Reviewer ’s report  
 
Title:  High expression of the Claudin -2 in esophageal carcinoma and precancerous lesions is 
significantly associated with the bile salt receptors VDR and TGR5  
 
Version: 0 Date: 29 Sep 2016 
 
Reviewer:  Christopher  Weber  
 
Reviewer’s report:  
 
The authors show a correlation between claudin -2 and esophageal carci noma and precancerous 
lesions. They also show that this correlates with VDR receptors VDR and TGR5. Data 
supporting these correlations are  convincing.  
 
One concern is tha t the results are correlative. In addition to the correlation with VDR and 
TRGR5, however, an additional possibility is that it that claudin -2 is a downstream effect of 
disrupted barrier (either due to the neoplastic process or vit D eff ects).  Whenever the barrier is 
disrupted in the GI tract, there is increased lamina propria inflammation, and this inflammation 
often in duces expression of claudin -2. To confirm if VDR and claudin2 are directly tied, I 
recommend performing some in vitro  studies to co mplement the staining studies.  The advantage 
of the in vitro studies is they can be performed with inflammatory cytokines strictly controlle d. If 
it is not possible to perform such studies, then it would be good to quantify inflammation i n some 
manner in the staining studies and/or expand the discussion to include this possibility.  
 
 
Regarding in vitro studies:  
 
Note that Caco2 cells have a VDR, so this might be an option  
 
Arch Biochem Biophys. 1991 Mar;285(2):261 -9. 
 
Also, note that IL13 has  been shown to specifically upregulate claudin2 expression.  
 
Another paper worth considering is: 
http://jid.oxfordjournals.org/content/early/2014/05/23/infdis.jiu235.full.pdf  
 
 
Minor comment:  
 
careful of wording throughout. For example, consider the followi ng statement in the abstract:  
 
"In contrast, Claudin -2 is a leaky protein that plays an opposing role and  
increases cell permeability."  
 
Claudins do not affect cell permeability...they a ffect epithelial permeability. Also, claudin -2 is 
not leaky. it for ms a charge selective and barrier. Leak implies no charge selectivity.    
 
Are the met hods 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 aut hors.  
 
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 statistical review is recommended, please specify what aspects require further 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
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 t hat may in any way gain or lose 
financially from the publication of this manuscript, either now or in the 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 content 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  
 
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 Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be include d 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:  High expression of the Claudin -2 in esophageal carcinoma and precancerous lesions is 
significantly associated with the bile salt receptors VDR and TGR5  
 
Version: 0 Date: 29 Sep 2016 
 
Reviewer:  Christopher  Weber  
 
Reviewer’s report:  
 
The authors show a correlation between claudin -2 and esophageal carci noma and precancerous 
lesions. They also show that this correlates with VDR receptors VDR and TGR5. Data 
supporting these correlations are  convincing.  
 
One concern is tha t the results are correlative. In addition to the correlation with VDR and 
TRGR5, however, an additional possibility is that it that claudin -2 is a downstream effect of 
disrupted barrier (either due to the neoplastic process or vit D eff ects).  Whenever the barrier is 
disrupted in the GI tract, there is increased lamina propria inflammation, and this inflammation 
often in duces expression of claudin -2. To confirm if VDR and claudin2 are directly tied, I 
recommend performing some in vitro  studies to co mplement the staining studies.  The advantage 
of the in vitro studies is they can be performed with inflammatory cytokines strictly controlle d. If 
it is not possible to perform such studies, then it would be good to quantify inflammation i n some 
manner in the staining studies and/or expand the discussion to include this possibility.  
 
 
Regarding in vitro studies:  
 
Note that Caco2 cells have a VDR, so this might be an option  
 
Arch Biochem Biophys. 1991 Mar;285(2):261 -9. 
 
Also, note that IL13 has  been shown to specifically upregulate claudin2 expression.  
 
Another paper worth considering is: 
http://jid.oxfordjournals.org/content/early/2014/05/23/infdis.jiu235.full.pdf  
 
 
Minor comment:  
 
careful of wording throughout. For example, consider the followi ng statement in the abstract:  
 
"In contrast, Claudin -2 is a leaky protein that plays an opposing role and  
increases cell permeability."  
 
Claudins do not affect cell permeability...they a ffect epithelial permeability. Also, claudin -2 is 
not leaky. it for ms a charge selective and barrier. Leak implies no charge selectivity.    
 
Are the met hods 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 aut hors.  
 
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 statistical review is recommended, please specify what aspects require further 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
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 t hat may in any way gain or lose 
financially from the publication of this manuscript, either now or in the 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 content 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  
 
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 Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be include d 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:  High expression of the Claudin -2 in esophageal carcinoma and precancerous lesions is 
significantly associated with the bile salt receptors VDR and TGR5  
 
Version: 0 Date: 29 Sep 2016 
 
Reviewer:  Christopher  Weber  
 
Reviewer’s report:  
 
The authors show a correlation between claudin -2 and esophageal carci noma and precancerous 
lesions. They also show that this correlates with VDR receptors VDR and TGR5. Data 
supporting these correlations are  convincing.  
 
One concern is tha t the results are correlative. In addition to the correlation with VDR and 
TRGR5, however, an additional possibility is that it that claudin -2 is a downstream effect of 
disrupted barrier (either due to the neoplastic process or vit D eff ects).  Whenever the barrier is 
disrupted in the GI tract, there is increased lamina propria inflammation, and this inflammation 
often in duces expression of claudin -2. To confirm if VDR and claudin2 are directly tied, I 
recommend performing some in vitro  studies to co mplement the staining studies.  The advantage 
of the in vitro studies is they can be performed with inflammatory cytokines strictly controlle d. If 
it is not possible to perform such studies, then it would be good to quantify inflammation i n some 
manner in the staining studies and/or expand the discussion to include this possibility.  
 
 
Regarding in vitro studies:  
 
Note that Caco2 cells have a VDR, so this might be an option  
 
Arch Biochem Biophys. 1991 Mar;285(2):261 -9. 
 
Also, note that IL13 has  been shown to specifically upregulate claudin2 expression.  
 
Another paper worth considering is: 
http://jid.oxfordjournals.org/content/early/2014/05/23/infdis.jiu235.full.pdf  
 
 
Minor comment:  
 
careful of wording throughout. For example, consider the followi ng statement in the abstract:  
 
"In contrast, Claudin -2 is a leaky protein that plays an opposing role and  
increases cell permeability."  
 
Claudins do not affect cell permeability...they a ffect epithelial permeability. Also, claudin -2 is 
not leaky. it for ms a charge selective and barrier. Leak implies no charge selectivity.    
 
Are the met hods 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 aut hors.  
 
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 statistical review is recommended, please specify what aspects require further 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
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 t hat may in any way gain or lose 
financially from the publication of this manuscript, either now or in the 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 content 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  
 
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 Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be include d 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