Reviewer's report
Title:Role of myosin light chain kinase in intestinal epithelial barrier defects in a
rat model of bowel obstruction
Version: 1Date:30 October 2009
Reviewer: Derek M McKay
Reviewer's report:
This manuscript by Wu et al describes the local effect of small bowel obstruction
due to ligation. The main finding being increases in ileal and proximal colonic
permeability as measured by ionic conductance, HRP fluxes and bacterial
translocation to the spleen and liver. Other data assess ileal structure and
apoptosis therein. Use of an MLCK inhibitor indicates that that MLC changes are
linked to the ionic conductance and HRP changes (but not the bacterial
translocation), and this is supported by western blotting showing increased
phosphorylation of MLC. The paper is well-written (there are a few grammatical
issues) and is clear and easy to follow (there is confusion in data interpretation
elating to apoptosis), and the in general the conclusions drawn are supported by
the data presented.
Major
1. Given the suggested role of TNFa (and IFNg) in the control of epithelial
permeability, it would be interesting to measure the local or systemic of one or
both of these cytokines by either PCR or ELISA.
Minor
2. The pMLC is assessed 24h after obstruction and one wonders if there is
evidence of MLC activation prior to obvious changes in barrier function.
3. Can ML7 kill bacteria or slow their growth? This is an easy in vitro experiment
that should be done.
4. It is interesting that ML7 reduced HRP fluxes (often considered a marker of
transcellular permeability) but not bacterial transcytosis. While I can accept the
data as is, a better explanation (speculation) for this should be offered in the
discussion.
5. Crypt hyperplasia has been associated with T cell activation (MacDonald and
colleagues in the early 1990s). Is there any evidence of increased T cells or T
cell activation in this model?
6. The positive control in Fig 1D is not mentioned in the methods. Some
reference to where this tissue comes from needs to be included.
7. The authors describe alterations in villus-crypt architecture; however, their
photomicrographs seem to some an increased cellularity and inflammatory
infiltrate. If this is correct, they should describe this.
8. Data are repeated in the manuscript (i.e. text on page 18 and table one; table
3 and figure 8) and the authors should choose one way, not two means, to show
the data.
9. TUNEL staining should be quantified to replace the subjective descriptors in
the text (e.g. “high numbers of apoptotic enterocytes” on page 20).
10. The text on page 22 “epithelial sloughing without apparent erosion” is
confusing. I don’t understand this. Indeed, it is unclear if the authors are
convinced that the increase in apoptosis that they see in the model is of
significance. See page 24, which alludes to apoptosis and “large sheets of
apoptotic” cells!
Level of interest: An article of importance 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 myosin light chain kinase in intestinal epithelial barrier defects in a
rat model of bowel obstruction
Version: 1Date:30 October 2009
Reviewer: Derek M McKay
Reviewer's report:
This manuscript by Wu et al describes the local effect of small bowel obstruction
due to ligation. The main finding being increases in ileal and proximal colonic
permeability as measured by ionic conductance, HRP fluxes and bacterial
translocation to the spleen and liver. Other data assess ileal structure and
apoptosis therein. Use of an MLCK inhibitor indicates that that MLC changes are
linked to the ionic conductance and HRP changes (but not the bacterial
translocation), and this is supported by western blotting showing increased
phosphorylation of MLC. The paper is well-written (there are a few grammatical
issues) and is clear and easy to follow (there is confusion in data interpretation
elating to apoptosis), and the in general the conclusions drawn are supported by
the data presented.
Major
1. Given the suggested role of TNFa (and IFNg) in the control of epithelial
permeability, it would be interesting to measure the local or systemic of one or
both of these cytokines by either PCR or ELISA.
Minor
2. The pMLC is assessed 24h after obstruction and one wonders if there is
evidence of MLC activation prior to obvious changes in barrier function.
3. Can ML7 kill bacteria or slow their growth? This is an easy in vitro experiment
that should be done.
4. It is interesting that ML7 reduced HRP fluxes (often considered a marker of
transcellular permeability) but not bacterial transcytosis. While I can accept the
data as is, a better explanation (speculation) for this should be offered in the
discussion.
5. Crypt hyperplasia has been associated with T cell activation (MacDonald and
colleagues in the early 1990s). Is there any evidence of increased T cells or T
cell activation in this model?
6. The positive control in Fig 1D is not mentioned in the methods. Some
reference to where this tissue comes from needs to be included.
7. The authors describe alterations in villus-crypt architecture; however, their
photomicrographs seem to some an increased cellularity and inflammatory
infiltrate. If this is correct, they should describe this.
8. Data are repeated in the manuscript (i.e. text on page 18 and table one; table
3 and figure 8) and the authors should choose one way, not two means, to show
the data.
9. TUNEL staining should be quantified to replace the subjective descriptors in
the text (e.g. “high numbers of apoptotic enterocytes” on page 20).
10. The text on page 22 “epithelial sloughing without apparent erosion” is
confusing. I don’t understand this. Indeed, it is unclear if the authors are
convinced that the increase in apoptosis that they see in the model is of
significance. See page 24, which alludes to apoptosis and “large sheets of
apoptotic” cells!
Level of interest: An article of importance 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 myosin light chain kinase in intestinal epithelial barrier defects in a
rat model of bowel obstruction
Version: 1Date:30 October 2009
Reviewer: Derek M McKay
Reviewer's report:
This manuscript by Wu et al describes the local effect of small bowel obstruction
due to ligation. The main finding being increases in ileal and proximal colonic
permeability as measured by ionic conductance, HRP fluxes and bacterial
translocation to the spleen and liver. Other data assess ileal structure and
apoptosis therein. Use of an MLCK inhibitor indicates that that MLC changes are
linked to the ionic conductance and HRP changes (but not the bacterial
translocation), and this is supported by western blotting showing increased
phosphorylation of MLC. The paper is well-written (there are a few grammatical
issues) and is clear and easy to follow (there is confusion in data interpretation
elating to apoptosis), and the in general the conclusions drawn are supported by
the data presented.
Major
1. Given the suggested role of TNFa (and IFNg) in the control of epithelial
permeability, it would be interesting to measure the local or systemic of one or
both of these cytokines by either PCR or ELISA.
Minor
2. The pMLC is assessed 24h after obstruction and one wonders if there is
evidence of MLC activation prior to obvious changes in barrier function.
3. Can ML7 kill bacteria or slow their growth? This is an easy in vitro experiment
that should be done.
4. It is interesting that ML7 reduced HRP fluxes (often considered a marker of
transcellular permeability) but not bacterial transcytosis. While I can accept the
data as is, a better explanation (speculation) for this should be offered in the
discussion.
5. Crypt hyperplasia has been associated with T cell activation (MacDonald and
colleagues in the early 1990s). Is there any evidence of increased T cells or T
cell activation in this model?
6. The positive control in Fig 1D is not mentioned in the methods. Some
reference to where this tissue comes from needs to be included.
7. The authors describe alterations in villus-crypt architecture; however, their
photomicrographs seem to some an increased cellularity and inflammatory
infiltrate. If this is correct, they should describe this.
8. Data are repeated in the manuscript (i.e. text on page 18 and table one; table
3 and figure 8) and the authors should choose one way, not two means, to show
the data.
9. TUNEL staining should be quantified to replace the subjective descriptors in
the text (e.g. “high numbers of apoptotic enterocytes” on page 20).
10. The text on page 22 “epithelial sloughing without apparent erosion” is
confusing. I don’t understand this. Indeed, it is unclear if the authors are
convinced that the increase in apoptosis that they see in the model is of
significance. See page 24, which alludes to apoptosis and “large sheets of
apoptotic” cells!
Level of interest: An article of importance 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