Reviewer ’s report  
 
Title: Histological, immunohistochemical and mRNA gene expression responses in coeliac  
disease patients challenged with gluten using PAXgene fixed paraffin -embedded duodenal  
biopsies  
 
Version: 0 Date: 13 Aug 2019  
 
Reviewer: Rong Jun Guo  
 
Reviewer's report:  
 
Please include all comments for the authors in this box rather than uploading your report as an 
attachment. Please only upload as attachments annotated versions of manuscr ipts, graphs, 
supporting materials or other aspects of your report which cannot be included in a text format.  
Please overwrite this text when adding your comments to the authors.  
 
This is an interesting technical study attempting a quantitative approach to  evaluating 
histological changes in the small intestine in the pathogenesis of celiac disease.   I have two 
concerns regarding this study:  
 
1) As we all know RNAs extracted from formalin -fixed tissues, in many cases, work pretty well.  
The authors argue th at, in some RNAs, formalin fixation do not work.  That was why the authors 
thought PAX gene was a better choice.  However, the authors did not perform a parallel study 
comparing these two fixatives when they performed their RNA study, raising the suspicion  that 
formalin fixed tissues might also work for the expression of the two genes they studied.  
 
2) In all of cases the authors studied, I assume the diagnosis had been established before the 
patients were registered.  The diagnosis of a celiac disease is b ased on clinical, serological and 
histological correlation.  The typical histological features are characteristic of , but not specific 
for, a celiac disease.  Several other entities can result in identical histological changes, such as 
Crohn disease, and medication -related injury.   The authors should clearly address this limitation.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
No 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional s tatistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I recommend additional statistical review  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Accept able 
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way  gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in th e future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the conte nt of the manuscript?  
 
5. Do you have any other financial competing interests?   
 
6. Do you have any non -financial competing interests in relation to this paper?   
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests' below  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Acc ess Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: Histological, immunohistochemical and mRNA gene expression responses in coeliac  
disease patients challenged with gluten using PAXgene fixed paraffin -embedded duodenal  
biopsies  
 
Version: 0 Date: 13 Aug 2019  
 
Reviewer: Rong Jun Guo  
 
Reviewer's report:  
 
Please include all comments for the authors in this box rather than uploading your report as an 
attachment. Please only upload as attachments annotated versions of manuscr ipts, graphs, 
supporting materials or other aspects of your report which cannot be included in a text format.  
Please overwrite this text when adding your comments to the authors.  
 
This is an interesting technical study attempting a quantitative approach to  evaluating 
histological changes in the small intestine in the pathogenesis of celiac disease.   I have two 
concerns regarding this study:  
 
1) As we all know RNAs extracted from formalin -fixed tissues, in many cases, work pretty well.  
The authors argue th at, in some RNAs, formalin fixation do not work.  That was why the authors 
thought PAX gene was a better choice.  However, the authors did not perform a parallel study 
comparing these two fixatives when they performed their RNA study, raising the suspicion  that 
formalin fixed tissues might also work for the expression of the two genes they studied.  
 
2) In all of cases the authors studied, I assume the diagnosis had been established before the 
patients were registered.  The diagnosis of a celiac disease is b ased on clinical, serological and 
histological correlation.  The typical histological features are characteristic of , but not specific 
for, a celiac disease.  Several other entities can result in identical histological changes, such as 
Crohn disease, and medication -related injury.   The authors should clearly address this limitation.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
No 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional s tatistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I recommend additional statistical review  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Accept able 
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way  gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in th e future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the conte nt of the manuscript?  
 
5. Do you have any other financial competing interests?   
 
6. Do you have any non -financial competing interests in relation to this paper?   
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests' below  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Acc ess Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: Histological, immunohistochemical and mRNA gene expression responses in coeliac  
disease patients challenged with gluten using PAXgene fixed paraffin -embedded duodenal  
biopsies  
 
Version: 0 Date: 13 Aug 2019  
 
Reviewer: Rong Jun Guo  
 
Reviewer's report:  
 
Please include all comments for the authors in this box rather than uploading your report as an 
attachment. Please only upload as attachments annotated versions of manuscr ipts, graphs, 
supporting materials or other aspects of your report which cannot be included in a text format.  
Please overwrite this text when adding your comments to the authors.  
 
This is an interesting technical study attempting a quantitative approach to  evaluating 
histological changes in the small intestine in the pathogenesis of celiac disease.   I have two 
concerns regarding this study:  
 
1) As we all know RNAs extracted from formalin -fixed tissues, in many cases, work pretty well.  
The authors argue th at, in some RNAs, formalin fixation do not work.  That was why the authors 
thought PAX gene was a better choice.  However, the authors did not perform a parallel study 
comparing these two fixatives when they performed their RNA study, raising the suspicion  that 
formalin fixed tissues might also work for the expression of the two genes they studied.  
 
2) In all of cases the authors studied, I assume the diagnosis had been established before the 
patients were registered.  The diagnosis of a celiac disease is b ased on clinical, serological and 
histological correlation.  The typical histological features are characteristic of , but not specific 
for, a celiac disease.  Several other entities can result in identical histological changes, such as 
Crohn disease, and medication -related injury.   The authors should clearly address this limitation.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
No 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional s tatistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I recommend additional statistical review  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Accept able 
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way  gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in th e future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the conte nt of the manuscript?  
 
5. Do you have any other financial competing interests?   
 
6. Do you have any non -financial competing interests in relation to this paper?   
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests' below  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Acc ess Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant