Reviewer’s report  
Title: Altered expression levels of occludin, claudin -1 and myosin light chain kinase in the 
common bile duct of pediatric patients with pancreaticobiliary maljunction  
 
Version: 0 Date:  13 Sep 2015  
 
Reviewer:  James  Tabibian  
Reviewer’s report:  
Dear XYZ,  
 
Please find below my review for this manuscript. I tried to login with my password to submit 
online, but it did not seem to work, and thus I'm sending via email instead. Of note, my 
recommendation for publication for this manuscript is "Major revisions".  
 
In this retrospective study, the authors aimed to examine whether PBM disrupts the epithelial 
cell barrier and identify possible molecular pathways involved.  This is an understudied disorder, 
and the efforts by the authors are laudable, and their manuscri pt is generally organized and 
logical.  My main concern is the paucity of mechanistic data and the somewhat unfounded 
conclusions.  Therefore, the overall implication of the study is unclear, which curbs enthusiasm 
for the manuscript.  
 
Specific points tha t should be considered, many of which are minor, are as follows:  
 
Abstract:  
 
- It would be helpful/more inviting if the authors could state the "why" portion of their 
objective and not solely what they did, i.e. something which will help the reader 
understan d how a comparison of expression profiles may be helpful for clinicians, 
researchers, or pediatric patients.  
 
- The following sentence does not need a comma before the "and" as the second clause is 
not independent: "Specimens of common bile duct were collect ed from 12 pediatric 
patients with PBM and 10 non -PBM controls, and analyzed by immunohistochemistry to 
examine expression of the tight junction -associated proteins occludin and claudin -1 in the 
epithelium."  
 
- If sufficient space, the type of statistical an alysis (e.g. Spearman correlation) should be 
noted in the methods and not the results.  
 
- What are "mean quick scores"?  If not defined in the abstract, would not use this verbage 
in the abstract.  
 
- It is unclear how the authors can substantiate the conclusio n "This suggests disruption of 
the common bile duct epithelial barrier, potentially mediated by MLCK." from the results 
described in the abstract.  The expression of epithelial barrier/junctional proteins was 
increased, not decreased, in PBM patients; are the authors assuming that the increase is 
secondary to a defect in permeability (i.e. compensatory)?  Also, a homogenate of 
common bile duct will contain much more than only biliary epithelium; therefore, the 
authors should use published techniques to isol ate cholangiocytes or revise the wording.   
 
Introduction:  
 
- The embryologic and etiopathogenetic events surrounding PBM are not well understood.  
While the authors statement, "The disorder arises when the common duct of the 
pancreatic and biliary ducts i s too long; as a result, the sphincter of Oddi on the duodenal 
papilla fails to control the union of the pancreatic and biliary ducts" is of practical value, 
it is oversimplified and should be rephrased (e.g. it is believed that…, may contribute 
to…, etc.) .  Also, a reference to the primary literature rather than Japanese clinical 
practice guidelines would be more useful.  
 
- The reference for the statement, "experiments in patients with primary biliary cirrhosis 
have shown that increasing the permeability of the biliary epithelial barrier leads to 
injury" is to a 20 year old review article.  A reference to the primary literature, and 
ideally one to that is more recent and more relevant to PBM (e.g. a non -microscopic 
cholangiopathy and/or one associated with in creased CCA risk, e.g. PSC or 
clonorochiasis or cryptosporidiosis) would be preferable.  
 
- The introduction is long (e.g. contains specific data from prior studies) and circuitous; a 
funnel approach would help facilitate understanding and flow for the reade r.  In addition, 
there are numerous errors such as "thatpreventstoxic" in the introduction and remainder 
of the manuscript.  
 
Methods:  
 
- The following key excerpt has several errors: "PBM was diagnosed based on the 
following criteria: (1) the union of pancr eatic and biliary duct was located outside the 
sphincter of Oddi, based onmagnetic resonance cholangiopancreatography (MRCP) or 
intraoperative cholangiography (IOC); (2) the common duct waslonger than 5 mm; and 
(3) the biliary amylase level was greater tha n 1000 U/L[24 -25]."  
 
- Do the authors mean pancreatic amylase instead of biliary amylase?  If not, how was the 
biliary origin of the amylase protein determined —IHC, IFM, other?  
 
Results:  
 
- The study would benefit tremendously from data on permeability, e.g. o f ductal explants 
or TEER on isolated biliary epithelial cells, as has been described in several recent 
studies.  Alternatively, or in addition, further study of the mechanisms of upregulation of 
junctional proteins would be valuable; is there a transcript ional event?  Post -
transcriptional event?  Ubiquitination and degradation of protein?  
 
Discussion:  
 
- The authors should clearly discuss the limitations of their study.  
 
Tables:  
 
- Z values are not necessary.  
 
Figures:  
 
- A conceptual schematic may help clarify the authors' speculations regarding the 
mechanisms and implications of junctional protein/permeability alterations.  
 
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 spe cify which controls are required in your comments to the authors.  
 
Yes 
 
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 o r 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 Engl ish 
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, consideri ng 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, e ither now or in the future?  
 
2. Do you hold any stocks or shares in an organis ation that may in any way gain or lose 
financially from the publication of this manuscript, e ither 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 fin ancial competing interests?  
 
6. Do you have any non -financial competing interests  in relation to this pa per? 
 
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 inc luded 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: Altered expression levels of occludin, claudin -1 and myosin light chain kinase in the 
common bile duct of pediatric patients with pancreaticobiliary maljunction  
 
Version: 0 Date:  13 Sep 2015  
 
Reviewer:  James  Tabibian  
Reviewer’s report:  
Dear XYZ,  
 
Please find below my review for this manuscript. I tried to login with my password to submit 
online, but it did not seem to work, and thus I'm sending via email instead. Of note, my 
recommendation for publication for this manuscript is "Major revisions".  
 
In this retrospective study, the authors aimed to examine whether PBM disrupts the epithelial 
cell barrier and identify possible molecular pathways involved.  This is an understudied disorder, 
and the efforts by the authors are laudable, and their manuscri pt is generally organized and 
logical.  My main concern is the paucity of mechanistic data and the somewhat unfounded 
conclusions.  Therefore, the overall implication of the study is unclear, which curbs enthusiasm 
for the manuscript.  
 
Specific points tha t should be considered, many of which are minor, are as follows:  
 
Abstract:  
 
- It would be helpful/more inviting if the authors could state the "why" portion of their 
objective and not solely what they did, i.e. something which will help the reader 
understan d how a comparison of expression profiles may be helpful for clinicians, 
researchers, or pediatric patients.  
 
- The following sentence does not need a comma before the "and" as the second clause is 
not independent: "Specimens of common bile duct were collect ed from 12 pediatric 
patients with PBM and 10 non -PBM controls, and analyzed by immunohistochemistry to 
examine expression of the tight junction -associated proteins occludin and claudin -1 in the 
epithelium."  
 
- If sufficient space, the type of statistical an alysis (e.g. Spearman correlation) should be 
noted in the methods and not the results.  
 
- What are "mean quick scores"?  If not defined in the abstract, would not use this verbage 
in the abstract.  
 
- It is unclear how the authors can substantiate the conclusio n "This suggests disruption of 
the common bile duct epithelial barrier, potentially mediated by MLCK." from the results 
described in the abstract.  The expression of epithelial barrier/junctional proteins was 
increased, not decreased, in PBM patients; are the authors assuming that the increase is 
secondary to a defect in permeability (i.e. compensatory)?  Also, a homogenate of 
common bile duct will contain much more than only biliary epithelium; therefore, the 
authors should use published techniques to isol ate cholangiocytes or revise the wording.   
 
Introduction:  
 
- The embryologic and etiopathogenetic events surrounding PBM are not well understood.  
While the authors statement, "The disorder arises when the common duct of the 
pancreatic and biliary ducts i s too long; as a result, the sphincter of Oddi on the duodenal 
papilla fails to control the union of the pancreatic and biliary ducts" is of practical value, 
it is oversimplified and should be rephrased (e.g. it is believed that…, may contribute 
to…, etc.) .  Also, a reference to the primary literature rather than Japanese clinical 
practice guidelines would be more useful.  
 
- The reference for the statement, "experiments in patients with primary biliary cirrhosis 
have shown that increasing the permeability of the biliary epithelial barrier leads to 
injury" is to a 20 year old review article.  A reference to the primary literature, and 
ideally one to that is more recent and more relevant to PBM (e.g. a non -microscopic 
cholangiopathy and/or one associated with in creased CCA risk, e.g. PSC or 
clonorochiasis or cryptosporidiosis) would be preferable.  
 
- The introduction is long (e.g. contains specific data from prior studies) and circuitous; a 
funnel approach would help facilitate understanding and flow for the reade r.  In addition, 
there are numerous errors such as "thatpreventstoxic" in the introduction and remainder 
of the manuscript.  
 
Methods:  
 
- The following key excerpt has several errors: "PBM was diagnosed based on the 
following criteria: (1) the union of pancr eatic and biliary duct was located outside the 
sphincter of Oddi, based onmagnetic resonance cholangiopancreatography (MRCP) or 
intraoperative cholangiography (IOC); (2) the common duct waslonger than 5 mm; and 
(3) the biliary amylase level was greater tha n 1000 U/L[24 -25]."  
 
- Do the authors mean pancreatic amylase instead of biliary amylase?  If not, how was the 
biliary origin of the amylase protein determined —IHC, IFM, other?  
 
Results:  
 
- The study would benefit tremendously from data on permeability, e.g. o f ductal explants 
or TEER on isolated biliary epithelial cells, as has been described in several recent 
studies.  Alternatively, or in addition, further study of the mechanisms of upregulation of 
junctional proteins would be valuable; is there a transcript ional event?  Post -
transcriptional event?  Ubiquitination and degradation of protein?  
 
Discussion:  
 
- The authors should clearly discuss the limitations of their study.  
 
Tables:  
 
- Z values are not necessary.  
 
Figures:  
 
- A conceptual schematic may help clarify the authors' speculations regarding the 
mechanisms and implications of junctional protein/permeability alterations.  
 
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 spe cify which controls are required in your comments to the authors.  
 
Yes 
 
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 o r 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 Engl ish 
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, consideri ng 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, e ither now or in the future?  
 
2. Do you hold any stocks or shares in an organis ation that may in any way gain or lose 
financially from the publication of this manuscript, e ither 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 fin ancial competing interests?  
 
6. Do you have any non -financial competing interests  in relation to this pa per? 
 
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 inc luded 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: Altered expression levels of occludin, claudin -1 and myosin light chain kinase in the 
common bile duct of pediatric patients with pancreaticobiliary maljunction  
 
Version: 0 Date:  13 Sep 2015  
 
Reviewer:  James  Tabibian  
Reviewer’s report:  
Dear XYZ,  
 
Please find below my review for this manuscript. I tried to login with my password to submit 
online, but it did not seem to work, and thus I'm sending via email instead. Of note, my 
recommendation for publication for this manuscript is "Major revisions".  
 
In this retrospective study, the authors aimed to examine whether PBM disrupts the epithelial 
cell barrier and identify possible molecular pathways involved.  This is an understudied disorder, 
and the efforts by the authors are laudable, and their manuscri pt is generally organized and 
logical.  My main concern is the paucity of mechanistic data and the somewhat unfounded 
conclusions.  Therefore, the overall implication of the study is unclear, which curbs enthusiasm 
for the manuscript.  
 
Specific points tha t should be considered, many of which are minor, are as follows:  
 
Abstract:  
 
- It would be helpful/more inviting if the authors could state the "why" portion of their 
objective and not solely what they did, i.e. something which will help the reader 
understan d how a comparison of expression profiles may be helpful for clinicians, 
researchers, or pediatric patients.  
 
- The following sentence does not need a comma before the "and" as the second clause is 
not independent: "Specimens of common bile duct were collect ed from 12 pediatric 
patients with PBM and 10 non -PBM controls, and analyzed by immunohistochemistry to 
examine expression of the tight junction -associated proteins occludin and claudin -1 in the 
epithelium."  
 
- If sufficient space, the type of statistical an alysis (e.g. Spearman correlation) should be 
noted in the methods and not the results.  
 
- What are "mean quick scores"?  If not defined in the abstract, would not use this verbage 
in the abstract.  
 
- It is unclear how the authors can substantiate the conclusio n "This suggests disruption of 
the common bile duct epithelial barrier, potentially mediated by MLCK." from the results 
described in the abstract.  The expression of epithelial barrier/junctional proteins was 
increased, not decreased, in PBM patients; are the authors assuming that the increase is 
secondary to a defect in permeability (i.e. compensatory)?  Also, a homogenate of 
common bile duct will contain much more than only biliary epithelium; therefore, the 
authors should use published techniques to isol ate cholangiocytes or revise the wording.   
 
Introduction:  
 
- The embryologic and etiopathogenetic events surrounding PBM are not well understood.  
While the authors statement, "The disorder arises when the common duct of the 
pancreatic and biliary ducts i s too long; as a result, the sphincter of Oddi on the duodenal 
papilla fails to control the union of the pancreatic and biliary ducts" is of practical value, 
it is oversimplified and should be rephrased (e.g. it is believed that…, may contribute 
to…, etc.) .  Also, a reference to the primary literature rather than Japanese clinical 
practice guidelines would be more useful.  
 
- The reference for the statement, "experiments in patients with primary biliary cirrhosis 
have shown that increasing the permeability of the biliary epithelial barrier leads to 
injury" is to a 20 year old review article.  A reference to the primary literature, and 
ideally one to that is more recent and more relevant to PBM (e.g. a non -microscopic 
cholangiopathy and/or one associated with in creased CCA risk, e.g. PSC or 
clonorochiasis or cryptosporidiosis) would be preferable.  
 
- The introduction is long (e.g. contains specific data from prior studies) and circuitous; a 
funnel approach would help facilitate understanding and flow for the reade r.  In addition, 
there are numerous errors such as "thatpreventstoxic" in the introduction and remainder 
of the manuscript.  
 
Methods:  
 
- The following key excerpt has several errors: "PBM was diagnosed based on the 
following criteria: (1) the union of pancr eatic and biliary duct was located outside the 
sphincter of Oddi, based onmagnetic resonance cholangiopancreatography (MRCP) or 
intraoperative cholangiography (IOC); (2) the common duct waslonger than 5 mm; and 
(3) the biliary amylase level was greater tha n 1000 U/L[24 -25]."  
 
- Do the authors mean pancreatic amylase instead of biliary amylase?  If not, how was the 
biliary origin of the amylase protein determined —IHC, IFM, other?  
 
Results:  
 
- The study would benefit tremendously from data on permeability, e.g. o f ductal explants 
or TEER on isolated biliary epithelial cells, as has been described in several recent 
studies.  Alternatively, or in addition, further study of the mechanisms of upregulation of 
junctional proteins would be valuable; is there a transcript ional event?  Post -
transcriptional event?  Ubiquitination and degradation of protein?  
 
Discussion:  
 
- The authors should clearly discuss the limitations of their study.  
 
Tables:  
 
- Z values are not necessary.  
 
Figures:  
 
- A conceptual schematic may help clarify the authors' speculations regarding the 
mechanisms and implications of junctional protein/permeability alterations.  
 
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 spe cify which controls are required in your comments to the authors.  
 
Yes 
 
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 o r 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 Engl ish 
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, consideri ng 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, e ither now or in the future?  
 
2. Do you hold any stocks or shares in an organis ation that may in any way gain or lose 
financially from the publication of this manuscript, e ither 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 fin ancial competing interests?  
 
6. Do you have any non -financial competing interests  in relation to this pa per? 
 
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 inc luded 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