Reviewer’s report  
Title: Synergistic effect of κ-carrageenan on oxazolone -induced inflammation in BALB/c mice  
 
Version: 0 Date: 08 Oct 2015  
 
Reviewer: Christine Delporte  
Reviewer’s report:  
Manuscript BMGE -D-15-00075 entitled 'Synergistic effect of k -carrageenan on oxazolone -
induced inflammation in BALB/c mice' by Wu et al. describes the effect of carrageenan in a 
model of colitis.  
Major concerns:  
The authors should clearly explain the rationale justifying the conc entration of oxazolone 
used to induce colitis, the time point chosen to investigate the measured parameters (1 
day post oxazolone injection), as well as the concentrations of carrageenan used in the 
study (low, medium and high). In addition, the authors sh ould discussed about the 
relevance of the carrageenan doses in relation to the human situation.  
Concerning the real -time quantitative PCR, the authors should follow the minimum 
information for publication of quantitative real -time PCR experiments guideline s in order 
to publish reliable and unequivocal interpretation of qPCR data (Bustin et al., 2009, Clin 
Chem, 55: 611 -612; Vandesompele, 2002, Genome Biology, 3: Research0034). The 
authors used only one reference gene (ß -actin) to normalize their data, this kind of 
normalization is nowadays considered as inappropriate proper data normalization should 
be done using at least 3 reference genes. The authors should include a positive and a 
negative control in each PCR experiment. Furthermore, the authors should pr ovide details 
concerning: primer efficiency, absence of genomic contamination (which methods were 
used to exclude it, even if RNA was treated with DNAse?) and absence of RNA 
degradation (which method was used to assess the RNA integrity?).  
Immunohistochem ical analyses were based on the determination of average integral 
density which relies on positive labelled area, optical density of labelling and total area 
(see page 10, lines 198 -200). However, modifications in optical density of labelling 
might result from technical variability and is thereby unlikely to represent an appropriate 
way to quantify labelling. Authors should rather quantity the expression of the protein by 
measuring positive labelled areas versus total area.  
The observed effects of carrageen an do not appear to respond to a traditional dose -
response curve for most measured parameters. What are the possible explanations for 
this?  
The authors claim that 'carrageenan alone results in the upregulation of the seven 
cytokines (Table 1)' (see page 17 , lines 336 -337). However, this sentence do not seems to 
be supported by the data presented in Table 1.  
Minor comments:  
The authors should express their data in similar way, ratio or percentage, when 
expressing the CD4+ CD25+/CD4+ ratio modifications (see page 16, lines 319 -329).  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
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 am able to assess the statistics  
 
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: 08 Oct 2015  
 
Reviewer: Christine Delporte  
Reviewer’s report:  
Manuscript BMGE -D-15-00075 entitled 'Synergistic effect of k -carrageenan on oxazolone -
induced inflammation in BALB/c mice' by Wu et al. describes the effect of carrageenan in a 
model of colitis.  
Major concerns:  
The authors should clearly explain the rationale justifying the conc entration of oxazolone 
used to induce colitis, the time point chosen to investigate the measured parameters (1 
day post oxazolone injection), as well as the concentrations of carrageenan used in the 
study (low, medium and high). In addition, the authors sh ould discussed about the 
relevance of the carrageenan doses in relation to the human situation.  
Concerning the real -time quantitative PCR, the authors should follow the minimum 
information for publication of quantitative real -time PCR experiments guideline s in order 
to publish reliable and unequivocal interpretation of qPCR data (Bustin et al., 2009, Clin 
Chem, 55: 611 -612; Vandesompele, 2002, Genome Biology, 3: Research0034). The 
authors used only one reference gene (ß -actin) to normalize their data, this kind of 
normalization is nowadays considered as inappropriate proper data normalization should 
be done using at least 3 reference genes. The authors should include a positive and a 
negative control in each PCR experiment. Furthermore, the authors should pr ovide details 
concerning: primer efficiency, absence of genomic contamination (which methods were 
used to exclude it, even if RNA was treated with DNAse?) and absence of RNA 
degradation (which method was used to assess the RNA integrity?).  
Immunohistochem ical analyses were based on the determination of average integral 
density which relies on positive labelled area, optical density of labelling and total area 
(see page 10, lines 198 -200). However, modifications in optical density of labelling 
might result from technical variability and is thereby unlikely to represent an appropriate 
way to quantify labelling. Authors should rather quantity the expression of the protein by 
measuring positive labelled areas versus total area.  
The observed effects of carrageen an do not appear to respond to a traditional dose -
response curve for most measured parameters. What are the possible explanations for 
this?  
The authors claim that 'carrageenan alone results in the upregulation of the seven 
cytokines (Table 1)' (see page 17 , lines 336 -337). However, this sentence do not seems to 
be supported by the data presented in Table 1.  
Minor comments:  
The authors should express their data in similar way, ratio or percentage, when 
expressing the CD4+ CD25+/CD4+ ratio modifications (see page 16, lines 319 -329).  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
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 am able to assess the statistics  
 
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: 08 Oct 2015  
 
Reviewer: Christine Delporte  
Reviewer’s report:  
Manuscript BMGE -D-15-00075 entitled 'Synergistic effect of k -carrageenan on oxazolone -
induced inflammation in BALB/c mice' by Wu et al. describes the effect of carrageenan in a 
model of colitis.  
Major concerns:  
The authors should clearly explain the rationale justifying the conc entration of oxazolone 
used to induce colitis, the time point chosen to investigate the measured parameters (1 
day post oxazolone injection), as well as the concentrations of carrageenan used in the 
study (low, medium and high). In addition, the authors sh ould discussed about the 
relevance of the carrageenan doses in relation to the human situation.  
Concerning the real -time quantitative PCR, the authors should follow the minimum 
information for publication of quantitative real -time PCR experiments guideline s in order 
to publish reliable and unequivocal interpretation of qPCR data (Bustin et al., 2009, Clin 
Chem, 55: 611 -612; Vandesompele, 2002, Genome Biology, 3: Research0034). The 
authors used only one reference gene (ß -actin) to normalize their data, this kind of 
normalization is nowadays considered as inappropriate proper data normalization should 
be done using at least 3 reference genes. The authors should include a positive and a 
negative control in each PCR experiment. Furthermore, the authors should pr ovide details 
concerning: primer efficiency, absence of genomic contamination (which methods were 
used to exclude it, even if RNA was treated with DNAse?) and absence of RNA 
degradation (which method was used to assess the RNA integrity?).  
Immunohistochem ical analyses were based on the determination of average integral 
density which relies on positive labelled area, optical density of labelling and total area 
(see page 10, lines 198 -200). However, modifications in optical density of labelling 
might result from technical variability and is thereby unlikely to represent an appropriate 
way to quantify labelling. Authors should rather quantity the expression of the protein by 
measuring positive labelled areas versus total area.  
The observed effects of carrageen an do not appear to respond to a traditional dose -
response curve for most measured parameters. What are the possible explanations for 
this?  
The authors claim that 'carrageenan alone results in the upregulation of the seven 
cytokines (Table 1)' (see page 17 , lines 336 -337). However, this sentence do not seems to 
be supported by the data presented in Table 1.  
Minor comments:  
The authors should express their data in similar way, ratio or percentage, when 
expressing the CD4+ CD25+/CD4+ ratio modifications (see page 16, lines 319 -329).  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
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 am able to assess the statistics  
 
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