Reviewer ’s report  
 
Title: Approaching treatment for immunological rejection of living -donor liver transplantation in 
rats 
 
Version: 1 Date: 24 Apr 2019  
 
Reviewer: Reviewer 2   
 
Reviewer's report:  
 
PEER REVIEWER ASSESSMENTS:  
 
OBJECTIVE - Full research articles: is there a clear objective that addresses a testable research 
question(s) (brief or other article types: is there a clear objective)?  
Yes - there is a clear objective  
 
DESIGN - Is the current approach (including controls  and analysis protocols) appropriate for the 
objective?  
No - there are minor issues  
 
EXECUTION - Are the experiments and analyses performed with technical rigor to allow 
confidence in the results?  
No - there are minor issues  
 
STATISTICS - Is the use of sta tistics in the manuscript appropriate?  
No - there are issues with the statistics in the study  
 
INTERPRETATION - Is the current interpretation/discussion of the results reasonable and not 
overstated?  
Yes - the author's interpretation is reasonable  
 
OVERALL MANUSCRIPT POTENTIAL - Is the current version of this work technically 
sound? If not, can revisions be made to make the work technically sound?  
Probably - with minor revisions  
 
PEER REVIEWER COMMENTS:  
 
GENERAL COMMENTS: The authors performed a pre clinical study in allograft transplantation 
using BN and Lewis rats, and performing small -for-size liver transplant (35 -42%). The 
manuscript is quite short and clearly written. Over interpretations and unnecessary long 
descriptions are avoided leading the reader straight to core of the study. However, the short 
Introduction may require a couple of additional sentences to better describe the tissutal graft 
(Fan-Kawasaki's guidelines) and the Tacrolimus altered treatment (drug adjustment based on 
biochemical parameters). Indeed, the first paragraph in the Discussion section is actually 
background (better moving in the Introduction). The large amount of Figures (8) should actually 
be revised. Most of the Figure are one panel only, and can be easily combined.  
 
 
REQUESTED REVISIONS:  
The authors described a significant higher amount TCR lymphos in allograft, please provide 
numbers (number of cells, and amount of fields counted to generate such conclusion in liver 
tissues; Figure 2). Similar request for PCNA and TU NEL analysis (Figure 6). In order to 
generate a proper statistical analysis, data must be provided.  
 
FACS analysis is a little bit confused. The region of interest is distant from the quadrant 
threshold. Isotype control should be provided (as a small pane l if necessary) and the mean 
fluorescence intensity considered, to better discriminated decreased CD4/CD25 cells in SFS iso - 
and allo -grafts.  
 
Data presented in Figure 4 have probably been generated by the analysis in one case only. As 
consequence, a prope r statistical analysis cannot be performed. However, allogenic graft is 
clearly resulting in higher amount f IL -17 compared to isograft transplant. Additional 
investigations are required to generate clear information regarding the SFS vs whole organ Tx.  
Reviewer's conclusion that one case only has been analyzed is based on the description in 
M&amp; M section (page 6), where the days after transplant should be specified for every 
sacrificed animal. Table 1 reports large variability between survival days in al l 7 groups, please 
motivate.  
 
In Tacrolimus analysis, the amount of samples analyzed is missing. Standard deviation bars are 
required for histogram in Figure 5 and 6 (Figure 5 and 6, and maybe also Figure 4, can also be 
combined in one Figure with 4 panels  without losing clearness)  
 
It's reviewer's opinion that "Survival analysis" section should be moved at the beginning of the 
Results section. Such change would help the reader to understand the biological results described 
later in the manuscript.  
 
CYP3A2 protein analysis requires instead a deeper consideration. Unfortunately, rat is not a 
good model for the most important human CYP (CYP3A4). The rat orthologous CYP3A1 (the 
main CYP3A form in the rat) is not as active in immunosuppressive drug metabolism as  
CYP3A4 in human. The authors' reason to select CYP3A2 as enzyme involved in tacrolimus 
metabolism seems to be based on a couple of manuscripts (refs 29 and 30) were interesting 
studies have been performed aimed to investigate the role in metabolism of xen obiotics and 
endogenous substances.  The rat orthologous CYP3A2 is not induced by prototypical CYP3A 
inducers. Thus, the authors in the cited publications selected an isoform that can be affected by 
inhibitory or inductive studies. The most abundant CYP sub family (in rat liver) is the CYP2C, 
which has a role closer to human CYP3A enzymes. Moreover, the amount of CYPs has been 
reported to increased with the size of the tissue (and level of maturation of the cells) and 
negatively correlated with the inflammati on (CYP activities decreased in inflammatory status). 
Maybe the authors should consider to analyze additional enzymes (such as urea cycle enzymes or  
additional CYPs) and add additional normalization, using not only actin but also cytoskeletal 
protein such as cytokeratin 8/18 (specific for hepatocytes)  
 
 
ADDITIONAL REQUESTS/SUGGESTIONS:  
 
Scale bars are missing  
Expression as "was more obvious" should be substituted with "was observed" or "was measured"  
 
 
Note: This reviewer report can be downloaded - see attached pdf file.  
 
 
 
Are the methods appropriate and well described?  
If not, please specify wha t is required in your comments to the authors.  
 
No 
 
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 manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require fu rther 
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, f ees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publi cation 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 curr ently 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.  
 
This reviewer has been recruited by a partner organization, Research Square. Reviewers with 
declared or apparent competing interests are not  utilized for these reviews. This reviewer has 
agreed to publication of their comments online under a Creative Commons Attribution License 
attributed to Research Square and was paid a small honorarium for completing the review within 
a specified timeframe.  Honoraria for reviews such as this are paid regardless of the reviewer 
recommendation.  
 
I agree to the open peer review policy of the journal. I understan d that my name will be included 
on my report to the authors and, if the manuscript is accepted f or publication, my named report 
including any att achments 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 in cluded 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: Approaching treatment for immunological rejection of living -donor liver transplantation in 
rats 
 
Version: 1 Date: 24 Apr 2019  
 
Reviewer: Reviewer 2   
 
Reviewer's report:  
 
PEER REVIEWER ASSESSMENTS:  
 
OBJECTIVE - Full research articles: is there a clear objective that addresses a testable research 
question(s) (brief or other article types: is there a clear objective)?  
Yes - there is a clear objective  
 
DESIGN - Is the current approach (including controls  and analysis protocols) appropriate for the 
objective?  
No - there are minor issues  
 
EXECUTION - Are the experiments and analyses performed with technical rigor to allow 
confidence in the results?  
No - there are minor issues  
 
STATISTICS - Is the use of sta tistics in the manuscript appropriate?  
No - there are issues with the statistics in the study  
 
INTERPRETATION - Is the current interpretation/discussion of the results reasonable and not 
overstated?  
Yes - the author's interpretation is reasonable  
 
OVERALL MANUSCRIPT POTENTIAL - Is the current version of this work technically 
sound? If not, can revisions be made to make the work technically sound?  
Probably - with minor revisions  
 
PEER REVIEWER COMMENTS:  
 
GENERAL COMMENTS: The authors performed a pre clinical study in allograft transplantation 
using BN and Lewis rats, and performing small -for-size liver transplant (35 -42%). The 
manuscript is quite short and clearly written. Over interpretations and unnecessary long 
descriptions are avoided leading the reader straight to core of the study. However, the short 
Introduction may require a couple of additional sentences to better describe the tissutal graft 
(Fan-Kawasaki's guidelines) and the Tacrolimus altered treatment (drug adjustment based on 
biochemical parameters). Indeed, the first paragraph in the Discussion section is actually 
background (better moving in the Introduction). The large amount of Figures (8) should actually 
be revised. Most of the Figure are one panel only, and can be easily combined.  
 
 
REQUESTED REVISIONS:  
The authors described a significant higher amount TCR lymphos in allograft, please provide 
numbers (number of cells, and amount of fields counted to generate such conclusion in liver 
tissues; Figure 2). Similar request for PCNA and TU NEL analysis (Figure 6). In order to 
generate a proper statistical analysis, data must be provided.  
 
FACS analysis is a little bit confused. The region of interest is distant from the quadrant 
threshold. Isotype control should be provided (as a small pane l if necessary) and the mean 
fluorescence intensity considered, to better discriminated decreased CD4/CD25 cells in SFS iso - 
and allo -grafts.  
 
Data presented in Figure 4 have probably been generated by the analysis in one case only. As 
consequence, a prope r statistical analysis cannot be performed. However, allogenic graft is 
clearly resulting in higher amount f IL -17 compared to isograft transplant. Additional 
investigations are required to generate clear information regarding the SFS vs whole organ Tx.  
Reviewer's conclusion that one case only has been analyzed is based on the description in 
M&amp; M section (page 6), where the days after transplant should be specified for every 
sacrificed animal. Table 1 reports large variability between survival days in al l 7 groups, please 
motivate.  
 
In Tacrolimus analysis, the amount of samples analyzed is missing. Standard deviation bars are 
required for histogram in Figure 5 and 6 (Figure 5 and 6, and maybe also Figure 4, can also be 
combined in one Figure with 4 panels  without losing clearness)  
 
It's reviewer's opinion that "Survival analysis" section should be moved at the beginning of the 
Results section. Such change would help the reader to understand the biological results described 
later in the manuscript.  
 
CYP3A2 protein analysis requires instead a deeper consideration. Unfortunately, rat is not a 
good model for the most important human CYP (CYP3A4). The rat orthologous CYP3A1 (the 
main CYP3A form in the rat) is not as active in immunosuppressive drug metabolism as  
CYP3A4 in human. The authors' reason to select CYP3A2 as enzyme involved in tacrolimus 
metabolism seems to be based on a couple of manuscripts (refs 29 and 30) were interesting 
studies have been performed aimed to investigate the role in metabolism of xen obiotics and 
endogenous substances.  The rat orthologous CYP3A2 is not induced by prototypical CYP3A 
inducers. Thus, the authors in the cited publications selected an isoform that can be affected by 
inhibitory or inductive studies. The most abundant CYP sub family (in rat liver) is the CYP2C, 
which has a role closer to human CYP3A enzymes. Moreover, the amount of CYPs has been 
reported to increased with the size of the tissue (and level of maturation of the cells) and 
negatively correlated with the inflammati on (CYP activities decreased in inflammatory status). 
Maybe the authors should consider to analyze additional enzymes (such as urea cycle enzymes or  
additional CYPs) and add additional normalization, using not only actin but also cytoskeletal 
protein such as cytokeratin 8/18 (specific for hepatocytes)  
 
 
ADDITIONAL REQUESTS/SUGGESTIONS:  
 
Scale bars are missing  
Expression as "was more obvious" should be substituted with "was observed" or "was measured"  
 
 
Note: This reviewer report can be downloaded - see attached pdf file.  
 
 
 
Are the methods appropriate and well described?  
If not, please specify wha t is required in your comments to the authors.  
 
No 
 
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 manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require fu rther 
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, f ees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publi cation 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 curr ently 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.  
 
This reviewer has been recruited by a partner organization, Research Square. Reviewers with 
declared or apparent competing interests are not  utilized for these reviews. This reviewer has 
agreed to publication of their comments online under a Creative Commons Attribution License 
attributed to Research Square and was paid a small honorarium for completing the review within 
a specified timeframe.  Honoraria for reviews such as this are paid regardless of the reviewer 
recommendation.  
 
I agree to the open peer review policy of the journal. I understan d that my name will be included 
on my report to the authors and, if the manuscript is accepted f or publication, my named report 
including any att achments 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 in cluded 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: Approaching treatment for immunological rejection of living -donor liver transplantation in 
rats 
 
Version: 1 Date: 24 Apr 2019  
 
Reviewer: Reviewer 2   
 
Reviewer's report:  
 
PEER REVIEWER ASSESSMENTS:  
 
OBJECTIVE - Full research articles: is there a clear objective that addresses a testable research 
question(s) (brief or other article types: is there a clear objective)?  
Yes - there is a clear objective  
 
DESIGN - Is the current approach (including controls  and analysis protocols) appropriate for the 
objective?  
No - there are minor issues  
 
EXECUTION - Are the experiments and analyses performed with technical rigor to allow 
confidence in the results?  
No - there are minor issues  
 
STATISTICS - Is the use of sta tistics in the manuscript appropriate?  
No - there are issues with the statistics in the study  
 
INTERPRETATION - Is the current interpretation/discussion of the results reasonable and not 
overstated?  
Yes - the author's interpretation is reasonable  
 
OVERALL MANUSCRIPT POTENTIAL - Is the current version of this work technically 
sound? If not, can revisions be made to make the work technically sound?  
Probably - with minor revisions  
 
PEER REVIEWER COMMENTS:  
 
GENERAL COMMENTS: The authors performed a pre clinical study in allograft transplantation 
using BN and Lewis rats, and performing small -for-size liver transplant (35 -42%). The 
manuscript is quite short and clearly written. Over interpretations and unnecessary long 
descriptions are avoided leading the reader straight to core of the study. However, the short 
Introduction may require a couple of additional sentences to better describe the tissutal graft 
(Fan-Kawasaki's guidelines) and the Tacrolimus altered treatment (drug adjustment based on 
biochemical parameters). Indeed, the first paragraph in the Discussion section is actually 
background (better moving in the Introduction). The large amount of Figures (8) should actually 
be revised. Most of the Figure are one panel only, and can be easily combined.  
 
 
REQUESTED REVISIONS:  
The authors described a significant higher amount TCR lymphos in allograft, please provide 
numbers (number of cells, and amount of fields counted to generate such conclusion in liver 
tissues; Figure 2). Similar request for PCNA and TU NEL analysis (Figure 6). In order to 
generate a proper statistical analysis, data must be provided.  
 
FACS analysis is a little bit confused. The region of interest is distant from the quadrant 
threshold. Isotype control should be provided (as a small pane l if necessary) and the mean 
fluorescence intensity considered, to better discriminated decreased CD4/CD25 cells in SFS iso - 
and allo -grafts.  
 
Data presented in Figure 4 have probably been generated by the analysis in one case only. As 
consequence, a prope r statistical analysis cannot be performed. However, allogenic graft is 
clearly resulting in higher amount f IL -17 compared to isograft transplant. Additional 
investigations are required to generate clear information regarding the SFS vs whole organ Tx.  
Reviewer's conclusion that one case only has been analyzed is based on the description in 
M&amp; M section (page 6), where the days after transplant should be specified for every 
sacrificed animal. Table 1 reports large variability between survival days in al l 7 groups, please 
motivate.  
 
In Tacrolimus analysis, the amount of samples analyzed is missing. Standard deviation bars are 
required for histogram in Figure 5 and 6 (Figure 5 and 6, and maybe also Figure 4, can also be 
combined in one Figure with 4 panels  without losing clearness)  
 
It's reviewer's opinion that "Survival analysis" section should be moved at the beginning of the 
Results section. Such change would help the reader to understand the biological results described 
later in the manuscript.  
 
CYP3A2 protein analysis requires instead a deeper consideration. Unfortunately, rat is not a 
good model for the most important human CYP (CYP3A4). The rat orthologous CYP3A1 (the 
main CYP3A form in the rat) is not as active in immunosuppressive drug metabolism as  
CYP3A4 in human. The authors' reason to select CYP3A2 as enzyme involved in tacrolimus 
metabolism seems to be based on a couple of manuscripts (refs 29 and 30) were interesting 
studies have been performed aimed to investigate the role in metabolism of xen obiotics and 
endogenous substances.  The rat orthologous CYP3A2 is not induced by prototypical CYP3A 
inducers. Thus, the authors in the cited publications selected an isoform that can be affected by 
inhibitory or inductive studies. The most abundant CYP sub family (in rat liver) is the CYP2C, 
which has a role closer to human CYP3A enzymes. Moreover, the amount of CYPs has been 
reported to increased with the size of the tissue (and level of maturation of the cells) and 
negatively correlated with the inflammati on (CYP activities decreased in inflammatory status). 
Maybe the authors should consider to analyze additional enzymes (such as urea cycle enzymes or  
additional CYPs) and add additional normalization, using not only actin but also cytoskeletal 
protein such as cytokeratin 8/18 (specific for hepatocytes)  
 
 
ADDITIONAL REQUESTS/SUGGESTIONS:  
 
Scale bars are missing  
Expression as "was more obvious" should be substituted with "was observed" or "was measured"  
 
 
Note: This reviewer report can be downloaded - see attached pdf file.  
 
 
 
Are the methods appropriate and well described?  
If not, please specify wha t is required in your comments to the authors.  
 
No 
 
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 manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require fu rther 
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, f ees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publi cation 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 curr ently 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.  
 
This reviewer has been recruited by a partner organization, Research Square. Reviewers with 
declared or apparent competing interests are not  utilized for these reviews. This reviewer has 
agreed to publication of their comments online under a Creative Commons Attribution License 
attributed to Research Square and was paid a small honorarium for completing the review within 
a specified timeframe.  Honoraria for reviews such as this are paid regardless of the reviewer 
recommendation.  
 
I agree to the open peer review policy of the journal. I understan d that my name will be included 
on my report to the authors and, if the manuscript is accepted f or publication, my named report 
including any att achments 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 in cluded as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant