PONE-D-24-53838Identification of potential inflammation markers for outgrowth of cow’s milk allergyPLOS ONE

Dear Dr. Clara Belzer

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PLOS ONE

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“CB, RGvdM and DMH were funded by the Dutch Research Council (NWO) and Danone Nutricia Research (grant no.16490, received by CB). The project was part of the partnership programme between NWO-TTW and Danone-Nutricia Research (https://www.nwo.nl/en/researchprogrammes/partnership/partnership-programmas/danone-nutricia-research ). The Dutch Research Council (NWO) played no role in the study design, data collection and analysis, the decision to publish and the preparation of the manuscript. Danone Nutricia Research provided the clinical data and saliva samples for this study. HW is an employee of Danone-Nutricia Research. HW reviewed the manuscript, provided comments and approved the article.”

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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: No

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: No

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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

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

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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: I read with great interest the manuscript “Supplementary Material for: Identification of potential inflammation markers for outgrowth of cow’s milk allergy”.

The topic is of interest; however many criticisms rise regarding the study design and the inclusion criteria. My comments, as following:

-Study design: Flowchart of the subject selection process (see also S1 Fig): 169 patients were randomized to the formulas (AAF-syn 80 vs AAF 89), however only 23/80 and 17/89 respectively remained after removing patients (no feces sample available, no 16S rRNA sequencing performed, plasma immunological data incomplete). This represents a main bias for the results interpretations and conclusions

- Inclusion criteria: I am puzzled to see that more than 60% of subjects resolved their IgE allergy at 12 months of life. This is even higher than that observed in Europrevall Study (57% of those with confirmed CMA at 12 months), which was among the highest reported in literature. Most of recent powered studies evaluating the natural history of IgE mediated CMA reported a lower percentage of tolerance at 12 months of that observed in Europrevall study. This rise doubt on the criteria used to diagnose IgE cow’s milk allergy (Line 59) in the PRESTO Study

Looking at Supplementary Material demonostration of IgE mediated CMA was defined as following: “ infants sensitized to CM, confirmed by CM-specific serum IgE >0.1 kU/L and/or CM skin prick test wheal size ≥3 mm, were diagnosed with CMA as follows. For some infants, CMA diagnosis was confirmed by an open or double-blind placebo-controlled CM challenge (DBPCFC). For others, diagnosis was confirmed by a history of anaphylaxis reaction to CM reported by two physicians. Furthermore, looking at Table S1 we can see that severity of atopic dermatitis has been reported; thus it means that some infants had concomitant AD, and it is well known that AD infants may be sensitized to trofoallergens, especially CM proteins but they are not real allergic to CM. This is even more true if a confirmation OFC is not performed to confirm the diagnosis of CMA. Another point of interest regards the IgE level which was the lowest which may be considered (0.1, instead of 0.35 as often used as cut off to consider IgE sensitization). The percentage of infants who underwent a confirmatory open OFC need to be explicited as well as the percentage of infants with concomitant AD. Clinical characteristics of infants at the enrollment, especially regarding symptoms at onset, are lacking also in supplementary materials should be provided (how many patients with anaphylaxis? How many with AD and so on..)

All the above mentioned issues, regarding the inclusion criteria and the CMA diagnosis itself, joined to the small sample size, represent important methodological lacks, which call in questions the observed results and limit the validity of the conclusions.

-Last but not least, patients were fed with AAF which usually should be reserved to infants/children with more severe symptoms. Again, considering the study population, 61.5% reached tolerance at 12 months, that implies that most of patients had no severe form of CMA (if we consider “true” the diagnosis of IgE CMA and not simply sensitization).

- In the Discussion section limitations regarding the above mentioned issues are lacking (except for the small sample size)

Overall, main methodological lacks hamper the initial eagerness about the scope of the manuscript.

Reviewer #2: This study investigates the natural resolution mechanisms of cow’s milk allergy by analyzing changes in multiple cytokines using saliva, a non-invasive specimen. The finding that specific cytokine trajectories differ between children with and without resolution is noteworthy and meaningful as a hypothesis-generating study. However, the study has several limitations, including a small sample size and the retrospective nature of a secondary analysis derived from an interventional trial.

The authors are requested to address the following points. If any of these are considered limitations, they should be clearly acknowledged in the “Limitations” section:

#1 Do salivary cytokine levels reliably reflect systemic (blood) cytokine concentrations?

#2 What criteria were used to select participants from the AAF and AAF-syn interventional trial for inclusion in the present analysis?

#3 Could the dietary interventions with AAF and AAF-syn have influenced the observed resolution of CMA? It is unclear whether the cytokine changes represent responses to the intervention or natural resolution.

#4 The study mainly analyzes individual cytokine trajectories but does not include between-group comparisons or integrative analyses. Is there a reason why integrative or multivariate approaches were not applied to compare cytokine profiles across groups?

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Reviewer #1: No

Reviewer #2: Yes:  Takao Fikosawa

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Identification of potential inflammation markers for outgrowth of cow’s milk allergy

PONE-D-24-53838R1

Dear Dr. Belzer,

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.

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Kind regards,

Dost Muhammad Khan, PhD

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: (No Response)

Reviewer #2: All comments have been addressed

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: N/A

Reviewer #2: 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

Reviewer #2: 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: Yes

Reviewer #2: Yes

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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 have properly addressed most of my concerns, also adding further study limitations in the Discussion section, as suggested.

In the revised version of the manuscript the authors state that “Infants were frequently on a CM elimination diet before enrollment and consequently had mild but persistent symptoms[11].”(lines 479-480)”.

It is not clear what type of CM elimination diet they did before enrolment (multiple cow’s milk elimination diets, I suppose different formulas before enrolment, but it should be clarified) and what it exactly means that they consequently had mild but persistent symptoms. The last question: it is still unclear how CMA is defined “severe” if symtoms was mild; did they mean severe as persisten? If so, please add clarification to this point

Reviewer #2: The authors have thoroughly and appropriately addressed all the points raised in the review. Each comment has been carefully considered, and the corresponding revisions made to the manuscript are clear and satisfactory. I appreciate the authors’ efforts in improving the quality and clarity of the manuscript based on the feedback provided.

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Reviewer #1: No

Reviewer #2: Yes:  Takao Fujisawa, MD, PhD

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