Epidermal Protein C Levels Correspond to Local Injury Severity and Increased Clinical Support in Burn Patients

Round 1

Reviewer 1 Report

Epidermal protein C levels predict outcomes in burn patients

The aims to evaluate the expression of PC and its receptor (EPCR) in tissue biopsies with severe burns, which are met in the study.

The authors' experience with both patients and with protein C and different cytokines that participate in the inflammatory and repair process can be observed in the manuscript.

Table 1 shows 101% of the sum of all patients with biopsy (Female / Male)

Is the scale used in (0-3) shown in tables 2 and 3 validated by any entity?

If the frequency of the same is shown in figure 1, I think it is convenient to explain the content with the figure, since it is redundant.

What are the variables that were evaluated for the outcome of the burn and taken into account, it is necessary to specify this section within the methods, to justify the measurement of the outcome?

Author Response

please see file attached

Author Response File: Author Response.pdf

Reviewer 2 Report

This is a very interresting manuscript on PC and EPCR levels in burned skin. Interstingly, skin damage and even the severity of the skin damage was correletaed to EPCR-levels and negatively correlated to PC levels. Moreover, PC-levels were associated to patients requiering incresed support. The mnuscript is well written, the study as well as statistics are well performed. 

I have only two minor comments. 

1. I would recommend to modify the title. The title suggests that PC-levels predict the outcome of burn patients (survival). However, the authors only demonstrated an association of PC levels with the likelihood of "increased support" (more fluids, more surgeries, longer hoslitalization). The new titlw should reflect this issue.
2. Discussion (page 8, line 262/263): "We found that skin tissue PC acted in a similar manner to plasma PC and was directly predictive for clinical outcome" => s. above not for outcome but for the need increased clinical support. 

Author Response

please see file attached

Author Response File: Author Response.pdf

Reviewer 3 Report

The paper submitted by Zhao and colleagues is the first to investigate the impact of severe thermal injury on the tissue expression of protein C (PC), and its receptor, endothelial protein C receptor (EPCR). Analysis of 34 tissue biopsies revealed a significant burn-induced reduction in PC expression that was accompanied by increased expression of EPCR. CD68 staining on the same tissue samples, to examine macrophage infiltration, revealed a negative association between PC expression and CD68 positive staining. Interestingly, the authors found no correlations between the tissue expression of PC or EPCR and the levels of these proteins in plasma samples, suggesting different processes are occurring at the local and systemic level. Finally, the paper reported that high PC tissue expression was significantly associated with a decreased likelihood of requiring increased hospital support.

My specific comments are:

Introduction

(1) The word "a" needs to be added before the word "vitamin" - Line 31.

(2) The authors could expand upon the term "biomarkers" when discussing how PC/APC is a biomarker in many diseases. What outcomes are PC/APC biomarkers for? Line 40.

(3) Examples of cytokines/chemokines that comprised the "inflammatory milieu" described on line 48 should be included.

(4) The authors should state that the correlation they observed between early changes in PC/APC and injury severity was positive in nature - line 48.

(5) A more specific description of the "clinical outcome" discussed on line 50 should be provided.

Materials and Methods

(1) The 2-year period during which samples were collected should be stated.

(2)  Are the authors able to provide the ranges for the numbers that were used to score regions of tissue as being negative, low positive, positive and high positive for their PC and EPCR staining?

(3) Could the authors clarify why in the study design section of the manuscript it states that 38 patients consented to punch biopsies, but in Table 1 of the results section, the number of biopsied patients is stated as 34?

(4) Whilst, mentioned in the results section, greater detail on the statistical analyses performed would improve the manuscript. For example, which normality tests were used? I assume p<0.05 was used as the level at which statistical significance was accepted? For the logistic regression analysis, did the authors consider examining whether the relationships observed were independent of such co-variates as TBSA given that PC levels correlated negatively with %TBSA and higher PC levels were associated with less hospital support?

Results

(1) Information in Table 1 relating to the biopsied patients could be split into those that received increased hospital support and those that had a standard admission. This would be important for the logistic regression analysis section where the authors investigated whether PC levels were associated with a higher likelihood of increased hospital care. It would also allow for information relating to ICU LOS, volumes of fluid administered and number of surgical interventions to be presented. These are the 3 factors that make up their composite outcome termed "increased support" so it would be good for the reader to have data on this presented in the manuscript to show the degree of differences in these factors between the 2 patient groups. 

(2) The number of tissue sections analysed should be included in all figure legends.

(3) A description of Figure 3C should be included in the accompanying legend.

Author Response

please see documents attached

• point by point response
• tracked changes

thank you

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

My suggestions and comments were properly resolved, the manuscript contains relevant and novel information to be accepted.

Author Response

no new changes from Reviewer 3 comments altered changes from Reviewer 1

Reviewer 2 Report

Thank you very much for the revisions. The reviewers recommendations have been sufficiently adressed. 

Author Response

no new changes from Reviewer 3 comments altered changes from Reviewer 2

Reviewer 3 Report

The authors have addressed all of my comments. The greater detail added into the revised manuscript will assist the reader in understanding the patient cohort and the background to the study.

I have no additional points/comments to make.