Review Reports - Hydrodynamic Delivery of IL-10 Gene for Local Immunomodulation in Human Crohn’s Disease Tissue: A Proof-of-Concept Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The present study is of great interest, bringing into attention the application of hydrodynamic gene transfer for localized delivery of naked plasmid bearing hIL-10 to colon segments affected by Crohn’s disease, as a continuation and deepening of previous studies. The purpose of this technique is to obtain a better modulation of localized IL-10 expression and inflammatory mediators, given the limitations of systemic IL-10 therapy. The authors addressed a current topic, with particular clinical importance, considering that IBD has witnessed a marked increase in its incidence over the last two decades, mainly but not only in Western countries. Overall, the manuscript is scientifically sound and presented in a well-structured manner. Nevertheless, the following issues should be addressed:

In the lines 157-158 there is a classification “proximal (A), intermediate (B), and distal (C)”. What is the relevance of this classification, because it can not be found in Results section
In lines 187-188 one colon segment (n=1) was injected with gold particles. Is it one of the twelve probes? It is not clear
In the Results section, the images from Figures 1 and 2 should be numbered or noted (1,2,3 or A,B,C) for ease of tracking.
Sections 3.3, 3.4 and 3.5 should be expanded and better described.
It is not well described what does exactly “control“ mean.

Author Response

In the lines 157-158 there is a classification “proximal (A), intermediate (B), and distal (C)”. What is the relevance of this classification, because it can not be found in Results section

The reviewer is right. We indicated it to inform about the tributary area of the vessel selected for gene transfer to distinguish the influence of proximity. We have removed it within the new version of the manuscript to avoid confusion.

In lines 187-188 one colon segment (n=1) was injected with gold particles. Is it one of the twelve probes? It is not clear

According to this comment, we have modified along the manuscript to inform that the segment with gold nanoparticles injection was one of those 12 segments employed.

In the Results section, the images from Figures 1 and 2 should be numbered or noted (1,2,3 or A,B,C) for ease of tracking.

We appreciate the reviewer’s comment. We have introduced the suggested changes within the new version of the manuscript.

Sections 3.3, 3.4 and 3.5 should be expanded and better described.

Thanks for the suggestion. We have introduced some changes accordingly.

It is not well described what does exactly “control“ mean.

According to the reviewer’s comment, we have introduced the following sentence in the material and methods section within the new version of the manuscript: were divided into two groups: control and treated (artery and vein). The control samples were those ob-tained from the ends of the segments, in regions without perfusion due to vascular ex-clusion mediated by clamps and confirmed by X-rays. All samples

Reviewer 2 Report

Comments and Suggestions for Authors

Sendra et al. conducted an important experiment to explore the use of hydrodynamic delivery of IL10 in colon samples from Crohn’s disease patients. This ex vivo experiment is part of a series of experiments the authors conducted to test this technique. The authors have demonstrated that IL10 levels can be increased while proinflammatory cytokine levels can be decreased in colon samples from patients with Crohn’s disease.

The following are the comments to improve this manuscript:

The authors published a similar study in 2017 with 19 patient samples. In that study, they obtained written consent from patients, which was waived in this study. This inconsistency is unclear despite their statement that consent is not required for these samples because they will be discarded.

In the introduction section, the paragraphs starting at L93 need to be rewritten, as the statements are not presented in an orderly manner.

The authors mentioned that they used the ProcartaPlex™ Porcine Cytokine & Chemokine Panel 1, 9plex to measure secretion of cytokines. Is this panel for human cytokine secretion? Since there are 9 cytokines in this panel, did the authors analyze all of them?

With the exception of IL6, the decrease in other cytokine levels followed a similar trend in control and treated experiments. Was this due to the ex vivo experimental condition?

The authors reported IL10 mRNA expression as mRNA copies per cell. How the authors performed this experiment is not clearly stated. Did the authors isolate the cells and perform this experiment? Please make it clear.

IL10 protein levels on day 1 appear to be lower in secretion from vein-injected tissue samples compared to controls. What could be the reason for this? How many samples were used in each of these experiments? Please provide n values for each experiment reported in this manuscript. Also, it is unclear whether the IL10 secretions were significantly higher, as the data were presented on a log scale without providing p-values.

The authors did not provide a statistical section. Hence, it is difficult to determine which statistical tests were applied and whether the authors considered the repeated-measures nature of the experiments in their analysis.

In several places, "colonic" was written as "colic". Please fix them all.

Author Response

The following are the comments to improve this manuscript:

The reviewer is right, we have checked it and patients did sign general informed consent in which they authorize the use of their sample for research purposes, without specifying the project. We have included the following sentence within the revised version of the manuscript: The Declaration of Helsinki protocols were followed, and patients gave their written informed consent. This research was approved by the clinical research ethics Committee of the University Hospital La Fe, Valencia (Spain).

In the introduction section, the paragraphs starting at L93 need to be rewritten, as the statements are not presented in an orderly manner.

We truly appreciate the reviewer’s suggestion. We have re-phrased and re-structured part of the introduction section in the revised version of the manuscript accordingly. We are sure this change improves the quality of this work.

We are so sorry, we dragged this information from another document and we did not realize. We used another panel in this work, the Cytokine 25-Plex Human Panel (Invitrogen, ThermoFisher scientific, Waltham, MA, USA; Cat. No. LHC0009M). The 25 cytokines or chemokines were simultaneously quantified, but we focused on those affecting inflammatory processes and among these, those that proved differences between treated and control samples. We have modified it within the new version of the manuscript.

With the exception of IL6, the decrease in other cytokine levels followed a similar trend in control and treated experiments. Was this due to the ex vivo experimental condition?

This is an interesting question. We agree with the reviewer that this may be due to the ex vivo experimental conditions, but we also believe that an inflammatory status is already established in the sample due to the pathological process affecting the patient, which is difficult to reverse. We have included the following sentence within the revised version of the manuscript to clarify this point: Although the concentrations are different, we observe that control and treated samples yield similar trend in cytokines expression. We do not know the exact reason but we cannot exclude that this could be due to the ex vivo conditions of our experiments.

The reviewer is right. We expressed the transcription of hIL-10 plasmid as copies per cell, but we did not isolate the cells. We made this calculation by assuming that regular mRNA content in a mammalian cell is estimated to be 20 pg [Alberts B (2002)]. This allows us deduce the number of cells from which the mRNA proceeds. We have included this clarification within the new version of the manuscript: The data were expressed as copies per cell considering the average content of RNA per cell in a mammalian cell (20 pg) for gene transcription.

This is an interesting question, we think that the reason for the decrease in protein concentration could be due to the first washing effect mediated by the gene solution injection. We have included the following sentence in the revised version of the manuscript to clarify this point: The levels of IL10 protein observed in venous treated samples were lower than in control on day 1. We believe that the reason for the decrease in protein concentration could be due to the first washing effect mediated by the gene solution injection. We do not know the exact reason why we do not observed that result when we use the arterial route of administration but we guess that this could be the limited flow rate in arterial route or the higher efficacy in gene transfer when employing it.

We agree with the reviewer in the relevance of including n values for each experiment. We have modified the text accordingly.

We have modified the axis in the new version of the manuscript aiming at offering an easier observation of data. Despite the different levels observed, no statistical difference was found. This may be due to the data dispersion.

The reviewer is right, we have included this section in the revised version of the manuscript. The repeated measures nature of the experiments was considered.

In several places, "colonic" was written as "colic". Please fix them all.

The reviewer is right. We are sorry for the mistake. We have corrected it accordingly.