Federal University of Ceara
INSTITUTE OF BIOMEDICINE Home page: http://www.upcibimed.ufc.br
Rua Coronel Nunes de Melo, 1315 CEP: 60430-270, Fortaleza, CE, Brazil
PHONE: +55 085 33668239 FAX: +55 085 33668445
To:
The editor of BMC Gastroenterology
Fortaleza, December 12th, 2011
Enclosed is our revised manuscript entitled “Apolipoprotein E COG 133 mimetic peptide improves
5-fluorouracil-induced intestinal mucositis.” for your consideration for publication in BMC
Gastroenterology by O.G.R. Azevedo, R.A.C. Oliveira, B.M. Oliveira, J.E. Sevilleja,
S. Zala-
Milatovic, C.V. Araújo , D.V.T. Wong, T. B. Costa, R.C.P. Lima- Júnior , R.A. Ribeiro, C.A.
Warren, A.A.M. Lima, M.P. Vitek, R.L. Guerrant, R. B. Oriá. All authors have read, approved and
contributed significantly to the manuscript. No portion of the manuscript other than the abstract has
been published or posted on the internet and is not under consideration for publication elsewhere. A
conflict of interest was added to the manuscript following the conclusion section.
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Reviewer: Jerzy-Roch Nofer
Reviewer's report:
The specific comments (major compulsary revision) are as follows:
1. The rationale for the study is unclear. Why did the authors use apoE mimetics and not other
apolipoprotein mimetics or - more generally - other anti-inflammatory compounds? Protective
effects of apoE mimetics seen in traumatic brain injury (as pointed by the authors in the
Introduction) certainly do not provide sufficient explanation.
Reply: ApoE mimetic peptides have been found to reduce inflammation in several models of brain
injury, acting as anti-inflammatory factor and recently it has been found to improve inflammation in a
model of colitis (Singh K et al, 2011), as COG 133 share the properties we tested in model of
mucositis, where an important inflammatory component is present. In addition, in our revised
manuscript we have added some experiments with the apoA-I peptide for comparison purpose. In
order to evaluate the benefit of the ApoE COG 133 peptide treatment in the absent of the apoE
endogenous peptide, we also used apoE knock-out mice in some experiments.
2. The major drawback of the study is that it does not provide any insight into mechanisms underlying
protective effects of apoE COG 133. In particular, it is not clear whether these effects are apoEspecific and/or apoE-receptor-specific or can be attributed to the amphipatic properties of apoE
mimetic, which are shared with other apolipoprotein mimetics such as apoA-I mimetics. To address
this question the authors should:
- provide more information about the structure of apoE COG 133 and its amphipaticity and
hydrophobic moment
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Reply: Since the apoE COG 133 mimetic peptide comprises only residues of the amino terminal
domain where the LDL-receptor binding site resides, and not contain residues of the carboxi-terminal
domain, where the amphipaticity property is located in ApoE full protein that property should not
interfere with the biological effects seen in our studies.
- assess the 5FU-induced epithelial damage in apoE deficient mice (which are commercially
available) and whether this damage can be reversed by apoE COG 133
Reply: We agree with the reviewer’s suggestion and to comply with this request, we ran some
experiments using apoE deficient mice and their respective wild-type controls and addressed whether
the apoE COG peptide could restitute the epithelial damage induced by the 5-FU treatment. Those
changes and additions are highlighted in yellow
- compare the effect of apoE COG 133 on the 5FU-induced epithelial damage in vivo with standard
apoA-I mimetic (i.e. D4-F)
Reply: We had tested D4-F as requested in some in vitro experiments and compare those findings
with ApoE COG mimetic peptide. We ran new migration assays, TUNEL, and Ki67 immunolabeling
in IEC-6 cells following 5-FU challenge and found similar protective role as seen in COG 133.
- assess anti-apoptotic and migration-stimulating effects of apoE COG 133 in vitro in the presence of
RAP, which inhibits apoE binding to receptors, as well as compare the effects of apoE COG 133 with
the effects of apoA-I mimetic. The assessment of effects of apoE COG 133 on the inflammatory
response is not sufficient. The study should also include:
- immunochemical characterization of inflammation in the intestine with particular focus on
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macrophages and lymphocytes, and – if possible (discretionary) – functional polarization of these
cells (M1 vs. M2; Th1 vs. Th2)
- determination of inflammation and polarization markers in blood - assessment of the activation of
NF-kB pathway in the intestine (discretionary)
- determination of IL-1ra levels in the intestine tissue and blood. The latter parameter seems to exert a
key role in the intestinal inflammatory response following 5FU treatment
Reply: Due to length constraints and time deadline of this paper, we could not perform all requested
assays, but we are currently running some more experiments for a future publication considering these
important points. However, we could update our manuscript with intestinal NF-kB
immunohistochemistry in apoE deficient mice and their controls. In addition, we could also include a
TUNEL protocol to identify apoptosis and we analyzed crypt mitotic index in our in vivo experiments
with ApoE knock-out mice. In addition, we run TUNEL experiments in our IEC-6 studies.
4. The discussion at present resembles a rather random collection of loose commentaries on previously
published papers. It has to be re-written to focus more on the results of the present study.
5. The manuscript seems to be put together rather hastily and is quite sloppily edited with several
inconsistencies in abbreviations and reference format, typos, etc. The authors should definitely pay
more attention to details!!!
Reply: This manuscript was revised and especially reviewed for inconsistencies in figures, legends
and other minor errors in the overall manuscript.
Reviewer: Wei Han
Reviewer's report:
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The manuscript is generally acceptable, but has many specific problems, some of which I have noted
as follows. So this manuscript needs some language and format corrections before being published.
And there are some recommended experiments to the author which are useful to clarify the effect of
apoE mimetic peptide in 5-Fu induced intestinal mucositis.
- Major Compulsory Revisions
1)Slight compliance was found between In vivo with in vitro studies. Detecting apoptosis and
proliferation after 5-Fu challenged with or without apoE mimetic peptide treatment in vivo, by
TUNEL, Brdu or PCNA respectively or other widely used methods. Then, the results become more
persuasive.
Reply: We have done a TUNEL protocol and crypt mitotic index in vivo and additionally we have
conducted TUNEL and Ki67 labeling in IEC-6 cells in order to comply with the reviewer request.
These changes are highlighted in yellow in the overall paper when appropriate.
2)Morphological Figures of IEC-6 after 5-Fu challenged with or without apoE treatment are needed to
show in the manuscript. Just a result from flow cytometry is not convincing.
Reply: We have added imaging to improve the quality of this manuscript as requested.
- Minor Essential Revisions
There are some minor mistakes in the manuscript, which the author should correct. Such as:
1)Abstract,”...Improvements were also found with IEC-6 necrosis and migration following apoE
treatment” According to the experiment results, necrosis should be replaced by apoptosis.
Reply: We corrected this and revise the overall abstract to cover our new findings.
2)The information about the role of apoE plays in 5-Fu induced mucositis in the background is
insufficient.
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Reply: To our knowledge this is the first study addressing the role of the apolipoprotein E in the 5-FU
induced intestinal mucositis and very few studies have tested apoE COG peptides in intestinal
diseases, therefore we could not expand the background as we would like to and as per requested.
3)Methods, ninth paragraph, 95#,60#are the correct writing; Twelfth paragraph, “After 24 h”, h is
missing.
Reply: Methods section was rewritten to cover new experiments added to this paper. Changes
requested were corrected.
4)Discussion, sixth paragraph, the quotation labeling is wrong, maybe “...suggesting that IL-1 peptides
are key players in the intestinal inflammatory response following 5-Fu treatment[21]” should be
changed to”...[22]”.
Reply: We have added that reference as requested to the quotation.
5)Please make sure the formats of the entire manuscript are consistency. Whether or not beginning
with two spaces in each paragraph; The tittles of FIGURE 6 A and B will be better if both are bold or
not; The figures should be showed with uniform format; The abbreviation of apolipoprotein E COG
133mimetic peptide should be consistent, and so on. 6)The descriptions of each figure should be more
clearly. 7)Specify references expression - Discretionary Revisions
Reply: We have thoroughly revised the manuscript to eliminate inconsistencies.
1)Using immunohistochemistry method or specific assay kit to detect iNOS expression in the intestine
after 5-Fu challenged with or without apoE mimetic peptide treatment (protein level detecting).
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Reply: Although we could not add iNOS western blot data to the paper, we have added NF-KB
immunohistochemistry since that would add to the understanding of the pathophysiology of the
intestinal mucositis and the role of the apoE COG peptide protection. Authorship was revised to
include the ones who contribute with these new experiments.
We appreciate yours and the reviewers’ comments that have helped us to further improve our
manuscript. Look forward to hearing your thoughts.
Sincerely,
Reinaldo Barreto Oriá
Corresponding author
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