Abstract

Childhood cerebral adrenoleukodystrophy (cALD) is a devastating manifestation of ALD accompanied by demyelination, inflammation, and blood brain barrier (BBB) disruption with shared characteristics of an auto-immune disease. We utilized plasma samples pre- and postdevelopment of cALD to determine the presence of specific auto-antibodies. Mass spectrometry of protein specifically bound with post-cALD plasma antibody identified Profilin1 (PFN1) as the target. In a screen of 94 boys with cALD 48 (51%) had anti-PFN1 antibodies, whereas only 2/29 boys with ALD but without cerebral disease, and 0/30 healthy controls showed anti-PFN1 immunoreactivity. Cerebral spinal fluid from those with cALD showed higher levels of PFN1 protein compared with non-cALD samples (324 +/- 634 versus 42 +/- 23 pg/mL, p = 0.04). Boys that were anti-PFN positive had a significant increase in the amount of gadolinium signal observed on MRI when compared to boys that were anti-PFN1 negative (p = 0.04) possibly indicating increased BBB disruption. Anti-PFN1 positivity was also associated with elevated levels of very long chain fatty acids (C26 of 1.12 +/- 0.41 versus 0.97 +/- 0.30 mg/dL, p = 0.03) and increased plasma BAFF (973 +/- 277 versus 733 +/- 269 pg/mL, p = 0.03). In conclusion, anti-PFN may be a novel biomarker associated with the development of cALD in boys with ALD.


Authors

Orchard, Paul J.;  Nascene, David R.;  Gupta, Ashish;  Taisto, Mandy E.;  Higgins, LeeAnn;  Markowski, Todd W.;  Lund, Troy C.

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    2020/08/23

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    Decision Letter
    2019/02/22

    Dear Dr. Lund,

    It is a pleasure to provisionally accept your manuscript entitled "Cerebral adrenoleukodystrophy is associated with loss of tolerance to profilin" for publication in the European Journal of Immunology. For final acceptance, please follow the instructions below and return the requested items as soon as possible as we cannot process your manuscript further until all items listed below are dealt with.

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    on behalf of Prof. Steffen Jung

    Dr. Nadja Bakocevic Editorial Office European Journal of Immunology e-mail: ejied@wiley.com www.eji-journal.eu


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    Decision letter by
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    Reviewer report
    2019/02/20

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    Reviewer report
    2019/02/20

    The authors have addressed the questions raised by the reviewers

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    Author Response
    2019/02/15

    We thank the reviewers for the time and effort. As a reviewer myself, I appreciate their undertakings. Our point by point response is listed below. Reviewer: 1 Comments to the Author Orchard et al have identified a biomarker in plasma (anti-profilin1) that shows a strong association with cerebral ALD. The included 94 boys with cerebral ALD (cALD), 29 without cerebral ALD and healthy controls. In the cerebral group 51% had anti-PFN1 antibody versus 7% in the non-cerebral group. It’s a very well-written and highly informative manuscript. The choice to combine Results and Discussion really benefited the story! The study highlights the importance of longitudinal good clinical follow-up of patients with a rare disease accompanied by (annual) sample collection. This has resulted in the identification of Profilin1 in plasma from a boy that converted to the cerebral phenotype. It was absent prior to pre-cALD, and present post-cALD. Whether PFN1 demonstrable in CSF is a predictor of cerebral disease, or a consequence of cerebral disease and BBB breakdown remains to be resolved. But, as the authors conclude themselves, the intriguing findings presented in this study will be explored in follow-up studies.

    Minor comments Page 4, line 54: (Figure 3D) should be (Figure 2D).

    Response: we have corrected this.

    Page 5, lines 49-52: I have read the following sentence several times and it remained confusing: “Conversely, plasma levels of anti-PFN1 between cALD patients and healthy controls were not statistically different (data not shown).” What am I missing here? What material was then measured in (p4, lines 8-15) “Boys without ALD, ALD without cALD, and boys with cALD were screened for antibodies to PFN1. The relative number of boys that screened positive for PFN1 antibodies were: 0/30 in those without ALD, 2/29 (6.9%) in those with ALD but without cALD, and 48/94 (51.1%) in those having cALD.”?? Because the authors, throughout the manuscript, switch between CSF and plasma and because the section prior to the lines p4, lines 8-15 is about plasma the reader (at least I) may think that the findings are from plasma. This confusion is easily resolved by starting the section p4, lines 8-15 with: “CSF samples derived from boys without ALD, ALD without ………”

    Response: page 5 has been corrected to read PFN levels were not different between cALD patients and controls in the plasma. (Not anti-PFN). The opening paragraph was also modified for clarity.

    Page 24. It’s unclear why this figure is presented. It doesn’t seem to have a number and I couldn’t find a reference to it in the text.

    Response: the figure on page 24 was meant to be a supplemental figure to show our FACS gating strategy. We have labeled and submitted it correctly upon revision.

    Reviewer: 2 Comments to the Author The Manuscript entitled “Cerebral adrenoleukodystrophy is associated with loss of tolerance to profiling” by Orchard et al if of highest interest and nicely describe the way how the authors identified profiling as an antigen in a significant proportion of cerebral ALD patients with inflammatory demyelination and opening of the BBB. It will be highly interesting to see if, in near future, those two non-inflammatory X-ALD patients with plasma immunoreactivity to PFN1 develop MRI abnormalities and thus anti-PFN1 antibody presence might serve as early sensitive biomarker for cALD.

    I would have only minor suggestions: In the abstract it is written ….higher levels of PFN1 protein compared wit hnon-cALD samples (944 versus 291 pg/ml, p=0.004). Within the entire manuscript, the standard deviation cannot be seen in numbers, not in the results not in the figure legend. Please, throughout the manuscript always provide the SD next to the mean.

    Response: we have added the standard deviation numbers throughout the text which reflects the error bars in the figures and denoted in the figure legend as SD.

    In the abstract it would be nicer if it is written: In a screen of 94 boys with cALD 48 (51%) had anti-PFN1 antibodies whereas only 2/29 boys with ALD but without cerebral disease and 0/30 healthy controls showed anti-PFN1 immunoreactivity.

    Response: we have made this change as suggested.

    On page 5 last sentence: Conversely, plasma levels of anti-PFN1 between cALD patients and healthy controls were not statistically different. This should probably be plasma levels of PFN1 …or anti-PFN1 reactivity….. (as it might we mixed up with anti-PFN1 AB)

    Response: the reviewer is correct and we clarified that it was levels of plasma PFN1 (not anti-PFN1).

    The used PFN1 protein is named differently throughout the paper: Immunization of mice: hPFN1; Immunobloting: rPFN1; and in the results discussion section: rhPFN1 and hPFN1 please uniform or write recombinant in cases you use the commercial recombinant protein.

    Response: we have made the nomenclature uniform and used rhPFN in cases when the commercial recombinant protein was used.

    Editorial comments…

    Response: we have answered and modified the manuscript according to the editorial comments made.



    Cite this author response
  • pre-publication peer review (ROUND 1)
    Decision Letter
    2019/02/14

    Dear Dr. Lund,

    Manuscript ID eji.201848043 entitled "Cerebral adrenoleukodystrophy is associated with loss of tolerance to profilin", which you submitted to the European Journal of Immunology, has been reviewed. The comments of the referees are included at the bottom of this letter. We are sorry about the delay in the peer review, but there was a delay receiving one of the reports.

    Although the referees have recommended publication, some revisions to your manuscript have been requested. Therefore, I invite you to respond to the comments of the referees and revise your manuscript accordingly. If revisions require further experiments please ensure that data shown in the revised manuscript are reproducible.

    You should also pay close attention to the editorial comments included below. In particular, please edit your figure legends to follow Journal standards as outlined in the editorial comments. Failure to do this will result in delays in the re-review process.

    If the revision of the paper is expected to take more than three months, please inform the editorial office. Revisions taking longer than six months may be assessed by new referees to ensure the relevance and timeliness of the data.

    Once again, thank you for submitting your manuscript to European Journal of Immunology. We look forward to receiving your revision.

    Yours sincerely, Nadja Bakocevic

    on behalf of Prof. Steffen Jung

    Dr. Nadja Bakocevic Editorial Office European Journal of Immunology e-mail: ejied@wiley.com

    www.eji-journal.eu


    Reviewer: 1

    Comments to the Author Orchard et al have identified a biomarker in plasma (anti-profilin1) that shows a strong association with cerebral ALD. The included 94 boys with cerebral ALD (cALD), 29 without cerebral ALD and healthy controls. In the cerebral group 51% had anti-PFN1 antibody versus 7% in the non-cerebral group.

    It’s a very well-written and highly informative manuscript. The choice to combine Results and Discussion really benefited the story!

    The study highlights the importance of longitudinal good clinical follow-up of patients with a rare disease accompanied by (annual) sample collection. This has resulted in the identification of Profilin1 in plasma from a boy that converted to the cerebral phenotype. It was absent prior to pre-cALD, and present post-cALD.

    Whether PFN1 demonstrable in CSF is a predictor of cerebral disease, or a consequence of cerebral disease and BBB breakdown remains to be resolved. But, as the authors conclude themselves, the intriguing findings presented in this study will be explored in follow-up studies.

    Minor comments Page 4, line 54: (Figure 3D) should be (Figure 2D).

    Page 5, lines 49-52: I have read the following sentence several times and it remained confusing: “Conversely, plasma levels of anti-PFN1 between cALD patients and healthy controls were not statistically different (data not shown).” What am I missing here? What material was then measured in (p4, lines 8-15) “Boys without ALD, ALD without cALD, and boys with cALD were screened for antibodies to PFN1. The relative number of boys that screened positive for PFN1 antibodies were: 0/30 in those without ALD, 2/29 (6.9%) in those with ALD but without cALD, and 48/94 (51.1%) in those having cALD.”?? Because the authors, throughout the manuscript, switch between CSF and plasma and because the section prior to the lines p4, lines 8-15 is about plasma the reader (at least I) may think that the findings are from plasma. This confusion is easily resolved by starting the section p4, lines 8-15 with: “CSF samples derived from boys without ALD, ALD without ………”

    Page 24. It’s unclear why this figure is presented. It doesn’t seem to have a number and I couldn’t find a reference to it in the text.

    Reviewer: 2

    Comments to the Author The Manuscript entitled “Cerebral adrenoleukodystrophy is associated with loss of tolerance to profiling” by Orchard et al if of highest interest and nicely describe the way how the authors identified profiling as an antigen in a significant proportion of cerebral ALD patients with inflammatory demyelination and opening of the BBB. It will be highly interesting to see if, in near future, those two non-inflammatory X-ALD patients with plasma immunoreactivity to PFN1 develop MRI abnormalities and thus anti-PFN1 antibody presence might serve as early sensitive biomarker for cALD.

    I would have only minor suggestions: In the abstract it is written ….higher levels of PFN1 protein compared wit hnon-cALD samples (944 versus 291 pg/ml, p=0.004). Within the entire manuscript, the standard deviation cannot be seen in numbers, not in the results not in the figure legend. Please, throughout the manuscript always provide the SD next to the mean. In the abstract it would be nicer if it is written: In a screen of 94 boys with cALD 48 (51%) had anti-PFN1 antibodies whereas only 2/29 boys with ALD but without cerebral disease and 0/30 healthy controls showed anti-PFN1 immunoreactivity.

    On page 5 last sentence: Conversely, plasma levels of anti-PFN1 between cALD patients and healthy controls were not statistically different. This should probably be plasma levels of PFN1 …or anti-PFN1 reactivity….. (as it might we mixed up with anti-PFN1 AB)

    The used PFN1 protein is named differently throughout the paper: Immunization of mice: hPFN1; Immunobloting: rPFN1; and in the results discussion section: rhPFN1 and hPFN1 please uniform or write recombinant in cases you use the commercial recombinant protein.


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    Decision letter by
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    Reviewer report
    2019/02/11

    The Manuscript entitled “Cerebral adrenoleukodystrophy is associated with loss of tolerance to profiling” by Orchard et al if of highest interest and nicely describe the way how the authors identified profiling as an antigen in a significant proportion of cerebral ALD patients with inflammatory demyelination and opening of the BBB. It will be highly interesting to see if, in near future, those two non-inflammatory X-ALD patients with plasma immunoreactivity to PFN1 develop MRI abnormalities and thus anti-PFN1 antibody presence might serve as early sensitive biomarker for cALD.

    I would have only minor suggestions:

    In the abstract it is written ….higher levels of PFN1 protein compared wit hnon-cALD samples (944 versus 291 pg/ml, p=0.004). Within the entire manuscript, the standard deviation cannot be seen in numbers, not in the results not in the figure legend. Please, throughout the manuscript always provide the SD next to the mean.

    In the abstract it would be nicer if it is written: In a screen of 94 boys with cALD 48 (51%) had anti-PFN1 antibodies whereas only 2/29 boys with ALD but without cerebral disease and 0/30 healthy controls showed anti-PFN1 immunoreactivity.

    On page 5 last sentence: Conversely, plasma levels of anti-PFN1 between cALD patients and healthy controls were not statistically different. This should probably be plasma levels of PFN1 …or anti-PFN1 reactivity….. (as it might we mixed up with anti-PFN1 AB)

    The used PFN1 protein is named differently throughout the paper: Immunization of mice: hPFN1; Immunobloting: rPFN1; and in the results discussion section: rhPFN1 and hPFN1 please uniform or write recombinant in cases you use the commercial recombinant protein.

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    Reviewer report
    2019/01/31

    Comments to the Author Orchard et al have identified a biomarker in plasma (anti-profilin1) that shows a strong association with cerebral ALD. The included 94 boys with cerebral ALD (cALD), 29 without cerebral ALD and healthy controls. In the cerebral group 51% had anti-PFN1 antibody versus 7% in the non-cerebral group.

    It’s a very well-written and highly informative manuscript. The choice to combine Results and Discussion really benefited the story!

    The study highlights the importance of longitudinal good clinical follow-up of patients with a rare disease accompanied by (annual) sample collection. This has resulted in the identification of Profilin1 in plasma from a boy that converted to the cerebral phenotype. It was absent prior to pre-cALD, and present post-cALD.

    Whether PFN1 demonstrable in CSF is a predictor of cerebral disease, or a consequence of cerebral disease and BBB breakdown remains to be resolved. But, as the authors conclude themselves, the intriguing findings presented in this study will be explored in follow-up studies.

    Minor comments Page 4, line 54: (Figure 3D) should be (Figure 2D).

    Page 5, lines 49-52: I have read the following sentence several times and it remained confusing: “Conversely, plasma levels of anti-PFN1 between cALD patients and healthy controls were not statistically different (data not shown).” What am I missing here? What material was then measured in (p4, lines 8-15) “Boys without ALD, ALD without cALD, and boys with cALD were screened for antibodies to PFN1. The relative number of boys that screened positive for PFN1 antibodies were: 0/30 in those without ALD, 2/29 (6.9%) in those with ALD but without cALD, and 48/94 (51.1%) in those having cALD.”?? Because the authors, throughout the manuscript, switch between CSF and plasma and because the section prior to the lines p4, lines 8-15 is about plasma the reader (at least I) may think that the findings are from plasma. This confusion is easily resolved by starting the section p4, lines 8-15 with: “CSF samples derived from boys without ALD, ALD without ………”

    Page 24. It’s unclear why this figure is presented. It doesn’t seem to have a number and I couldn’t find a reference to it in the text.

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