An Elevated IL10 mRNA Combined with Lower TNFA mRNA Level in Active Rheumatoid Arthritis Peripheral Blood
, Viktoria Vasileva 3,4,*,†, Mariana Ivanova 5,6, Spaska Stanilova 3, Irena Manolova 3 and Lyuba Miteva 3Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsComments
1. Lines 107-109: “We analysed data from 31 patients diagnosed with rheumatoid arthritis who attended the Rheumatology Clinic of University Hospital “St. Ivan. Rilski” in Sofia compared to 21 healthy subjects.” – the fact that you tested 31 patients and 21 controls is a result, not a method. Nobody plans in their study to include exactly 52 cases, but this happened after you applied your methods. So please report the number of groups in the Results section, since these numbers are a consequence of the Methods.
2. Lines 107-109: “We analysed data from 31 patients diagnosed with rheumatoid arthritis who attended the Rheumatology Clinic of University Hospital “St. Ivan. Rilski” in Sofia compared to 21 healthy subjects.” – when? Please give a timeframe or any indication of time.
3. Lines 107-109: “We analysed data from 31 patients diagnosed with rheumatoid arthritis who attended the Rheumatology Clinic of University Hospital “St. Ivan. Rilski” in Sofia compared to 21 healthy subjects.” – how did you choose the 31 RA patients? Certainly, the Rheumatology Clinic of University Hospital “St. Ivan. Rilski” from Sofia has more than 31 RA patients. Please briefly explain in the manuscript how you achieved randomness of inclusion.
4. Please recheck abbreviation management in the entire manuscript: at first appearance in the text abbreviate (for example “rheumatoid arthritis (RA)”), then use only the abbreviation (for example, “RA”).
5. Line 113: you excluded obese patients (“BMI ≥ 29.9”), why? Please briefly justify your decision in the manuscript.
6. Lines 115-119: “The mean age of the RA patients was 43.1 years (SD ±14.2), ranging from 18 to 73 years old. The average disease duration was 9.6 ± 8.5 years. The disease onset was below 40 years in 52 % of the patients and above 40 years in 48 %. 71% of patients who have rheumatoid arthritis were anti-CCP positive, and 81% were rheumatoid factor (RF-IgM) positive.” – these are results, please move them from the Methods section to the Results section.
7. Line 119: “Disease activity in RA patients was measured using an online calculator” – confessing to using an online calculator is a bit childish. In calculating DAS28 everybody uses some sort of calculator since it includes square roots and logarithms. Please omit the online calculator and add a literature reference to the DAS28.
8. Lines 124-131: “Regarding drug therapy, 9 (29%) of patients received only symptomatic medications (long-term low-dose prednisolone 10 mg/day), while 22 (71%) were treated with the conventional synthetic disease-modifying anti-rheumatic drug [csDMARD) - methotrexate (MTX) 15”mg once weekly orally] with folic acid supplementation 5 mg weekly. For the control group, 21 healthy individuals (3 men and 18 women) were selected. Their ages ranged from 24 to 73 years, with an average age of 39.3 and a standard 130 deviation of 12.2.” - these are results, please move them from the Methods section to the Results section.
9. Line 124: regarding therapy, we read that 29% of your patients had only prednisolone and 71% had methotrexate. This is a biased sample in terms of treatment, since in real life the fraction of patients needing more that prednisolone is far greater, in real life patients have also other csDMARDs (leflunomide, sulfasalazine, hydroxychloroquine) and in real life patients also have bDMARDs or JAK inhibitors (which for sure are available in Bulgaria). Please discuss/explain this in the Discussion section and mention it in the limitations section, since it is highly relevant. No wonder the poor souls had an average CRP of 4 times the upper limit of normal and only a third were in the therapeutic target according to Table 1. Please also consider recalling these patients and augmenting their therapeutic scheme were appropriate.
10. Line 134: “All participants were Caucasian” – this is a result, please move it from the Methods section to the Results section.
11. Line 138: Table 1 is a result, please move it from the Methods section to the Results section.
12. Line 138: Please add normal ranges for blood tests in Table 1.
13. Line 316: Figure 6 is missing the image, it has only its caption.
minor issues
Author Response
Dear Reviewers,
Thank you for reviewing our paper entitled "AN ELEVATED IL10 mRNA COMBINED WITH LOWER TNFA mRNA LEVEL IN ACTIVE RHEUMATOID ARTHRITIS PERIPHERAL BLOOD" with manuscript ID cimb-2910868
We thank the reviewers for their valuable suggestions on improving our manuscript further.
We revised the manuscript extensively and hope our efforts will satisfy the reviewers.
The corrections in the revised version of the manuscript are marked in red text.
We provide a point-by-point response to the reviewer's comments.
Reviewer 1
Comments
- Lines 107-109: “We analysed data from 31 patients diagnosed with rheumatoid arthritis who attended the Rheumatology Clinic of University Hospital “St. Ivan. Rilski” in Sofia compared to 21 healthy subjects.” – the fact that you tested 31 patients and 21 controls is a result, not a method. Nobody plans in their study to include exactly 52 cases, but this happened after you applied your methods. So please report the number of groups in the Results section, since these numbers are a consequence of the Methods.
Author Response: Thank you for the comment. This sentence has been moved to the Results section.
Lines 107-109: “We analysed data from 31 patients diagnosed with rheumatoid arthritis who attended the Rheumatology Clinic of University Hospital “St. Ivan. Rilski” in Sofia compared to 21 healthy subjects.” – when? Please give a timeframe or any indication of time.
Author Response: Thank you for the comment. Patients and controls were recruited between October 2021 and October 2022.
Lines 107-109: “We analysed data from 31 patients diagnosed with rheumatoid arthritis who attended the Rheumatology Clinic of University Hospital “St. Ivan. Rilski” in Sofia compared to 21 healthy subjects.” – how did you choose the 31 RA patients? Certainly, the Rheumatology Clinic of University Hospital “St. Ivan. Rilski” from Sofia has more than 31 RA patients. Please briefly explain in the manuscript how you achieved randomness of inclusion.
Author Response: The patients and healthy controls were selected based on inclusion and exclusion criteria. The number of study participants was determined based on the research project's funding and is consistent with that of other similar studies reported in the scientific literature. We believe that by studying the mRNAs from 8 target genes and the circulating levels of their final functional gene products, the proteins IL-6, IL-10, IL-12p40, IL-17A, IL-18, IL-23, TNF-α and TGF-β1 in association with disease-specific clinical measures, we would contribute to a more complex understanding of the genetic and immunological basis of the disease, although the testing was performed on a relatively small number of patients.
- Please recheck abbreviation management in the entire manuscript: at first appearance in the text abbreviate (for example “rheumatoid arthritis (RA)”), then use only the abbreviation (for example, “RA”).
Author Response: Thank you for the comment. This was done.
Line 113: you excluded obese patients (“BMI ≥ 29.9”), why? Please briefly justify your decision in the manuscript.
Author Response: We focused on patients with normal body weight to avoid the possible influence of obesity on the studied biomarkers, as obesity is associated with low-grade systemic inflammation.
- Lines 115-119: “The mean age of the RA patients was 43.1 years (SD ±14.2), ranging from 18 to 73 years old. The average disease duration was 9.6 ± 8.5 years. The disease onset was below 40 years in 52 % of the patients and above 40 years in 48 %. 71% of patients who have rheumatoid arthritis were anti-CCP positive, and 81% were rheumatoid factor (RF-IgM) positive.” – these are results, please move them from the Methods section to the Results section.
Author Response: Thanks for the comment. This paragraph has been moved to the Results section.
- Line 119: “Disease activity in RA patients was measured using an online calculator” – confessing to using an online calculator is a bit childish. In calculating DAS28 everybody uses some sort of calculator since it includes square roots and logarithms. Please omit the online calculator and add a literature reference to the DAS28.
Author Response: Thanks for the comment. We added a literature reference to the DAS28 [ ref #24].
Lines 124-131: “Regarding drug therapy, 9 (29%) of patients received only symptomatic medications (long-term low-dose prednisolone 10 mg/day), while 22 (71%) were treated with the conventional synthetic disease-modifying anti-rheumatic drug [csDMARD) - methotrexate (MTX) 15”mg once weekly orally] with folic acid supplementation 5 mg weekly. For the control group, 21 healthy individuals (3 men and 18 women) were selected. Their ages ranged from 24 to 73 years, with an average age of 39.3 and a standard 130 deviation of 12.2.” - these are results, please move them from the Methods section to the Results section.
Author Response: Thanks for the comment. This paragraph has been moved to the Results section.
- Line 124: regarding therapy, we read that 29% of your patients had only prednisolone and 71% had methotrexate. This is a biased sample in terms of treatment, since in real life the fraction of patients needing more that prednisolone is far greater, in real life patients have also other csDMARDs (leflunomide, sulfasalazine, hydroxychloroquine) and in real life patients also have bDMARDs or JAK inhibitors (which for sure are available in Bulgaria). Please discuss/explain this in the Discussion section and mention it in the limitations section, since it is highly relevant. No wonder the poor souls had an average CRP of 4 times the upper limit of normal and only a third were in the therapeutic target according to Table 1. Please also consider recalling these patients and augmenting their therapeutic scheme were appropriate.
Author Response: The majority of patients have an inadequate therapeutic response to Methotrexate, for whom an upgrade to b/tsDMARDs treatment has been planned and was subsequently done. We deliberately selected patients on treatment with Methotrexate, which is a “gold standard” for the treatment of RA, in order to avoid the possible influence of therapeutic diversity on the results obtained.
10. Line 134: “All participants were Caucasian” – this is a result, please move it from the Methods section to the Results section.
Author Response: Thanks for the comment. This sentence has been moved to the Results section.
Line 138: Table 1 is a result, please move it from the Methods section to the Results section.
Author Response: Thanks for the comment. Table 1 has been moved to the Results section.
- Line 138: Please add normal ranges for blood tests in Table 1.
Author Response: Thanks for the comment. The normal ranges for the included blood tests are added in Table 1.
Line 316: Figure 6 is missing the image, it has only its caption.
Author Response: We apologise for our omission; the figure is now in the manuscript.
Sincerely yours,
On behalf of all authors
Georgi Vasilev, MD, PhD
Viktoria Vasileva, MD
Reviewer 2 Report
Comments and Suggestions for AuthorsManuscript Title: An Elevated IL10 mRNA Combined with Lower Tnfa mRNA Level in Active Rheumatoid Arthritis Peripheral Blood
Major Comments:
Authors have talked about T cells cytokines and discussed the correlation among them. It would be interesting how Th1/Th2/Th17/T reg cells and their secreted cytokines are expressed in active RA when analyzed through Flow Cytometry assay.
ELISA and Real Time PCR should also be performed for Th2 cytokine IL-4
Minor Comments:
Abbreviations and Acronyms: Ensure that all abbreviations are defined upon initial use.
Abstract: A slight disparity in describing purpose in the abstract can leave readers bewildered.
The authors should discuss each section of the results and limit the conclusion.
Introduction: Line 39-41 “This activation leads to a breakdown of immune tolerance, abnormal autoantigen presentation, and activation of antigen-specific T and B cells” Cite a proper reference for B cells involvement in RA.
Line 41-42 “The inflammation inevitably progresses to irreversible articular cartilage and bone damage that, regardless of therapeutic regimen, can lead to lifelong disability”. It should be restructured. Irreversible damage comes if the patients have not followed therapeutic regimen instructed to them.
The whole para from line 51-84 does not correlate with the title, abstract, results and most importantly structure of the article. This section is long with much of no needed information. Consider restructuring the content.
Spelling mistakes should be corrected, line 91- R?
For the immunoregulatory role of IL-10 cytokine, more references should be cited.
The authors have not included Th2 cytokines, specifically IL-4 as they also contribute to anti-inflammatory cytokine production. It should be included in the introduction, results and discussion.
Line 95-97 should be revised, as they directly cannot be used as prognostic marker for RA.
Line 103, authors have mentioned protein levels. However, they have not carried out any immunoblotting experiments and neither mentioned in introduction. This should be removed.
Materials and Methods: All demographic and clinical data should be restricted to a table format for better navigation and readability.
Authors should cite a reference for Reverse transcription PCR and Real-Time Quantitative PCR.
In the Quantification of serum cytokines concentrations section line should be introduced as per manufactures protocol.
Table 2 (supplementary) should be included in the main article. Table 2 can be moved to supplementary section. Wherever authors have given reference of supplementary table in the text of main article, it should be written supplementary.
Figure 6 is not included in the manuscript.
Discussion: Section needs to be re-written. It should be crisp, well explanatory, and emphasize the importance of the discussed topic. I would suggest a diagram in the discussion explaining involvement of T cells, cytokines and therapeutic regimen regulation on the mRNA levels.
Limitation section should be separately mentioned.
Editing of the English language is needed.
Citation style should be kept constant.
Comments on the Quality of English LanguageModerate editing needed.
Author Response
Dear Reviewers,
Thank you for reviewing our paper entitled "AN ELEVATED IL10 mRNA COMBINED WITH LOWER TNFA mRNA LEVEL IN ACTIVE RHEUMATOID ARTHRITIS PERIPHERAL BLOOD" with manuscript ID cimb-2910868
We thank the reviewers for their valuable suggestions on improving our manuscript further.
We revised the manuscript extensively and hope our efforts will satisfy the reviewers.
The corrections in the revised version of the manuscript are marked in red text.
We provide a point-by-point response to the reviewer's comments.
Reviewer 2
Comments and Suggestions for Authors
Manuscript Title: An Elevated IL10 mRNA Combined with Lower TNFA mRNA Level in Active Rheumatoid Arthritis Peripheral Blood
Major Comments:
Authors have talked about T cells cytokines and discussed the correlation among them. It would be interesting how Th1/Th2/Th17/T reg cells and their secreted cytokines are expressed in active RA when analyzed through Flow Cytometry assay.
ELISA and Real Time PCR should also be performed for Th2 cytokine IL-4
Author Response: Thanks for the comment. We entirely agree that Th2 cytokines also play a significant role in RA immunopathogenesis. A recent review [https://doi.org/10.3390/cells10113000] summarizes the results obtained from animal models as well as in vitro analysis of PB and SF of patients with arthritis and suggests that the IL-4/IL-13 anti-inflammatory properties might be beneficial in the context of inflammatory arthritis treatment. The role of Th2 cytokines, including IL-4, in RA pathogenesis was added in the revised version of the manuscript. Moreover, Th1, Th2, Th17, Treg and some other T cells secrete a lot of cytokines involved in the pathogenesis and severity of rheumatoid arthritis, and it is challenging to be a purpose of one study. The set of genes and cytokines explored in our study was determined based on the research project's funding, which is why our current study focused on the Th1/Th17/Treg axis. In the future, we may extend our study by exploring the Th2 profile. Thank you for your valuable suggestion!
Minor Comments:
Abbreviations and Acronyms: Ensure that all abbreviations are defined upon initial use.
Author Response: Thank you for the comment. We checked for all abbreviations used.
Abstract: A slight disparity in describing purpose in the abstract can leave readers bewildered.
The authors should discuss each section of the results and limit the conclusion.
Author Response: Thank you for the comment. The abstract was revised according to your recommendations.
Introduction: Line 39-41 “This activation leads to a breakdown of immune tolerance, abnormal autoantigen presentation, and activation of antigen-specific T and B cells” Cite a proper reference for B cells involvement in RA.
Author Response: Thanks for your suggestion. We have changed the reference.
Line 41-42 “The inflammation inevitably progresses to irreversible articular cartilage and bone damage that, regardless of therapeutic regimen, can lead to lifelong disability”. It should be restructured. Irreversible damage comes if the patients have not followed therapeutic regimen instructed to them.
Author Response: Thanks for your suggestion. We have rewritten the sentence as follows: “In patients with flares in disease activity, the inflammation inevitably progresses to irreversible articular cartilage and bone damage that can lead to lifelong disability and require an adequate treatment strategy”.
The whole para from line 51-84 does not correlate with the title, abstract, results and most importantly structure of the article. This section is long with much of no needed information. Consider restructuring the content.
Author Response: Thanks for your suggestion. We agree with your notion, and the whole paragraph from lines 51 to 70 was removed.
Spelling mistakes should be corrected, line 91- R?
Author Response: Thank you for the comment. The spelling mistakes were corrected - “R” means RA.
For the immunoregulatory role of IL-10 cytokine, more references should be cited.
Author Response: Thanks for the comment. We added several additional references for the immunoregulatory role of IL-10 [ref # 14;15;16; 17].
The authors have not included Th2 cytokines, specifically IL-4 as they also contribute to anti-inflammatory cytokine production. It should be included in the introduction, results and discussion.
Author Response: Thank you for the comment. We have added new information about the role of Th2 cytokines and IL-4 in the RA pathogenesis.[ref.#17;18]
Line 95-97 should be revised, as they directly cannot be used as prognostic marker for RA.
Author Response: Thank you for the comment. We have revised this sentence as follows:” Blood-based mRNA gene expression has the potential to identify patients suitable for a given treatment regimen.”
Line 103, authors have mentioned protein levels. However, they have not carried out any immunoblotting experiments and neither mentioned in introduction. This should be removed.
Author Response: Thank you for the comment. We agree, and we have removed "at the protein level."
Materials and Methods: All demographic and clinical data should be restricted to a table format for better navigation and readability.
Author Response: Thanks for the comment. According to the reviewer's recommendations, this paragraph was moved to the Results section.
Authors should cite a reference for Reverse transcription PCR and Real-Time Quantitative PCR.
Author Response: Thanks for the comment. We have added the references for the reverse transcription PCR and real-time quantitative PCR. [ ref # 26]
In the Quantification of serum cytokines concentrations section line should be introduced as per manufactures protocol.
Author Response: Thank you for the comment. We have added the required information.
Table 2 (supplementary) should be included in the main article. Table 2 can be moved to supplementary section. Wherever authors have given reference of supplementary table in the text of main article, it should be written supplementary.
Author Response: Thank you for the comment. The supplementary Table was included in the main text, while Table 2 was moved to the supplementary section.
Figure 6 is not included in the manuscript.
Author Response: Thank you for the comment. We apologise for our omission. Figure 6 was included in the manuscript.
Discussion: Section needs to be re-written. It should be crisp, well explanatory, and emphasize the importance of the discussed topic. I would suggest a diagram in the discussion explaining involvement of T cells, cytokines and therapeutic regimen regulation on the mRNA levels.
Author Response: Thank you for the comment. The diagram involving different T cell subsets, cytokines produced, and the effect of different treatment agents would provide additional information. Unfortunately, reviewing all this information will be challenging because of the heterogeneous nature and fluctuating course of such chronic autoimmune disease. In addition, different cell types and cytokines are involved in the different stages of RA progression. Thus, we consider such a diagram and a review of the topic much more appropriate for a separate review article, which is beyond the current study's aim.
Limitation section should be separately mentioned.
Author Response: Thank you for the comment. The study's limitations were separately mentioned in the manuscript.
Editing of the English language is needed.
Author Response: English Language editing was performed.
Citation style should be kept constant.
Author Response: Thank you for the comment. All citations were re-edited using the “Cite this for me” platform.
Sincerely yours,
On behalf of all authors
Georgi Vasilev, MD, PhD
Viktoria Vasileva, MD
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsNo Comments
