Reviewer's report
Title:Intestinal Epithelial Serum Amyloid A Modulates Bacterial Growth and
Protects from Experimental Colitis
Version: 1Date:23 September 2010
Reviewer: Christian Jobin
Reviewer's report:
This study investigates the function of SAA in intestinal homeostasis. The
authors report that SAA is expressed in IEC and that this expression increased in
patients with CD. In vitro data using an overexpression system showed that SAA
impact bacterial survival. DSS-induced colitis worsens in SAA1/2 KO mice
compared to WT mice. The authors conclude that SAA protects against intestinal
inflammation and regulates bacterial growth.
This is an interesting study that highlights potentially novel functions for SAA in
the gut. However, a number of weaknesses at both the experimental and
interpretation level impede on the quality of the manuscript. First, it appears that
the SAA1/2 DKO mice were not fully backcrossed to the C57BL/6 background
(method section). This is a major concern as DSS susceptibility is highly
dependent on the genetic background of a given mouse. Similarly, it appears that
the investigators have utilized C57BL/6 from a vendor (Taconic) as WT control
for their studies. Again, this experimental approach is flawed since susceptibility
to DSS is highly influenced by microbial composition, not to mention that the
C57BL/6 from Taconic are genetically different from the SAA1/2 DKO used by
the investigators. Finally, the impact of SAA1/2 on DSS-induced colitis is
marginal because two of the most important parameters (histological score and
body weight) are not significantly affected (Fig.6). Other weaknesses are listed
below:
1) The study on intestinal SAA expression (Fig.1) would be more convincing by
staining intestinal section obtained from SAA1/2 DKO mice. This will show
specificity of the protein signal.
2) Fig2A should be presented in the result section and not in the method section.
3) It is unclear why the SAA proteins detected in the stools (Fig.2B) migrate at
the different molecular weights than the protein detected in the plasma SAA
control.
4) The authors should correlate their SAA mRNA analysis (Fig.3A) with SAA
protein. Basically, is there a direct relationship between SAA mRNA and protein
levels?
5) Although SAA1/2 mRNA levels do not appear to be induced by LPS, the
authors have used a relatively small amount of endotoxin in this assay
(100ng/ml). It is not unusual to utilize LPS in the microgram range for this kind of
cellular stimulation, as there are high amounts of this bacterial product in the
intestine.
6) There is wide variation in SAA protein expression level in the Ad5SAA-infected
cells. How were these variations accounted for in the assay?
7) The impact of SAA on E.coli survival is an interesting finding. However, the
result could be improved by demonstrating bacterial specificity (other gram –
and/or gram + bacteria). Also, the physiological relevance of E.coli DH5 is
questionable and the authors could have utilized a typical commensal intestinal
bacteria in this assay.
8) The manuscript would have gained in impact and significance by testing the
microbial composition between WT and SAA1/2 DKO mice. One would predict a
change in microbial composition if SAA impacts bacterial survival.
Level of interest: An article of limited interest
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
Reviewer's report
Title:Intestinal Epithelial Serum Amyloid A Modulates Bacterial Growth and
Protects from Experimental Colitis
Version: 1Date:23 September 2010
Reviewer: Christian Jobin
Reviewer's report:
This study investigates the function of SAA in intestinal homeostasis. The
authors report that SAA is expressed in IEC and that this expression increased in
patients with CD. In vitro data using an overexpression system showed that SAA
impact bacterial survival. DSS-induced colitis worsens in SAA1/2 KO mice
compared to WT mice. The authors conclude that SAA protects against intestinal
inflammation and regulates bacterial growth.
This is an interesting study that highlights potentially novel functions for SAA in
the gut. However, a number of weaknesses at both the experimental and
interpretation level impede on the quality of the manuscript. First, it appears that
the SAA1/2 DKO mice were not fully backcrossed to the C57BL/6 background
(method section). This is a major concern as DSS susceptibility is highly
dependent on the genetic background of a given mouse. Similarly, it appears that
the investigators have utilized C57BL/6 from a vendor (Taconic) as WT control
for their studies. Again, this experimental approach is flawed since susceptibility
to DSS is highly influenced by microbial composition, not to mention that the
C57BL/6 from Taconic are genetically different from the SAA1/2 DKO used by
the investigators. Finally, the impact of SAA1/2 on DSS-induced colitis is
marginal because two of the most important parameters (histological score and
body weight) are not significantly affected (Fig.6). Other weaknesses are listed
below:
1) The study on intestinal SAA expression (Fig.1) would be more convincing by
staining intestinal section obtained from SAA1/2 DKO mice. This will show
specificity of the protein signal.
2) Fig2A should be presented in the result section and not in the method section.
3) It is unclear why the SAA proteins detected in the stools (Fig.2B) migrate at
the different molecular weights than the protein detected in the plasma SAA
control.
4) The authors should correlate their SAA mRNA analysis (Fig.3A) with SAA
protein. Basically, is there a direct relationship between SAA mRNA and protein
levels?
5) Although SAA1/2 mRNA levels do not appear to be induced by LPS, the
authors have used a relatively small amount of endotoxin in this assay
(100ng/ml). It is not unusual to utilize LPS in the microgram range for this kind of
cellular stimulation, as there are high amounts of this bacterial product in the
intestine.
6) There is wide variation in SAA protein expression level in the Ad5SAA-infected
cells. How were these variations accounted for in the assay?
7) The impact of SAA on E.coli survival is an interesting finding. However, the
result could be improved by demonstrating bacterial specificity (other gram –
and/or gram + bacteria). Also, the physiological relevance of E.coli DH5 is
questionable and the authors could have utilized a typical commensal intestinal
bacteria in this assay.
8) The manuscript would have gained in impact and significance by testing the
microbial composition between WT and SAA1/2 DKO mice. One would predict a
change in microbial composition if SAA impacts bacterial survival.
Level of interest: An article of limited interest
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
Please review this paper.
user
Reviewer's report
Title:Intestinal Epithelial Serum Amyloid A Modulates Bacterial Growth and
Protects from Experimental Colitis
Version: 1Date:23 September 2010
Reviewer: Christian Jobin
Reviewer's report:
This study investigates the function of SAA in intestinal homeostasis. The
authors report that SAA is expressed in IEC and that this expression increased in
patients with CD. In vitro data using an overexpression system showed that SAA
impact bacterial survival. DSS-induced colitis worsens in SAA1/2 KO mice
compared to WT mice. The authors conclude that SAA protects against intestinal
inflammation and regulates bacterial growth.
This is an interesting study that highlights potentially novel functions for SAA in
the gut. However, a number of weaknesses at both the experimental and
interpretation level impede on the quality of the manuscript. First, it appears that
the SAA1/2 DKO mice were not fully backcrossed to the C57BL/6 background
(method section). This is a major concern as DSS susceptibility is highly
dependent on the genetic background of a given mouse. Similarly, it appears that
the investigators have utilized C57BL/6 from a vendor (Taconic) as WT control
for their studies. Again, this experimental approach is flawed since susceptibility
to DSS is highly influenced by microbial composition, not to mention that the
C57BL/6 from Taconic are genetically different from the SAA1/2 DKO used by
the investigators. Finally, the impact of SAA1/2 on DSS-induced colitis is
marginal because two of the most important parameters (histological score and
body weight) are not significantly affected (Fig.6). Other weaknesses are listed
below:
1) The study on intestinal SAA expression (Fig.1) would be more convincing by
staining intestinal section obtained from SAA1/2 DKO mice. This will show
specificity of the protein signal.
2) Fig2A should be presented in the result section and not in the method section.
3) It is unclear why the SAA proteins detected in the stools (Fig.2B) migrate at
the different molecular weights than the protein detected in the plasma SAA
control.
4) The authors should correlate their SAA mRNA analysis (Fig.3A) with SAA
protein. Basically, is there a direct relationship between SAA mRNA and protein
levels?
5) Although SAA1/2 mRNA levels do not appear to be induced by LPS, the
authors have used a relatively small amount of endotoxin in this assay
(100ng/ml). It is not unusual to utilize LPS in the microgram range for this kind of
cellular stimulation, as there are high amounts of this bacterial product in the
intestine.
6) There is wide variation in SAA protein expression level in the Ad5SAA-infected
cells. How were these variations accounted for in the assay?
7) The impact of SAA on E.coli survival is an interesting finding. However, the
result could be improved by demonstrating bacterial specificity (other gram –
and/or gram + bacteria). Also, the physiological relevance of E.coli DH5 is
questionable and the authors could have utilized a typical commensal intestinal
bacteria in this assay.
8) The manuscript would have gained in impact and significance by testing the
microbial composition between WT and SAA1/2 DKO mice. One would predict a
change in microbial composition if SAA impacts bacterial survival.
Level of interest: An article of limited interest
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
assistant