Review for 10.1186/s12876-015-0305-3 (2015)

Reviewer's report
Title:Lamina propria macrophage phenotypes in relation to Escherichia coli in
Crohn's disease
Version:2Date:25 February 2015
Reviewer: Sanna Edelman
Reviewer's report:
Manucript submitted by Elliot et al. is an important research article reporting gene
expression profiles and phenotypic characterization of Lamina propria
macrophages in situ in patients of Crohn’s disease (CD). It provides important
new information of involvement of E. coli-laden macrophages in the pathogenesis
of CD. The experimental design has been challenging baring in mind the number
of available cells to be analyzed from patient biopsy samples. The data will be
useful to the field and will lead to further research identifying the potential
causative role of E. coli in disease progression of CD.
Comments:
Major Compulsory Revisions
1. The normalization of the RT-qPCR data has been done against total RNA from
a fresh frozen tissue biopsy. This does not take into account the varying degrees
of RNA degradation in the samples that significantly influences on detection of
gene expression. An endogenous control reference gene would be essential to
be included for the normalization of the data. (For more detailed information
about optimized normalization methods, please see Heidi S. Erickson and the
coworker’s article in Nature Protocols: 4, 902-922 (2009) Quantitative RT-PCR
gene expression analysis of laser microdissected tissue samples).
Minor Essential Revisions
1. Page 5 row 105. References for the validated antibodies used in the study
could be included.
2. Page 6 row 123. A range of RIN values (or RQNs) and a cutoff value for
sufficient integrity of RNA should be stated out.
3. Page 6 rows 141-142. ‘Cq values of <0.5’ refers supposedly to Standard
Deviation of Cq values.
4. Page 7 rows 1511-152. I did not find a recommendation for RT-qPCR data
normalization nor gene expression copy number representation on reference #14
(Mosser and Edwards, 2008)
5. Page 8 rows 182-185. Results part indicates that mean mRNA expressions of
cytokines are shown in the figures. The figure show Y-axis titled with ug/ul RNA
throughout the result figures 3 and 4. It is unclear to a reader how ug/ul RNA
correlates to the mean mRNA expression.
6. Page 10 row 243. The prevalence percentage 11% of E.coli-laden
macrophages in UC patients is misleading as n=9 is so low. 1/9 should be used
instead to indicate to the reader that only one patient was identified with
E.coli-laden macrophages among UC patients
7. Page 10 row 245. Authors state here 14 healthy subjects. In other parts of the
manuscript 18 healthy individuals are indicated.
8. Page 18. Table 3. The lines around the heading row need to be adjusted.
9. Figure 1. A size bars should be included in the microscopic images. Images
lacking primary antibodies showing assay specificity could be included.
10. Figures 3 and 4. Gene expression copy number (mRNA/ug of total RNA) not
clear on plotted Y-axis of ug/ul of RNA. Normalization to a housekeeping
reference gene necessary for reporting gene expression.
Discretionary Revisions
1. Additional comparative analysis of immunomodulatory medication on presence
of intracellular E.coli load on macrophages and their functional profiles could be
interesting.
Level of interest: An article of importance in its field
Quality of written English: Acceptable
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
I declare that I have no competing interests.