to prevent entry (entry assay), no inhibition was observed for two EV preparations, and a weak inhibition was observed at high doses for the sample 3-derived EVs  to evaluate the ability of EVs to affect the interaction between the viral particles and the cell surface proteins. To this end, the EVs were subjected to trypsin digestion in order to shave the proteins that were present on the surface of the EVs. As a control, an aliquot of each EV preparation was subjected to the same shaving protocol but without digestion with trypsin. The shaved EVs were characterized using NanoSight to evaluate their size, which showed a reduction in diameter  compared with intact EVs  , while n50 values of the intact EVs were around 3.65 × 1010 particles/mL, while the EC50 values of the shaved EVs were in a range from 1.47 × 1011 to 4.61 × 1011 particles/mL. In order to validate these data, antiviral experiments were also performed against HCMV strain AD169—another laboratory reference strain. Again, the clarified colostrum exhibited a net anti-HCMV-AD169 activity (EC50 163 µg/mL), and the intact colostrum-derived EVs exerted a high antiviral activity (EC50 24.2 µg/mL), which was significantly reduced after the shaving experiments . Our results indicate that the proteins on the surface of EVs play a role in the antiviral mechanism of action of EVs.After shaving, the expected decrease in the number of EVs was observed, and the inhibition test data were therefore normalized to the decreased number of vesicles. Antiviral assays against the HCMV Towne strain were then performed by treating cells with intact, shaved and control EVs. As reported in The shaved peptide mixture derived from the EV membrane surface proteins (surfaceome) was analyzed by means of LC–MS/MS in order to investigate the proteins that could be responsible for the EV antiviral properties .The total number of identified proteins in the three analyzed samples was 258. Of these proteins, 45 were found to be shared between at least two samples out of three, and 16 were found in all the analyzed samples A. SampleThe surfaceome proteomic analysis showed that 91% of the identified proteins had already been found in the HM EVs ,30. SomeThe components of the immunoglobulin superfamily—and IgAs in particular—are already known to be prominent in human breast colostrum and to provide newborns with passive immune protection. Among the complement proteins, complement component C9—which has here been identified for the first time in the HM EV fraction—is the major actor of the membrane attack complex (MAC), a multi-protein complex that forms pores in the plasma membrane of target pathogens or virally infected cells . C9 polyMost of the proteins identified in our study are phosphorylated and/or glycosylated and thus function as soluble receptors that inhibit pathogen binding to the mucosal cell surface. Caseins, for instance, can exert their antiviral activity with both phospho-groups and glycans. Κ-casein has been reported to have anti-human rotavirus activity via the direct binding of glycans to the virus . Among tRegarding milk fat membrane associated proteins, the involvement of human milk mucins in protection against virus infection has already been established. Purified mucin components (MUC1 and MUC4) are able to inhibit HIV-1 in vitro and prevent the transmission of HIV-1 from dendritic cells to CD4+ T cells ,39. MoreThe macrophage mannose receptor (MRC1)—a single-pass transmembrane glycoprotein belonging to the C-type lectin family—has been investigated in great detail. It is found on the surface of most tissues—macrophages, dendritic cells and some lymphatic or liver endothelial cells—where it plays the role of capturing pathogens and removing them from circulation in the bloodstream . As alreThrombospondin (TSP1) is an extracellular matrix glycoprotein with functional domains that is able to bind calcium, heparin, collagens, other matrix components and cell surface receptors, including CD36, heparan sulfate proteoglycans (HSPG) and integrins. It has been demonstrated that HCMV infection suppresses the expression of TSP1 and 2 in human fibroblasts, in human retinal glial cells and in primary fetal astrocytes, and this action is mainly mediated by HCMV immediate–early proteins ,55. ConsTenascin is a large, hexameric extracellular matrix glycoprotein that is heavily sialylated: each monomer contains an assembly domain, a region of epidermal growth factor-like repeats, a region of fibronectin type III-like domains and a fibrinogen-like globe . Fouda eAnother protein involved in the anti-HIV-1 mechanism of action is gelsolin, which belongs to the actin binding protein superfamily and is known to promote actin cytoskeleton remodeling. García-Expósito et al. (2013) demonstrated that gelsolin can compromise HIV-induced actin reorganization and the viral receptor capping events required for HIV-1 Env-mediated fusion, entry and infection. Therefore, gelsolin can constitute a barrier that restricts the HIV-1 infection of CD4+ lymphocytes in a pre-fusion step . Finally, osteopontin is a multifunctional protein that is involved in both innate and adaptive immunity. Zhao et al. (2016) demonstrated that osteopontin is an essential positive regulator that protects the host from virus infection through the stabilization of the tumor necrosis factor receptor (TNFR)-associated factor 3, induction of interferon regulatory factor 3 activation and interferon beta (IFN-β) production .The peptide mixtures derived from the EV shaving of preparations 12 and 13 were investigated for their anti-HCMV Towne potency. Pooled peptides were dissolved in 420 µL DMEM medium, and two different volumes of the samples were assessed in triplicate by means of an attachment assay. As reported in Overall, our results provide novel insights into the protective role of human colostrum against HCMV infection in reducing the risk of viral transmission from mother to child through breastfeeding. We have confirmed the anti-HCMV activity of human colostrum in vitro, and we have identified extracellular vesicles as additional antiviral effectors, together with well-known non-specific bioactive and immune components of human colostrum. The investigation of the mechanism of action has indicated that the anti-HCMV activity of EVs is not mediated by a direct inactivation of the viral particle, but rather by the inhibition of the viral attachment to the cell surface. The significant reduction in the antiviral activity of EVs, after shaving experiments, suggests a crucial role of EV surface proteins in inhibiting viral infection. The proteomic analysis of the EV surfaceome revealed the contribution of immune components including antibodies (mainly IgA) and complement component proteins (C9 and C3) to antiviral activity. Moreover, we identified several proteins which have proven antiviral properties against HIV infection but which have scarcely been characterized so far for their activity against HCMV. We found both milk proteins  and proteins shared by different tissue cells and biological fluids. The common feature of the identified proteins is that their glycomoieties seem to play a crucial role in the interference mechanism between a virus and the host cells, acting as a point of cell-specific recognition for the virus particle and inhibiting its attachment on the cell surface. Further studies are necessary to explore the involvement of the identified proteins and their glycan chain composition in the protective role of EVs against HCMV.


After the first case of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV2) was observed in China in December 2019, coronavirus infection 2019 (COVID-19) was declared as a pandemic by WHO on 11 March 2020 . At thisChest CT has been used widely not only suspected but also confirmed COVID-19 cases. According to the American College of Radiology guideline, it should not be used for COVID-19 screening and diagnosis because they emphasized that chest CT findings of COVID-19 might be confused with the other infectious diseases such as influenza. Furthermore, if the patient does not have a positive viral test result for COVID-19, it might affect the patient’s management and treatment due to false chest CT findings . Despiteet al. showed that 1 out of 25 patients having cancer diagnosis was diagnosed by incidentally while performing blood tests or imaging due to other pre-existing conditions [et al. estimated that 4.8 million people had chest CT scan in the USA and 63,000 of them  had malignant nodules in the lung parenchyma [In a study, the number of incidental cancer diagnosis was 4% among cancer patients. Koo nditions . Furtherrenchyma . In thisWe do not have information about the total daily numbers of chest CT scans during the pandemic in the USA and Europe yet. However, the number of chest CT scans are expected to increase during the pandemic. Despite the recommendations of American College of Radiology that chest CT should not be used as a diagnostic tool for COVID-19, it plays a crucial role in determining the severity of the infection in hospitalized patients in particular . Thus, iThere is still a debate regarding decrement in cancer screening and new cancer diagnoses during the pandemic. However, the number of cancer cases diagnosed incidentally may increase and there is no clear advice on how we manage these patients during and most importantly, after COVID-19 infection. We have to take into account these patients and plan concerning the diagnosis and treatment of cancer in these patients because it seems that we might need more evidence-based recommendations for the management of them in the near future.


Early diagnosis of head and neck cancer (HNC) will improve patient outcomes. The low incidence of HNC renders its detection challenging for a general practitioner (GP) in primary health care (PHC). To examine these challenges, our cohort consisted of all patients visiting PHC centres in the City of Helsinki in 2016. We chose 57 ICD-10 codes representing a sign or symptom resulting from a possible HNC and compared data for all new HNC patients. A total of 242,211 patients  visited PHC centres, 11,896 (5%) of whom presented with a sign or symptom possibly caused by HNC. Altogether, 111 new HNCs were diagnosed within the Helsinki area, of which 40 (36%) were referred from PHC. The median delay from the initial PHC visit to the referral to specialist care was 5 days, whereby 88% of patients were referred within one month.For every head and neck cancer (HNC) patient encountered in the primary health care, a general practitioner (GP) will meet approximately 6000 other patients, 100 of whom exhibit a sign or a symptom potentially caused by a HNC.Despite the low incidence of HNC, GPs referred patients to specialist care effectively, limiting the median delay from the initial appointment to referral to only 5 days. Despite the low incidence of HNC and the large number of patients presenting with HNC-related symptoms, GPs working in PHC sort out potential HNC patients from the general patient group in most cases remarkably effectively. Any delay in the diagnosis and treatment of head and neck cancer (HNC) might affect patient outcome . The worThe European Head and Neck Society launched the Make Sense Campaign (MSC) in 2013 in order to inform the general public and PHC personnel about HNC . The MSCThe background population of patients who contact a GP with a symptom potentially caused by HNC and among whom a GP needs to identify suspected HNC patients for further referral remains largely unexamined. Here, we aimed to study this background population in order to investigate the challenges encountered by a GP related to detecting HNC patients. We hypothesised that due to the rarity of HNC, their diagnostics in PHC is difficult. We also aimed to provide a rough estimate for the likelihood that a PHC patient of a certain age with a specific symptom has HNC.We retrospectively investigated the total number of patients attending outpatient visits at all 28 PHC centres in the City of Helsinki, with a population of 635,181 in 2016, excluding emergencies. Data were collected from the centralised electronic database managed by the Social Services and Health Care Division of Helsinki, which records every visit to a nurse or a GP. The database contains all patient information recorded through the patient record system Pegasos, that was used in all 28 PHC centres. From this database, it is possible to retrieve anonymous data from patient appointments that include the patients’ age, sex and the ICD-10 diagnosis code used. In Finland, it is compulsory for a GP to select at least one diagnosis using the ICD-10 codes most accurately describing the reason for each PHC visit. However, it is not possible to automatically gather any information written in text form.We selected 57 ICD-10 codes we identified as representing a sign or symptom potentially caused by HNC, examining how often they were used in our patient population . Data wen = 40), we collected their data from the patient record system that was used in the PHC centres (Pegasos). Patient-related variables included age and sex.In Helsinki, all HNC patients are referred to and treated at our university hospital . The treatment modality for all HNC patients in the Helsinki and the surrounding region (Uusimaa), is assessed in multidisciplinary tumour board meetings, which are held weekly. We investigated all 111 new HNC patients diagnosed from the population of Helsinki in 2016. This area covers about one-third of the total referral population of our university hospital. We manually collected data on patients’ health-seeking behaviour, the number of visits to a doctor before the referral, the place from which the referral was made to specialist care (SC), the delay from the initial PHC visit until the referral to SC and the ICD-10 code for the appointment from hospital records. Additionally, regarding patients who were referred from PHC  and an institutional permit was granted. The Helsinki Social and Health Care Services also approved the study protocol.A total of 242,211 patients visited a GP in PHC in the City of Helsinki in 2016, resulting in a total of 499,542 outpatient visits. Among these patients, 55.0% were over 40  years old and 60.2% were female. In December 2016, 380 GPs were concurrently working at PHC centres in the City of Helsinki.The total number of patients who visited a GP and had a symptom potentially caused by HNC reached 11,896 . Among tn = 23; 20.7%), a public dentist , other hospital specialties , a private dentist , occupational health care  or a hospital emergency unit . One patient visited a public PHC centre in a neighbouring city.In 2016, altogether 111 new HNCs were diagnosed among residents of the City of Helsinki. Public PHC centres referred 40 (36.0%) of them to SC . The remn = 15; 37.5%), oral cavity , hypopharynx , nose and nasal cavity , larynx , unknown primary  and major salivary glands . All cancer sites according to the place of referral appear in A total of 40 new HNC patients were referred from a public PHC centre to SC. Among these patients, 27 (67.5%) were male and 13 (32.5%) were female with a mean age of 64 . The most common cancer sites among these patients were the oropharynx . Other symptoms listed in order of frequency included: non-healing mouth ulcers and/or red or white patches in the mouth ; pain in the throat ; painful and/or difficulty swallowing ; blocked nose and/or bloody discharge from the nose ; difficulties breathing and/or haemoptysis ; and hoarseness . Three patients (7.5%) did not have any diagnosis code registered at their initial visit to PHC and 4 (10%) patients had an ICD-10 diagnosis code that was not relevant or was too general compared to their symptoms.The most common symptom among the 40 patients referred from PHC (according to the ICD-10 diagnosis code used at the visit) was a lump in the head and neck site  of the patients were referred to SC during the initial visit to the PHC centre, 17 patients at the second visit and 3 patients during the third visit. Two thirds of these additional visits (65%) were related to an ultrasound and fine-needle sample, while the remaining were clinical follow-up or other visits. Only one patient who initially visited a public PHC centre subsequently sought medical advice from the private sector.Half  was 5 days . More than half (57.5%) of the patients were referred to SC within a week, 72.5% within 2 weeks and 87.5% within 1 month. Only three patients had a PHC delay of more than 2 months.In the present patient population consisting of patients residing in the Helsinki area, a new HNC was detected roughly once in every 12,500 visits or in every 6000 patients seen in PHC. Yet, the median doctor-related PHC delay was no more than 5 days, and half of the patients were referred to SC during the initial visit. The number of patients who presented with a similar symptom potentially caused by HNC remains largely unexamined. In our study, we collected data from all patients who visited a PHC centre in the City of Helsinki in 2016, amounting to a total of 242,211 patients. Among these patients, we identified 40 new HNC cases. To our knowledge, this is the only study that has thoroughly investigated the background population of patients among whom a GP needed to identify potential HNC patients for further referral.In Finland, the management of all HNC patients is regulated by governmental authorities, organized by the public health care system and centralized at five university hospitals. Patients can either seek medical care through public PHC centres or private sector. Additionally, all employers must provide occupational health care services to their employees which are organized either through public or private sector. Public health care is almost entirely funded through tax collection by the municipality that the patient belongs to. Total payment limit for any health service is 683 euros during a calendar year, after which all care is entirely free for the patient. Private health insurances are not widely used because of good availability and low cost of public PHC and occupational health care. All sectors can similarly refer patients to public SC unit for further investigation and treatment of potential HNC.In Finland, all head and neck malignancies are discussed at weekly multidisciplinary tumour board meetings held at the five university hospitals. Private treatment in the field of HNC does not exist; even the most minor malignancies are referred to the public SC unit.If we assume that the patient population was distributed evenly across all 380 GPs working in PHC centres, each GP would have had 1315 appointments with 637 patients each year. Based on our series, with this patient volume in an unselected patient material, a single GP would diagnose an average of 3.2 HNCs during a 30-year career. Compared to the extremely low number of new HNC patients identified by a GP and the vast number of other patients a single GP sees each year, the median doctor-related PHC delay of 5 days seems extremely low. Most notably in this study setting, we were only able to study the doctor-related delay from the initial PHC visit until the referral to SC. Any system-related delay between the first contact with PHC and the initial visit remains unknown. The median of 1.5 visits before the referral to SC is similar to two other studies regarding HNC and oral cancer ,12, but In our patient population, the annual number of newly diagnosed HNC cases was 111, which is in line with the annual number reported by the Finnish Cancer Registry . Howevern = 40) of the HNC patients were referred from PHC. Oral cancers were underrepresented in this patient group, since the majority of these patients (64%) were referred from a public or private dentist. In a systematic review consisting of 16 studies examining the referral of oral cancer patients in PHC, fewer patients (44%) were referred by dentists [Regarding the division of labour in HNC between medical doctors and dentists, the majority of HNC patients were referred to SC by a medical doctor (72%), which agrees with a study by Ligier et al. . Around dentists . This diAccording to our evaluation, a new HNC was detected in 1 of every 6000 patients or 12,500 visits to PHC centres, whereby 5% of patients had HNC-related symptoms. This proportion is significantly higher than in another study by Alho et al., which investigated the incidence of HNC in Northern Finland . In theiAccording to our analysis, stratification according to certain clinical characteristics results in a higher HNC detection ratio. If patients younger than 40 years old are ruled out, the ratio rises to 1:3300. The majority of HNC patients were male (67.5%), indicating that the likelihood of a ≥40-year-old man visiting a GP in PHC would have a HNC stood at 1:2000. Luckily, patients often present with various symptoms, which point clinicians towards suspicion of a possible malignant aetiology. Among all patients who presented with a symptom possibly caused by HNC , the ratn = 4611), the prevalence of HNC reached nearly 3% [In addition, certain cancer risk groups emerged. HNC was particularly prevalent among males ≥60 years old who presented with one of the six symptoms mentioned in the MSC. The likelihood of cancer among this patient group varied between 0.5 and 3%, whereby ICD-10 codes associated with a lump in the head and neck region represented the most malicious symptom . A canceearly 3% . Given tearly 3% .One limitation of this study lies in the likely heterogeneity in the use of ICD-10 codes. Because determining the ICD-10 diagnosis code relies on a subjective decision-making process from the treating physician, the comparability of diagnoses remains limited. In 17.5% of our cases, the ICD-10 diagnosis code was not used at all or was non-descriptive. This generates a bias when assessing the relative cancer risks among different patient groups. Importantly, the number of doctor visits with a certain symptom-related ICD-10 diagnosis code does not reflect the prevalence of specific symptoms in the general population. That is, a distinctive difference exists between a patient with a symptom and a patient seeking medical advice because of a symptom, which should alarm GPs, particularly if the symptom is potentially cancer related and the patient has risk factors for a malignancy. Furthermore, it is important to notice that usually only a single ICD-10 code is used per visit; if patient visits PHC with multiple symptoms, only the primary symptom is recorded. Therefore, calculations which are too precise or far-fetched conclusions should not be made. Another apparent limitation is that the codes include a large variety of different and often benign conditions, particularly in the most commonly used codes, such as difficulties breathing, pain in the throat or painful swallowing and a blocked nose. These ICD-10 codes can be used to describe anything from cardiac arrest to a viral upper respiratory tract infection. In addition, further limitations include the lack of data on tobacco use and alcohol consumption. Despite these obvious limitations related to the use of ICD-10 codes, they are currently the only practical way to study symptom epidemiology in a large population series before the emergence of structured patient record systems.To conclude, we identified one HNC patient for every 6000 patients visiting PHC. If patients’ symptoms, age and sex were taken into account, this ratio climbed to as high as 1:33 . Despite the challenges in identifying HNC patients from the normal patient flow, the median PHC delay was only 5 days and delays of over a month occurred only in 12.5% of all cases.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee  and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.