Calcitriol Modulates Both the Vitamin D Receptor and the Calcium-Sensing Receptor in Blood Mononuclear Cells in Elderly Female Patients with Hip Osteoporotic Fractures

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The topic of this research is interesting and relevant for the readership of Biomolecules. However, the main drawback of the manuscript is the extremely small amount of generated data, which does not justify the publication of this research article. The Results section contains only 14 lines of text, which is absolutely insufficient for a research article. Part of the obtained results is presented in tabular form, and it would be preferable if the results were instead presented as figures. In addition, Figures 1 and 2 are excessively large, and their size is inappropriate for publication.

I believe that, prior to publication, this study should be expanded and the description of the obtained results significantly extended. On the other hand, the Introduction as well as the Materials and Methods sections are appropriate and provide a good overview of the field and a clear description of the applied research methodology.

Minor comments: The use of abbreviations in the title should be avoided.

Author Response

The topic of this research is interesting and relevant for the readership of Biomolecules. However, the main drawback of the manuscript is the extremely small amount of generated data, which does not justify the publication of this research article. The Results section contains only 14 lines of text, which is absolutely insufficient for a research article.

Part of the obtained results is presented in tabular form, and it would be preferable if the results were instead presented as figures.

In addition, Figures 1 and 2 are excessively large, and their size is inappropriate for publication.

I believe that, prior to publication, this study should be expanded and the description of the obtained results significantly extended.

On the other hand, the Introduction as well as the Materials and Methods sections are appropriate and provide a good overview of the field and a clear description of the applied research methodology.

Thank you very much for your kind and accurate comments. We have tried to modify our comment as best we can, based on your insightful observations and suggestions. We have highlighted the changes and modifications in red (deletions are not highlighted).

We believe that the results produced may be interesting to potential readers of ‘Biomolecules’ journal. We have chosen a model of vitamin D deficiency (commonly measured as 25-hydroxyvitamin D) in women with fractures, who due to a faulty innate immune response, suffer from a high rate of infections as a complication of the natural history of their condition.

Vitamin D deficiency through the regulation of vitamin D receptor (VDR) and calcium-sensing receptor (CaSR) expression could influence the apoptotic functioning of PBMCs, despite its importance in the immune response has not been enough explored.

In order to show the results following the reviewer’s comments, we have expanded and modified the figures to make them more understandable.

 

Minor comments: The use of abbreviations in the title should be avoided.

Done. We have removed the abbreviations.

We would like to point out that abbreviations should generally be avoided in the title (and also in the abstract, if possible). However, in cases of standardised and accepted acronyms, it may be more informative to include them. This is recommended for authors/researchers who carry out bibliographic searches limited to the title. If, for example, a search was carried out for a systematic review using the criteria VDR[title] OR CaSR[title], our manuscript would not be retrieved for inclusion in the systematic review (or scoping review, or whatever).

However, since the reviewer has pointed out this modification to us, we are doing as suggested.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript investigates the effects of calcitriol on VDR and CaSR regulation, as well as monocyte apoptosis, in patients with aging-associated osteoporosis and hip fracture using clinical blood samples. The findings suggest that immune dysfunction is associated with vitamin D deficiency, and that activation of VDR by vitamin D or its structural analogs may represent a potential therapeutic strategy for infectious, inflammatory, or autoimmune diseases. Overall, the topic is interesting and clinically relevant. However, the experimental approaches employed are relatively limited. The rigor of the study would be significantly strengthened by incorporating additional molecular and mechanistic analyses to more thoroughly elucidate the relationship between VDR/CaSR signaling and apoptosis. In addition, the writing of "result section" could be further improved.  

Author Response

The manuscript investigates the effects of calcitriol on VDR and CaSR regulation, as well as monocyte apoptosis, in patients with aging-associated osteoporosis and hip fracture using clinical blood samples. The findings suggest that immune dysfunction is associated with vitamin D deficiency, and that activation of VDR by vitamin D or its structural analogues may represent a potential therapeutic strategy for infectious, inflammatory, or autoimmune diseases. Overall, the topic is interesting and clinically relevant.

Thank you very much for your kind and accurate comments. We have tried to modify our comment as best we can, based on your insightful observations and suggestions. We have highlighted the changes and modifications in red (deletions are not highlighted).

 

However, the experimental approaches employed are relatively limited. The rigor of the study would be significantly strengthened by incorporating additional molecular and mechanistic analyses to more thoroughly elucidate the relationship between VDR/CaSR signalling and apoptosis. In addition, the writing of "result section" could be further improved.

The authors thank the reviewer a lot for their appropriate comments. Indeed, as the reviewer points out, we have chosen a model of vitamin D deficiency: that of elderly women with osteoporotic hip fractures, who are commonly deficient in vitamin D. As the reviewer told us, we have used clinical blood samples, which is a strength of our study.

Vitamin D deficiency through the regulation of vitamin D receptor (VDR) and calcium-sensing receptor (CaSR) expression could influence the apoptotic functioning of PBMCs, which, despite its importance in the immune response and its involvement in the natural history of infectious, inflammatory, or autoimmune diseases, has not been enough studied.

Therefore, we believe that our results may be of interest to potential readers of ‘Biomolecules’ journal.

Following the reviewer's comments, we have modified in the manuscript the presentation of the results to make them more appealing and understandable.

We fully agree with the comment that ‘The rigour of the study would be significantly enhanced if additional molecular and mechanical analyses were incorporated to further elucidate the relationship between VDR/CaSR signalling and PBMCs apoptosis’, but unfortunately, we do not have the appropriate biological material to carry out these studies. We indicate it in the new version of manuscript (at the end of DISCUSSION, admitting weaknesses of the study.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This is a quite well-organized clinical study with sufficient novelty. Nevertheless, some points should be addressed.

• In the abstract, before the aim of the study, 1-2 introductory section should be added.
• The subheading "Methods" is missing in the abstract.
• In the title, the gender of patients should be specifief, e.g. elderly female patients.
• The p-values in the conclusion section of the abstract should be avoided.
• Please, report one only time the introduction subheading (lines 50-51).
• The sentence in lines 70-75 "Organization..." is quite complex and it should be split into two smaller sentences.
• In the sentence, in lines 77-78, please specify the gender of patients.
• The sentence in lines 79-80 "Agining ..." needs more analysis.
• At the end of the introduction section, the literature gap that the present study will cover should be emphasized.
• Please check, if all abbreviations used in the introduction section are explained.
• In section 2.1, the inclusion and exclusion criteria should be emphasized.
• In section 2.4, relevant references should be added.
• Ethic concerns (section 2.6) should be transferred at the beggining of the methods section.
• A table with the descriptive statistics of the study population should be added in the results section. A relevant description of the descriptive statistics should be also incuded into the text.
• The authors should try to perform a comparative analysis of their results with previous studies.
• At the end of the discussion section "The Strengths and the Limitations" of their study should be repored.
• In the conclusion section, the authors should include their critical proposals concerning what future studies could be performed based on their results.

Author Response

This is a quite well-organized clinical study with sufficient novelty. Nevertheless, some points should be addressed.

Thank you very much for your kind and accurate comments. We have tried to modify our comment as best we can, based on your insightful observations and suggestions. We have highlighted the changes and modifications in red (deletions are not highlighted).

 

• In the abstract, before the aim of the study, 1-2 introductory section should be added.

In the abstract, we have inserted an explanatory text as an exordium.

Peripheral blood mononuclear cells (PBMCs) constitute a diverse population of cells involved in adaptive and innate immunity, playing an essential role in pathogen recognition, immune signaling, and immune response modulation. Vitamin D deficiency through the regulation of vitamin D receptor (VDR) and calcium-sensing receptor (CaSR) gene expression could influence the apoptotic functioning of PBMCs, despite its importance in immune response has not been enough studied.

 

• The subheading "Methods" is missing in the abstract.

Thanks a lot for your observation. The subheading ‘methods’ has been added to the abstract of the manuscript.

 

• In the title, the gender of patients should be specifief, e.g. elderly female patients.

The patients’ gender has been specified in the title. Thank you for your appropriate comment.

 

• The p-values in the conclusion section of the abstract should be avoided.

The p-values have been removed from the abstract according to the reviewer's suggestion.

 

• Please, report one only time the introduction subheading (lines 50-51).

It’s been corrected (one of them has been deleted). Thank you for your accurate comment.

 

• The sentence in lines 70-75 "Organization..." is quite complex and it should be split into two smaller sentences.

Done. We have rewritten that sentence and it is now more concise.

 

• In the sentence, in lines 77-78, please specify the gender of patients.

Done. We have determined that they are female.

 

• The sentence in lines 79-80 "Agining ..." needs more analysis.

Ageing is a process commonly recognized as a cause of decreased serum levels of 25-hydroxyvitamin D. In other words, it is well known that the older you are, the lower your serum vitamin D levels are.

 

• At the end of the introduction section, the literature gap that the present study will cover should be emphasized.

Following the reviewer’s advice, we have rewritten the end of the introduction to emphasize the importance of the study

 

• Please check, if all abbreviations used in the introduction section are explained.

Done. It’s been checked and we have adapted it to the new text we have added.

 

• In section 2.1, the inclusion and exclusion criteria should be emphasized.

The inclusion and exclusion criteria have been modified and we think that now this text is more clearly explained.

 

• In section 2.4, relevant references should be added.

Following your advice, we have added two references to the text:

• Blüher M, Krohn K, Wallaschofski H, Braverman LE, Paschke R. Cytokine gene expression in autoimmune thyroiditis in BioBreeding/Worcester rats. Thyroid. 1999 Oct;9(10):1049-55. doi: 10.1089/thy.1999.9.1049. PMID: 10560963.
• Fernandez PM, Pluta LJ, Fransson-Steen R, Goldsworthy TL, Fox TR. Reverse transcription-polymerase chain reaction-based methodology to quantify differential gene expression directly from microdissected regions of frozen tissue sections. Mol Carcinog. 1997 Nov;20(3):317-26. doi: 10.1002/(sici)1098-2744(199711)20:3<317::aid-mc8>3.0.co;2-h. PMID: 9397192.

 

• Ethic concerns (section 2.6) should be transferred at the beginning of the methods section.

Done. Now it is now in first place.

 

• A table with the descriptive statistics of the study population should be added in the results section. A relevant description of the descriptive statistics should be also included into the text.

Done. We have described the statistical study in greater detail, as well as the results obtained from it.

 

• The authors should try to perform a comparative analysis of their results with previous studies.

Done. This has been shown explicitly.

 

• At the end of the discussion section "The Strengths and the Limitations" of their study should be reported.

Done. At the end of the discussion, we report on the strengths and limitations of the study. Thanks a lot for your suggestion.

 

 

• In the conclusion section, the authors should include their critical proposals concerning what future studies could be performed based on their results.

Done. Critical proposals for future studies that could be carried out based on the results obtained have been included in the conclusion section of the manuscript.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Dear authors,

Thank you for submitting your article to Biomolecules.

Please find my comments below.

Title: The study design needs to be considered in the title of the paper

Line 50: Introduction

There are too many paragraphs in this section. I would recommend you merge some of them and present 3-4 paragraphs only.

Line 58: I would recommend you reduce the number of citations here.

Line 209: Please describe the fact that you have reported the corresponding  95% CIs in the graph. Same for figure 2.

Discussion: line 222

Please note that the first sentence needs to be the opening sentence of the paragraph and describe the findings of your own study only.

In the subsequent paragraphs you will need to report and discuss the results of the outcomes of the study in relation to the relevant literature.

Also a limitations paragraph needs to be added onto the discussion section as well.

Author Response

Dear authors,

Thank you for submitting your article to Biomolecules.

Please find my comments below.

Thank you very much for your kind and accurate comments. We have tried to modify our comment as best we can, based on your insightful observations and suggestions. We have highlighted the changes and modifications in red (deletions are not highlighted).

 

Title: The study design needs to be considered in the title of the paper

We have modified the title. However, we believe that if we also include the type of design it has, it would be too long (since it is in fact a mixed design), and this is clarified in the materials and methods section. In any case, if you think it is essential, we would be willing to change it.

 

Line 50: Introduction

There are too many paragraphs in this section. I would recommend you merge some of them and present 3-4 paragraphs only.

Done according to your instructions in the Introduction of the new manuscript.

 

Line 58: I would recommend you reduce the number of citations here.

Done in the revised manuscript. We have thirteen fewer references, despite having had to add four references at the suggestion of other reviewers who asked us to clarify other sections by providing scientific literature support.

 

Line 209: Please describe the fact that you have reported the corresponding 95% CIs in the graph. Same for figure 2.

Done, and included in the figures 1 and 2, which have also been reduced.

 

Discussion: line 222

Please note that the first sentence needs to be the opening sentence of the paragraph and describe the findings of your own study only.

We have considered the reviewer's recommendation (which we appreciate) indicating that the first sentence of the opening paragraph should describe only the results of our own study,

 

In the subsequent paragraphs you will need to report and discuss the results of the outcomes of the study in relation to the relevant literature.

Done. This has been discussed afterwards.

 

Also, a limitations paragraph needs to be added onto the discussion section as well.

Done. At the end of the discussion, we report on the strengths and limitations of the study. Thanks a lot for your suggestion.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have taken the reviewers’ comments into account and, within their possibilities, revised the manuscript accordingly. However, I believe that the fundamental concern has not been addressed, namely that this paper does not contain a sufficient amount of results to justify publication.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed the major concerns. I have no further comments. 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have significantly revised and improved their manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

Dear authors,

You have revised the paper according the my original comments and I believe that it merits publication.