Reviewer's report 
 Loss of interleukin-12 modifies the pro-inflammatory response but does not prevent duct Title: 
obstruction in experimental biliary atresia 
 1  30 December 2005 Version: Date: 
 Claus Petersen Reviewer: 
Reviewer's report: 
Summary of the study 
Based on the previous observation that IFN-? is a key factor in the development of biliary obstruction in the balb/c-RRV model, the authors hypothesized that loss of IL-12 would hamper the development of RRV-induced bile duct obstruction in mice. They compared IL-12-knock-out and 
conventional Balb/c-mice with regard to clinical and histomorphological chances, as well as 
differences of the cellular components, virus clearance and regulation of several cytokines. The 
results suggest that IL-12 does not play a crucial role in clearance of RRV and development of BA in 
this model. On the other hand, in RRV-infected IL-12-ko-mice, the expression of TNF-a and IFNs 
Type I was increased and the inflammatory cells were found to be predominantly neutrophils. 
Specific aspects of the study 
This is another excellent study from this group. The hypothesis of investigating the role of IL-12 in 
the murine model of BA is well taken and based on previous data from the authors, as well as 
others. Therefore, the negative main result is of particular interest for research in this field. 
Moreover, it is supported by various data, which also opens intriguing new aspects and raises more 
questions. 
Nonetheless, some issues need to be addressed: 
1. The authors use mice lacking the p40 subunit of the IL-12 in this experiment. As this subunit is 
also part of IL-23 and IL-27, the lack of these cytokines of the IL-12 family shown in the real time 
PCR should be discussed, as well as its impact on the outcome. 
2. The authors cite unpaired t-test as statistical analysis. The use of this test should be explained as 
sometimes more than two groups are being compared. Please also mention whether normal 
distribution has been postulated or tested and give standard deviation, or better standard error of the 
mean. 
3. It is astonishing that, days after the intraperitoneal injection of a large virus dose, there should be no sign of the viruses in the liver. Please discuss the lack of viral mRNA in the liver at 3 days after 
inoculation, also with regard to other methods of viral detection, such as plague-forming units. 
Moreover, data regarding the viral clearance in WT mice is missing; therefore, it is not possible to 
judge the effects of IL12-p40 depletion on this parameter. 
4. The addition of the TH2 cytokines is a very interesting and important part of the paper. Please 
give some details as to why you chose these cytokines and the potential interaction with the IL-12 
family. Unfortunately, the data regarding the WT is missing. This data should be included. We feel you should also address the relevance of the visible changes in the relative levels, e.g. of IL-4 after saline injection between 7d and 14d, as well as the amplitude of the change of the relative 
expression of IL-18 between ~ 1.1 and 1.7 on the overall conclusion that RRV infection leads to a 
decrease in the expression of IL-4 and IL-18. 5. Please give the FACS data in more detail. We assume that a defined number of events was counted in order to allow an intergroup comparison. This number must be given. Alternatively, the 
number of the counted cell populations could be given as percentage of total cells. 
6. In our opinion, a clear structure between the results and discussion could help understanding of 
this very complex manuscript 
As already mentioned, this excellent study is BA-related basic research and IFN-related immune 
response in newborns and, therefore, of great interest and should be – after consideration of the 
abovementioned points - recommended for publication. 
 Accept after minor essential revisions What next?: 
 An article of outstanding merit and interest in its field Level of interest: 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
I declare that I have no competing interests 
Reviewer's report 
 Loss of interleukin-12 modifies the pro-inflammatory response but does not prevent duct Title: 
obstruction in experimental biliary atresia 
 1  30 December 2005 Version: Date: 
 Claus Petersen Reviewer: 
Reviewer's report: 
Summary of the study 
Based on the previous observation that IFN-? is a key factor in the development of biliary obstruction in the balb/c-RRV model, the authors hypothesized that loss of IL-12 would hamper the development of RRV-induced bile duct obstruction in mice. They compared IL-12-knock-out and 
conventional Balb/c-mice with regard to clinical and histomorphological chances, as well as 
differences of the cellular components, virus clearance and regulation of several cytokines. The 
results suggest that IL-12 does not play a crucial role in clearance of RRV and development of BA in 
this model. On the other hand, in RRV-infected IL-12-ko-mice, the expression of TNF-a and IFNs 
Type I was increased and the inflammatory cells were found to be predominantly neutrophils. 
Specific aspects of the study 
This is another excellent study from this group. The hypothesis of investigating the role of IL-12 in 
the murine model of BA is well taken and based on previous data from the authors, as well as 
others. Therefore, the negative main result is of particular interest for research in this field. 
Moreover, it is supported by various data, which also opens intriguing new aspects and raises more 
questions. 
Nonetheless, some issues need to be addressed: 
1. The authors use mice lacking the p40 subunit of the IL-12 in this experiment. As this subunit is 
also part of IL-23 and IL-27, the lack of these cytokines of the IL-12 family shown in the real time 
PCR should be discussed, as well as its impact on the outcome. 
2. The authors cite unpaired t-test as statistical analysis. The use of this test should be explained as 
sometimes more than two groups are being compared. Please also mention whether normal 
distribution has been postulated or tested and give standard deviation, or better standard error of the 
mean. 
3. It is astonishing that, days after the intraperitoneal injection of a large virus dose, there should be no sign of the viruses in the liver. Please discuss the lack of viral mRNA in the liver at 3 days after 
inoculation, also with regard to other methods of viral detection, such as plague-forming units. 
Moreover, data regarding the viral clearance in WT mice is missing; therefore, it is not possible to 
judge the effects of IL12-p40 depletion on this parameter. 
4. The addition of the TH2 cytokines is a very interesting and important part of the paper. Please 
give some details as to why you chose these cytokines and the potential interaction with the IL-12 
family. Unfortunately, the data regarding the WT is missing. This data should be included. We feel you should also address the relevance of the visible changes in the relative levels, e.g. of IL-4 after saline injection between 7d and 14d, as well as the amplitude of the change of the relative 
expression of IL-18 between ~ 1.1 and 1.7 on the overall conclusion that RRV infection leads to a 
decrease in the expression of IL-4 and IL-18. 5. Please give the FACS data in more detail. We assume that a defined number of events was counted in order to allow an intergroup comparison. This number must be given. Alternatively, the 
number of the counted cell populations could be given as percentage of total cells. 
6. In our opinion, a clear structure between the results and discussion could help understanding of 
this very complex manuscript 
As already mentioned, this excellent study is BA-related basic research and IFN-related immune 
response in newborns and, therefore, of great interest and should be – after consideration of the 
abovementioned points - recommended for publication. 
 Accept after minor essential revisions What next?: 
 An article of outstanding merit and interest in its field Level of interest: 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
I declare that I have no competing interests 
Please review this paper.
user
Reviewer's report 
 Loss of interleukin-12 modifies the pro-inflammatory response but does not prevent duct Title: 
obstruction in experimental biliary atresia 
 1  30 December 2005 Version: Date: 
 Claus Petersen Reviewer: 
Reviewer's report: 
Summary of the study 
Based on the previous observation that IFN-? is a key factor in the development of biliary obstruction in the balb/c-RRV model, the authors hypothesized that loss of IL-12 would hamper the development of RRV-induced bile duct obstruction in mice. They compared IL-12-knock-out and 
conventional Balb/c-mice with regard to clinical and histomorphological chances, as well as 
differences of the cellular components, virus clearance and regulation of several cytokines. The 
results suggest that IL-12 does not play a crucial role in clearance of RRV and development of BA in 
this model. On the other hand, in RRV-infected IL-12-ko-mice, the expression of TNF-a and IFNs 
Type I was increased and the inflammatory cells were found to be predominantly neutrophils. 
Specific aspects of the study 
This is another excellent study from this group. The hypothesis of investigating the role of IL-12 in 
the murine model of BA is well taken and based on previous data from the authors, as well as 
others. Therefore, the negative main result is of particular interest for research in this field. 
Moreover, it is supported by various data, which also opens intriguing new aspects and raises more 
questions. 
Nonetheless, some issues need to be addressed: 
1. The authors use mice lacking the p40 subunit of the IL-12 in this experiment. As this subunit is 
also part of IL-23 and IL-27, the lack of these cytokines of the IL-12 family shown in the real time 
PCR should be discussed, as well as its impact on the outcome. 
2. The authors cite unpaired t-test as statistical analysis. The use of this test should be explained as 
sometimes more than two groups are being compared. Please also mention whether normal 
distribution has been postulated or tested and give standard deviation, or better standard error of the 
mean. 
3. It is astonishing that, days after the intraperitoneal injection of a large virus dose, there should be no sign of the viruses in the liver. Please discuss the lack of viral mRNA in the liver at 3 days after 
inoculation, also with regard to other methods of viral detection, such as plague-forming units. 
Moreover, data regarding the viral clearance in WT mice is missing; therefore, it is not possible to 
judge the effects of IL12-p40 depletion on this parameter. 
4. The addition of the TH2 cytokines is a very interesting and important part of the paper. Please 
give some details as to why you chose these cytokines and the potential interaction with the IL-12 
family. Unfortunately, the data regarding the WT is missing. This data should be included. We feel you should also address the relevance of the visible changes in the relative levels, e.g. of IL-4 after saline injection between 7d and 14d, as well as the amplitude of the change of the relative 
expression of IL-18 between ~ 1.1 and 1.7 on the overall conclusion that RRV infection leads to a 
decrease in the expression of IL-4 and IL-18. 5. Please give the FACS data in more detail. We assume that a defined number of events was counted in order to allow an intergroup comparison. This number must be given. Alternatively, the 
number of the counted cell populations could be given as percentage of total cells. 
6. In our opinion, a clear structure between the results and discussion could help understanding of 
this very complex manuscript 
As already mentioned, this excellent study is BA-related basic research and IFN-related immune 
response in newborns and, therefore, of great interest and should be – after consideration of the 
abovementioned points - recommended for publication. 
 Accept after minor essential revisions What next?: 
 An article of outstanding merit and interest in its field Level of interest: 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
I declare that I have no competing interests 
assistant