Reviewer ’s report  
 
Title: Transplantation, gene therapy and intestinal pathology in MNGIE patients and mice  
 
Version: 1 Date: 21 Feb 2018  
 
Reviewer: Loris Pironi  
 
Reviewer's report:  
 
The gastrointestinal (GI) tract is usually one of the main target of mitochondrial 
neurogastrointestinal encephalomyo pathy (MNGIE). Patients with end -stage MNGIE often die 
because of GI complications. Since it is unclear whether current gold -standard treatment, e.g. 
hematopoietic stem cell transplantation, may restore (at least in part) the structural integrity of 
the gu t, the present study was aimed to assess the enteric neuro -muscular pathology of gut tissue 
specimens from patients with MNGIE and a transgenic mouse model reminiscent of the disease.  
In human tissues (n= 3 MNGIE cases), the authors confirmed that the pre dominant 
histopathological feature is a myopathy of the small bowel along with significant loss of Kit -
labeled interstitial cells of Cajal (ICCs). Apparently there were no changes of neurons. Intestinal 
pathology did not change as a result of hematopoietic  stem cell transplantation (at least in the 
short -term).  
In MNGIE -like mouse model, H&E staining revealed smooth muscle atrophy and myenteric 
ganglion cell loss; these features were more readily detectable in old KO vs. young Tymp -/-
Upp1 -/- and old WT mi ce.  Following hematopoietic stem cell gene therapy (HCSGT), the 
authors showed recovery of the muscularis propria thickness in old treated mice vs. Tymp -/-
Upp1 -/-mice, but no significant changes were observed in the number of myenteric ganglion 
cells.  
Even though this study is largely confirmatory of previous data in humans, the authors should be 
commended for tackling this important aspect of MNGIE.  
 
Major comments  
1. It is unclear how the authors have established that there are no changes in the human gut 
tissues of MNGIE patients vs. controls.  Did they perform an accurate neuronal marker 
analysis (similarly to that of KO mice)? Please provide detailed info about n euronal 
quantitative analysis in both myenteric and submucosal plexus.  
 
2. The origin of the human control tissues should be specified?  From autopsy material or from 
surgical resection  (there are clearly huge differences in terms of tissue analysis depe nding 
on the origin of specimens)?  
 
3. Mouse phenotype. Clearly the gut histopathological phenotype of Tymp -/-Upp1 -/-mice is 
different from human tissues as neuronal abnormalities could be demonstrated in addition to 
smooth muscle atrophy. A thorough and accurate quantitative analysis of the number of 
neuronal cell bodies in both plexuses is lacking. How did the authors established changes in 
the myenteric (and also submucosal) plexus ?  Which criteria were used to establish the 
existence of a neuropathy i n addition to muscle abnormalities ?  
 
4. In the Tymp -/-Upp1 -/- mice vs WT, the assessment of neurons was performed by using 
calretinin which is a marker of a subset of myenteric neurons (particularly in the mouse 
enteric nervous system calretinin is detect able in a subset of cholinergic neurons). The 
author should comment the reason why they did not use PGP9.5 or NSE or HuD pan -
neuronal markers.  
 
5. Information about the glial cells both in human and mouse tissues should be provided.  
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.  
 
Unable to assess  
 
Are you able to assess any statistics in the manuscr ipt 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.  
 
Not relevant to this manuscript  
 
  
Quality of written Engl ish 
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 reimbursemen ts, 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 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 com peting 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 t o 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 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: Transplantation, gene therapy and intestinal pathology in MNGIE patients and mice  
 
Version: 1 Date: 21 Feb 2018  
 
Reviewer: Loris Pironi  
 
Reviewer's report:  
 
The gastrointestinal (GI) tract is usually one of the main target of mitochondrial 
neurogastrointestinal encephalomyo pathy (MNGIE). Patients with end -stage MNGIE often die 
because of GI complications. Since it is unclear whether current gold -standard treatment, e.g. 
hematopoietic stem cell transplantation, may restore (at least in part) the structural integrity of 
the gu t, the present study was aimed to assess the enteric neuro -muscular pathology of gut tissue 
specimens from patients with MNGIE and a transgenic mouse model reminiscent of the disease.  
In human tissues (n= 3 MNGIE cases), the authors confirmed that the pre dominant 
histopathological feature is a myopathy of the small bowel along with significant loss of Kit -
labeled interstitial cells of Cajal (ICCs). Apparently there were no changes of neurons. Intestinal 
pathology did not change as a result of hematopoietic  stem cell transplantation (at least in the 
short -term).  
In MNGIE -like mouse model, H&E staining revealed smooth muscle atrophy and myenteric 
ganglion cell loss; these features were more readily detectable in old KO vs. young Tymp -/-
Upp1 -/- and old WT mi ce.  Following hematopoietic stem cell gene therapy (HCSGT), the 
authors showed recovery of the muscularis propria thickness in old treated mice vs. Tymp -/-
Upp1 -/-mice, but no significant changes were observed in the number of myenteric ganglion 
cells.  
Even though this study is largely confirmatory of previous data in humans, the authors should be 
commended for tackling this important aspect of MNGIE.  
 
Major comments  
1. It is unclear how the authors have established that there are no changes in the human gut 
tissues of MNGIE patients vs. controls.  Did they perform an accurate neuronal marker 
analysis (similarly to that of KO mice)? Please provide detailed info about n euronal 
quantitative analysis in both myenteric and submucosal plexus.  
 
2. The origin of the human control tissues should be specified?  From autopsy material or from 
surgical resection  (there are clearly huge differences in terms of tissue analysis depe nding 
on the origin of specimens)?  
 
3. Mouse phenotype. Clearly the gut histopathological phenotype of Tymp -/-Upp1 -/-mice is 
different from human tissues as neuronal abnormalities could be demonstrated in addition to 
smooth muscle atrophy. A thorough and accurate quantitative analysis of the number of 
neuronal cell bodies in both plexuses is lacking. How did the authors established changes in 
the myenteric (and also submucosal) plexus ?  Which criteria were used to establish the 
existence of a neuropathy i n addition to muscle abnormalities ?  
 
4. In the Tymp -/-Upp1 -/- mice vs WT, the assessment of neurons was performed by using 
calretinin which is a marker of a subset of myenteric neurons (particularly in the mouse 
enteric nervous system calretinin is detect able in a subset of cholinergic neurons). The 
author should comment the reason why they did not use PGP9.5 or NSE or HuD pan -
neuronal markers.  
 
5. Information about the glial cells both in human and mouse tissues should be provided.  
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.  
 
Unable to assess  
 
Are you able to assess any statistics in the manuscr ipt 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.  
 
Not relevant to this manuscript  
 
  
Quality of written Engl ish 
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 reimbursemen ts, 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 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 com peting 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 t o 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 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: Transplantation, gene therapy and intestinal pathology in MNGIE patients and mice  
 
Version: 1 Date: 21 Feb 2018  
 
Reviewer: Loris Pironi  
 
Reviewer's report:  
 
The gastrointestinal (GI) tract is usually one of the main target of mitochondrial 
neurogastrointestinal encephalomyo pathy (MNGIE). Patients with end -stage MNGIE often die 
because of GI complications. Since it is unclear whether current gold -standard treatment, e.g. 
hematopoietic stem cell transplantation, may restore (at least in part) the structural integrity of 
the gu t, the present study was aimed to assess the enteric neuro -muscular pathology of gut tissue 
specimens from patients with MNGIE and a transgenic mouse model reminiscent of the disease.  
In human tissues (n= 3 MNGIE cases), the authors confirmed that the pre dominant 
histopathological feature is a myopathy of the small bowel along with significant loss of Kit -
labeled interstitial cells of Cajal (ICCs). Apparently there were no changes of neurons. Intestinal 
pathology did not change as a result of hematopoietic  stem cell transplantation (at least in the 
short -term).  
In MNGIE -like mouse model, H&E staining revealed smooth muscle atrophy and myenteric 
ganglion cell loss; these features were more readily detectable in old KO vs. young Tymp -/-
Upp1 -/- and old WT mi ce.  Following hematopoietic stem cell gene therapy (HCSGT), the 
authors showed recovery of the muscularis propria thickness in old treated mice vs. Tymp -/-
Upp1 -/-mice, but no significant changes were observed in the number of myenteric ganglion 
cells.  
Even though this study is largely confirmatory of previous data in humans, the authors should be 
commended for tackling this important aspect of MNGIE.  
 
Major comments  
1. It is unclear how the authors have established that there are no changes in the human gut 
tissues of MNGIE patients vs. controls.  Did they perform an accurate neuronal marker 
analysis (similarly to that of KO mice)? Please provide detailed info about n euronal 
quantitative analysis in both myenteric and submucosal plexus.  
 
2. The origin of the human control tissues should be specified?  From autopsy material or from 
surgical resection  (there are clearly huge differences in terms of tissue analysis depe nding 
on the origin of specimens)?  
 
3. Mouse phenotype. Clearly the gut histopathological phenotype of Tymp -/-Upp1 -/-mice is 
different from human tissues as neuronal abnormalities could be demonstrated in addition to 
smooth muscle atrophy. A thorough and accurate quantitative analysis of the number of 
neuronal cell bodies in both plexuses is lacking. How did the authors established changes in 
the myenteric (and also submucosal) plexus ?  Which criteria were used to establish the 
existence of a neuropathy i n addition to muscle abnormalities ?  
 
4. In the Tymp -/-Upp1 -/- mice vs WT, the assessment of neurons was performed by using 
calretinin which is a marker of a subset of myenteric neurons (particularly in the mouse 
enteric nervous system calretinin is detect able in a subset of cholinergic neurons). The 
author should comment the reason why they did not use PGP9.5 or NSE or HuD pan -
neuronal markers.  
 
5. Information about the glial cells both in human and mouse tissues should be provided.  
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.  
 
Unable to assess  
 
Are you able to assess any statistics in the manuscr ipt 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.  
 
Not relevant to this manuscript  
 
  
Quality of written Engl ish 
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 reimbursemen ts, 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 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 com peting 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 t o 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 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