Reviewer's report 
 Mice with targeted disruption of p8 gene show increased sensitivity to lipopolysaccharide and Title: 
DNA microarray analysis of livers reveals an aberrant gene expression response. 
Authors: 
Sophie Vasseur ( vasseur@marseille.inserm.fr )
Albrecht Hoffmeister ( hofa@medizin.uni-leipzig.de )
Andres Garcia-Montero ( angarmon@gugu.usal.es )
Dr Marc Barthet ( MBARTHET@mail.ap-hm.fr )
Laure Saint-Michel ( stmichel@inserm-adr.univ-mrs.fr )
Patrice Berthezene ( berthezene@marseille.inserm.fr )
Fritz Fiedler ( fritz.fiedler@urz.uni-heidelberg.de )
Daniel Closa ( dcabam@iibb.csic.es )
Dr Jean Charles Dagorn ( dagorn@marseille.inserm.fr )
Dr Juan L Iovanna ( iovanna@marseille.inserm.fr )
 2  7 Jul 2003 Version: Date: 
 Peter M C Wong Reviewer: 
 not specified Level of interest: 
 Other (see below) Advice on publication: 
Comments from Reviewer 1: 
Authors really have done a lot of interesting and important work on p8. The p8-/- mice should have a 
lot of interesting results that can be obtained. For the purpose of this manuscript, more in-depth 
analyses and thorough comments should be incorporated between HMG-1 and p8, as italicized and 
bolded below. 
Author's response to reviews 
http://www.biomedcentral.com/imedia/8566497471788012_comment.pdf 
Title: Mice with targeted disruption of p8 gene show increased sensitivity to lipopolysaccharide and 
DNA microarray analysis of livers reveals an aberrant gene expression response. 
Authors: 
Sophie Vasseur (vasseur@marseille.inserm.fr) 
Albrecht Hoffmeister (hofa@medizin.uni-leipzig.de) 
Andres Garcia-Montero (angarmon@gugu.usal.es) 
Dr Marc Barthet (MBARTHET@mail.ap-hm.fr) 
Laure Saint-Michel (stmichel@inserm-adr.univ-mrs.fr) 
Patrice Berthezene (berthezene@marseille.inserm.fr) 
Fritz Fiedler (fritz.fiedler@urz.uni-heidelberg.de) Daniel Closa (dcabam@iibb.csic.es) Dr Jean Charles Dagorn (dagorn@marseille.inserm.fr) 
Dr Juan L Iovanna (iovanna@marseille.inserm.fr) 
2 23 Jun 2003 Version: Date: 
MS: 3086498041503774 
Answer to reviewers 
Reviewer #1 
(1) Introduction - The investigators have pointed... 
This is a very relevant comment. We added to the Background section a brief description of the role 
of the p8-related HMG1 as a pro-inflammatory factor. 
(2) Include HMG1 analysis... 
This point has also been considered. In fact, HMG1 was absent from the microarray that we used. 
Therefore, we performed semiquantitative RT-PCR analysis on RNA purified from liver after 6 and 18 hours of LPS treatment and found that HMG1 mRNA remains unchanged. This observation is described in the Discussion section. 
>>Authors should present the semi-quantitative RT-PCR data in a figure, not as data not shown. 
Authors also indicated that upon LPS stimulation, p8 was elevated and HMG-1 was not in normal 
mice. HMG-1 was elevated in studies reported by Tracey and associates. They should provide 
comparative explanation for the discrepancy. 
(3) Questionable significance of DNA microarray data... 
This is an important remark. As suggested by this reviewer, microarray analysis is generally made to 
identify candidate genes deserving further analysis. In this work, however, the microarray study was 
carried out to validate the hypothesis by which p8 acts as a co-factor of transcription over-expressed 
in response to LPS to adapt the transcriptional response of the insulted tissue. In fact, expression of 
a large number of genes was found modified in liver from p8-deficient mice compared to 
p8-expressing mice, strongly supporting that hypothesis. p8 expression seems to regulate the 
expression of several genes that improve mouse resistance to LPS. 
>>In Discussions, authors indicated that HMG-1 was elevated in p8-/- mice, and interpreted that 
they compensated each other by function. It would be interesting and important if they perform 
microarray on liver of HMG-/- mice and compare it with that of p8-/-. According to their expection, the pattern should be similar. 
Liver is an important organ in sepsis and multiorgan failure syndrome. However, it really should not 
be the only organ investigators should look at. Description of claims based on only liver data therefore should be much softer. 
For any gene that has potential effects on sepsis, which involves multiple cell types and different 
signaling pathways, transcriptional modification should produce a whole host of up-stream and down-stream of chain reactions and biological consequences. Authors should really critically comment on it. 
Liver was chosen for microarray analysis because it is known to be an important target of LPS and 
because its response to LPS has been thoroughly characterized. On the contrary, although the pancreas is also a target tissue for LPS, its response to the endotoxin is less known and 
interpretation of similar microarray analysis would, in this context, be very difficult. 
(4) Figure 1 sample size - The most interesting data... 
The correct value is 20. This mistake was corrected in the legend of Figure 1. 
(5) Figure 2 Serum TNF level - Upon LPS... 
This is an important observation. We are also very surprised to see that the TNF serum level peaked 
between 6 and 12 hours after LPS challenge. Although we present in Figure 2 data from only 4 
mice/group, TNF was also measured in an independent experiment including groups of 2 or 3 mice, 
with similar results. It is possible that, as suggested by the reviewer, the difference is due to 
the mouse strain. 
>>Authors really should include at least 6 mice for each experimental point. We all know that in vivo 
studies, especially those for sepsis and MODS, the results are highly variable. Collection of these 
data should be a matter of 2 weeks or so. 
Reviewer #2 
The major question that remains is which... This is an interesting comment. We measured serum levels of the anti-inflammatory cytokine IL-10 and found not significant differences between p8-expressing and p8-deficient mice. We also 
monitored IFNa since it is involved in LPS response, but levels were increased to similar extents in 
p8+/+ and p8-/- mice. Finally, to evaluate pancreatic and liver injury we measured serum levels of amylase and GOT respectively, but again increases were similar in p8-expressing and p8-deficient mice. Corresponding data were added to the Results section. 
The time course of TNFa after LPS... 
This point was also underscored by the first reviewer and a possible explanation was already given. 
TNF-alpha mRNA does not show up... 
The microarray study was performed three years ago using the first generation of Affimetrix chips 
with about 6000 mouse genes, more than 50% of them corresponding to ESTs. TNFa, as well as NO 
synthase and cyclooxygenase were indeed absent from the chip, as noticed by this reviewer. 
However, many other cytokines, their receptors and their intracellular mediators as well as several 
cytokine-induced genes, particularly IFN-induced genes, were available and were found activated upon LPS treatment as expected. Data are presented in tables 3 & 4 and 5 & 6. 
In light of all the above plus the fact... 
This point has been considered in the response to point 3 of the first reviewer 
There is an impressive number of genes altered... 
This is not really surprising because we know that p8 is a co-factor of transcription activated in 
response to tissue stress. It is therefore very likely that over-expression of p8 upon LPS treatment 
will modulate the activity of several transcription factors and, consequently, of a large number of 
genes. 
Finally, although the LPS model used in this paper... 
We are very surprise by this comment because endotoxin challenge is a widely used model in studies on the cellular and molecular mechanisms involved in sepsis. Clinically, endotoxin is the 
main mediator of lesions induced by Gram negative microorganisms, which can be lethal. 
Competing interests: 
None declared. 
Reviewer's report 
 Mice with targeted disruption of p8 gene show increased sensitivity to lipopolysaccharide and Title: 
DNA microarray analysis of livers reveals an aberrant gene expression response. 
Authors: 
Sophie Vasseur ( vasseur@marseille.inserm.fr )
Albrecht Hoffmeister ( hofa@medizin.uni-leipzig.de )
Andres Garcia-Montero ( angarmon@gugu.usal.es )
Dr Marc Barthet ( MBARTHET@mail.ap-hm.fr )
Laure Saint-Michel ( stmichel@inserm-adr.univ-mrs.fr )
Patrice Berthezene ( berthezene@marseille.inserm.fr )
Fritz Fiedler ( fritz.fiedler@urz.uni-heidelberg.de )
Daniel Closa ( dcabam@iibb.csic.es )
Dr Jean Charles Dagorn ( dagorn@marseille.inserm.fr )
Dr Juan L Iovanna ( iovanna@marseille.inserm.fr )
 2  7 Jul 2003 Version: Date: 
 Peter M C Wong Reviewer: 
 not specified Level of interest: 
 Other (see below) Advice on publication: 
Comments from Reviewer 1: 
Authors really have done a lot of interesting and important work on p8. The p8-/- mice should have a 
lot of interesting results that can be obtained. For the purpose of this manuscript, more in-depth 
analyses and thorough comments should be incorporated between HMG-1 and p8, as italicized and 
bolded below. 
Author's response to reviews 
http://www.biomedcentral.com/imedia/8566497471788012_comment.pdf 
Title: Mice with targeted disruption of p8 gene show increased sensitivity to lipopolysaccharide and 
DNA microarray analysis of livers reveals an aberrant gene expression response. 
Authors: 
Sophie Vasseur (vasseur@marseille.inserm.fr) 
Albrecht Hoffmeister (hofa@medizin.uni-leipzig.de) 
Andres Garcia-Montero (angarmon@gugu.usal.es) 
Dr Marc Barthet (MBARTHET@mail.ap-hm.fr) 
Laure Saint-Michel (stmichel@inserm-adr.univ-mrs.fr) 
Patrice Berthezene (berthezene@marseille.inserm.fr) 
Fritz Fiedler (fritz.fiedler@urz.uni-heidelberg.de) Daniel Closa (dcabam@iibb.csic.es) Dr Jean Charles Dagorn (dagorn@marseille.inserm.fr) 
Dr Juan L Iovanna (iovanna@marseille.inserm.fr) 
2 23 Jun 2003 Version: Date: 
MS: 3086498041503774 
Answer to reviewers 
Reviewer #1 
(1) Introduction - The investigators have pointed... 
This is a very relevant comment. We added to the Background section a brief description of the role 
of the p8-related HMG1 as a pro-inflammatory factor. 
(2) Include HMG1 analysis... 
This point has also been considered. In fact, HMG1 was absent from the microarray that we used. 
Therefore, we performed semiquantitative RT-PCR analysis on RNA purified from liver after 6 and 18 hours of LPS treatment and found that HMG1 mRNA remains unchanged. This observation is described in the Discussion section. 
>>Authors should present the semi-quantitative RT-PCR data in a figure, not as data not shown. 
Authors also indicated that upon LPS stimulation, p8 was elevated and HMG-1 was not in normal 
mice. HMG-1 was elevated in studies reported by Tracey and associates. They should provide 
comparative explanation for the discrepancy. 
(3) Questionable significance of DNA microarray data... 
This is an important remark. As suggested by this reviewer, microarray analysis is generally made to 
identify candidate genes deserving further analysis. In this work, however, the microarray study was 
carried out to validate the hypothesis by which p8 acts as a co-factor of transcription over-expressed 
in response to LPS to adapt the transcriptional response of the insulted tissue. In fact, expression of 
a large number of genes was found modified in liver from p8-deficient mice compared to 
p8-expressing mice, strongly supporting that hypothesis. p8 expression seems to regulate the 
expression of several genes that improve mouse resistance to LPS. 
>>In Discussions, authors indicated that HMG-1 was elevated in p8-/- mice, and interpreted that 
they compensated each other by function. It would be interesting and important if they perform 
microarray on liver of HMG-/- mice and compare it with that of p8-/-. According to their expection, the pattern should be similar. 
Liver is an important organ in sepsis and multiorgan failure syndrome. However, it really should not 
be the only organ investigators should look at. Description of claims based on only liver data therefore should be much softer. 
For any gene that has potential effects on sepsis, which involves multiple cell types and different 
signaling pathways, transcriptional modification should produce a whole host of up-stream and down-stream of chain reactions and biological consequences. Authors should really critically comment on it. 
Liver was chosen for microarray analysis because it is known to be an important target of LPS and 
because its response to LPS has been thoroughly characterized. On the contrary, although the pancreas is also a target tissue for LPS, its response to the endotoxin is less known and 
interpretation of similar microarray analysis would, in this context, be very difficult. 
(4) Figure 1 sample size - The most interesting data... 
The correct value is 20. This mistake was corrected in the legend of Figure 1. 
(5) Figure 2 Serum TNF level - Upon LPS... 
This is an important observation. We are also very surprised to see that the TNF serum level peaked 
between 6 and 12 hours after LPS challenge. Although we present in Figure 2 data from only 4 
mice/group, TNF was also measured in an independent experiment including groups of 2 or 3 mice, 
with similar results. It is possible that, as suggested by the reviewer, the difference is due to 
the mouse strain. 
>>Authors really should include at least 6 mice for each experimental point. We all know that in vivo 
studies, especially those for sepsis and MODS, the results are highly variable. Collection of these 
data should be a matter of 2 weeks or so. 
Reviewer #2 
The major question that remains is which... This is an interesting comment. We measured serum levels of the anti-inflammatory cytokine IL-10 and found not significant differences between p8-expressing and p8-deficient mice. We also 
monitored IFNa since it is involved in LPS response, but levels were increased to similar extents in 
p8+/+ and p8-/- mice. Finally, to evaluate pancreatic and liver injury we measured serum levels of amylase and GOT respectively, but again increases were similar in p8-expressing and p8-deficient mice. Corresponding data were added to the Results section. 
The time course of TNFa after LPS... 
This point was also underscored by the first reviewer and a possible explanation was already given. 
TNF-alpha mRNA does not show up... 
The microarray study was performed three years ago using the first generation of Affimetrix chips 
with about 6000 mouse genes, more than 50% of them corresponding to ESTs. TNFa, as well as NO 
synthase and cyclooxygenase were indeed absent from the chip, as noticed by this reviewer. 
However, many other cytokines, their receptors and their intracellular mediators as well as several 
cytokine-induced genes, particularly IFN-induced genes, were available and were found activated upon LPS treatment as expected. Data are presented in tables 3 & 4 and 5 & 6. 
In light of all the above plus the fact... 
This point has been considered in the response to point 3 of the first reviewer 
There is an impressive number of genes altered... 
This is not really surprising because we know that p8 is a co-factor of transcription activated in 
response to tissue stress. It is therefore very likely that over-expression of p8 upon LPS treatment 
will modulate the activity of several transcription factors and, consequently, of a large number of 
genes. 
Finally, although the LPS model used in this paper... 
We are very surprise by this comment because endotoxin challenge is a widely used model in studies on the cellular and molecular mechanisms involved in sepsis. Clinically, endotoxin is the 
main mediator of lesions induced by Gram negative microorganisms, which can be lethal. 
Competing interests: 
None declared. 
Please review this paper.
user
Reviewer's report 
 Mice with targeted disruption of p8 gene show increased sensitivity to lipopolysaccharide and Title: 
DNA microarray analysis of livers reveals an aberrant gene expression response. 
Authors: 
Sophie Vasseur ( vasseur@marseille.inserm.fr )
Albrecht Hoffmeister ( hofa@medizin.uni-leipzig.de )
Andres Garcia-Montero ( angarmon@gugu.usal.es )
Dr Marc Barthet ( MBARTHET@mail.ap-hm.fr )
Laure Saint-Michel ( stmichel@inserm-adr.univ-mrs.fr )
Patrice Berthezene ( berthezene@marseille.inserm.fr )
Fritz Fiedler ( fritz.fiedler@urz.uni-heidelberg.de )
Daniel Closa ( dcabam@iibb.csic.es )
Dr Jean Charles Dagorn ( dagorn@marseille.inserm.fr )
Dr Juan L Iovanna ( iovanna@marseille.inserm.fr )
 2  7 Jul 2003 Version: Date: 
 Peter M C Wong Reviewer: 
 not specified Level of interest: 
 Other (see below) Advice on publication: 
Comments from Reviewer 1: 
Authors really have done a lot of interesting and important work on p8. The p8-/- mice should have a 
lot of interesting results that can be obtained. For the purpose of this manuscript, more in-depth 
analyses and thorough comments should be incorporated between HMG-1 and p8, as italicized and 
bolded below. 
Author's response to reviews 
http://www.biomedcentral.com/imedia/8566497471788012_comment.pdf 
Title: Mice with targeted disruption of p8 gene show increased sensitivity to lipopolysaccharide and 
DNA microarray analysis of livers reveals an aberrant gene expression response. 
Authors: 
Sophie Vasseur (vasseur@marseille.inserm.fr) 
Albrecht Hoffmeister (hofa@medizin.uni-leipzig.de) 
Andres Garcia-Montero (angarmon@gugu.usal.es) 
Dr Marc Barthet (MBARTHET@mail.ap-hm.fr) 
Laure Saint-Michel (stmichel@inserm-adr.univ-mrs.fr) 
Patrice Berthezene (berthezene@marseille.inserm.fr) 
Fritz Fiedler (fritz.fiedler@urz.uni-heidelberg.de) Daniel Closa (dcabam@iibb.csic.es) Dr Jean Charles Dagorn (dagorn@marseille.inserm.fr) 
Dr Juan L Iovanna (iovanna@marseille.inserm.fr) 
2 23 Jun 2003 Version: Date: 
MS: 3086498041503774 
Answer to reviewers 
Reviewer #1 
(1) Introduction - The investigators have pointed... 
This is a very relevant comment. We added to the Background section a brief description of the role 
of the p8-related HMG1 as a pro-inflammatory factor. 
(2) Include HMG1 analysis... 
This point has also been considered. In fact, HMG1 was absent from the microarray that we used. 
Therefore, we performed semiquantitative RT-PCR analysis on RNA purified from liver after 6 and 18 hours of LPS treatment and found that HMG1 mRNA remains unchanged. This observation is described in the Discussion section. 
>>Authors should present the semi-quantitative RT-PCR data in a figure, not as data not shown. 
Authors also indicated that upon LPS stimulation, p8 was elevated and HMG-1 was not in normal 
mice. HMG-1 was elevated in studies reported by Tracey and associates. They should provide 
comparative explanation for the discrepancy. 
(3) Questionable significance of DNA microarray data... 
This is an important remark. As suggested by this reviewer, microarray analysis is generally made to 
identify candidate genes deserving further analysis. In this work, however, the microarray study was 
carried out to validate the hypothesis by which p8 acts as a co-factor of transcription over-expressed 
in response to LPS to adapt the transcriptional response of the insulted tissue. In fact, expression of 
a large number of genes was found modified in liver from p8-deficient mice compared to 
p8-expressing mice, strongly supporting that hypothesis. p8 expression seems to regulate the 
expression of several genes that improve mouse resistance to LPS. 
>>In Discussions, authors indicated that HMG-1 was elevated in p8-/- mice, and interpreted that 
they compensated each other by function. It would be interesting and important if they perform 
microarray on liver of HMG-/- mice and compare it with that of p8-/-. According to their expection, the pattern should be similar. 
Liver is an important organ in sepsis and multiorgan failure syndrome. However, it really should not 
be the only organ investigators should look at. Description of claims based on only liver data therefore should be much softer. 
For any gene that has potential effects on sepsis, which involves multiple cell types and different 
signaling pathways, transcriptional modification should produce a whole host of up-stream and down-stream of chain reactions and biological consequences. Authors should really critically comment on it. 
Liver was chosen for microarray analysis because it is known to be an important target of LPS and 
because its response to LPS has been thoroughly characterized. On the contrary, although the pancreas is also a target tissue for LPS, its response to the endotoxin is less known and 
interpretation of similar microarray analysis would, in this context, be very difficult. 
(4) Figure 1 sample size - The most interesting data... 
The correct value is 20. This mistake was corrected in the legend of Figure 1. 
(5) Figure 2 Serum TNF level - Upon LPS... 
This is an important observation. We are also very surprised to see that the TNF serum level peaked 
between 6 and 12 hours after LPS challenge. Although we present in Figure 2 data from only 4 
mice/group, TNF was also measured in an independent experiment including groups of 2 or 3 mice, 
with similar results. It is possible that, as suggested by the reviewer, the difference is due to 
the mouse strain. 
>>Authors really should include at least 6 mice for each experimental point. We all know that in vivo 
studies, especially those for sepsis and MODS, the results are highly variable. Collection of these 
data should be a matter of 2 weeks or so. 
Reviewer #2 
The major question that remains is which... This is an interesting comment. We measured serum levels of the anti-inflammatory cytokine IL-10 and found not significant differences between p8-expressing and p8-deficient mice. We also 
monitored IFNa since it is involved in LPS response, but levels were increased to similar extents in 
p8+/+ and p8-/- mice. Finally, to evaluate pancreatic and liver injury we measured serum levels of amylase and GOT respectively, but again increases were similar in p8-expressing and p8-deficient mice. Corresponding data were added to the Results section. 
The time course of TNFa after LPS... 
This point was also underscored by the first reviewer and a possible explanation was already given. 
TNF-alpha mRNA does not show up... 
The microarray study was performed three years ago using the first generation of Affimetrix chips 
with about 6000 mouse genes, more than 50% of them corresponding to ESTs. TNFa, as well as NO 
synthase and cyclooxygenase were indeed absent from the chip, as noticed by this reviewer. 
However, many other cytokines, their receptors and their intracellular mediators as well as several 
cytokine-induced genes, particularly IFN-induced genes, were available and were found activated upon LPS treatment as expected. Data are presented in tables 3 & 4 and 5 & 6. 
In light of all the above plus the fact... 
This point has been considered in the response to point 3 of the first reviewer 
There is an impressive number of genes altered... 
This is not really surprising because we know that p8 is a co-factor of transcription activated in 
response to tissue stress. It is therefore very likely that over-expression of p8 upon LPS treatment 
will modulate the activity of several transcription factors and, consequently, of a large number of 
genes. 
Finally, although the LPS model used in this paper... 
We are very surprise by this comment because endotoxin challenge is a widely used model in studies on the cellular and molecular mechanisms involved in sepsis. Clinically, endotoxin is the 
main mediator of lesions induced by Gram negative microorganisms, which can be lethal. 
Competing interests: 
None declared. 
assistant