Reviewer’s report  
Title: Synergistic effect of κ-carrageenan on oxazolone -induced inflammation in BALB/c mice  
 
Version: 0 Date: 13 Oct 2015  
 
Reviewer: Philippe Lehours  
Reviewer’s report:  
The paper submitted by Wu W et al., aimed at determining the effect of k -carrageenan on 
oxazolone -induced colonic and systemic inflammation in BALB/c mice.  
 
The paper is based on  macroscopic analyses, immunochemistry and molecular approaches.  
The manuscript is easy to read and clearly presented.  
 
The authors conclude that aggravated OXA -induced intestinal inflammation in association with 
the activation of TLR4 -NFkB pathway, a decre ased ratio of Tregs and the induction of a Th2 
response.  
 
Minor and in particular major points can be formulated which considered together make this 
paper not clear and in a large way preliminary. Moreover some data are overinterpreted.  
 
1- Introduction: clea r and well presented.  
 
- page 5, line 75: reformulate the sentence "we found that the…"  
 
- page 5, line 82: a reference should be added at the end of the sentence.  
 
2- Material and methods  
 
- analysis of cytokine levels (end of a page 10 and beginning of page 11):  reduce this 
part if the CBA cytokine kit was used according to manufacturer recommendations.  
 
3- Results  
 
- Effect of k -carrageenan on weight loss and survival ratio (line 242, page 12): Data on 
weight loss should be reviewed by an independent statistician be cause the differences 
between the different conditions appear to be rather minimal. It is not obvious that k -
carrageenan aggravated the weight loss, there is also no correlation with the dose 
which seem to be in contradiction with some data presented furth er. 
 
In the paragraph from line 255 to 261, there was no statistical difference (except in the 
MED/OXA -treated group) in the survival ratio. Therefore the authors have 
overinterpreted their results.  
 
- Effect of k -carrageenan on the severity of colonic damage (line 262, page 13)  
Figure 2 is difficult to understand for a non specialist. Add arrows or other symbols on 
the different macroscopic lesions. Line 277 replaces Figs. 1F -G by Figs. 2F -G. 
 
- Effects of k-carrageenan on the histological changes of OXA -induced colonic damage 
(line 283, page 14)  
 
The presence of lymphocyte infiltration is not obvious, neither the presence of 
granulocytes. Higher magnifications of the pictures should be provided. Histologi cal 
analysis should also be complemented by IHC analysis. How can the authors be sure that 
eosinophils are infiltrating the lesions?  
 
- Scanning electron microscopy (line 302 - page 15): Figure 4 present only selected 
slides however these data are not accompa nied by any statistical analysis.  
 
- k-carrageenan effect on the OXA -induced decrease in the ratio of Tregs (line 317, 
page 16): again the authors have overinterpreted their results. They  found a 
significant decrease of Tregs in the MED -OXA treated group o nly. These data should 
be reviewed by an independent statistician.  
 
- k-carrageenan promotes cytokines production in OXA -induced mice colonic 
inflammation (line330, page 17):  
 
IL-10 is not a Th2 -type cytokine but rather than a Treg marker. Therefore all the 
interpretation of the results should be reconsidered.  
 
There is not obvious augmentation of the seven cytokines in the carrageenan alone 
groups. The most significant changes were found in the MED -OXA group. Therefore 
again the authors overinterpreted their r esults.  
 
What is the rational to investigate the systemic inflammatory response? It would have 
been more interesting to investigate the local production of several cytokines by using 
protein extracts from digestive biopsies.  
 
Remove all the sentences from li ne 343 to 353.  
 
- Synergistic effects of k -carrageenan on OXA -induced protein expression in colonic 
mucosa (line 354, page 17): There is no unit indication for the data presented in Table 
2. The authors refer in line 358 to concentrations, whereas to number of positive cells 
in line 369. The upregulation of NF -kB and TLR4 should be verified by western blot.  
 
- Expression levels of TLR4, NFkB and Foxp3 (line 372, page 18): these data should 
be presented before the previous paragraph. Again data, especially those  concerning 
foxp3 mRNA expression, should be controlled by an independent statistician.  
 
4- Discussion: the discussion could be shortened and rewriten according to all the majors 
comments formulated above.  
 
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 con trols 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 or would you r ecommend 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 recommend additional statistical review  
 
Quality of written English  
Please in dicate the quality of language in the manuscript:  
 
Acceptable  
 
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, fun ding, 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 organisation that may in any way gain or lose 
financially from t he 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 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 m y 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: Synergistic effect of κ-carrageenan on oxazolone -induced inflammation in BALB/c mice  
 
Version: 0 Date: 13 Oct 2015  
 
Reviewer: Philippe Lehours  
Reviewer’s report:  
The paper submitted by Wu W et al., aimed at determining the effect of k -carrageenan on 
oxazolone -induced colonic and systemic inflammation in BALB/c mice.  
 
The paper is based on  macroscopic analyses, immunochemistry and molecular approaches.  
The manuscript is easy to read and clearly presented.  
 
The authors conclude that aggravated OXA -induced intestinal inflammation in association with 
the activation of TLR4 -NFkB pathway, a decre ased ratio of Tregs and the induction of a Th2 
response.  
 
Minor and in particular major points can be formulated which considered together make this 
paper not clear and in a large way preliminary. Moreover some data are overinterpreted.  
 
1- Introduction: clea r and well presented.  
 
- page 5, line 75: reformulate the sentence "we found that the…"  
 
- page 5, line 82: a reference should be added at the end of the sentence.  
 
2- Material and methods  
 
- analysis of cytokine levels (end of a page 10 and beginning of page 11):  reduce this 
part if the CBA cytokine kit was used according to manufacturer recommendations.  
 
3- Results  
 
- Effect of k -carrageenan on weight loss and survival ratio (line 242, page 12): Data on 
weight loss should be reviewed by an independent statistician be cause the differences 
between the different conditions appear to be rather minimal. It is not obvious that k -
carrageenan aggravated the weight loss, there is also no correlation with the dose 
which seem to be in contradiction with some data presented furth er. 
 
In the paragraph from line 255 to 261, there was no statistical difference (except in the 
MED/OXA -treated group) in the survival ratio. Therefore the authors have 
overinterpreted their results.  
 
- Effect of k -carrageenan on the severity of colonic damage (line 262, page 13)  
Figure 2 is difficult to understand for a non specialist. Add arrows or other symbols on 
the different macroscopic lesions. Line 277 replaces Figs. 1F -G by Figs. 2F -G. 
 
- Effects of k-carrageenan on the histological changes of OXA -induced colonic damage 
(line 283, page 14)  
 
The presence of lymphocyte infiltration is not obvious, neither the presence of 
granulocytes. Higher magnifications of the pictures should be provided. Histologi cal 
analysis should also be complemented by IHC analysis. How can the authors be sure that 
eosinophils are infiltrating the lesions?  
 
- Scanning electron microscopy (line 302 - page 15): Figure 4 present only selected 
slides however these data are not accompa nied by any statistical analysis.  
 
- k-carrageenan effect on the OXA -induced decrease in the ratio of Tregs (line 317, 
page 16): again the authors have overinterpreted their results. They  found a 
significant decrease of Tregs in the MED -OXA treated group o nly. These data should 
be reviewed by an independent statistician.  
 
- k-carrageenan promotes cytokines production in OXA -induced mice colonic 
inflammation (line330, page 17):  
 
IL-10 is not a Th2 -type cytokine but rather than a Treg marker. Therefore all the 
interpretation of the results should be reconsidered.  
 
There is not obvious augmentation of the seven cytokines in the carrageenan alone 
groups. The most significant changes were found in the MED -OXA group. Therefore 
again the authors overinterpreted their r esults.  
 
What is the rational to investigate the systemic inflammatory response? It would have 
been more interesting to investigate the local production of several cytokines by using 
protein extracts from digestive biopsies.  
 
Remove all the sentences from li ne 343 to 353.  
 
- Synergistic effects of k -carrageenan on OXA -induced protein expression in colonic 
mucosa (line 354, page 17): There is no unit indication for the data presented in Table 
2. The authors refer in line 358 to concentrations, whereas to number of positive cells 
in line 369. The upregulation of NF -kB and TLR4 should be verified by western blot.  
 
- Expression levels of TLR4, NFkB and Foxp3 (line 372, page 18): these data should 
be presented before the previous paragraph. Again data, especially those  concerning 
foxp3 mRNA expression, should be controlled by an independent statistician.  
 
4- Discussion: the discussion could be shortened and rewriten according to all the majors 
comments formulated above.  
 
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 con trols 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 or would you r ecommend 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 recommend additional statistical review  
 
Quality of written English  
Please in dicate the quality of language in the manuscript:  
 
Acceptable  
 
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, fun ding, 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 organisation that may in any way gain or lose 
financially from t he 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 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 m y 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: Synergistic effect of κ-carrageenan on oxazolone -induced inflammation in BALB/c mice  
 
Version: 0 Date: 13 Oct 2015  
 
Reviewer: Philippe Lehours  
Reviewer’s report:  
The paper submitted by Wu W et al., aimed at determining the effect of k -carrageenan on 
oxazolone -induced colonic and systemic inflammation in BALB/c mice.  
 
The paper is based on  macroscopic analyses, immunochemistry and molecular approaches.  
The manuscript is easy to read and clearly presented.  
 
The authors conclude that aggravated OXA -induced intestinal inflammation in association with 
the activation of TLR4 -NFkB pathway, a decre ased ratio of Tregs and the induction of a Th2 
response.  
 
Minor and in particular major points can be formulated which considered together make this 
paper not clear and in a large way preliminary. Moreover some data are overinterpreted.  
 
1- Introduction: clea r and well presented.  
 
- page 5, line 75: reformulate the sentence "we found that the…"  
 
- page 5, line 82: a reference should be added at the end of the sentence.  
 
2- Material and methods  
 
- analysis of cytokine levels (end of a page 10 and beginning of page 11):  reduce this 
part if the CBA cytokine kit was used according to manufacturer recommendations.  
 
3- Results  
 
- Effect of k -carrageenan on weight loss and survival ratio (line 242, page 12): Data on 
weight loss should be reviewed by an independent statistician be cause the differences 
between the different conditions appear to be rather minimal. It is not obvious that k -
carrageenan aggravated the weight loss, there is also no correlation with the dose 
which seem to be in contradiction with some data presented furth er. 
 
In the paragraph from line 255 to 261, there was no statistical difference (except in the 
MED/OXA -treated group) in the survival ratio. Therefore the authors have 
overinterpreted their results.  
 
- Effect of k -carrageenan on the severity of colonic damage (line 262, page 13)  
Figure 2 is difficult to understand for a non specialist. Add arrows or other symbols on 
the different macroscopic lesions. Line 277 replaces Figs. 1F -G by Figs. 2F -G. 
 
- Effects of k-carrageenan on the histological changes of OXA -induced colonic damage 
(line 283, page 14)  
 
The presence of lymphocyte infiltration is not obvious, neither the presence of 
granulocytes. Higher magnifications of the pictures should be provided. Histologi cal 
analysis should also be complemented by IHC analysis. How can the authors be sure that 
eosinophils are infiltrating the lesions?  
 
- Scanning electron microscopy (line 302 - page 15): Figure 4 present only selected 
slides however these data are not accompa nied by any statistical analysis.  
 
- k-carrageenan effect on the OXA -induced decrease in the ratio of Tregs (line 317, 
page 16): again the authors have overinterpreted their results. They  found a 
significant decrease of Tregs in the MED -OXA treated group o nly. These data should 
be reviewed by an independent statistician.  
 
- k-carrageenan promotes cytokines production in OXA -induced mice colonic 
inflammation (line330, page 17):  
 
IL-10 is not a Th2 -type cytokine but rather than a Treg marker. Therefore all the 
interpretation of the results should be reconsidered.  
 
There is not obvious augmentation of the seven cytokines in the carrageenan alone 
groups. The most significant changes were found in the MED -OXA group. Therefore 
again the authors overinterpreted their r esults.  
 
What is the rational to investigate the systemic inflammatory response? It would have 
been more interesting to investigate the local production of several cytokines by using 
protein extracts from digestive biopsies.  
 
Remove all the sentences from li ne 343 to 353.  
 
- Synergistic effects of k -carrageenan on OXA -induced protein expression in colonic 
mucosa (line 354, page 17): There is no unit indication for the data presented in Table 
2. The authors refer in line 358 to concentrations, whereas to number of positive cells 
in line 369. The upregulation of NF -kB and TLR4 should be verified by western blot.  
 
- Expression levels of TLR4, NFkB and Foxp3 (line 372, page 18): these data should 
be presented before the previous paragraph. Again data, especially those  concerning 
foxp3 mRNA expression, should be controlled by an independent statistician.  
 
4- Discussion: the discussion could be shortened and rewriten according to all the majors 
comments formulated above.  
 
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 con trols 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 or would you r ecommend 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 recommend additional statistical review  
 
Quality of written English  
Please in dicate the quality of language in the manuscript:  
 
Acceptable  
 
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, fun ding, 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 organisation that may in any way gain or lose 
financially from t he 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 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 m y 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