References
infectious diseases, toxicity, infections, Newcastle disease, diabetic, infectious bursal disease
ADVERSE REACTIONS, INFECTIOUS DISEASES, VIRUS, HYPERVITAMINOSIS, PROLIFERATION, MINOR, AUTOIMMUNE DISEASES, INFECTIONS, INFECTIOUS BURSAL DISEASE, NEWCASTLE DISEASE, PROLIFERATIVE, BLIND, GESTATIONAL DIABETES

I am fine with changes. All are OKIs the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
I have read the revised version of the manuscript.
Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Limited sample size and gender imbalance: The study includes only 52 healthy subjects, with a significant gender imbalance (48 males and 4 females). This small sample size and gender disparity may limit the generalizability of the findings and raise concerns about the representativeness of the population.Lack of long-term follow-up: The trial's duration is only four weeks, which may not be sufficient to capture the full range of effects on immune parameters. Longer-term follow-up would provide a better understanding of the sustained effects and potential adverse reactions associated with N. sativa supplementation."Nigella sativa is one of the most commonly used herbal medicines worldwide nowadays.""It has been used for more than 2000 years as a natural remedy for various illnesses.""Research has documented its therapeutic potential as an antimicrobial, anti-inflammatory, antioxidant, antidiabetic, antihypertensive, antitumor, and immunomodulatory agent [1,2].""The immune system consists of multiple interconnected networks of cells, proteins, and lymphoid organs that provide protection against millions of microbes and infections.""The immune system includes innate immunity and adaptive immunity. Innate immunity provides immediate protection to the body, and its effect is similar in different individuals, while adaptive immunity takes more time to develop and is more specific and effective against invasive pathogens [3].""The immunomodulatory effect of N. sativa has been extensively studied on both innate and adaptive immunity, as well as related messengers and mediators [4–6].""Several in vivo studies have shown significant effects of N. sativa on immunity, autoimmune diseases, and toxicity.""A study demonstrated that the aqueous extract of N. sativa resulted in a significant increase (62.3% ± 6.4%) in natural killer (NK) cytotoxic cell activity against YAC-1 cells after 1 week of oral administration in 10-week-old BALB/c female mice [7].""Another in vitro study on the effect of thymoquinone (TQ) on immunity showed that TQ injected directly into cells at low concentrations (10, 2.5, or 0.62 μg mL−1) increased the survival of activated T-cells and the ability of CD8+ T-cells to generate IFN-γ, indicating the potential benefits of TQ against infectious diseases and immune enhancement [8].""N. sativa supplementation at levels of 4% or 5% positively enhanced immunity against Newcastle disease virus in newly evolved crossbred laying hens [9].""In another study on Newcastle virus vaccinated broilers, N. sativa supplementation at three doses (5, 10, and 20 g kg−1) for 42 days significantly increased antibodies against Newcastle virus on day 35 [10].""A diet supplemented with 40 g kg−1 N. sativa fed to broiler chicks improved antibody production against both Newcastle virus and infectious bursal disease [11].""The phagocytic index and rate were significantly higher in STZ-diabetic hamsters treated with N. sativa oil (NSO) at a dose of 400 mg·kg−1 for 4 weeks compared to untreated diabetic animals, as demonstrated by fluorescence microscopy [12].""N. sativa extract has a stimulatory effect on cellular immunity in vitro by increasing the proliferative capacity of T lymphocytes and splenocytes, as well as the response to different mitogens of human peripheral blood mononuclear cells (PBMC) [13].""NSO was shown to possess a protective role against vitamin A hypervitaminosis. Rats treated orally with 800 mg·kg−1 NSO showed higher serum levels of IgG and IgM than the control group or those receiving high doses of vitamin A [14].""Gestational diabetes rats showed improvement in the immune status of their offspring after oral antenatal feeding with 20 mg kg−1 TQ, reversing the decreased levels of IL-2, T-cell reproduction, and improving both circulating and thymus-homing T-cell proliferation [15].""The literature above demonstrates a very promising immunomodulating effect of N. sativa.""However, the immunopotentiation effect of this remarkable plant has not been investigated in normal humans.""Hence, this study was designed to evaluate the impact of different doses of N. sativa on the immune system in young, healthy humans." The conclusion could be improved by providing more specific details, addressing the limitations of the study, suggesting specific research questions for future trials, considering the generalizability of the findings, and discussing potential risks or side effects associated with the use of N. sativa.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:PharmacologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
The study is well designed and conducted a placebo controlled double blind clinical trial that confirms the immunopotentiation effect of
 Perhaps, the higher doses trigger some inhibitory mechanisms. The evidence in favour is that the
 It is desired that the authors find such examples (Human or animals studies) and describe the possible reasons for the absence of elevation in helper T cells by increasing the dose of
 Some other minor alterations have been suggested in the text (see attached and annotated report).Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
We would like to thank Professor Akram for his valuable comments. We have actually discussed the point of losing the significant increase in helper T cells when
 We agree that the most possible cause is the presence of some ingredients that possess negative effect at higher doses.
