PONE-D-22-12868Post-translational modifications glycosylation and phosphorylation of the major hepatic plasma protein fetuin-A are associated with severity of CNS inflammation in childrenPLOS ONE

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: 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: The authors of this manuscript have done an exceptional job describing the complexities of CNS inflammation and demonstrated the usefulness of Fetuin-A as a potential biomarker of inflammation. Readers will be well equipped with the experimental details given in this manuscript to study Fetuin-A and its glycosylation and phosphorylation.

I would like to recommend the following additional information that could be added to the manuscript:

1. Age and sex distribution of the study cohorts including controls be provided if not already

2. Provide an explanation of dietary influence on Fetuin-A and how your study controlled that influence

3. Provide CSF sample collection details. Explain how the effects of rostrocaudal gradient on CSF Fetuin-A was controlled in this study.

4. Provide details on the inflammatory conditions and controls. A table with patient demographics and clinical conditions would be helpful.

5. Explain if Fetuin-A can discriminate between different CNS inflammatory/autoimmune conditions as well as Systemic inflammation Vs CNS inflammation.

6. Age specific reference intervals for Fetuin-A should be the next step once a larger data set is available

Reviewer #2: The Authors studied CSF and serum concentrations of total as well as post-translationally modified fetuin-A in a group of 66 children (including controls and various CNS inflammatory diseases). The aim of the study is excellently described on p. 8, rows 140-144. They came to the conclusion that 1) fetuin-A in the CSF is blood-derived rather than being synthesised intrathecally (probably the most important result of the study); 2) asialofetuin-A was found more frequently in patients with CNS inflammation; 3) phosphofetuin-A was more abundant in serum samples than in CSF, possibly indicating a regulation of influx of fetuin-A over the blood-CSF barrier by phosphorylation.

In my opinion, the study has been executed very carefully and is of significant theoretical importance. The main strength of the study lies in the quantitative assessment (within the methodological limits of the assays applied) of both total and phosphorylated protein forms, a concept that should perhaps be applied to several other proteins (and maybe other post-translational modifications) when studying the origin of CSF proteins (blood-derived vs. intrathecally synthesised). Qualitative analysis of glycosylation patterns in CSF and serum is also very interesting. Many interesting aspects of fetuin-A physiology and possible relevance for CNS inflammation are carefully reviewed by the Authors that apparently are experts on fetuin-A biology. However, I have not been convinced by their arguments that CSF fetuin-A measurement can be of any significant clinical value in routine practice (there is no intrathecal fetuin-A synthesis; decreased concentrations and relatively increased asialofetuin-A were found by the Authors also in serum, so why to investigate CSF for this protein?) In this context, I would suggest to reconsider an appropriate manuscript title (also see below under Major criticisms, point 1).

In my opinion, the Manuscript deserves Major revision before acceptance. Major criticisms are:

1) clinical data of the subjects are not provided, except for partial information on page 17 (lines 339-343) concerning samples for glycosylation analysis. This should be definitely corrected. Definition of control group and diagnoses of patients in the inflammatory group should be provided in the Methods section under the Heading Probands. Since number of samples analyzed by various assays (total fetuin-A, glycosylation, phosphorylation) differs, number of patients in both groups (controls and CNS inflammation) should be provided separately for each assay. Looking at diagnoses of a subgroup mentioned at p.17, this is a mix of various CNS inflammatory diseases with substantially different CSF profiles and different immunopathogenesis, most of them usually associated with normal serum CRP levels. However, this is acceptable in a pilot study.

How was the severity of CNS inflammation assessed? This is important if the Authors wish to retain in the Article title that post-translational modifications of fetuin-A are associated with SEVERITY (rather than presence) of CNS inflammation in children.

Basic information about methods used for CSF analysis, especially total protein, albumin and IgG should be provided as well (method - e.g. turbidimetric or dye-binding for total protein, reagent kit, and instrument).

2) Throughout the text, the Authors should consider using the term "Blood-CSF barrier" instead of "Blood-brain barrier", especially in the context of CSF/Serum protein quotients.

3) The term "ratio" could possibly be replaced by "quotient" as the latter is used by most authorities in CSF analysis, including prof. H. Reiber and prof. E.Thompson. In Thompson´s book Proteins of the cerebrospinal fluid (Elsevier 2005, ISBN 0-12-369369-1), a review of appropriate terms is provided on pp. 10-11: Ratio is defined as the result of dividing the amount of one protein (e.g. IgG) in CSF by the amount of another (e.g. albumin) also in the CSF, whereas Quotient is the result of dividing the amount of a given protein (e.g. albumin) in the CSF by the amount of the same protein again (e.g. albumin) in the serum.

4) Throughout the Text and Figures, axis label for CSF/Serum quotients should contain x10^3 and NOT 10^-3 E.g., for CSF fetuin-A 0,6 mg/L and Serum fetuin-A 0,3 g/L = 300 mg/L, the CSF/S quotient is 0,002 - this should be multiplied by a factor of 1000 if we want to get convenient numbers (in this case, 2) on the x-axis.

5) Figure 1G (CSF/S Q of total Fetuin-A on the y axis versus Q Alb on the x axis) seems to me to be perhaps the most important. Please try to indicate 2 patient subgroups (controls vs. CNS inflammation) in this Figure by using different marks or colours for these 2 patient groups. The same might be of value for Fig. 3E. (Concerning Fig. 3E, I am not sure whether absolute values of phosphofetuin-A CSF/Serum concentration quotients could be more appropriate than relative values on the y-axis.)

Although not obligatory, I would recommend at least briefly discuss in the Discussion section: 1) why CSF/serum fetuin A quotients are somewhat lower than anticipated based on the molecular weight of this protein (compared with albumin with M.w. of 67 kDa, fetuin-A should have CSF/serum quotients close to Q albumin or somewhat higher). Since the CSF/S quotients depend rather on hydrodynamic radius than on M.w., have the Authors any information about the hydrodynamic radius of fetuin-A compared to albumin? (partly discussed on p. 25 rows 516-523, but many Q fetuin-A values are much lower than corresponding Q Alb values also in patients with normal Q Alb), 2) the Authors tried to perform some kind of quantitation of the phosphorylated fetuin-A; what about the precision of these data (%CV in repeated experiments or from the literature etc.), and do the Authors think such quantitative attempts are possible and worthwhile for their glycosylation studies? 3) The Authors used enzyme digestion to study post-translational modifications of fetuin-A. It would be interesting if they wish at least to mention the use of mass spectrometry for such a purpose and why they did not choose this analytical tool in their study (not suitable? not available? other reason?)

Minor Criticisms:

Abstract, p.2, row 23: MS - abbreviation not explained (multiple sclerosis?), I would recommend to drop it from the Abstract since the role of fetuin-A as a biomarker for MS is far from being firmly established.

p.4 row 52 and p.22 row 449: Greek symbols or letters should be used consistenly for TNF-alpha and IL-1beta throughout the text

p.10 row 188 - "multiple" instead of "multipe"

p.10 - subscript should be used appropriately in the formulas of chemical compounds (e.g. for hydrogen peroxide)

p. 11 row 192: "sodium dodecyl sulfate" instead of "sodiumdodecylsulfat"; row 208: "polyacrylamide" instead of "polyacrylamid"

p.13 row 237: "2+" should be in superscript

p.13 row. 246 please provide brief description of the in-house AS5359 anti-human fetuin-A antibody, or provide an appropriate reference in the Reference list

p. 16 rows 307-308: it is not clear what the Authors wished to say, and it seems to be related to Fig. 1F rather than Fig. 1G; please try to formulate in a clearer way

p. 16 row 317: Fig. 1G rather than 1H; row 321: Fig. 1H rather than 1G

p. 17 rows 339-343: this should be stated in the Methods section (see above)

p.19 row 392: incorrect unit for the range of CSF phospho fetuin-A concentrations (mg/ml instead of micrograms/ml)

p. 22 row 444: please use "CSF" instead of "liquor"

p. 26 row 533: "in the context" rather than "in the contexts"

Tables S6 and S7 have identical Table title; I would recommend either to use different Table titles or to merge in one Table

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Reviewer #1: No

Reviewer #2: No

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Post-translational modifications glycosylation and phosphorylation of the major hepatic plasma protein fetuin-A are associated with CNS inflammation in children.

PONE-D-22-12868R1

Dear Dr. Jahnen-Dechent,

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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Pavel Strnad

Academic Editor

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Additional Editor Comments (optional): Thank you for submitting this nice work to PLoS One!

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

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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: Yes

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: I congratulate the authors on their excellent research in a very complex field of neurology. Hopefully this work eventually leads to translational diagnostic and prognostic biomarkers in the management of CNS inflammatory conditions.

There is a great clinical need for acute markers of neuro-inflammation in both paediatric and adult patients that can diagnose inflammatory conditions at a very early stage of disease progression. This would assist neurologists in the differential diagnoses of inflammation due to autoimmunity, infection, cancers and anatomical causes as well as in following the response to treatment and the course of the disease.

Existing inflammatory biomarkers such as CSF Neopterin and Cytokines/Chemokines are highly sensitive for neuro-inflammation and they correlate very well with the state of inflammation following treatment with immunotherapy; but unfortunately they are nonspecific markers of inflammation. The hunt for a differential biomarker of CNS inflammation continues.

Due to the acute nature of neuroinflammation, it is also vital for the specialist laboratory to be able to test and report these biomarkers within a day or two to limit the damage that ensues CNS inflammation. This would require assays which can be fully automated, are random access and fit for clinical purposes.

Wish you the best for your future research.

Reviewer #2: The Authors did a tremendous amount of work, and although their manuscript is long and not easy to follow, I believe it paves new and promising ways to analyse not only fetuin A but possibly also other CSF proteins.

I appreciate exhausting answers to the Rewievers´ questions and meticulous revision of the Manuscript.

I would only recommend to check/correct the following issues in Tables 1 and 5: Serum protein is given in g/l and not g/dl. Also, "Blood-CSF barrier (BCB) disorder" might be preferable to the term "Blood-CSF border ..." in these Tables. Finally, on Page 11, row 234, there is missing "d" in the word "sialidase-Au". In a legend to Fig. 1(G) (page 16, row 382), "scatter plot" should be used instead of "scatter blot".

The Reviewer would like to take this opportunity to wish the Authors lasting enthusiasm and many success in their further research.

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Reviewer #1: Yes: Sushil Bandodkar

Reviewer #2: No

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