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 )
Jean Charles Dagorn ( dagorn@marseille.inserm.fr )
Dr Juan L Iovanna ( iovanna@marseille.inserm.fr )
 1  29 Apr 2003 Version: Date: 
 Peter M C Wong Reviewer: 
 not specified Level of interest: 
 Other (see below) Advice on publication: 
General comments: 
The investigators have previously isolated, identified and characterized that p8 gene is highly 
expressed during acute phase of pancreatitis, and that p8 protein is structurally similar to HMG1, a 
molecule shown by Tracey and co-workers that it is a late phase modulator of inflammatory 
response. Based on these strong track records of investigations, they proceeded further by making p8 knockout mice and comparing their physiological responses to those of wild type. The found that although p8 null mice appeared normal physiologically, there were intriguing differences. 
Specifically, p8 null mice appeared more sensitive to endotoxin challenge, which correlated with 
increased levels of serum TNFa, and different profiles of MPO and HPO levels in livers and pancreas but not lungs of these mice. The also presented DNA microarray data, and suggested that liver response to endotoxin in p8 null mice is different than in wild type. These data are very 
intriguing. If organized and discussed thoroughly, with additional data, the work would represent a 
significant contribution to the field of septic shock research. 
Specific comments: 
(1) Introduction - The investigators have pointed out interesting characteristics of p8 protein in that it 
can reside in the nuclear as a transcription factor and in the cytoplasm to carry out certain biological functions; it is similar to HMG1, which has physiological significance in response to stress, and is 
identical to human Com1, which mediates growth of tumor cells after metastasis. It would be very 
helpful to the readers if they briefly review the characteristics of HMG1 as opposed to p8 for the purpose of their studies. 
(2) Include HMG1 analysis - The knockout phenotype of p8 highly suggests the existence of 
redundant complementary signaling pathways in response to stress. Since p8 is structurally 
homologous to HMG1, it would be very logical to see if HMG1 level has increased in p8 null mice 
upon endotoxin challenge. Provision of this type of data would add strength to the study. 
(3) Questionable significance of DNA microarray data - The power of DNA microarray analysis 
increases greatly if it is combined with functional genomic analysis on the genes of interest. Alone it 
usually gives suggestive directional studies and the significance of the candidate molecule that may 
or may not breed true, especially when the comparison parameter is set at low stringency (2 fold to 64 fold). This is especially true when this study is related to the investigation of a multi-organ failure syndrome. If the investigators insist on inclusion of these data, they should include similar data on 
pancreas, as well as extensive discussions on those molecules whose expression is highest upon 
endotoxin stimulation. 
(4) Figure 1 sample size - The most interesting data are the lethality experiment shown in Figure 1. 
Numbers of mice in each group increase statistical power to the conclusion. In the text, it was stated 
groups of 20 mice were analyzed. In legend of Figure 1, it was stated groups of 10 mice. Which is 
correct? 
(5) Figure 2 Serum TNF level - Upon LPS stimulation, serum TNF levels of these mice appeared 
extremely high, compared to many other studies. Is this because of strain difference? The investigators should include TNF levels in unstimulated mice as well. More than 4 mice per group 
will strengthen the claim more. The reviewer recognizes that it is very labor intensive. Perhaps the 
investigators can include analysis of more mice on a critical time point in a separate table - such as 
12 hours (10 mice/ group). Also, serum TNF usually peaks 1-2 hours after endotoxin stimulation. Figure 2 shows the peak at 12 hours. Strain difference also? 
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 )
Jean Charles Dagorn ( dagorn@marseille.inserm.fr )
Dr Juan L Iovanna ( iovanna@marseille.inserm.fr )
 1  29 Apr 2003 Version: Date: 
 Peter M C Wong Reviewer: 
 not specified Level of interest: 
 Other (see below) Advice on publication: 
General comments: 
The investigators have previously isolated, identified and characterized that p8 gene is highly 
expressed during acute phase of pancreatitis, and that p8 protein is structurally similar to HMG1, a 
molecule shown by Tracey and co-workers that it is a late phase modulator of inflammatory 
response. Based on these strong track records of investigations, they proceeded further by making p8 knockout mice and comparing their physiological responses to those of wild type. The found that although p8 null mice appeared normal physiologically, there were intriguing differences. 
Specifically, p8 null mice appeared more sensitive to endotoxin challenge, which correlated with 
increased levels of serum TNFa, and different profiles of MPO and HPO levels in livers and pancreas but not lungs of these mice. The also presented DNA microarray data, and suggested that liver response to endotoxin in p8 null mice is different than in wild type. These data are very 
intriguing. If organized and discussed thoroughly, with additional data, the work would represent a 
significant contribution to the field of septic shock research. 
Specific comments: 
(1) Introduction - The investigators have pointed out interesting characteristics of p8 protein in that it 
can reside in the nuclear as a transcription factor and in the cytoplasm to carry out certain biological functions; it is similar to HMG1, which has physiological significance in response to stress, and is 
identical to human Com1, which mediates growth of tumor cells after metastasis. It would be very 
helpful to the readers if they briefly review the characteristics of HMG1 as opposed to p8 for the purpose of their studies. 
(2) Include HMG1 analysis - The knockout phenotype of p8 highly suggests the existence of 
redundant complementary signaling pathways in response to stress. Since p8 is structurally 
homologous to HMG1, it would be very logical to see if HMG1 level has increased in p8 null mice 
upon endotoxin challenge. Provision of this type of data would add strength to the study. 
(3) Questionable significance of DNA microarray data - The power of DNA microarray analysis 
increases greatly if it is combined with functional genomic analysis on the genes of interest. Alone it 
usually gives suggestive directional studies and the significance of the candidate molecule that may 
or may not breed true, especially when the comparison parameter is set at low stringency (2 fold to 64 fold). This is especially true when this study is related to the investigation of a multi-organ failure syndrome. If the investigators insist on inclusion of these data, they should include similar data on 
pancreas, as well as extensive discussions on those molecules whose expression is highest upon 
endotoxin stimulation. 
(4) Figure 1 sample size - The most interesting data are the lethality experiment shown in Figure 1. 
Numbers of mice in each group increase statistical power to the conclusion. In the text, it was stated 
groups of 20 mice were analyzed. In legend of Figure 1, it was stated groups of 10 mice. Which is 
correct? 
(5) Figure 2 Serum TNF level - Upon LPS stimulation, serum TNF levels of these mice appeared 
extremely high, compared to many other studies. Is this because of strain difference? The investigators should include TNF levels in unstimulated mice as well. More than 4 mice per group 
will strengthen the claim more. The reviewer recognizes that it is very labor intensive. Perhaps the 
investigators can include analysis of more mice on a critical time point in a separate table - such as 
12 hours (10 mice/ group). Also, serum TNF usually peaks 1-2 hours after endotoxin stimulation. Figure 2 shows the peak at 12 hours. Strain difference also? 
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 )
Jean Charles Dagorn ( dagorn@marseille.inserm.fr )
Dr Juan L Iovanna ( iovanna@marseille.inserm.fr )
 1  29 Apr 2003 Version: Date: 
 Peter M C Wong Reviewer: 
 not specified Level of interest: 
 Other (see below) Advice on publication: 
General comments: 
The investigators have previously isolated, identified and characterized that p8 gene is highly 
expressed during acute phase of pancreatitis, and that p8 protein is structurally similar to HMG1, a 
molecule shown by Tracey and co-workers that it is a late phase modulator of inflammatory 
response. Based on these strong track records of investigations, they proceeded further by making p8 knockout mice and comparing their physiological responses to those of wild type. The found that although p8 null mice appeared normal physiologically, there were intriguing differences. 
Specifically, p8 null mice appeared more sensitive to endotoxin challenge, which correlated with 
increased levels of serum TNFa, and different profiles of MPO and HPO levels in livers and pancreas but not lungs of these mice. The also presented DNA microarray data, and suggested that liver response to endotoxin in p8 null mice is different than in wild type. These data are very 
intriguing. If organized and discussed thoroughly, with additional data, the work would represent a 
significant contribution to the field of septic shock research. 
Specific comments: 
(1) Introduction - The investigators have pointed out interesting characteristics of p8 protein in that it 
can reside in the nuclear as a transcription factor and in the cytoplasm to carry out certain biological functions; it is similar to HMG1, which has physiological significance in response to stress, and is 
identical to human Com1, which mediates growth of tumor cells after metastasis. It would be very 
helpful to the readers if they briefly review the characteristics of HMG1 as opposed to p8 for the purpose of their studies. 
(2) Include HMG1 analysis - The knockout phenotype of p8 highly suggests the existence of 
redundant complementary signaling pathways in response to stress. Since p8 is structurally 
homologous to HMG1, it would be very logical to see if HMG1 level has increased in p8 null mice 
upon endotoxin challenge. Provision of this type of data would add strength to the study. 
(3) Questionable significance of DNA microarray data - The power of DNA microarray analysis 
increases greatly if it is combined with functional genomic analysis on the genes of interest. Alone it 
usually gives suggestive directional studies and the significance of the candidate molecule that may 
or may not breed true, especially when the comparison parameter is set at low stringency (2 fold to 64 fold). This is especially true when this study is related to the investigation of a multi-organ failure syndrome. If the investigators insist on inclusion of these data, they should include similar data on 
pancreas, as well as extensive discussions on those molecules whose expression is highest upon 
endotoxin stimulation. 
(4) Figure 1 sample size - The most interesting data are the lethality experiment shown in Figure 1. 
Numbers of mice in each group increase statistical power to the conclusion. In the text, it was stated 
groups of 20 mice were analyzed. In legend of Figure 1, it was stated groups of 10 mice. Which is 
correct? 
(5) Figure 2 Serum TNF level - Upon LPS stimulation, serum TNF levels of these mice appeared 
extremely high, compared to many other studies. Is this because of strain difference? The investigators should include TNF levels in unstimulated mice as well. More than 4 mice per group 
will strengthen the claim more. The reviewer recognizes that it is very labor intensive. Perhaps the 
investigators can include analysis of more mice on a critical time point in a separate table - such as 
12 hours (10 mice/ group). Also, serum TNF usually peaks 1-2 hours after endotoxin stimulation. Figure 2 shows the peak at 12 hours. Strain difference also? 
Competing interests: 
None declared. 
assistant