Reviewer's report 
 Integrative roles of transforming growth factor-alpha in the cytoprotection mechanisms of Title: 
gastric mucosal injury 
 1  29 March 2006 Version: Date: 
 Giuliano Ramadori Reviewer: 
Reviewer's report: 
General 
Kosone et al. studied effect of TGF-a on protection against gastric mucosal injury in a TGF-a- expressing mouse line on a CD1 background. The authors stated that this mouse line, when compared to a mouse line on a FVB background do not show hypertrophic gastropathy. In their 
model the authors studied the effect of ethanol on induction of gastric injury. As main result the 
authors found that TGF-a expressing mice maintains gastric mucosal defense against gastric injury induced by ethanol. 
Concerns: 
1.) For induction of gastric injury the authors administrated acidified ethanol. Firstly it is not stated 
how ethanol was administrated. Secondly it is unclear whether or how controls where performed. 
2.) It is unclear whether or how negative controls for their immunostainings and the TUNEL assay 
have been performed. 
3.) For their cell culture studies the authors added 5-100ng/ml TGF-a. However their cell culture 
medium contains 10% FCS. Firstly it is unknown how much TGF-a is already present in their culture 
medium. Secondly FCS could be capable for binding of TGF-a. Therefore real TGF-a concentration 
in their culture medium is unknown. The authors are encouraged to reduce FCS- concentration in their culture medium for their experiments. 
4.) It would be appreciable to state the number of independent experiments used for statistical 
analysis. 
5.) In figure 1B immunostainings are presented. Are these stainings of TGF-a-transgenic mice? This 
should be stated. In addition presentation of wild type mice would be interesting to give an 
impression of TGF-a expression in these mice. 
6.) The panels in figure 1E are too small. Bigger panels with a higher resolution should be 
presented. However it is unclear why in single stainings in the left panel of the TG mice a yellow 
color appears when this is only an FITC- staining and why the white dots in the middle staining of the 
TG mice appear when it is only a single staining with Cy3. This should be explained. 
7.) As noted in point 1 controls are missing. It is unclear whether the diluent of the ethanol or 
personal stress of the mice induced by the administration itself already causes the lesions. In the middle and the lower left region of the right panel of figure 2A also small lesions are visible. Are 
these as extensive as in those visible in the left panel? In figure 2C statistical analysis and number 
of independent experiments are missing. 
8.) Figure 2D is not convincing. TUNEL data are presented. The panels are too small to provide a 
good impression. However there are also many dark brown nuclei in the lower glandular area. This could mean that the lesions observed in the WT- mice only precede lesions in TG- mice. Has this 
been tested? Are the injured areas 12h after administration of ethanol (fig. 2C) significantly different 
in WT- and TG- mice? Is there any difference after a longer period of time? 
9.) In figure 3 it is not stated at which time point after administration of ethanol the blood flow has 
been evaluated. How was the blood flow during the other time points evaluated? 
10.) In figure 5 Western Blot data for COX-2 expression are given. Firstly, also longer time points 
should also be given. Secondly, for normalization the authors used actin. It would be appreciable if 
all lanes would have run on one blot. Secondly, the blots of COX-2 do not seem to fit to the blots of 
the actin since the COX-2 blots are not even and the distance of the lanes are not the same. The original blots should be given at least to the editors. Also the Blots presented for WT mice in figure 7A for Bcl-2 and Bax do not seem to fit to the same original Blot. However here additional data for 
12h and even longer periods of time (12h, 24h) would be appreciable. 
11.) In figure 7C Caspase 3 data are given. Firstly, again longer time periods should be given. 
Secondly, since for both, detection of procaspase3 and active caspase 3 the same antibody is used, the lanes presented should be from the same blot. This should be presented and not single lanes. 
Above that normalization on actin should also be presented. 
12.) In figure 8A and 8B additional questions occur. Normally. actin is expressed in very high levels. 
Why is a background level visible in actin but not in the HSP70, COX-2 and Bcl-2 lanes? And why 
have the lanes for HSP70, COX-2 and Bcl-2 a different, more curved appearance compared to the 
actin (fig 8A)? Are these from the same blot. This has to be stated. In figure 8B the actin lanes show 
rectangular pixels seeming that a higher resolution has been used. However especially the bands for 
Cox-2 show backgrounds which are sharpely interrupted to the others. This is strange. More 
convincing Blots should be given. 
13.) There are questions about the cell line used. The GSM06 cell line is a SV40 large T- antigen 
transformed cell line. Why has this cell line been used? This cell line is known, that dependent on 
furin expression, which is dependant on the culture temperature, exhibits an immature state of 
surface mucous cells whereas suppression of furin induces a differentiated penotype. Therefore characteization of the cell used should be presented. Have other temeperatures been investigated? In addition concentration dependency studies as well as negative controls for the experiments using 
specific inhibitors are missing. 
Taken together, the study by Kosone et al. is interesting but leaves many methodical questions to be 
answered. Furthermore blocking experiments using anti- human TGF-a- antibodies or human TGF-a- antisenese- and/or siRNA- techniques would help to significantly improve the authors´ suggestions. In the current version especially the data about regulation of the intracellular defense 
mechanisms are rather descriptive and should be supported by more convincing data e.c. by using 
different concentrations of the inhibitors and primary isolation of different cell types in their models. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the 
author can be trusted to correct) 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
Reviewer's report 
 Integrative roles of transforming growth factor-alpha in the cytoprotection mechanisms of Title: 
gastric mucosal injury 
 1  29 March 2006 Version: Date: 
 Giuliano Ramadori Reviewer: 
Reviewer's report: 
General 
Kosone et al. studied effect of TGF-a on protection against gastric mucosal injury in a TGF-a- expressing mouse line on a CD1 background. The authors stated that this mouse line, when compared to a mouse line on a FVB background do not show hypertrophic gastropathy. In their 
model the authors studied the effect of ethanol on induction of gastric injury. As main result the 
authors found that TGF-a expressing mice maintains gastric mucosal defense against gastric injury induced by ethanol. 
Concerns: 
1.) For induction of gastric injury the authors administrated acidified ethanol. Firstly it is not stated 
how ethanol was administrated. Secondly it is unclear whether or how controls where performed. 
2.) It is unclear whether or how negative controls for their immunostainings and the TUNEL assay 
have been performed. 
3.) For their cell culture studies the authors added 5-100ng/ml TGF-a. However their cell culture 
medium contains 10% FCS. Firstly it is unknown how much TGF-a is already present in their culture 
medium. Secondly FCS could be capable for binding of TGF-a. Therefore real TGF-a concentration 
in their culture medium is unknown. The authors are encouraged to reduce FCS- concentration in their culture medium for their experiments. 
4.) It would be appreciable to state the number of independent experiments used for statistical 
analysis. 
5.) In figure 1B immunostainings are presented. Are these stainings of TGF-a-transgenic mice? This 
should be stated. In addition presentation of wild type mice would be interesting to give an 
impression of TGF-a expression in these mice. 
6.) The panels in figure 1E are too small. Bigger panels with a higher resolution should be 
presented. However it is unclear why in single stainings in the left panel of the TG mice a yellow 
color appears when this is only an FITC- staining and why the white dots in the middle staining of the 
TG mice appear when it is only a single staining with Cy3. This should be explained. 
7.) As noted in point 1 controls are missing. It is unclear whether the diluent of the ethanol or 
personal stress of the mice induced by the administration itself already causes the lesions. In the middle and the lower left region of the right panel of figure 2A also small lesions are visible. Are 
these as extensive as in those visible in the left panel? In figure 2C statistical analysis and number 
of independent experiments are missing. 
8.) Figure 2D is not convincing. TUNEL data are presented. The panels are too small to provide a 
good impression. However there are also many dark brown nuclei in the lower glandular area. This could mean that the lesions observed in the WT- mice only precede lesions in TG- mice. Has this 
been tested? Are the injured areas 12h after administration of ethanol (fig. 2C) significantly different 
in WT- and TG- mice? Is there any difference after a longer period of time? 
9.) In figure 3 it is not stated at which time point after administration of ethanol the blood flow has 
been evaluated. How was the blood flow during the other time points evaluated? 
10.) In figure 5 Western Blot data for COX-2 expression are given. Firstly, also longer time points 
should also be given. Secondly, for normalization the authors used actin. It would be appreciable if 
all lanes would have run on one blot. Secondly, the blots of COX-2 do not seem to fit to the blots of 
the actin since the COX-2 blots are not even and the distance of the lanes are not the same. The original blots should be given at least to the editors. Also the Blots presented for WT mice in figure 7A for Bcl-2 and Bax do not seem to fit to the same original Blot. However here additional data for 
12h and even longer periods of time (12h, 24h) would be appreciable. 
11.) In figure 7C Caspase 3 data are given. Firstly, again longer time periods should be given. 
Secondly, since for both, detection of procaspase3 and active caspase 3 the same antibody is used, the lanes presented should be from the same blot. This should be presented and not single lanes. 
Above that normalization on actin should also be presented. 
12.) In figure 8A and 8B additional questions occur. Normally. actin is expressed in very high levels. 
Why is a background level visible in actin but not in the HSP70, COX-2 and Bcl-2 lanes? And why 
have the lanes for HSP70, COX-2 and Bcl-2 a different, more curved appearance compared to the 
actin (fig 8A)? Are these from the same blot. This has to be stated. In figure 8B the actin lanes show 
rectangular pixels seeming that a higher resolution has been used. However especially the bands for 
Cox-2 show backgrounds which are sharpely interrupted to the others. This is strange. More 
convincing Blots should be given. 
13.) There are questions about the cell line used. The GSM06 cell line is a SV40 large T- antigen 
transformed cell line. Why has this cell line been used? This cell line is known, that dependent on 
furin expression, which is dependant on the culture temperature, exhibits an immature state of 
surface mucous cells whereas suppression of furin induces a differentiated penotype. Therefore characteization of the cell used should be presented. Have other temeperatures been investigated? In addition concentration dependency studies as well as negative controls for the experiments using 
specific inhibitors are missing. 
Taken together, the study by Kosone et al. is interesting but leaves many methodical questions to be 
answered. Furthermore blocking experiments using anti- human TGF-a- antibodies or human TGF-a- antisenese- and/or siRNA- techniques would help to significantly improve the authors´ suggestions. In the current version especially the data about regulation of the intracellular defense 
mechanisms are rather descriptive and should be supported by more convincing data e.c. by using 
different concentrations of the inhibitors and primary isolation of different cell types in their models. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the 
author can be trusted to correct) 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
Please review this paper.
user
Reviewer's report 
 Integrative roles of transforming growth factor-alpha in the cytoprotection mechanisms of Title: 
gastric mucosal injury 
 1  29 March 2006 Version: Date: 
 Giuliano Ramadori Reviewer: 
Reviewer's report: 
General 
Kosone et al. studied effect of TGF-a on protection against gastric mucosal injury in a TGF-a- expressing mouse line on a CD1 background. The authors stated that this mouse line, when compared to a mouse line on a FVB background do not show hypertrophic gastropathy. In their 
model the authors studied the effect of ethanol on induction of gastric injury. As main result the 
authors found that TGF-a expressing mice maintains gastric mucosal defense against gastric injury induced by ethanol. 
Concerns: 
1.) For induction of gastric injury the authors administrated acidified ethanol. Firstly it is not stated 
how ethanol was administrated. Secondly it is unclear whether or how controls where performed. 
2.) It is unclear whether or how negative controls for their immunostainings and the TUNEL assay 
have been performed. 
3.) For their cell culture studies the authors added 5-100ng/ml TGF-a. However their cell culture 
medium contains 10% FCS. Firstly it is unknown how much TGF-a is already present in their culture 
medium. Secondly FCS could be capable for binding of TGF-a. Therefore real TGF-a concentration 
in their culture medium is unknown. The authors are encouraged to reduce FCS- concentration in their culture medium for their experiments. 
4.) It would be appreciable to state the number of independent experiments used for statistical 
analysis. 
5.) In figure 1B immunostainings are presented. Are these stainings of TGF-a-transgenic mice? This 
should be stated. In addition presentation of wild type mice would be interesting to give an 
impression of TGF-a expression in these mice. 
6.) The panels in figure 1E are too small. Bigger panels with a higher resolution should be 
presented. However it is unclear why in single stainings in the left panel of the TG mice a yellow 
color appears when this is only an FITC- staining and why the white dots in the middle staining of the 
TG mice appear when it is only a single staining with Cy3. This should be explained. 
7.) As noted in point 1 controls are missing. It is unclear whether the diluent of the ethanol or 
personal stress of the mice induced by the administration itself already causes the lesions. In the middle and the lower left region of the right panel of figure 2A also small lesions are visible. Are 
these as extensive as in those visible in the left panel? In figure 2C statistical analysis and number 
of independent experiments are missing. 
8.) Figure 2D is not convincing. TUNEL data are presented. The panels are too small to provide a 
good impression. However there are also many dark brown nuclei in the lower glandular area. This could mean that the lesions observed in the WT- mice only precede lesions in TG- mice. Has this 
been tested? Are the injured areas 12h after administration of ethanol (fig. 2C) significantly different 
in WT- and TG- mice? Is there any difference after a longer period of time? 
9.) In figure 3 it is not stated at which time point after administration of ethanol the blood flow has 
been evaluated. How was the blood flow during the other time points evaluated? 
10.) In figure 5 Western Blot data for COX-2 expression are given. Firstly, also longer time points 
should also be given. Secondly, for normalization the authors used actin. It would be appreciable if 
all lanes would have run on one blot. Secondly, the blots of COX-2 do not seem to fit to the blots of 
the actin since the COX-2 blots are not even and the distance of the lanes are not the same. The original blots should be given at least to the editors. Also the Blots presented for WT mice in figure 7A for Bcl-2 and Bax do not seem to fit to the same original Blot. However here additional data for 
12h and even longer periods of time (12h, 24h) would be appreciable. 
11.) In figure 7C Caspase 3 data are given. Firstly, again longer time periods should be given. 
Secondly, since for both, detection of procaspase3 and active caspase 3 the same antibody is used, the lanes presented should be from the same blot. This should be presented and not single lanes. 
Above that normalization on actin should also be presented. 
12.) In figure 8A and 8B additional questions occur. Normally. actin is expressed in very high levels. 
Why is a background level visible in actin but not in the HSP70, COX-2 and Bcl-2 lanes? And why 
have the lanes for HSP70, COX-2 and Bcl-2 a different, more curved appearance compared to the 
actin (fig 8A)? Are these from the same blot. This has to be stated. In figure 8B the actin lanes show 
rectangular pixels seeming that a higher resolution has been used. However especially the bands for 
Cox-2 show backgrounds which are sharpely interrupted to the others. This is strange. More 
convincing Blots should be given. 
13.) There are questions about the cell line used. The GSM06 cell line is a SV40 large T- antigen 
transformed cell line. Why has this cell line been used? This cell line is known, that dependent on 
furin expression, which is dependant on the culture temperature, exhibits an immature state of 
surface mucous cells whereas suppression of furin induces a differentiated penotype. Therefore characteization of the cell used should be presented. Have other temeperatures been investigated? In addition concentration dependency studies as well as negative controls for the experiments using 
specific inhibitors are missing. 
Taken together, the study by Kosone et al. is interesting but leaves many methodical questions to be 
answered. Furthermore blocking experiments using anti- human TGF-a- antibodies or human TGF-a- antisenese- and/or siRNA- techniques would help to significantly improve the authors´ suggestions. In the current version especially the data about regulation of the intracellular defense 
mechanisms are rather descriptive and should be supported by more convincing data e.c. by using 
different concentrations of the inhibitors and primary isolation of different cell types in their models. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the 
author can be trusted to correct) 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
assistant