Inflammation and Liver Cell Death in Patients with Hepatitis C Viral Infection
Round 1
Reviewer 1 Report
This paper is well designed and discussed.
The authors reported the mechanisms of hepatitis C viral infection and they focused on not only immunological reactions but inflammatory mediators using blood samples from HCV patients. In addition, the schima is nice.
Author Response
Thank you very much for reviewing our paper.
Reviewer 2 Report
In this article, the authors examine levels of cytokines and apoptotic proteins in the sera of patients with Hepatitis C viral (HCV) infection and compare their results with a control cohort.
The authors claim that their data reveal the inflammatory status in HCV and in HCV disease kinetics.
Unfortunately, several claims regarding what their paper shows do not appear to be supported by the data currently shown.
Perhaps the most important point is that the aim of the paper is a little unclear.
Many authors have examined serum cytokines in HCV. It has been known for over 20 years that e.g., serum levels of IL10 are elevated in HCV.
Is the paper to compare this cohort with the others that have been published? If so, then how does this cohort compare with the cohorts that have been published? How do their data enable personalised medicine? How do their data support clinical decisions?
Abstract
Requires extensive rewriting to reflect the data shown.
The abstract claims that the paper "discusses the potential role of biomarkers in monitoring cell death due to inflammatory cascade leading to the severity of liver damage." (page 1, lines 20-22) This claim is not fair: No data has been provided on inflammatory markers based upon disease stage. No data has been provided correlating inflammatory markers with liver biopsy analysis - another claim made in page 1, line 23-24.
Furthermore, a biomarker is a marker that changes with disease status. No data is provided that could be used as a biomarker as the patient group is analysed as a group. For example, Metavir F2 patients could potentially be compared with Metavir F3 patients.
The abstract states that many TEM changes were observed in biopsies from HCV patients. How representative are these data? No comparison with previously published data are provided for context and no control data are shown.
Introduction
The introduction is too long. It should be shortened to make it more focused on the aim of the paper.
Figure 1.
Please revise this figure extensively to clarify it or please delete.
Mf@ does not appear to be present.
Please provide a legend to the cartoons.
"Transmigration" is not clear.
The star on the right of the normal hepatocyte is not meaningful as presented.
TGF-b - please use consistent "beta" versus "b".
Aims
1 - not shown as no association data was provided.
The paper aims to improve quality indicators for clinical indicators - however, no sensitivity, specificity or test-retest data are provided with respect to disease (although some sensitivity and specificity data are discussed relative to particular ELISAs (see below)). Similarly, if the authors aim is to improve collaboration between clinicians and laboratory specialists, then I would encourage the authors to provide evidence to show how they achieved this goal.
Methods
Patient and control epidemiological data should be provided in results.
Please clarify alcohol consumption (page 5, line 194 versus 202).
Please clarify sensitivity and specificity of ELISAs (page 5 line 225 versus line 227).
Histological analysis - This section appears repetitive. Please edit to provide more clear methods for processing tissue, light microscopy and EM.
Results
Table 1 - please confirm samples (serum?).
Please explain the numbers in parentheses in the column titles. "N=280" (e.g.,) is more typical when describing group sizes.
Figure 2 please provide the Y axis title. Please remove the red colour from between control and HCV data in the TNF column.
All EM images - please annotate to show precisely each item of interest (e.g., rough ER). Only one mitochondrion is shown in Figure 4. Please provide control images.
Discussion
Requires extensive re-writing to reflect the data that were actually shown in the article.
For example, no data is provided on correlations to pathological data, to disease status, to age, to BMI etc. The data that are provided are therefore not strictly biomarkers (page 12, line 408).
Moreover, these data do not enable personalised medicine as no correlations to disease status are provided (page 12, line 408-409).
Please delete page 12, lines 411-413 as these data have not been shown.
Small point
I would recommend editing sentence structures for English. Please also do a spell check.
Author Response
Please check the attachment.
Author Response File: 
Author Response.pdf
Round 2
Reviewer 2 Report
Unfortunately, the authors have failed to address several points in the original review. Please see some examples below.
E.g., The paragraph at the end of the current Discussion states: "We described the immunoregulaory (sic) events, the T helper response profile (Th1, Th2) 440 and the different inflamasome (sic) and apoptosome profiles associated with different stages
of liver disease severity can be observed throughout the evolution of symptoms".
Unfortunately, I again note that no such data has been provided because the patients have not been grouped based upon disease progression. This is important because the authors state in the Discussion "By comparing these levels we show the importance of
using non-invasive biomarkers in monitoring the disease progression."
Finally, a new sentence at the end of the current Discussion states "The clinician understands the importance of using the specific biomarkers." Again, as no biomarkers of disease progression have been demonstrated this sentence is at odds with demonstrated results.
E.g., In their response, the authors state "We grouped the 2 fibrosis states in one" but an explanation as to how this enables analysis of disease progression is not apparent.
E.g., Table 3 - the authors' response states "We introduce the requested data (table 3)". This statement does not appear to be accurate as this table appears to be of correlations of pathological data with ELISA data in patients.
E.g., Amended manuscript, Fig 01: It is not clear which figure is the final version. Mf remains in the figure, Mf@ remains in the legend.
E.g., Response of the authors was "The images have been adnotated (sic) as requested." However, I note that none of the EM photos have been annotated to show items discussed in the text - this was requested in the original review.
E.g., Authors provided no response to this question in the original review "Please clarify alcohol consumption (page 5, line 194 versus 202)." It seems that the exclusion criteria was no alcohol consumption?
E.g., Authors provide no response to this question: Please clarify sensitivity and specificity of ELISAs (page 5 line 225 versus line 227).
E.g., Authors provide no response to this point in the original review "Discussion Requires extensive re-writing to reflect the data that were actually shown in the article."
Author Response
My reply is in italics (MN)..
"Unfortunately, the authors have failed to address several points in the original review. Please see some examples below.
E.g., The paragraph at the end of the current Discussion states: "We described the immunoregulaory (sic) events, the T helper response profile (Th1, Th2) 440 and the different inflamasome (sic) and apoptosome profiles associated with different stages of liver disease severity can be observed throughout the evolution of symptoms".
Unfortunately, I again note that no such data has been provided because the patients have not been grouped based upon disease progression. This is important because the authors state in the Discussion "By comparing these levels we show the importance of using non-invasive biomarkers in monitoring the disease progression."
Finally, a new sentence at the end of the current Discussion states "The clinician understands the importance of using the specific biomarkers." Again, as no biomarkers of disease progression have been demonstrated this sentence is at odds with demonstrated results.
E.g., In their response, the authors state "We grouped the 2 fibrosis states in one" but an explanation as to how this enables analysis of disease progression is not apparent."
Thank you for reviewing the revised manuscript. You are right in order to make a comparison with a different stage of the disease we introduce a group of cirrhotic patients with the same genotype 1 HCV. The patients have been treated by the same clinician and I performed all the analysis stated in the material and methods. In all the tables I introduced the biomarkers for the new group of 25 cirrhotic HCV. I made the statistic analysis versus controls and versus HCV cirrhotic.
I thank you for asking me to provide the biomarker differences between the severity of the liver injury.
E.g., Table 3 - the authors' response states
"We introduce the requested data (table 3)".
This statement does not appear to be accurate as this table appears to be of correlations of pathological data with ELISA data in patients.
It is true, I interpreted incorrectly your request. I deleated table 3.
E.g., Amended manuscript, Fig 01: It is not clear which figure is the final version. Mf remains in the figure, Mf@ remains in the legend.
Mf - It is in the figure and in the legend
E.g., Response of the authors was "The images have been adnotated (sic) as requested." However, I note that none of the EM photos have been annotated to show items discussed in the text - this was requested in the original review.
Please look at the new TEM figures 3,4,5
E.g., Authors provided no response to this question in the original review "Please clarify alcohol consumption (page 5, line 194 versus 202)." It seems that the exclusion criteria was no alcohol consumption?
In the original and in the revised forms it is clearly stated that alcohol and drugs of misuse are excluded
E.g., Authors provide no response to this question: Please clarify sensitivity and specificity of ELISAs (page 5 line 225 versus line 227).
We corrected the statement.
E.g., Authors provide no response to this point in the original review "Discussion Requires extensive re-writing to reflect the data that were actually shown in the article."
We change the discussion as suggested
Round 3
Reviewer 2 Report
Please amend Fig 1, which is unacceptable in its current form.
Please delete the irrelevant figure in Fig 2.
Please insert data on HCV-infected patients that were not cirrhotic into the Methods.
The mitochondrion shown in Fig 4 shows no double outer membrane or cristae. Please provide the definition criteria for EM analysis of mitochondria versus members of the autophagic compartment.
Discussion lines 371-372, the authors state "By comparing these levels we show the importance of using non-invasive biomarkers in monitoring the disease progression."
The authors provide no discussion on the differences in TNF, IFN, RANTES and NFKB between HCV and HCV cirrhotics (Fig 2). It seems that the only large difference is with VEGF (although no variance is provided). Please discuss in the discussion (e.g., the statistical power of these outcome measures to monitor disease progression). Please also insert variances into Fig 2.
Author Response
Please amend Fig 1, which is unacceptable in its current form.
I agree that the form was presented to you is unacceptable. However in the PDF uploaded to the journal the figure was well preserved.
See it in the new version too.
We wanted to delete Figure 2 but at the end you asked to keep It and to add the standard deviation in the legend
Please insert data on HCV-infected patients that were not cirrhotic into the Methods.
We inserted the requested data
The mitochondrion shown in Fig 4 shows no double outer membrane or cristae.
In the figure 4 the noted is a mithochondrion that lost the double membrane and the cristae are only seen in the remaining membrane of the organelle. We explained in the legend.
Please provide the definition criteria for EM analysis of mitochondria versus members of the autophagic compartment.
We did not introduce the aspect of autophagy in this specific paper. We describe specifically autophagy in other papers..
Discussion lines 371-372, the authors state
"By comparing these levels we show the importance of using non-invasive biomarkers in monitoring the disease progression."
The authors provide no discussion on the differences in TNF, IFN, RANTES and NFKB between HCV and HCV cirrhotics (Fig 2). It seems that the only large difference is with VEGF (although no variance is provided).
"Please discuss in the discussion (e.g., the statistical power of these outcome measures to monitor disease progression).""
We discussed.
"Please also insert variances into Fig 2.
We inserted the variances in the legend
Round 4
Reviewer 2 Report
No comments
