PONE-D-24-22098NOMO-1 combination with the Luciferase reporter gene offers a simple and rapid Monocyte Activation Test that can detect a wide range of pyrogen.PLOS ONE

Dear Dr. Nanao,

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.

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We have received one thorough and insightful review of your work. After careful consideration, I have determined that this single review provides sufficient basis for a decision. The reviewer's comments are comprehensive and align well with our publication criteria. Please note that this approach is not our standard practice. However, given the circumstances and the substantive nature of the review received, we believe this decision serves both the authors and the journal well.

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Please submit your revised manuscript by Aug 31 2024 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 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 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). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Rui Tada, Ph.D.

Academic Editor

PLOS ONE

Journal requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

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2. Thank you for stating the following financial disclosure: 

 [T.O. was supported by FUJIFILM Corporation, Japan.].  

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.

3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

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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

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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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

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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: No

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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: In the presented work, Nanao and colleagues demonstrated the establishment of a NF-kB gene reporter assay employing the NOMO-1 monocyte like cell line as a suitable model for pyrogen detection. In their work, the authors showcased the capacity of the established reporter cell line to detect both enodotoxin and non-endotoxin based pyrogens at relatively short incubation period (3 h) and low LOD, relying on the transcription factor NF-kB activation. Hence, the illustrated model provides an attractive tool to further advance the monocyte activation test replacing the tedious and time intensive ELISA based MAT with a more sensitive and automated readout.

Overall Comment: The authors have demonstrated a tool to further advance the field of pyrogenicity testing, nevertheless the novelty of the presented work is majorly lacking due to the abundance of similar and more advanced models already published with gene reporter assays employing more relevant immune cells (e.g. more primary like cells, iPSC- derived reporter macrophages). Moreover, the presentation of the work requires drastic revision with regard to language and profound/ critical result description and discussion. We therefore have strong concern to publish the work in the PLOS ONE journal due to lack of sufficient merit and novelty for the presented work.

Below the authors may find helpful comments to improve the overall quality of their work,

Comment 1 Given the fact that the introduced NOMO-1 cell line is not yet approved by respective regulators or introduced to the MAT context, the authors are strongly advised to start with detailed phenotyping of the used cell line with respect to monocyte/macrophage markers, maturity, and functional analysis in comparison to proper standard primary monocytes. This should be the first critical analysis that can determine further the suitability of the line for the MAT assay.

Comment 2 While the assessment of TLR expression is a critical factor to allow for the cell line inclusion in the MAT context, nevertheless the performed and demonstrated comparison of the NOMO-1 TLR expression profile with respect to MM6 or THP1 cells is not a useful or an informative comparison. As the illustrated reference lines (MM6/THP1) are highly debatable and doubted for their suitability in the MAT, with the fact that THP1 cells already failed the MAT validation studies, and MM6 validation status is incomplete. Hence, the TLR expression analysis needs to be compared to and evaluated against primary monocytes expression. In this line, a functional comparison to established MAT cell lines will add more reliable data than comparing only the expression levels.

Comment 3 The authors need to be more accurate in their introduction concerning properly defining NEPs, and the approved cell sources for MAT that also included whole blood MAT.

Comment 4 The value of the data needs to be better highlighted throughout the manuscript by conducting the indicated statistical analysis. This is particularly needed when it comes to evaluating the LOD, the authors need to show whether the observed reading at the indicated LOD is of significant difference compared to the untreated control reading.

Comment 5 The authors need to better highlight (perhaps in a separate bar chart) the observed background/noise reading/luminescence at the chosen incubation period at different LPS concentrations. It is a bit confusing why the authors didn’t choose the incubation period of the highest S/N ratio for their assays?

Comment 6 While the authors highlighted in their methods a section for cryopreservation, they didn’t show any data for successful freezing/thawing of functional reporter cells. Such data can add valuable input, to further support the use of the chosen cancer cell line. For example, data for consistency and viability and functionality post thawing is needed.

Comment 7 In their method part the authors described two versions of virus production with nearly the same wording. At that point it is not clear whether and where two different viruses (lentivirus and undefined virus) were used. If not my mistake, it is strongly recommended to not use the exact wording for both descriptions.

Comment 8: Given the affiliation of the authors and the commercialization interest of Fuji Film in the space of stem cells, the authors should state clearly any potential conflict of interest and whether the work presented will/is planed to be commercialized.

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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.

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

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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 PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

PONE-D-24-22098R1NOMO-1 combination with the Luciferase reporter gene offers a simple and rapid Monocyte Activation Test that can detect a wide range of pyrogen.PLOS ONE

Dear Dr. Nanao,

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.

Please submit your revised manuscript by Mar 31 2025 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 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 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). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Rui Tada, Ph.D.

Academic Editor

PLOS ONE

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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)

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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

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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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

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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: No

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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 presented a revised version of their original draft in which they included important data comparing the expression levels of different TLRs between NOMO-1 cells and primary macrophages, which correspond to the current standard of MAT. In addition, they presented confirmatory data comparing the results of their reporter-based approach with standard ELISA measurements. Several questions raised during the review process were answered satisfactorily. However, their work as a whole still lacks sufficient depth and profound language revision.

It is important that the authors clarify whether their results are based on four technical or biological replicates. Biological replicates would include at least four different cell batches that were thawed and seeded, while technical replicates would mean that one frozen cell batch was distributed to four wells for simultaneous measurement. At this point, the present description does not allow an evaluation of whether the NOMO-1 cell line is a reliable and reproducible source for the MAT. Given the importance of the assay and the regulatory requirements it is of utmost importance to demonstrate reproducibility and standardization.

Overall, the manuscript requires a more thorough and critical discussion of its results, including an open acknowledgement of all limitations. In addition, a fundamental revision of the wording used in the text is strongly suggested. Amongst others, lines 23, 32, 175, 276, 290-295, 324-326, 334 and 429, were particularly difficult to comprehend or inadequately phrased. These are just exemplary lines! We therefore recommend revising the submitted work prior to publishing in the PLOS ONE journal.

Below, the authors may find additional helpful comments to improve the overall quality of their work:

Comment 1 – It should be noted that normalizing TLR or CD14 expression to β-actin levels is not an adequate approach to generally compare and draw conclusions about expression levels between different cell types, which may express β-actin to very different degrees. The authors could give information on the number of replicates for this assay to emphasize their conclusions.

Comment 2 – The authors referred to other established monocytic cell lines that included a reporter-based approach and stated that they performed a more thorough validation with more different TLR targets compared to them. However, a more functional comparison would have been informative, considering the availability of THP1 and MM6 cells in their first draft.

Comment 3 – The authors should include more up-to-date information regarding the European Pharmacopeia. They are no longer just committed to replacing RPT, but have officially announced in July 2024 that they will abolish the RPT.

Comment 4 – The authors display financial disclosures only for TO, although TN, YM, KS, TM and TN are also assigned to FUJIFILM Corporation.

Line 4 – pyrogens should be used as plural

Line 22 – pyrogens compromise endotoxins and non-endotoxins, as also later described

Line 23 – pyrogens do not necessarily enter immune cells to induce fever and endotoxic shock

32 – although it became clear later, “NF-kB reporter gene introduced cells” is hard to understand

345 – The detectability “of” NEPs

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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

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

PONE-D-24-22098R2NOMO-1 cells expressing an NF-κB luciferase reporter gene facilitate a simple, rapid monocyte activation test that can detect a wide range of pyrogensPLOS ONE

Dear Dr. Nanao,

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.

Please submit your revised manuscript by May 30 2025 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 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 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). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Rui Tada, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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)

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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: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

**********

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: (No Response)

**********

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: (No Response)

**********

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 addressed most of the comments. I still ask to disclose any finical relationship for this scientific work.

It is not sufficient to disclose this in the cover letter nor I can see a financial disclose given to the PLOS one editorial office. The authors just say "yes", however a disclosure statement is requested and missing.

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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

**********

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

NOMO-1 cells expressing an NF-κB luciferase reporter gene facilitate a simple, rapid monocyte activation test that can detect a wide range of pyrogens

PONE-D-24-22098R3

Dear Dr. Nanao,

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.

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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,

Rui Tada, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: