Reviewer ’s report  
 
Title: Plasma levels of TNF -α, IL-6, IFN -γ, IL-12, IL -17, IL-22, and IL -23 in Achalasia, 
Eosinophilic Esophagitis (EoE), and Gastroesophageal Reflux Disease (GERD)  
 
Version: 0 Date: 17 May 2018  
 
Reviewer: J Furuzawa -Carballeda  
 
Reviewer's report:  
 
After reading the paper "Plasma levels of TNF -α, IL -6, IFN -γ, IL -12, IL -17, IL -22, and IL -23 in 
Achalasia, Eosinophilic Esophagitis (EoE), and Gastroesophageal Reflux Disease (GERD)" 
where  the main objective of the study was to evaluate serum concentrations of proinflammatory 
cytokines in some esophageal motility disorders, this reviewer has considered the following:  
 
 
The study is multi -institutional, and the authors collected plasma sampl es for more than 2 years. 
The number of sera is not negligible. However, this reviewer finds that the work has poor results 
and contributes weakly to the pathophysiology of achalasia. The authors could perform a more 
extensive serological profile including  the evaluation of hsCRP, concentration of C3 and C4, 
ANAs, etc.  
 
 
The authors do not describe deeply the method to obtain the plasma (Standard operating 
procedures for collection, processing, handling and storage of plasma). Small differences in the 
proce ssing or handling of a specimen can have dramatic effects in analytical reliability and 
reproducibility, especially when multiplex methods are used, e.g.,  
 
 
o How long did it take from when the sample was taken until it was centrifuged?  
 
 
o Have you used hemol yzed samples?  
 
 
o What kind of anticoagulant was used?  
 
 
o At what speed and temperature were the samples centrifuged?  
 
 
o At what temperature were samples transported?  
 
 
o Were the plasmas obtained in sterile conditions?  
o Are the samples protected from the light?  
 
 
They also do not describe whether the immunoenzymatic assays were performed in duplicate, in 
triplicate, etc.  
 
 
The authors do not present the clinical characteristics, laboratory data, time of evolution of the  
disease, disease activity, and if the patients were under treatment. All these data are important, 
since they could directly affect the expression of cytokines.  
 
 
Another important point includes matching the samples by age and gender and preferably 
selec ting those with early disease.  
 
 
On the other hand, in a previous study it was shown that patients with achalasia have 
autoimmune and allergic comorbidities, thus the exclusion of those patients is a bias (Romero -
Hernández F, Furuzawa -Carballeda J, Hernánd ez-Molina G, et al. comorbidity in achalasia 
patients J Gastroenterol Hepatol 2018 Jan; 33 (1): 203 -208).  
 
 
Studies of plasma/serum cytokines concentration in patients with esophageal motility disorders 
are scarce, so it is interesting to study not only pr o-inflammatory cytokines, but also anti -
inflammatory cytokines.  
 
 
Likewise, the authors do not evaluate cytokines related to EoE, such as IL -4, IL -5, IL -13, 
eotaxin, etc.  
 
 
It is important to calculate a sample size with a formula for calculating sample for proportion 
based on the work of Cauble et al, to determine the number of plasma needed to obtain 
statistically significant differences between study groups.  
 
 
Finally, additional statistical analysis is required such as, comparing cytokine levels betw een 
EoE, GERD and type I achalasia; EoE, GERD and achalasia type II; EoE, GERD and achalasia 
type III.  
 
 
 
 
 
 
 
Level of interest  
Please indicate how interesting you found the manuscript:  
 
 
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
 
 
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 financial ly from the  publication 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 currently applying for any patents  relating to the content of the 
manuscript?  
 
4. Have you recei ved 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.  
 
I declare that I have no competing interests  
 
I agree to the open peer r eview 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 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 a s confidential comments 
to the editor s, which will not be published.  
 
 
 
Reviewer ’s report  
 
Title: Plasma levels of TNF -α, IL-6, IFN -γ, IL-12, IL -17, IL-22, and IL -23 in Achalasia, 
Eosinophilic Esophagitis (EoE), and Gastroesophageal Reflux Disease (GERD)  
 
Version: 0 Date: 17 May 2018  
 
Reviewer: J Furuzawa -Carballeda  
 
Reviewer's report:  
 
After reading the paper "Plasma levels of TNF -α, IL -6, IFN -γ, IL -12, IL -17, IL -22, and IL -23 in 
Achalasia, Eosinophilic Esophagitis (EoE), and Gastroesophageal Reflux Disease (GERD)" 
where  the main objective of the study was to evaluate serum concentrations of proinflammatory 
cytokines in some esophageal motility disorders, this reviewer has considered the following:  
 
 
The study is multi -institutional, and the authors collected plasma sampl es for more than 2 years. 
The number of sera is not negligible. However, this reviewer finds that the work has poor results 
and contributes weakly to the pathophysiology of achalasia. The authors could perform a more 
extensive serological profile including  the evaluation of hsCRP, concentration of C3 and C4, 
ANAs, etc.  
 
 
The authors do not describe deeply the method to obtain the plasma (Standard operating 
procedures for collection, processing, handling and storage of plasma). Small differences in the 
proce ssing or handling of a specimen can have dramatic effects in analytical reliability and 
reproducibility, especially when multiplex methods are used, e.g.,  
 
 
o How long did it take from when the sample was taken until it was centrifuged?  
 
 
o Have you used hemol yzed samples?  
 
 
o What kind of anticoagulant was used?  
 
 
o At what speed and temperature were the samples centrifuged?  
 
 
o At what temperature were samples transported?  
 
 
o Were the plasmas obtained in sterile conditions?  
o Are the samples protected from the light?  
 
 
They also do not describe whether the immunoenzymatic assays were performed in duplicate, in 
triplicate, etc.  
 
 
The authors do not present the clinical characteristics, laboratory data, time of evolution of the  
disease, disease activity, and if the patients were under treatment. All these data are important, 
since they could directly affect the expression of cytokines.  
 
 
Another important point includes matching the samples by age and gender and preferably 
selec ting those with early disease.  
 
 
On the other hand, in a previous study it was shown that patients with achalasia have 
autoimmune and allergic comorbidities, thus the exclusion of those patients is a bias (Romero -
Hernández F, Furuzawa -Carballeda J, Hernánd ez-Molina G, et al. comorbidity in achalasia 
patients J Gastroenterol Hepatol 2018 Jan; 33 (1): 203 -208).  
 
 
Studies of plasma/serum cytokines concentration in patients with esophageal motility disorders 
are scarce, so it is interesting to study not only pr o-inflammatory cytokines, but also anti -
inflammatory cytokines.  
 
 
Likewise, the authors do not evaluate cytokines related to EoE, such as IL -4, IL -5, IL -13, 
eotaxin, etc.  
 
 
It is important to calculate a sample size with a formula for calculating sample for proportion 
based on the work of Cauble et al, to determine the number of plasma needed to obtain 
statistically significant differences between study groups.  
 
 
Finally, additional statistical analysis is required such as, comparing cytokine levels betw een 
EoE, GERD and type I achalasia; EoE, GERD and achalasia type II; EoE, GERD and achalasia 
type III.  
 
 
 
 
 
 
 
Level of interest  
Please indicate how interesting you found the manuscript:  
 
 
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
 
 
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 financial ly from the  publication 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 currently applying for any patents  relating to the content of the 
manuscript?  
 
4. Have you recei ved 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.  
 
I declare that I have no competing interests  
 
I agree to the open peer r eview 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 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 a s confidential comments 
to the editor s, which will not be published.  
 
 
 
Please review this paper.
user
Reviewer ’s report  
 
Title: Plasma levels of TNF -α, IL-6, IFN -γ, IL-12, IL -17, IL-22, and IL -23 in Achalasia, 
Eosinophilic Esophagitis (EoE), and Gastroesophageal Reflux Disease (GERD)  
 
Version: 0 Date: 17 May 2018  
 
Reviewer: J Furuzawa -Carballeda  
 
Reviewer's report:  
 
After reading the paper "Plasma levels of TNF -α, IL -6, IFN -γ, IL -12, IL -17, IL -22, and IL -23 in 
Achalasia, Eosinophilic Esophagitis (EoE), and Gastroesophageal Reflux Disease (GERD)" 
where  the main objective of the study was to evaluate serum concentrations of proinflammatory 
cytokines in some esophageal motility disorders, this reviewer has considered the following:  
 
 
The study is multi -institutional, and the authors collected plasma sampl es for more than 2 years. 
The number of sera is not negligible. However, this reviewer finds that the work has poor results 
and contributes weakly to the pathophysiology of achalasia. The authors could perform a more 
extensive serological profile including  the evaluation of hsCRP, concentration of C3 and C4, 
ANAs, etc.  
 
 
The authors do not describe deeply the method to obtain the plasma (Standard operating 
procedures for collection, processing, handling and storage of plasma). Small differences in the 
proce ssing or handling of a specimen can have dramatic effects in analytical reliability and 
reproducibility, especially when multiplex methods are used, e.g.,  
 
 
o How long did it take from when the sample was taken until it was centrifuged?  
 
 
o Have you used hemol yzed samples?  
 
 
o What kind of anticoagulant was used?  
 
 
o At what speed and temperature were the samples centrifuged?  
 
 
o At what temperature were samples transported?  
 
 
o Were the plasmas obtained in sterile conditions?  
o Are the samples protected from the light?  
 
 
They also do not describe whether the immunoenzymatic assays were performed in duplicate, in 
triplicate, etc.  
 
 
The authors do not present the clinical characteristics, laboratory data, time of evolution of the  
disease, disease activity, and if the patients were under treatment. All these data are important, 
since they could directly affect the expression of cytokines.  
 
 
Another important point includes matching the samples by age and gender and preferably 
selec ting those with early disease.  
 
 
On the other hand, in a previous study it was shown that patients with achalasia have 
autoimmune and allergic comorbidities, thus the exclusion of those patients is a bias (Romero -
Hernández F, Furuzawa -Carballeda J, Hernánd ez-Molina G, et al. comorbidity in achalasia 
patients J Gastroenterol Hepatol 2018 Jan; 33 (1): 203 -208).  
 
 
Studies of plasma/serum cytokines concentration in patients with esophageal motility disorders 
are scarce, so it is interesting to study not only pr o-inflammatory cytokines, but also anti -
inflammatory cytokines.  
 
 
Likewise, the authors do not evaluate cytokines related to EoE, such as IL -4, IL -5, IL -13, 
eotaxin, etc.  
 
 
It is important to calculate a sample size with a formula for calculating sample for proportion 
based on the work of Cauble et al, to determine the number of plasma needed to obtain 
statistically significant differences between study groups.  
 
 
Finally, additional statistical analysis is required such as, comparing cytokine levels betw een 
EoE, GERD and type I achalasia; EoE, GERD and achalasia type II; EoE, GERD and achalasia 
type III.  
 
 
 
 
 
 
 
Level of interest  
Please indicate how interesting you found the manuscript:  
 
 
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
 
 
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 financial ly from the  publication 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 currently applying for any patents  relating to the content of the 
manuscript?  
 
4. Have you recei ved 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.  
 
I declare that I have no competing interests  
 
I agree to the open peer r eview 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 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 a s confidential comments 
to the editor s, which will not be published.  
 
 
 
assistant