A novel assay for improved detection of sputum periostin in patients with asthma

Junya Ono; Masayuki Takai; Ayami Kamei; Shoichiro Ohta; Parameswaran Nair; Kenji Izuhara; Sven-Erik Dahlén; Anna James; on behalf of the BIOAIR consortium

doi:10.1371/journal.pone.0281356

Peer Review History

Original SubmissionFebruary 8, 2022
10 May 2022 Decision Letter - Fabio Luigi Massimo Ricciardolo, Editor PONE-D-22-03930A novel assay for improved detection of sputum periostin in patients with asthmaPLOS ONE Dear Dr. Anna James, 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 manuscript is interesting but it needs additional improvement following the reviewers' suggestions. Please submit your revised manuscript by 60 days. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at 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. 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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: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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: Yes Reviewer #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: No Reviewer #2: Yes ****** 5. Review Comments to the Author Please 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. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors tested two ELISA assays that could be used to detect either whole or cleaved serum and/or sputum periostin in BIOAIR population. Furthermore, this study aimed to reveal the appropriateness of the sputum periostin as a biomarker in airway disease. The paper is of interest, however, several issues need to be resolved. Major comments: 1) In my opinion, the Authors explored several aspects related to periostin assessment. I think that they should focus the study on a singular point and explore it more deeply (i.e. validation of the Assay B as a feasible tool for the detection of cleaved sputum periostin). In this way, the article will be clear. 2) In my opinion the Material and Methods section must be rewritten. For example, in this section, the Immunoprecipitation and Western blotting paragraph can be put together with other paragraphs. Furthermore, I suggested moving all the figures and tables cited in the Result section. 3) The authors did not indicate in the Statistical Analysis paragraph for what analysis the author used the Chi2 test. Minor comments: 1) Table 1 and 2 abbreviation: IL, interleukin this abbreviation, is not necessary. 2) Please insert in Figure 2 (panel A) the Spearman correlation rank and P-value. 3) In Table 1 the authors did not indicate the inhaled corticosteroid considered for the analyses (beclomethasone or fluticasone). Please provide this information. 4) I suggest changing the word “Atopic” with “Atopy” in Table 1. Reviewer #2: The paper of Ono and colleagues compared two ELISA methods, assay A and Asssay B, and showed a higher efficacy of the latter for the detection of sputum periostin in samples from asthmatic patients. The Authors showed also a greater usefulness of sputum periostin as a biomarker of airway inflammation than serum periostin. Despite the paper gives interesting information, it should be improved as it takes into account several facets which deserve a deeper investigation. The authors showed different sputum periostin levels according to different asthma inflammatory phenotypes, being highest in mixed granulocytic asthma. I think it is interesting to assess whether sputum periostin correlates with inflammatory cytokines other than Type-2 mediators (i.e. IL-8, IL-17A/F). It is not clear what test was used to measure serum periostin. Have the authors performed the ELISA tests on the whole sputum? ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. 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. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. 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Revision 1
8 Nov 2022 Author Response Dear PLOS One Editors, We thank the reviewers for their helpful feedback and guidance, and we are pleased to resubmit our manuscript entitled “A novel assay for improved detection of sputum periostin in patients with asthma” for consideration for publication in PLOS One, along with our responses to comments from the editors and reviewers below. As described in more detail below, our original plan was to add more data regarding sputum cytokines as suggested by reviewer 2, before our extended deadline of 11th October 2022. However, for reasons beyond our control we have not been able to do this. We have however addressed all other suggestions made by the reviewers including the addition of important new data relating to the clinical utility of sputum periostin as a potential biomarker in asthma. Your sincerely, Anna James, corresponding author Editors’ comments 1.PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. Response: Original blot or gel results have now been uploaded as a Supporting Information file. In order to meet the PLOS One requirements for the presentation of gel/blot results we have also slightly adjusted the figures which contain such data (figs 1, 5 and S3). 2. Please include a complete copy of PLOS’ questionnaire on inclusivity in global research in your revised manuscript. Response: A completed version of the “Inclusivity in global research questionnaire” has been uploaded as Supporting Information. 3. Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. Response: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 4. Thank you for stating the following in the Competing Interests section: [I have read the journal's policy and the authors of this manuscript have the following competing interests: JO, MT and AK are employees of Shino-Test Corporation. PN reports grants and/or personal fees from AstraZeneca, Teva, Sanofi, BI, Methapharm, Cyclomedica, GSK, Equillium, Foresee and Knopp. KI reports grants from Shino-test Corporation, AstraZeneca and Sanofi. SED reports personal fees from AstraZeneca, Cayman Chemicals, GSK, Novartis, Sanofi-Regeneron and TEVA. SO and AJ have no relevant financial disclosures.]. Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. Response: This does not alter our adherence to PLOS ONE policies on sharing data and materials. Reviewers’ comments Reviewer #1: The authors tested two ELISA assays that could be used to detect either whole or cleaved serum and/or sputum periostin in BIOAIR population. Furthermore, this study aimed to reveal the appropriateness of the sputum periostin as a biomarker in airway disease. The paper is of interest, however, several issues need to be resolved. Major comments: 1. In my opinion, the Authors explored several aspects related to periostin assessment. I think that they should focus the study on a singular point and explore it more deeply (i.e. validation of the Assay B as a feasible tool for the detection of cleaved sputum periostin). In this way, the article will be clear. We fully agree with reviewer 1 and in order to improve the clarity of our manuscript we have therefore made the following changes: 1. The first paragraph of the Results section concerning development of the methodology used and entitled “Development of ELISAs detecting whole and cleaved periostin” has been moved to the Methods section and shortened somewhat. This will hopefully improve the focus of the results section which now describes associations between the sputum periostin levels measured, asthma and airway inflammation (line 97-166). 2. To further validate and investigate the clinical relevance of sputum periostin levels measured using assay B, as suggested, we have added new data regarding the effects of anti-inflammatory glucocorticoid therapy, an important factor often overlooked when novel biomarkers are proposed (lines 44, 85, 171, 262-272, 368). In addition we have added information regarding assay performance at repeat visits (lines 173, 209). 2. In my opinion the Material and Methods section must be rewritten. For example, in this section, the Immunoprecipitation and Western blotting paragraph can be put together with other paragraphs. Furthermore, I suggested moving all the figures and tables cited in the Result section. The Material and Methods section has now been adjusted as suggested. The Immunoprecipitation and Western blotting paragraph has been merged with the first paragraph on antibodies (line 96). Furthermore, and as described above in point 1, the first paragraph of the Results section along with all figures and tables concerning development of the methodology used have been moved to the Methods section to improve the distinction between the two aspects of this manuscript, firstly the development of a new method and secondly its validation in well-characterised patients with asthma. 3. The authors did not indicate in the Statistical Analysis paragraph for what analysis the author used the Chi2 test. This Chi2 test was used to compare percentages of males/females, those taking oral corticosteroids and those with a positive skin prick test in the table showing patient characteristics. This has been clarified in the Statistics section line 196. Minor comments: 1) Table 1 and 2 abbreviation: IL, interleukin this abbreviation, is not necessary. This abbreviation has been removed. 2) Please insert in Figure 2 (panel A) the Spearman correlation rank and P-value. The figure actually shows a linear regression, but we have added more information both about the Spearman correlation rank and P-value, as well as the linear regression correlation coefficient and associated p value, into the figure legend (line 238) as well as on figure 2. 3) In Table 1 the authors did not indicate the inhaled corticosteroid considered for the analyses (beclomethasone or fluticasone). Please provide this information. This information has been provided (table at line 177). 4) I suggest changing the word “Atopic” with “Atopy” in Table 1. This has been changed (table at line 177). Reviewer #2: The paper of Ono and colleagues compared two ELISA methods, assay A and Asssay B, and showed a higher efficacy of the latter for the detection of sputum periostin in samples from asthmatic patients. The Authors showed also a greater usefulness of sputum periostin as a biomarker of airway inflammation than serum periostin. Despite the paper gives interesting information, it should be improved as it takes into account several facets which deserve a deeper investigation. The authors showed different sputum periostin levels according to different asthma inflammatory phenotypes, being highest in mixed granulocytic asthma. I think it is interesting to assess whether sputum periostin correlates with inflammatory cytokines other than Type-2 mediators (i.e. IL-8, IL-17A/F). It is not clear what test was used to measure serum periostin. Regarding the last point, we have now added more information to describe how serum periostin was analysed in the current investigation, line 123-125. Serum periostin levels measured using this method (assay A) have been extensively reported in the literature (see reference 4 in manuscript for summary, Izuhara et al.). We fully agree that it would be of interest to examine how sputum periostin relates to other inflammatory cytokines and began to plan for such an investigation after receiving this relevant comment. We decided to extract sputum and matched plasma samples from our biobanks where samples from the BIOAIR study are stored, and perform an OLINK multiplex analysis of 96 inflammatory mediators in these samples which would enable us to answer several important questions including those raised by reviewer #2, but also comparisons between local and circulating biomarker levels. However, due to general delays caused by the COVID pandemic, the biobank and waiting times for OLINK analysis at our core facility, we will not be able to add this information before the 6-month resubmission deadline at PLOS One (which led us to enquire about an extension). As this investigation has already been scheduled, the best we can do is follow up the current, initial investigation with a later publication, where we delve more deeply into the inflammatory pathways that relate to sputum periostin levels. Have the authors performed the ELISA tests on the whole sputum? The ELISA tests were performed on sputum supernatants, the fraction containing soluble mediators after the cell pellet has been separated away in the process of sputum handling and this has been clarified in the text (line 190). https://doi.org/10.1371/journal.pone.0281356.r002
23 Jan 2023 Decision Letter - Shama Ahmad, Editor A novel assay for improved detection of sputum periostin in patients with asthma PONE-D-22-03930R1 Dear Dr. James, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at 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. 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 onepress@plos.org. Kind regards, Shama Ahmad, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. 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 addressed Reviewer #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: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. 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: Yes Reviewer #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: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please 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. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors replied exhaustively to the Reviewer's comments and is now considered to be suitable for publication in Plos One. Reviewer #2: The authors amended the manuscript appropriately and provided satifactory answers. Themanuscript is suitable for publication. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. 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. Reviewer #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0281356.r003
Formally Accepted
2 Feb 2023 Acceptance Letter - Shama Ahmad, Editor PONE-D-22-03930R1 A novel assay for improved detection of sputum periostin in patients with asthma Dear Dr. James: 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. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Shama Ahmad Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0281356.r004

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