Reviewer's report
Title:Role of tight junction proteins in gastroesophageal reflux disease
Version: 1Date:3 July 2012
Reviewer: Ricard Farre
Reviewer's report:
The manuscript: “Role of tight junction proteins in gastroesophageal reflux
disease”. by Mönkemüller et al., addresses the interesting concept that molecular
changes in the tight junction complex at the mucosa of the distal esophagus are
present in GERD.
The present study is well designed and the relevance is unquestionable.
Nevertheless, I have some points that I would like to discuss.
My major concern is about the specificity of the antibodies used for
immunohistochemistry (Occludin, claudin-1 and -2 and ZO-1 and -2)
These tight junction proteins are present at the surface of the epithelial cells, one
expects a very intense staining at this location, as is already published. I suggest
to the authors improve the resolution of the actual pictures (better resolution and
larger size) and include a larger magnification to show the expression pattern.
-Another important point is if authors tested the size of the PCR products
(amplicon) in an agarose gel. Then they can be very sure that they amplified the
right fragment of the c-DNA.
-Authors should express RT-PCR data as fold change using the 2-##Ct method
as previously described (Schmittgen T et al, Nat Protoc 2008) not just only gene
expression/B actin. This way is much more accurate.
-Patients are 2 weeks off PPI, results are the reflection of the disease, or a
response after stopping medication? Authors should discuss this point and
mention more clearly that GERD patients are not naïve and stopped medication.
-Another limitation of this study that should be discussed is the fact that IHC
evaluation is semi-quantitative and maybe is not showing accurately that the
protein is more expressed. RT-PCR data do not mean increased protein
expression, only m-RNA.
-Authors correlate gene expression of different TJ proteins with
histomorphological changes but is not clear if they corrected for multiple
comparisons. There are higher changes to have significant correlations when
several comparisons are done. I think that correcting for this some correlations
will not be significant anymore.
Minor comments:
-Pag 4: “The widened ICS are supposed to permit the diffusion of molecules to
the submucosa and submucosal plexus and nerve fibers”. This sentence is
wrong. It is hard to believe, impossible in my opinion, that molecules can reach
the submucosa crossing also the muscularis mucosa. Maybe authors wanted to
say lamina propia, where sensory nerve ending are located. Please, rephrase the
sentence.
-Last sentence pag. 5: “Patients with Barrett´s esophagus were not investigated”.
Please, erase this sentence is already included as exclusion criteria.
-What LA classification means?
-Pag.8. Please, use arbitrary units instead artificial units.
-Authors used the non-parametric Kruskal-Wallis test for the analysis of m-RNA
expression levels when the 3 groups were compared and the Mann-Whitney post
hoc analyses for pair wise comparison. I assume that authors checked first that
the data was not normally distributed to use non-parametric tests.
-Authors should make more clear at the results section (pag. 11) if gene
expression is related to RT-PCR data or IHQ? Why authors do not use both
comparisons?
Level of interest: An article of outstanding merit and interest in its field
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
'I declare that I have no competing interests'
Reviewer's report
Title:Role of tight junction proteins in gastroesophageal reflux disease
Version: 1Date:3 July 2012
Reviewer: Ricard Farre
Reviewer's report:
The manuscript: “Role of tight junction proteins in gastroesophageal reflux
disease”. by Mönkemüller et al., addresses the interesting concept that molecular
changes in the tight junction complex at the mucosa of the distal esophagus are
present in GERD.
The present study is well designed and the relevance is unquestionable.
Nevertheless, I have some points that I would like to discuss.
My major concern is about the specificity of the antibodies used for
immunohistochemistry (Occludin, claudin-1 and -2 and ZO-1 and -2)
These tight junction proteins are present at the surface of the epithelial cells, one
expects a very intense staining at this location, as is already published. I suggest
to the authors improve the resolution of the actual pictures (better resolution and
larger size) and include a larger magnification to show the expression pattern.
-Another important point is if authors tested the size of the PCR products
(amplicon) in an agarose gel. Then they can be very sure that they amplified the
right fragment of the c-DNA.
-Authors should express RT-PCR data as fold change using the 2-##Ct method
as previously described (Schmittgen T et al, Nat Protoc 2008) not just only gene
expression/B actin. This way is much more accurate.
-Patients are 2 weeks off PPI, results are the reflection of the disease, or a
response after stopping medication? Authors should discuss this point and
mention more clearly that GERD patients are not naïve and stopped medication.
-Another limitation of this study that should be discussed is the fact that IHC
evaluation is semi-quantitative and maybe is not showing accurately that the
protein is more expressed. RT-PCR data do not mean increased protein
expression, only m-RNA.
-Authors correlate gene expression of different TJ proteins with
histomorphological changes but is not clear if they corrected for multiple
comparisons. There are higher changes to have significant correlations when
several comparisons are done. I think that correcting for this some correlations
will not be significant anymore.
Minor comments:
-Pag 4: “The widened ICS are supposed to permit the diffusion of molecules to
the submucosa and submucosal plexus and nerve fibers”. This sentence is
wrong. It is hard to believe, impossible in my opinion, that molecules can reach
the submucosa crossing also the muscularis mucosa. Maybe authors wanted to
say lamina propia, where sensory nerve ending are located. Please, rephrase the
sentence.
-Last sentence pag. 5: “Patients with Barrett´s esophagus were not investigated”.
Please, erase this sentence is already included as exclusion criteria.
-What LA classification means?
-Pag.8. Please, use arbitrary units instead artificial units.
-Authors used the non-parametric Kruskal-Wallis test for the analysis of m-RNA
expression levels when the 3 groups were compared and the Mann-Whitney post
hoc analyses for pair wise comparison. I assume that authors checked first that
the data was not normally distributed to use non-parametric tests.
-Authors should make more clear at the results section (pag. 11) if gene
expression is related to RT-PCR data or IHQ? Why authors do not use both
comparisons?
Level of interest: An article of outstanding merit and interest in its field
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
'I declare that I have no competing interests'
Please review this paper.
user
Reviewer's report
Title:Role of tight junction proteins in gastroesophageal reflux disease
Version: 1Date:3 July 2012
Reviewer: Ricard Farre
Reviewer's report:
The manuscript: “Role of tight junction proteins in gastroesophageal reflux
disease”. by Mönkemüller et al., addresses the interesting concept that molecular
changes in the tight junction complex at the mucosa of the distal esophagus are
present in GERD.
The present study is well designed and the relevance is unquestionable.
Nevertheless, I have some points that I would like to discuss.
My major concern is about the specificity of the antibodies used for
immunohistochemistry (Occludin, claudin-1 and -2 and ZO-1 and -2)
These tight junction proteins are present at the surface of the epithelial cells, one
expects a very intense staining at this location, as is already published. I suggest
to the authors improve the resolution of the actual pictures (better resolution and
larger size) and include a larger magnification to show the expression pattern.
-Another important point is if authors tested the size of the PCR products
(amplicon) in an agarose gel. Then they can be very sure that they amplified the
right fragment of the c-DNA.
-Authors should express RT-PCR data as fold change using the 2-##Ct method
as previously described (Schmittgen T et al, Nat Protoc 2008) not just only gene
expression/B actin. This way is much more accurate.
-Patients are 2 weeks off PPI, results are the reflection of the disease, or a
response after stopping medication? Authors should discuss this point and
mention more clearly that GERD patients are not naïve and stopped medication.
-Another limitation of this study that should be discussed is the fact that IHC
evaluation is semi-quantitative and maybe is not showing accurately that the
protein is more expressed. RT-PCR data do not mean increased protein
expression, only m-RNA.
-Authors correlate gene expression of different TJ proteins with
histomorphological changes but is not clear if they corrected for multiple
comparisons. There are higher changes to have significant correlations when
several comparisons are done. I think that correcting for this some correlations
will not be significant anymore.
Minor comments:
-Pag 4: “The widened ICS are supposed to permit the diffusion of molecules to
the submucosa and submucosal plexus and nerve fibers”. This sentence is
wrong. It is hard to believe, impossible in my opinion, that molecules can reach
the submucosa crossing also the muscularis mucosa. Maybe authors wanted to
say lamina propia, where sensory nerve ending are located. Please, rephrase the
sentence.
-Last sentence pag. 5: “Patients with Barrett´s esophagus were not investigated”.
Please, erase this sentence is already included as exclusion criteria.
-What LA classification means?
-Pag.8. Please, use arbitrary units instead artificial units.
-Authors used the non-parametric Kruskal-Wallis test for the analysis of m-RNA
expression levels when the 3 groups were compared and the Mann-Whitney post
hoc analyses for pair wise comparison. I assume that authors checked first that
the data was not normally distributed to use non-parametric tests.
-Authors should make more clear at the results section (pag. 11) if gene
expression is related to RT-PCR data or IHQ? Why authors do not use both
comparisons?
Level of interest: An article of outstanding merit and interest in its field
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
'I declare that I have no competing interests'
assistant