PONE-D-19-23084

SERUM GALECTINS AS POTENTIAL BIOMARKERS OF INFLAMMATORY BOWEL DISEASES

PLOS ONE

Dear Dr Subramanian,

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 very constructive points raised during the review process by the two reviewers.

We would appreciate receiving your revised manuscript by Nov 04 2019 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

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We look forward to receiving your revised manuscript.

Kind regards,

Roger Chammas, M.D, Ph.D

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: Partly

Reviewer #2: Partly

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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

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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

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 in this study determine the potential for serum galectin-1 and galectin-3 to act as a biomarker for either IBD screening or IBD activity. Previously, tissue levels of galectin-1 and galectin 3 have been shown to be elevated in IBD patients but a systematic comparison of galectin-1 and galectin-3 with other biomarkers has not occurred.

I am concerned by the analysis that uses a CRP threshold of 5mg/dL. The reference that they cite to provide this actually provides data to support a threshold of 5mg/L. This does not negate the findings that serum levels of galectin-1 and -3 are increased in IBD patients but it does affect much of the data analysis.

They do not really describe the issue with these markers being elevated in IBD but not acting as markers of disease activity. Discussion should center around the possibilities that either it is only an early step in the inflammatory cascade or that treatment effect also could cause this to falsely rise (rather than be a marker of inflammation).

Minor points include a few spelling errors and that much of the data provided in the introduction is not appropriately referenced.

Reviewer #2: GENERAL COMMENTS:

The authors presented a relevant work for the field of inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis. They studied whether circulating galectins can be associated with C-reactive protein in diagnosis and/or biomarkers of IBD. In this study, it was evident that serum galectin-1 and -3 levels were increased in serum of patients with IBD in comparison with healthy individuals. On the other hand, the levels of galectin-1, -2, -3, -4, -7 and -8 did not modified in these IBD patients. However, the high levels of galectins were not able of separate active and inactive IBD. The potential to use galectin levels as biomarkers was clearly demonstrated in this manuscript, but the authors need to improve the analysis to try to validate their proposal. Current format is not recommended to publish, although the authors should be encouraged to resubmit after major revisions.

SPECIFIC POINTS PER SECTIONS:

INTRODUCTION

1. The first paragraph needs some references.

2. Some references about galectins and IBD were neglected in the manuscript, such as Sundblad et al., 2018 and Cibor et al., 2019. Please, explore this theme in the introduction section. Moreover, Cibor and colleagues concluded that serum galectin-3 (and Gal-9) should not be considered biomarkers of IBD. Please, make a discussion regarding to galectins as biomarkers of IBD using more recent papers.

MATERIALS AND METHODS

1. Subtitle of the first paragraph is absent

RESULTS

1. Table 1 was appropriately used as base to correlate galectins levels with disease activity and C-reactive protein. However, to amplify the spectrum of analyzed data, the authors should explore underestimated data, such as disease location, disease duration, and medications:

1.1 - Galectin expression and synthesis vary throughout the small and large intestines. Then, disease location can be a relevant factor to be included in the analysis.

1.2 – Disease duration can also be important to study of biomarkers. Galectins have differential expression depending on stage of diseases, such as inflammation and fibrosis.

1.3 – Medications can alter the inflammatory and immunological responses and consequently the expression, synthesis and/or secretion of galectins. Medications can be critical modulators of biomarkers and it is impossible to be neglected in study of biomarkers.

1.4 - Values of serum levels of galectins are missed without a concentration unit as a reference (e.g. ng/mL).

2. Table 2 was excellent to detect serum levels of galectins, in special galectin-1 and -3. However, some information is missing:

2.1 - The insertion of concentration unit (possibly ng/mL) can improve to readers.

2.2 – In table legend, please, include the meaning of “Gal-1 conc”, “Gal-2 conc”, etc.

3. Table 3 was critical to compare active and inactive UC. Again, the insertion of concentration unit (possibly ng/mL) can improve to readers.

4. Table 4 was important to compare active and inactive CD. To better comprehension:

4.1 – Insert the concentration unit (possibly ng/mL) in values of serum galectins;

4.2 – The third column (“All”) shows an asterisk (*). Please, describe the meaning of this symbol.

5. Table 5 showed a comparison with different protocols of analysis using Gal-1 and Gal-3 independently combined with CRP.

5.1 – Describe the unit (possibly ng/mL);

5.2 – It was not clear the strategy to obtain the values (“mean” and “median”). Please, describe in the text or in materials and methods section

6. Table 6 showed a ROC analysis. However, the text subsequent (below of table) can be reorganized to explain the values (unit) and symbols (“>=” is equivalent to “≥”?).

DISCUSSION

1. Selection criteria for using galectin-1, -2, -3, -4, -7 and -8 should be discussed here. Why other galectins were not investigated in IBD patients? At least, these arguments can be detailed in materials and methods section.

2. The authors discussed about immunomodulatory roles of galectin-3 inducing pro-inflammatory signals (such as IL-6 dependent manner). These aspects can be explored with more details, including galectin-1 in the context. Several cytokines are associated with galectin-1 and galectin-3 levels in IBD. Imbalances between pro and anti-inflammatory responses seem relevant to insert both galectins as biomarkers.

3. There are several neglected works using galectins to study the pathogenesis and progression of IBD. The insertion in discussion section will improve the quality of this manuscript.

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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.

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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 to be viewed.]

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. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-19-23084R1

SERUM GALECTINS AS POTENTIAL BIOMARKERS OF INFLAMMATORY BOWEL DISEASES

PLOS ONE

Dear Dr Subramanian,

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.

We would appreciate receiving your revised manuscript by Dec 20 2019 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Roger Chammas, M.D, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Reviewer 1 still has some concerns that need to be addressed before the ms is considered acceptable. Please, address the constructive issues raised and submit a revised version of your ms.

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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: (No Response)

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: Partly

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: In the materials and methods section, there is a statement that "CRP>=5mg/L was used to define active disease" although I believe the clinical indices were actually used to define activity or inactivity.

I'm a bit concerned about the methods to add CRP and galectin-1 or galectin-3 to create a composite score to analyze against active or inactive IBD as it might underestimate the potential finding regarding an improvement in sensitivity for active IBD if you simply use the dichotomous variable of "galectin-1 normal or abnormal" and similarly for galectin-3.

With regard to the interesting finding that neither galectin-1 or galectin-3 associate with disease activity, it's not clear what the spectrum of disease activity (based on HBI and SCCAI) was. If there is a predominance of mildly active patients, that may explain this finding as patients with moderate-severe activity might be more likely to have higher serum biomarkers than patients in the inactive state.

Reviewer #2: The authors followed all suggestions. Current form is extremely relevant to review possible correlation between galectins and inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis. This version contemplates an extensive analysis of data regarding to circulating galectins and biomarkers of IBD. Discussion about increase of galectin-1 and -3 serum levels in patients with IBD was substantially improved in comparison with first version of the review. The potential to use galectin levels as biomarkers was clearly demonstrated in this manuscript. Given that authors satisfactorily responded the reviewer's comments, added the relevance of the theme, current format can be recommended to 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

[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 to be viewed.]

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. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-19-23084R2

SERUM GALECTINS AS POTENTIAL BIOMARKERS OF INFLAMMATORY BOWEL DISEASES

PLOS ONE

Dear Dr Subramanian,

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, consider the points raised by the reviewer 1 which points to some limitations and concerns about the score suggested and the relation amon CRP and galectins -1 and -3.

We would appreciate receiving your revised manuscript by Jan 23 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Roger Chammas, M.D, Ph.D

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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 have responded to all of my concerns in a satisfactory manner. I have no additional concerns.

Reviewer #2: This current version is appropriate to be published. The second revision improved the quality of manuscript and contemplated an extensive analysis of data regarding to circulating galectins and biomarkers of IBD. Possible correlation between galectins and inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, is extremely relevant. Moreover, the potential to use galectin levels as biomarkers was clearly demonstrated in this manuscript. Given that authors satisfactorily responded the reviewer's comments, current format is recommended to 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: Yes: Felipe Leite de Oliveira

[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 to be viewed.]

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. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: Reviewer Recommendation and Comments for Manuscript Number PONE-D-19-23084R2.pdf

SERUM GALECTINS AS POTENTIAL BIOMARKERS OF INFLAMMATORY BOWEL DISEASES

PONE-D-19-23084R3

Dear Dr. Subramanian,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Roger Chammas, M.D, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: