Reviewer's report
Title:Liver fibrosis secondary to bile duct injury: correlation of Smad7 with TGF-b
(beta)and fibrotic proteins
Version: 2Date:15 April 2009
Reviewer: Ralf Weiskirchen
Reviewer's report:
General:
The authors analysed if Smad7 gene expression correlates with expression of
TGF-beta, collagen (type I, III and IV) and PAI-1 in patients suffering from liver
fibrosis secondary to bile duct injury. Therefore, quantitative real time PCR was
performed for respective genes and TGF-beta serum concentration were
measured in fourteen patients with bile duct injury and four controls.
In summary, the authors found that TGF-beta concentrations were higher in
patients with bile duct injury and that Smad7 mRNA expression correlated with
expression of TGF-beta, collagen type I/III and the amount of hepatic fibrosis as
determined in morphometric analysis.
Major Compulsory Revisions
• The authors measured serum TGF-beta concentration with a commercially
available ELISA kit system following the manufacturer’s protocol. Using this
technique the authors determined mean concentrations of 39296 pg/mL
(patients) and 9008 pg/mL (controls). There are several critical issues that should
be mentioned: (i) It is well known that the measurement of TGF-beta in blood
samples is a complex problem (for critical review see Grainger et al., Cytokine
Growth Factor Rev 11 (2000):133-145). In particular there are several
pre-analytical requirements on the samples that must be fulfilled to allow
appropriate measurements. Based on the statements (see page 6), the samples
from the patients and control subjects were recruited from different locations.
Therefore, it is hardly to believe that the blood samples are comparable. (ii)
Moreover, the given absolute concentrations for the controls are somewhat
doubtful. According to the sample values provided by R&D (see R&d data sheet),
the mean concentrations of TGF-beta in 23 samples was found to be 39592
pg/mL. In contrast the mean value of the four controls was determined to 9008
pg/mL in the present study. (iii) It is generally known that the expression and
serum concentration of TGF-beta is associated with several non-coding
(-509C/T) and coding gene polymorphisms (codon 10, codon 25, codon 263).
Therefore, it might be possible that the observed differences in TGF-beta
concentrations are simply reflecting gene polymorphisms rather than alteration
that are induced by bile duct injury. Please critically comment on all three issues.
• The authors stated “Dooley et al. and Tahashi et al demonstrated that Smad7 is
not expressed in experimental liver fibrosis, and this event allows liver scar
formation” (page 5). In my view the opposite is stated in the mentioned paper of
Dooley and co-workers. He stated that “Expression levels of endogenous Smad7
mRNA were low in AdSmad7-infected and control livers”. Please correct
respective text passage. In addition, low expression of Smad7 is not the reason
for development of liver fibrosis.
• Commonly, stage and grade of hepatic injury are given in well-accepted scores
(METAVIR; Desmet Scheuer etc.). In the present study the authors used %
fibrosis. This morphometric analysis is somewhat obscure. Would it be possible
to provide more reliable data on hepatic lesions than the morphometric analysis?
• Abstract: “The 6-fold increase in Smad7 expression was not enough to inhibit
the expression of TGF-beta, collagens, and PAI-1”. Smad7 is known to interfere
with the activity (phosphorylation) of Smad2 and Smad3. Therefore, it would be
more interesting to analyse the status of Smad phosphorylation than the
expression of collagen and PAI-1 to predict Smad7 activity.
Minor Essential Revisions
• Terminology: There are several phrases that are scientifically unusual or even
misleading (e.g. “fibrotic proteins” in the title; “Liver fibrosis percentage “ on page
6; “collagen surface density” on page 7). Please rephrase respective passages.
• Relative quantification (page 8): The authors provided relative expression units
(REU) for the different genes. These values were normalized to the content of
18S rRNA. However, a clear statement of what is set to 1 is missing. Was it the
mean of the four individual controls or was it the lowest expression in one of the
controls that was set to 1? Please add a short comment.
• It must read 18S rRNA instead of 18S RNA on page 8 and in Table 1.
• page 10: The authors classified the patients with the Bismuth classification
method. One patient was staged to type 1, two patients to type 2 and eleven to
type 3. It makes no sense to provide the values in percentages when only 14
patients were classified. Please remove the values 7.14, 14.28 and 78.57% and
provide number of cases.
• The speculation “This might indicate that the type or severity of BDI does not
influence the liver fibrosis process” (page 14) is over-interpreted. The authors
analysed only 14 patients which is far beyond to allow any general conclusion.
• The statement “… or through a TGF-beta-independent pathway, such as
connective tissue growth factor (CTGF)….” (page 14) is scientifically wrong. It is
general known that CTGF is a target of TGF-beta in most cells and, moreover, a
protein that stimulates and enhances TGF-beta signalling by increasing the
affinity of TGF-beta for the type II receptor. Please rephrase the sentence
accordingly.
• Please provide a reference for the statement “…. and the posttransductional
regulation is mediated through the effects of Smurf and Jab1 proteins, which
promote Smad7 degradation” (page 14).
• Please combine the two parts of Figure 1 into one Figure (lettered by A and B).
• Legend to Figure 4: Col II must read Col I
• Table 2: (i) BDI patient no. 10: What is the significance of the value 0.01 mg/dl
for direct bilirubin? (ii) What essentially is meant with the “term of lesion”?
Discretionary Revisions
no recommendations for improvement
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
Reviewer's report
Title:Liver fibrosis secondary to bile duct injury: correlation of Smad7 with TGF-b
(beta)and fibrotic proteins
Version: 2Date:15 April 2009
Reviewer: Ralf Weiskirchen
Reviewer's report:
General:
The authors analysed if Smad7 gene expression correlates with expression of
TGF-beta, collagen (type I, III and IV) and PAI-1 in patients suffering from liver
fibrosis secondary to bile duct injury. Therefore, quantitative real time PCR was
performed for respective genes and TGF-beta serum concentration were
measured in fourteen patients with bile duct injury and four controls.
In summary, the authors found that TGF-beta concentrations were higher in
patients with bile duct injury and that Smad7 mRNA expression correlated with
expression of TGF-beta, collagen type I/III and the amount of hepatic fibrosis as
determined in morphometric analysis.
Major Compulsory Revisions
• The authors measured serum TGF-beta concentration with a commercially
available ELISA kit system following the manufacturer’s protocol. Using this
technique the authors determined mean concentrations of 39296 pg/mL
(patients) and 9008 pg/mL (controls). There are several critical issues that should
be mentioned: (i) It is well known that the measurement of TGF-beta in blood
samples is a complex problem (for critical review see Grainger et al., Cytokine
Growth Factor Rev 11 (2000):133-145). In particular there are several
pre-analytical requirements on the samples that must be fulfilled to allow
appropriate measurements. Based on the statements (see page 6), the samples
from the patients and control subjects were recruited from different locations.
Therefore, it is hardly to believe that the blood samples are comparable. (ii)
Moreover, the given absolute concentrations for the controls are somewhat
doubtful. According to the sample values provided by R&D (see R&d data sheet),
the mean concentrations of TGF-beta in 23 samples was found to be 39592
pg/mL. In contrast the mean value of the four controls was determined to 9008
pg/mL in the present study. (iii) It is generally known that the expression and
serum concentration of TGF-beta is associated with several non-coding
(-509C/T) and coding gene polymorphisms (codon 10, codon 25, codon 263).
Therefore, it might be possible that the observed differences in TGF-beta
concentrations are simply reflecting gene polymorphisms rather than alteration
that are induced by bile duct injury. Please critically comment on all three issues.
• The authors stated “Dooley et al. and Tahashi et al demonstrated that Smad7 is
not expressed in experimental liver fibrosis, and this event allows liver scar
formation” (page 5). In my view the opposite is stated in the mentioned paper of
Dooley and co-workers. He stated that “Expression levels of endogenous Smad7
mRNA were low in AdSmad7-infected and control livers”. Please correct
respective text passage. In addition, low expression of Smad7 is not the reason
for development of liver fibrosis.
• Commonly, stage and grade of hepatic injury are given in well-accepted scores
(METAVIR; Desmet Scheuer etc.). In the present study the authors used %
fibrosis. This morphometric analysis is somewhat obscure. Would it be possible
to provide more reliable data on hepatic lesions than the morphometric analysis?
• Abstract: “The 6-fold increase in Smad7 expression was not enough to inhibit
the expression of TGF-beta, collagens, and PAI-1”. Smad7 is known to interfere
with the activity (phosphorylation) of Smad2 and Smad3. Therefore, it would be
more interesting to analyse the status of Smad phosphorylation than the
expression of collagen and PAI-1 to predict Smad7 activity.
Minor Essential Revisions
• Terminology: There are several phrases that are scientifically unusual or even
misleading (e.g. “fibrotic proteins” in the title; “Liver fibrosis percentage “ on page
6; “collagen surface density” on page 7). Please rephrase respective passages.
• Relative quantification (page 8): The authors provided relative expression units
(REU) for the different genes. These values were normalized to the content of
18S rRNA. However, a clear statement of what is set to 1 is missing. Was it the
mean of the four individual controls or was it the lowest expression in one of the
controls that was set to 1? Please add a short comment.
• It must read 18S rRNA instead of 18S RNA on page 8 and in Table 1.
• page 10: The authors classified the patients with the Bismuth classification
method. One patient was staged to type 1, two patients to type 2 and eleven to
type 3. It makes no sense to provide the values in percentages when only 14
patients were classified. Please remove the values 7.14, 14.28 and 78.57% and
provide number of cases.
• The speculation “This might indicate that the type or severity of BDI does not
influence the liver fibrosis process” (page 14) is over-interpreted. The authors
analysed only 14 patients which is far beyond to allow any general conclusion.
• The statement “… or through a TGF-beta-independent pathway, such as
connective tissue growth factor (CTGF)….” (page 14) is scientifically wrong. It is
general known that CTGF is a target of TGF-beta in most cells and, moreover, a
protein that stimulates and enhances TGF-beta signalling by increasing the
affinity of TGF-beta for the type II receptor. Please rephrase the sentence
accordingly.
• Please provide a reference for the statement “…. and the posttransductional
regulation is mediated through the effects of Smurf and Jab1 proteins, which
promote Smad7 degradation” (page 14).
• Please combine the two parts of Figure 1 into one Figure (lettered by A and B).
• Legend to Figure 4: Col II must read Col I
• Table 2: (i) BDI patient no. 10: What is the significance of the value 0.01 mg/dl
for direct bilirubin? (ii) What essentially is meant with the “term of lesion”?
Discretionary Revisions
no recommendations for improvement
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
Please review this paper.
user
Reviewer's report
Title:Liver fibrosis secondary to bile duct injury: correlation of Smad7 with TGF-b
(beta)and fibrotic proteins
Version: 2Date:15 April 2009
Reviewer: Ralf Weiskirchen
Reviewer's report:
General:
The authors analysed if Smad7 gene expression correlates with expression of
TGF-beta, collagen (type I, III and IV) and PAI-1 in patients suffering from liver
fibrosis secondary to bile duct injury. Therefore, quantitative real time PCR was
performed for respective genes and TGF-beta serum concentration were
measured in fourteen patients with bile duct injury and four controls.
In summary, the authors found that TGF-beta concentrations were higher in
patients with bile duct injury and that Smad7 mRNA expression correlated with
expression of TGF-beta, collagen type I/III and the amount of hepatic fibrosis as
determined in morphometric analysis.
Major Compulsory Revisions
• The authors measured serum TGF-beta concentration with a commercially
available ELISA kit system following the manufacturer’s protocol. Using this
technique the authors determined mean concentrations of 39296 pg/mL
(patients) and 9008 pg/mL (controls). There are several critical issues that should
be mentioned: (i) It is well known that the measurement of TGF-beta in blood
samples is a complex problem (for critical review see Grainger et al., Cytokine
Growth Factor Rev 11 (2000):133-145). In particular there are several
pre-analytical requirements on the samples that must be fulfilled to allow
appropriate measurements. Based on the statements (see page 6), the samples
from the patients and control subjects were recruited from different locations.
Therefore, it is hardly to believe that the blood samples are comparable. (ii)
Moreover, the given absolute concentrations for the controls are somewhat
doubtful. According to the sample values provided by R&D (see R&d data sheet),
the mean concentrations of TGF-beta in 23 samples was found to be 39592
pg/mL. In contrast the mean value of the four controls was determined to 9008
pg/mL in the present study. (iii) It is generally known that the expression and
serum concentration of TGF-beta is associated with several non-coding
(-509C/T) and coding gene polymorphisms (codon 10, codon 25, codon 263).
Therefore, it might be possible that the observed differences in TGF-beta
concentrations are simply reflecting gene polymorphisms rather than alteration
that are induced by bile duct injury. Please critically comment on all three issues.
• The authors stated “Dooley et al. and Tahashi et al demonstrated that Smad7 is
not expressed in experimental liver fibrosis, and this event allows liver scar
formation” (page 5). In my view the opposite is stated in the mentioned paper of
Dooley and co-workers. He stated that “Expression levels of endogenous Smad7
mRNA were low in AdSmad7-infected and control livers”. Please correct
respective text passage. In addition, low expression of Smad7 is not the reason
for development of liver fibrosis.
• Commonly, stage and grade of hepatic injury are given in well-accepted scores
(METAVIR; Desmet Scheuer etc.). In the present study the authors used %
fibrosis. This morphometric analysis is somewhat obscure. Would it be possible
to provide more reliable data on hepatic lesions than the morphometric analysis?
• Abstract: “The 6-fold increase in Smad7 expression was not enough to inhibit
the expression of TGF-beta, collagens, and PAI-1”. Smad7 is known to interfere
with the activity (phosphorylation) of Smad2 and Smad3. Therefore, it would be
more interesting to analyse the status of Smad phosphorylation than the
expression of collagen and PAI-1 to predict Smad7 activity.
Minor Essential Revisions
• Terminology: There are several phrases that are scientifically unusual or even
misleading (e.g. “fibrotic proteins” in the title; “Liver fibrosis percentage “ on page
6; “collagen surface density” on page 7). Please rephrase respective passages.
• Relative quantification (page 8): The authors provided relative expression units
(REU) for the different genes. These values were normalized to the content of
18S rRNA. However, a clear statement of what is set to 1 is missing. Was it the
mean of the four individual controls or was it the lowest expression in one of the
controls that was set to 1? Please add a short comment.
• It must read 18S rRNA instead of 18S RNA on page 8 and in Table 1.
• page 10: The authors classified the patients with the Bismuth classification
method. One patient was staged to type 1, two patients to type 2 and eleven to
type 3. It makes no sense to provide the values in percentages when only 14
patients were classified. Please remove the values 7.14, 14.28 and 78.57% and
provide number of cases.
• The speculation “This might indicate that the type or severity of BDI does not
influence the liver fibrosis process” (page 14) is over-interpreted. The authors
analysed only 14 patients which is far beyond to allow any general conclusion.
• The statement “… or through a TGF-beta-independent pathway, such as
connective tissue growth factor (CTGF)….” (page 14) is scientifically wrong. It is
general known that CTGF is a target of TGF-beta in most cells and, moreover, a
protein that stimulates and enhances TGF-beta signalling by increasing the
affinity of TGF-beta for the type II receptor. Please rephrase the sentence
accordingly.
• Please provide a reference for the statement “…. and the posttransductional
regulation is mediated through the effects of Smurf and Jab1 proteins, which
promote Smad7 degradation” (page 14).
• Please combine the two parts of Figure 1 into one Figure (lettered by A and B).
• Legend to Figure 4: Col II must read Col I
• Table 2: (i) BDI patient no. 10: What is the significance of the value 0.01 mg/dl
for direct bilirubin? (ii) What essentially is meant with the “term of lesion”?
Discretionary Revisions
no recommendations for improvement
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
assistant