PONE-D-24-16624Modulatory Action of Bryonia alba on the Immune System in Cyclophosphamide Induced Immunosuppression in BALB/c MicePLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: I suggest that the author pays attention to the bibliographical references in the introduction. I consider the references used to be somewhat outdated for the publication, I try citations that have been published for more than 14 years.

Reviewer #2: Some points to improve

In the introduction, some improvements can be implemented to strengthen the scientific basis and clarity of the text. Initially, it would be beneficial to update the references used to support claims about homeopathy and Bryonia alba, incorporating more recent and robust studies to increase the credibility of the information presented. Furthermore, it is important to detail the evidence mentioned in the introduction. Although several studies on the use of BA are cited, there is a lack of specifications on study design, sample sizes, and specific results. Including more details could strengthen the claims and provide a clearer view of the current scientific landscape.

To improve the accessibility of the text, it could be useful to briefly explain some technical terms, such as "immunosuppression" and "homeopathic potencies", making it more understandable for non-specialist readers. In addition, small grammatical and concordance errors must be reviewed to ensure the fluidity and accuracy of the text.

Regarding the mechanisms of action of BA, the introduction mentions its immunomodulatory and antioxidant characteristics, but does not offer a detailed explanation of the possible underlying biological mechanisms. A deeper analysis of these mechanisms would not only increase understanding but also reinforce the credibility of the conclusions presented. The rationale for the study can also be expanded. While the introduction states that the immunostimulating properties of BA in situations of immunosuppression have not been adequately explored, it would be useful to explain in more detail why this gap in knowledge is relevant and what the potential clinical impacts of this research are. Finally, discussing the challenges inherent in researching homeopathic treatments, such as the variability of results and the difficulty of replication, would provide a more balanced and critical view of the study, offering a more complete perspective for readers.

In the methodology, some improvements can be suggested to strengthen the robustness of the study. First, it would be beneficial to supplement the references used (e.g., Singh et al., 2019; Jahnavi et al., 2019) with more recent studies or updated literature reviews that solidify the scientific basis for the doses and procedures employed. Additionally, the list of reagents and chemicals could be expanded to include all critical materials used in the study, such as ELISA kits, reagents for RNA and cDNA, and the specific equipment used in PCR and microscopy analyses. The justification for the choice of dose, although it mentioned the conversion of the dose from albino rats to albino mice, could be more robust. It would be helpful to provide better reasoning, perhaps with additional references or a more detailed explanation of the conversion process used to determine the final volume (40 μL/100 g). Furthermore, some minor procedural details, such as the exact protocol for retro-orbital blood collection, could be more thoroughly described. This would ensure that all procedures can be replicated accurately, promoting the consistency and reliability of the results obtained.

The discussion recognizes the limitation of using an animal model to simulate chemotherapy-induced immunosuppression, which may not fully replicate human immune responses. This recognition is important as it highlights the need for clinical studies in humans to validate these findings. Research could benefit from further exploration of the molecular mechanisms by which BA exerts its immunomodulatory effects. Investigating cellular signaling pathways and molecular interactions would provide a more complete understanding of BA's modes of action. The discussion addresses the use of alcohol as a vehicle for BA and its lack of impact on the observed immunomodulatory properties, which is relevant to ensure that the beneficial effects are due to the BA and not the vehicle. Comparison with Levamisole, a known immunomodulator, strengthens the results, demonstrating that BA has comparable efficacy, which is encouraging for the validation of BA as a potential therapeutic agent.

The conclusion effectively summarizes the main results, such as increased levels of RBC, WBC, spleen indices and cytokines (IL-2, IL-4, TNF-α, IFN-γ) in serum and spleen, reinforcing the evidence that BA has a relevant immunomodulatory action. While recognizing that the mechanism of action of BA is still unknown, it would be useful to suggest specific methods or approaches to explore these mechanisms in future research, adding value and direction to studies. It is important to reinforce caution when generalizing results from animal models to humans, contextualizing the findings within the limits of preclinical research. The conclusion could include a brief reference to previous studies on immunomodulation, better situating the findings within the broader body of scientific literature. Furthermore, explicitly suggesting clinical trials to validate the effects in humans would be valuable, showing a clear path to practical application of the results.

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Reviewer #1: No

Reviewer #2: Yes: Jéssica Rabelo Bezerra

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Modulatory Action of Bryonia alba on the Immune System in Cyclophosphamide Induced Immunosuppression in BALB/c Mice

PONE-D-24-16624R1

Dear Dr. Kumar,

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Kind regards,

Wesley L. C. Ribeiro, Ph.D.

Academic Editor

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Additional Editor Comments (optional):

Reviewers' comments:

The authors have adequately considered all reviewers' comments.