Correlations between components of the immune system

Yehudit Shabat; Yaron Ilan

doi:10.12688/f1000research.54487.3

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Research Article

Revised

Correlations between components of the immune system

[version 3; peer review: 2 approved, 1 not approved]

Yehudit Shabat¹, Yaron Ilan ¹

PUBLISHED 12 Mar 2024

Author details Author details

¹ Hadassah University Hospital, Jerusalem, Jerusalem, Israel, Israel

Yehudit Shabat
Roles: Data Curation, Formal Analysis, Methodology, Writing – Original Draft Preparation

Yaron Ilan
Roles: Conceptualization, Data Curation, Formal Analysis, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background

No evidence of the possibility of altering a constituent of the immune system without directly affecting one of its associated components has yet been shown.

Methods

A schematic model was developed in which two triggers, fasting and splenectomy, were studied for their ability to affect the expression of cell membrane epitopes and the cytokine secretion of out-of-body autogeneic and syngeneic lymphocytes.

Results

The effect of fasting and/or splenectomy on promoting correlations between immune systems was studied by determining the alterations in expressions of cell membrane epitopes and in cytokine secretion by out-of-body autogeneic and syngeneic lymphocytes. The effect of fasting as a trigger decreased expression of CD8 and CD25 and increased TNFα levels. The effect of splenectomy as a trigger was investigated in non-fasting mice by comparing splenectomized and non-splenectomized mice. An increase in the CD8 expression and in TNFα, IFNg, and IL10 secretion was noted. The effect of splenectomy as a trigger in fasting mice was determined by comparing splenectomized and non-splenectomized mice. Splenectomy significantly affected the expression of CD25 and CD4 CD25 and on secretion of TNFα, IFNg, and IL10. To determine the effect of keeping the cells in an out-of-body location on the expression of lymphocyte epitopes, tubes kept on top of the cages of the fasting mice were compared with tubes kept on top of empty cages. The results showed a significant change in the CD8 expression was noted. To determine the effect of keeping cells in an out-of-body location on cytokine secretion, tubes kept on cages were tested for cytokine levels significant decrease was noted in the secretion of TNFα and IFNg.

Conclusions

The study showed that a mouse could affect cells at a distance and alter the expression of surface markers and cytokine secretion following two types of triggers: fasting and/or splenectomy. The data characterized a system for the induction of correlations between two’s immune system components without a transfer of mediators. It suggests that an out-of-body correlation can be induced between two components of the immune system.

Keywords

complex systems; immune memory; information transfer; immune response; randomness

Corresponding author: Yaron Ilan

Competing interests: No competing interests were disclosed.

Grant information: This work was supported by the Roaman-Epstein Research Foundation
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Shabat Y and Ilan Y. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Shabat Y and Ilan Y. Correlations between components of the immune system [version 3; peer review: 2 approved, 1 not approved]. F1000Research 2024, 10:1174 (https://doi.org/10.12688/f1000research.54487.3) First published: 22 Nov 2021, 10:1174 (https://doi.org/10.12688/f1000research.54487.1) Latest published: 12 Mar 2024, 10:1174 (https://doi.org/10.12688/f1000research.54487.3)

Revised Amendments from Version 2

Changes made to the new version:

The Abstract, Introduction, Reuslts, and part of the Discussion sections were revised.
A new set of figures is included.
Table 2 was included to add a schematic description of the experiments conducted.
A conclusion section was added.

See the authors' detailed response to the review by Suprabhat Mukherjee
See the authors' detailed response to the review by Rohit Malik
See the authors' detailed response to the review by Biswadeep Das

Abbreviations

IFNγ: interferon gamma

TGFβ: transforming growth factor beta

TNFα: tumor necrosis alpha

IL10: interleukin 10

OL: an independent control laboratory

Introduction

Living systems carry information, and the more complex a system is, the higher the likelihood it contains information. Like other biological systems, the immune system may not have a perfect structure or symmetry, but it optimizes functionality. The system’s irregular pathways offer an opportunity to apply physics concepts to the biological system.¹

Correlations between immune system components always involve reactions mediated by a direct association between different system elements.² Current paradigms of immune crosstalk between cells or at a subcellular molecular level are based on direct contact or the transfer of mediators in both health and disease.³^–⁵ These elements serve as links for relocating associations between immune system constituents. No evidence for correlations between components of the immune system that alter a constituent of this system without directly affecting one of its associated parts has been reported.

This study aimed to develop a system where a correlation exists between two immune system components without either a direct interaction or a transfer of mediators. The data support the concept that out-of-body correlations can occur between immune system components in isolated structures.

Methods

Animals

24 Male C57BL/6 healthy mice (11–12 weeks old, 30 gr.) were obtained from Harlan Laboratories (Jerusalem, Israel) and maintained in the Animal Core of the Hadassah-Hebrew University Medical School. Mice were administered standard laboratory chow and water ad libitum and kept in a 12-hour light/dark cycle. Mice were administered standard laboratory chow and water ad libitum and kept in a 12-hour light/dark cycle. Animal experiments were carried out according to the guidelines of the Institutional Committee for Care and Use of Laboratory Animals and with the committee's approval. All efforts were made to ameliorate any suffering of animals by using mild anesthesia.

Experimental groups

Six groups of mice (Table 1) were studied, with four mice per group in each of the four experiments. The number of animals was determined based on statistical analysis. Data on all mice in each of the studies is included in the Underlying data.²¹ Groups were selected based on the different parameters tested with the appropriate controls. Table 2 provides a representation of the research protocol.

Table 1. Experimental groups (n = 4 for each group).

Group	Mouse in cage	Splenectomy	Fasting	Autogeneic cells on top of the cage	Syngeneic cells on top of the cage
A	+	+	+	+	-
B	+	+	+	-	+
C	+	+	-	+	-
D	+	+	-	-	+
E	+	-	+	-	+
F	+	-	-	-	+
G/H	-	-	-	-	-

Table 2. A representation of the research protocol.

Step A

Step B

Step C

Trigger:

• Splenectomy
• Start fasting (21-24 hours)

Mouse kept in a cage for 24 hours with a tube containing autogenic or syngeneic cells on top of the cage.
Blood tested for cytokines and epitopes expression on lymphocytes are taken at time 0.

Blood taken and blood in tube tested after 24 hours:

• The serum was tested for cytokines.
• Lymphocytes were analyzed for epitope expression.

The mice in groups A and B underwent splenectomy followed by fasting. Each mouse was kept in a cage with a tube containing its own autogeneic (group A) or syngeneic (group B) lymphocytes on top. The mice in groups C and D underwent splenectomy but did not fast, and they were kept in cages with tubes containing autogeneic (group C) or syngeneic (group D) lymphocytes on top. The mice in groups E and F did not undergo splenectomy and were kept in cages with a tube containing syngeneic lymphocytes on top. The mice in group E fasted, while those in group F did not. Tubes marked G and H contained lymphocytes harvested from syngeneic donors and were kept on top of empty cages. The study design determined the number of animals to enable significance. Controls were used for each of the experiments based on the parameters tested. Studies were repeated in an outside laboratory for verification of the effect. Studies were conducted under the Arrive guidelines.

Study design

The described system enabled us to study two types of triggers for the associations between two immune system constituents. Mice underwent splenectomy using standard procedure⁶ or fasting (21-24 hours). Each mouse was kept in a separate cage for the duration of the experiment. Autogeneic or syngeneic splenocytes were prepared from splenectomized mice and kept for 24 hours in a tube on top of the cage at a distance of 10-20 cm from the mouse itself. Control cells were kept in a tube placed on top of the cages containing either non-splenectomized or non-fasting naïve mice or were kept on top of empty cages without a mouse. In splenectomized mice, we tested the effect of splenectomy alone or combined with fasting on either autogeneic or syngeneic cells. In naïve, non-splenectomized mice, we tested the effect of fasting on syngeneic cells. The effects of these triggers on the associations between immune components were examined by determining the expression of surface markers and cytokine secretion by the out-of-body lymphocytes. The study was replicated three times, and for each study, four mice per group were analyzed (marked 11A, 11B, and 11C). For epitope parameters, the experiment was repeated for the fourth time by an independent control laboratory (marked OL) for several parameters. Cytokine study was performed once in the controlled laboratory.

Flow cytometry

Splenocytes were isolated, as described previously.⁷ Flow cytometry was performed on splenocytes resuspended in 1 mL of Flow Cytometry Staining (FACS) buffer. Cells were stained with diluted antibodies (50 μL/sample). Cells were examined using FACS at times 0 and 24 hours for the expressions of epitopes, CD3, CD4, CD8, CD25, FoxP3, and NK1.1, on the lymphocytes (antibodies by BD Biosciences). Flow cytometry was performed using an LSR-II flow cytometer (BD Biosciences, DeNovo software). The following subsets were analyzed: CD4, CD25, CD4 CD25, Foxp3, CD4 Foxp3, CD4 CD25 Foxp3, CD4 CD25 Foxp3, CD8, CD8 Foxp3, CD8 CD25, CD8 CD25 Foxp3, CD8 CD25 Foxp3, CD3, NK1.1, and CD3 NK1.1. Full data of the study can be accessed from the Underlying Data²¹ section.

Cytokine measurement

One set of mice marked OL (n = 4) was analyzed in the control laboratory for IFNγ, TNFα, IL10, and TGFβ serum levels, IL10, and TGFβ. Determination of IFNγ, TNFα, and IL10 in supernatants was performed concurrently, using Flow Cytomix (Thermo Fisher Scientific (eBioscience)). TGFβ levels were determined separately using ELISA Quantikine (MB100B) (R&D Systems). Cytokine levels were determined according to the manufacturer's instructions.

Statistical analysis

The Mann-Whitney SPSS test (software version 1.0) was performed for each experiment. Data of all individual mice in all studies is provided in the Underlying data.²¹ Only parameters that showed significant changes in two experiments or more, including changes in opposing directions, were used for the final analysis.

Results

The effect of fasting as a trigger for altering the association that can modify epitope expression on out-of-body lymphocytes kept in tubes on top of cages was investigated in splenectomized mice. The effect on autogeneic cells was determined by comparing groups A (fasting) and C (non-fasting).

Figure 1A shows the effect of fasting on autogeneic lymphocytes; decreased expression of CD8 and CD25 was found in the mice in group A compared with mice in group C in experiments 11B (p = 0.02) and 11C (p = 0.019).

Fasting significantly affected cytokine secretion by the out-of-body syngeneic lymphocytes in non-splenectomized mice.

Figure 1B shows a significant increase in the TNFα levels (p = 0.047) when comparing group E (fasting) to group F (non-fasting, the TNFα levels were undetectable in-group F).

The data suggested that correlations between immune system components can be induced after fasting in splenectomized and non-splenectomized mice targeting out-of-body autogeneic and syngeneic lymphocytes. Fasting did not affect the syngeneic lymphocytes kept in a tube on top of the cages of splenectomized mice, as indicated by comparing groups B (fasting) and D (non-fasting).

Figure 1. The effect of fasting as a trigger for inducing associations between components of the immune system of splenectomized mice was determined by comparing the autogeneic lymphocytes from groups A (fasting) vs. C (non-fasting); there was a significant effect on the expression of CD8 and CD25 epitopes.

The effect of fasting as a trigger on splenectomized mice was determined by comparing the syngeneic lymphocytes kept in tubes on top of the cages of the mice in groups B (fasting) vs. D (non-fasting). The effect of fasting as a trigger for an association that may alter the epitope expression and/or cytokine secretion of out-of-body lymphocytes kept in tubes on top of cages was determined by comparing the syngeneic lymphocytes of the mice in groups E (fasting) vs. F (non-fasting); there was a significant effect on the TNFα secretion.

The effect of splenectomy as a trigger for altering the association that can modify epitope expression and/or cytokine secretion from out-of-body syngeneic lymphocytes kept in tubes on top of cages was investigated in non-fasting mice by comparing groups D (splenectomized) and F (non-splenectomized).

Figure 2A shows a significant increase in the CD8 expression (between time points 0 and 24 h) in experiments 11A (p = 0.021) and OL (p = 0.021) in group D compared with that in group F; however, the changes in CD8 expression in experiment 11A were opposite to those in experiment OL. The secretion of three cytokines, TNFα (p = 0.014), IFNγ (p = 0.021), and IL10 (p = 0.013), significantly increased in group D compared with group F, as shown in Figure 2B.

Figure 2. The effect of splenectomy as a trigger for an association that may alter the epitope expression and/or cytokine secretion of out-of-body lymphocytes kept in tubes on top of cages was determined by comparing the syngeneic lymphocytes kept in the tubes on top of the cages containing the non-fasting mice from groups D (splenectomized) vs. F (non-splenectomized).

The effect was significant for CD8 expression and for TNFα, IFNγ, and IL10 secretion. The effect was also determined by comparing the syngeneic lymphocytes that were kept in tubes on top of the cages containing fasting mice from groups B (splenectomized) vs. E (non-splenectomized). It was significant for CD25 and CD4 CD25 expressions and TNFα, IFNγ, and IL10 secretion.

The effect of splenectomy as a trigger for modifying the epitope expression and/or cytokine secretion of out-of-body syngeneic lymphocytes was investigated in fasting mice by comparing groups B (splenectomized) and E (non-splenectomized).

Figure 2C shows that splenectomy significantly affected several parameters, as indicated by comparing the two groups in experiments 11B and 11C; however, the effects in group B were opposite to those in group E. The expression of CD25 (p = 0.02 and p = 0.03 for 11B and 11C, respectively) and CD4 CD25 (p = 0.021 and p = 0.034 for 11B and 11C, respectively) differed significantly between groups B and E.

The secretion of TNFα (p = 0.021), IFNγ (p = 0.02), and IL10 (p = 0.02) were significantly higher in group B compared with that in group E, as shown in Figure 2D. The results suggested that a link can be induced between immune system components after splenectomy in fasting and non-fasting mice in syngeneic lymphocytes.

No significant differences were observed when assessing differences between the effects on out-of-body autogeneic vs. syngeneic cells; these differences were determined by performing a group A (autogeneic) vs. B (syngeneic) comparison of the lymphocytes harvested from the fasting-splenectomized mice and by performing a group C (autogeneic) vs. D (syngeneic) comparison of the non-fasting splenectomized mice.

The results suggested that the associations made for autogeneic cells did not differ significantly from that made for syngeneic cells.

To determine the effect of keeping the cells in an out-of-body location on the expression of lymphocyte epitopes, tubes kept on top of the cages of the fasting mice from groups A, B, and E were compared with tubes kept on top of empty cages (G), as shown in Figure 3. A significant change in the CD8 expression was noted by comparing groups B and G in 11B (p = 0.034) and OL (p = 0.043), as shown in Figure 3A; however, the changes in group B were opposite to those in group G. No significant effect was observed when comparing groups A and G.

Figure 3. The effect of the presence of a mouse in a cage on altering the expression of membrane epitopes and cytokine secretion was determined by analyzing the differences between the tubes from all groups and the tubes kept on empty cages (G and H).

The effect was significant for CD8 expression when comparing groups B and G (3A); for the secretion of TNFα and IFNγ when comparing groups A and G (3B); for the secretion of TNFα, IFNγ, and IL10 when comparing groups B and G (3C); for the expression of Foxp3 when comparing groups F and H (3D); for TNFα, IFNγ, and TGFβ secretion when comparing groups C and H (3E, 3F); for IFNγ, TNFα, and IL10 secretion when comparing groups D and H (3G); and for TNFα secretion when comparing groups F and H (3H).

To determine the effect of keeping cells in an out-of-body location on cytokine secretion, tubes kept on cages were tested for cytokine levels. A significant decrease was noted for the secretion of TNFα (p = 0.021) and IFNγ (p = 0.028) between groups A and G, as shown in Figure 3B. Similarly, a significant decrease was noted between groups B and G for the secretion of TNFα (p = 0.021), IFNγ (p = 0.021), and IL10 (p = 0.018), as shown in Figure 3C. No significant difference was observed between groups E and G for the epitope expression or cytokine secretion.

To determine the effect of keeping cells in an out-of-body location on the expression of lymphocyte epitopes, tubes kept on top of cages containing the non-fasting mice from groups C, D, and F were compared with tubes kept on top of empty cages (H). The Foxp3 expression significantly increased between groups F and H in all three experiments, as shown in Figure 3D (for 11A, p = 0.083; for 11B, p = 0.081; and 11C, p = 0.083; p = 0.006 for all three; this parameter was not tested in OL). No significant difference was observed when comparing groups C vs. H and D vs. H.

To determine the effect of keeping cells in an out-of-body location for cytokine secretion, tubes kept on top of cages were assessed for cytokine levels. A significant decrease in IFNγ, TNFα, and TGFβ secretion was observed when comparing groups C and H, as shown in Figure 3E (p = 0.020) and Figure 3F (p = 0.43). A significant decrease in IFNγ, TNFα, and IL10 secretion was observed between groups D and H, as shown in Figure 3G (p = 0.021). A significant increase in the TNFα secretion was observed between groups F and H, as shown in Figure 3H (p = 0.047). The data suggested that a fasting or non-fasting mouse in a cage affects its connections with syngeneic cells.

Discussion

The present study presents measurements of parameters over a set of mutually unbiased states, which demonstrated an ability to induce a correlation between components of the immune system through an indirect effect in an isolated system. The described experiment showed that a mouse could affect cells at a distance and alter the expression of surface markers and cytokine secretion following two triggers: fasting and/or splenectomy. In the described laboratory setting, only the measured observables resulting from this effect can be detected. The effects were compared for cells kept on top of cages containing mice that did not undergo a splenectomy and did not fast with cells kept in empty cages (Figures 1 and 2 vs. Figure 3).

In biological systems, different levels of associated states may be present. These levels might depend on the degree of comparison and/or correlation between donors and recipients that act simultaneously on various immune system components.³^–⁵ In the present schematic model, a correlation between two systems, a cell or other immune system component, allows for the recovery of a new state of a lymphocyte or cytokine-secreting cell.

The lymphocyte expression of cell surface markers and cytokine secretion involved different pathways.⁷^–¹⁰ The classical transportation of immune signals is based on messages delivered by molecules (e.g., chemokines) secreted by different subsets of cells that transmit a direct signal to a target cell or a subcellular organelle (e.g., receptor) through physical contact or a messenger molecule. The present data demonstrated that a correlation can be induced independent of a direct interaction in an isolated system. The observed effects cannot be explained by classical immunology suggesting a correlation-dependent phenomenon in the described system (Figures 1 and 2).

The effect that underlines the outcome observed in the present study on autogeneic cells can be explained by an inherent association between two immune system components, suggesting an undefined historical correlation between the two parts of the system. At the same time, the effects observed on syngeneic cells may imply a wave-dependent phenomenon between foreign components of the immune system.

The fidelity obtained in the current schematic model shows excellent adherence correlations properties when targeting the expression of cell membrane epitopes and cytokine secretion of lymphocytes. The results from each mouse in the described system suggest an inherent pattern that repeats itself. Each attempt for separate mice is viewed as independent of all others. Using a standard trigger, fasting or splenectomy, enables our protocol to succeed without filtering results, and the effects are relatively reliable. The experiments were repeated in an independent laboratory, further supporting the feasibility of setting up isolated conditions under which these effects can be measured (Figuers 1-3).

The lack of effects for all parameters in all experiments (see Underlying data²¹) and changes in opposing directions for some of the measured observables may result from the multiple confounding factors that act simultaneously, which are hard to control, or from the disordered nature of the immune pathways and networks. These are inherent to any biological system.¹¹^–¹⁴ The out-of-body correlation was significant for several investigated parameters in repeated experiments. For some of the observables, the changes were small and could be claimed to result from intra-test variability. However, each mouse was tested in a separate cage, which can be considered independent of the others. Only parameters that significantly changed in two separate experiments were used for the final data analysis.

The results described in the present study may support built-in memory in an isolated system, which is mandatory for such correlations to occur. Although memory in immune systems is based on the direct delivery of mediators or messenger molecules secreted by immune cells,¹⁵^,¹⁶ a “wave type of memory” is required at both the transmitting and receiving sites for such a correlation to occur. This type of immune memory may explain some of the observations in the present study.

The effects observed in the present study may occur at a cellular or subcellular level and may act per theories that apply some of the principles of quantum physics to biological systems.¹¹^,¹⁷^–¹⁹

A model using a non-natural computer-brain interface to induce an out-of-body effect, which showed non-invasive information transfer between the brains of different species, has been reported.²⁰ A translation of the intention of a human volunteer to stimulate the rat brain motor area responsible for tail movement has also been demonstrated. The data suggested the feasibility of a computer-mediated brain–brain interface that can link the neural functions between two biological entities.

The system described here provides a robust model for biological applications of correlations in immunology and biology. The model demonstrated here could also be used as an elementary constituent of a wave-type repeater in other biological systems. The state implemented in this protocol is based on complex systems, which enables modifying the surface markers and cytokine secretion of lymphocytes. Even with a relatively low success probability, this system can be scaled to more complex biological systems.

The optimal function of the immune system may require an alliance between classical and correlative immunology. The present data shed light on several ununderstood biological phenomena, including inter-immune cell interactions, brain–immune component connections, environmental impacts, genotype-phenotype interfaces, and organism-host interactions. By enabling diagnostic and therapeutic procedures to be performed on out-of-body autogeneic or syngeneic tissues or organisms, these concepts may apply to developing improved methods for diagnosing and treating immune-associated disorders.

Study limitations include using the defined model and extrapolations to other biological systems that may require further studies.

Conclusions

This study measured parameters on a group of mutually unbiased states, demonstrating the ability to induce a correlation between immune system components through an indirect effect in an isolated system. The experiment revealed that a mouse could affect cells at a distance and alter the expression of surface markers and cytokine secretion following two triggers: fasting and/or splenectomy. The findings of this research characterized a system for establishing a correlation between two components of the immune system without the need for mediators. The data suggests that two immune system components could induce an out-of-body correlation.

Data availability

Underlying data

Dryad: Correlations between components of the immune system, https://doi.org/10.5061/dryad.prr4xgxn3.²¹

This project contains the following underlying data:

- LDB_EM_Supplementary_File_1.5.xls

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

Reporting guidelines

PLoS Biol: ARRIVE checklist for ‘Correlations between components of the immune system’, https://doi.org/10.1371/journal.pbio.3000411.²²

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Version 3

VERSION 3 PUBLISHED 22 Nov 2021

Author details Author details

¹ Hadassah University Hospital, Jerusalem, Jerusalem, Israel, Israel

Yehudit Shabat
Roles: Data Curation, Formal Analysis, Methodology, Writing – Original Draft Preparation

Yaron Ilan
Roles: Conceptualization, Data Curation, Formal Analysis, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

This work was supported by the Roaman-Epstein Research Foundation
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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© 2024 Shabat Y and Ilan Y. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Shabat Y and Ilan Y. Correlations between components of the immune system [version 3; peer review: 2 approved, 1 not approved]. F1000Research 2024, 10:1174 (https://doi.org/10.12688/f1000research.54487.3)

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Reviewer Report 28 Feb 2024

Rohit Malik, Department of Pharmacology, Gurugram Global College of Pharmacy, SRM Modi Nagar College of Pharmacy, SRMIST, Delhi-NCR Campus, Ghaziabad, Gurugram, Haryana, India

Not Approved

https://doi.org/10.5256/f1000research.156314.r245655

The manuscript necessitates significant revisions:
1) The Abstract's Results section is unclear, as it merely presents outcomes without clearly articulating the determination process as stated.
2) Reconstruct the Conclusion section of the Abstract to align with your findings.
3) Add the complete abbreviations as mentioned in the manuscript.
4) Major revisions are needed in the Introduction section to justify the aspects of your study adequately.
5) Why n=4 in animals groupings, instead of n=6 Justify.
6) Insert a graphical representation of the research protocol with the grouping of animals and day-wise plan.
7) Cross-check the sequence of references in the manuscript.
8) Why was SPSS used instead of GraphPad Prism?
9) Regenerate the figures to include standard deviation (SD), standard error of the mean (SEM), degrees of freedom (Df) values, comparisons among all groups, and R-squared values for a clearer understanding of the results.
10) In the discussion part, mention the findings with figure numbers when compared with previous studies.
11) Add the conclusion part to the manuscript.

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

No

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Ethnopharmacology and Natural Product Chemistry

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

CITE

Report a concern

Author Response 13 Apr 2024

Yaron Ilan, Hadassah University Hospital, Jerusalem, Jerusalem, Israel

13 Apr 2024

Author Response
Response to reviewers
1. The Abstract's Results section is unclear, as it merely presents outcomes without clearly articulating the determination process as stated.
Response: The paragraph was rewritten ... Continue reading
Response to reviewers

The Abstract's Results section is unclear, as it merely presents outcomes without clearly articulating the determination process as stated.

Response: The paragraph was rewritten for improved clarity.

2) Reconstruct the Conclusion section of the Abstract to align with your findings.
Response: We accepted the comment, and the paragraph was revised.

3) Add the complete abbreviations as mentioned in the manuscript.
Response: Corrected

4) Major revisions are needed in the Introduction section to justify the aspects of your study adequately.
Response: The Introduction section was revised for improved clarity.

5) Why n=4 in animals groupings, instead of n=6 Justify.
Response: The study design determined the number of animals to enable significance. We have revised the relevant section for improved accuracy.

6) Insert a graphical representation of the research protocol with the grouping of animals and day-wise plan.
Response: We have revised Table 1 and added Table 2 with a graphical representation of the research protocol as suggested.

7) Cross-check the sequence of references in the manuscript.
Response: Corrected.

8) Why was SPSS used instead of GraphPad Prism?
Response: The method was recommended by one of the reviewers.

9) Regenerate the figures to include standard deviation (SD), standard error of the mean (SEM), degrees of freedom (Df) values, comparisons among all groups, and R-squared values for a clearer understanding of the results.
Response: A new set of figures is included with the SD. We have revised the relevant paragraphs in the results to include the statistical data.

10) In the discussion part, mention the findings with figure numbers when compared with previous studies.
Response: Corrected as suggested.

11) Add the conclusion part to the manuscript.
Response: A conclusion section was added as suggested.
Response to reviewers

The Abstract's Results section is unclear, as it merely presents outcomes without clearly articulating the determination process as stated.

Response: The paragraph was rewritten for improved clarity.

2) Reconstruct the Conclusion section of the Abstract to align with your findings.
Response: We accepted the comment, and the paragraph was revised.

3) Add the complete abbreviations as mentioned in the manuscript.
Response: Corrected

4) Major revisions are needed in the Introduction section to justify the aspects of your study adequately.
Response: The Introduction section was revised for improved clarity.

5) Why n=4 in animals groupings, instead of n=6 Justify.
Response: The study design determined the number of animals to enable significance. We have revised the relevant section for improved accuracy.

6) Insert a graphical representation of the research protocol with the grouping of animals and day-wise plan.
Response: We have revised Table 1 and added Table 2 with a graphical representation of the research protocol as suggested.

7) Cross-check the sequence of references in the manuscript.
Response: Corrected.

8) Why was SPSS used instead of GraphPad Prism?
Response: The method was recommended by one of the reviewers.

9) Regenerate the figures to include standard deviation (SD), standard error of the mean (SEM), degrees of freedom (Df) values, comparisons among all groups, and R-squared values for a clearer understanding of the results.
Response: A new set of figures is included with the SD. We have revised the relevant paragraphs in the results to include the statistical data.

10) In the discussion part, mention the findings with figure numbers when compared with previous studies.
Response: Corrected as suggested.

11) Add the conclusion part to the manuscript.
Response: A conclusion section was added as suggested.
Competing Interests: None Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 13 Apr 2024

Yaron Ilan, Hadassah University Hospital, Jerusalem, Jerusalem, Israel

13 Apr 2024

Author Response
Response to reviewers
1. The Abstract's Results section is unclear, as it merely presents outcomes without clearly articulating the determination process as stated.
Response: The paragraph was rewritten ... Continue reading
Response to reviewers

The Abstract's Results section is unclear, as it merely presents outcomes without clearly articulating the determination process as stated.

Response: The paragraph was rewritten for improved clarity.

2) Reconstruct the Conclusion section of the Abstract to align with your findings.
Response: We accepted the comment, and the paragraph was revised.

3) Add the complete abbreviations as mentioned in the manuscript.
Response: Corrected

4) Major revisions are needed in the Introduction section to justify the aspects of your study adequately.
Response: The Introduction section was revised for improved clarity.

5) Why n=4 in animals groupings, instead of n=6 Justify.
Response: The study design determined the number of animals to enable significance. We have revised the relevant section for improved accuracy.

6) Insert a graphical representation of the research protocol with the grouping of animals and day-wise plan.
Response: We have revised Table 1 and added Table 2 with a graphical representation of the research protocol as suggested.

7) Cross-check the sequence of references in the manuscript.
Response: Corrected.

8) Why was SPSS used instead of GraphPad Prism?
Response: The method was recommended by one of the reviewers.

9) Regenerate the figures to include standard deviation (SD), standard error of the mean (SEM), degrees of freedom (Df) values, comparisons among all groups, and R-squared values for a clearer understanding of the results.
Response: A new set of figures is included with the SD. We have revised the relevant paragraphs in the results to include the statistical data.

10) In the discussion part, mention the findings with figure numbers when compared with previous studies.
Response: Corrected as suggested.

11) Add the conclusion part to the manuscript.
Response: A conclusion section was added as suggested.
Response to reviewers

The Abstract's Results section is unclear, as it merely presents outcomes without clearly articulating the determination process as stated.

Response: The paragraph was rewritten for improved clarity.

2) Reconstruct the Conclusion section of the Abstract to align with your findings.
Response: We accepted the comment, and the paragraph was revised.

3) Add the complete abbreviations as mentioned in the manuscript.
Response: Corrected

4) Major revisions are needed in the Introduction section to justify the aspects of your study adequately.
Response: The Introduction section was revised for improved clarity.

5) Why n=4 in animals groupings, instead of n=6 Justify.
Response: The study design determined the number of animals to enable significance. We have revised the relevant section for improved accuracy.

6) Insert a graphical representation of the research protocol with the grouping of animals and day-wise plan.
Response: We have revised Table 1 and added Table 2 with a graphical representation of the research protocol as suggested.

7) Cross-check the sequence of references in the manuscript.
Response: Corrected.

8) Why was SPSS used instead of GraphPad Prism?
Response: The method was recommended by one of the reviewers.

9) Regenerate the figures to include standard deviation (SD), standard error of the mean (SEM), degrees of freedom (Df) values, comparisons among all groups, and R-squared values for a clearer understanding of the results.
Response: A new set of figures is included with the SD. We have revised the relevant paragraphs in the results to include the statistical data.

10) In the discussion part, mention the findings with figure numbers when compared with previous studies.
Response: Corrected as suggested.

11) Add the conclusion part to the manuscript.
Response: A conclusion section was added as suggested.
Competing Interests: None Close
Report a concern

Views

Reviewer Report 06 Oct 2023

Suprabhat Mukherjee, Integrative Biochemistry & Immunology Laboratory, Department of Animal Science, Kazi Nazrul University, Asansol, West Bengal, India

Approved

https://doi.org/10.5256/f1000research.156314.r210454

Authors have addressed ... Continue reading

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Version 1

VERSION 1

PUBLISHED 22 Nov 2021

Views

Reviewer Report 11 Sep 2023

Biswadeep Das, School of Biotechnology, Kalinga Institute of Industrial Technology, KIIT Deemed to Be University, Bhubaneswar, Odisha, India

Not Approved

https://doi.org/10.5256/f1000research.57980.r197558

The manuscript depicts the correlation of different components of the immune system in fasting and splenectomy scenarios. Though the topic is moderately interesting, and in context to metabolism and immune system cross-talk, the overall manuscript and the methods are inadequate ... Continue reading

The introduction is too small and does not involve the rationale behind selecting such a research work. Need to elaborate significantly.
Methods are the major limitation: The selection of mice need to be well defined. Flow cytometry is very subtly written. The authors say that, "The following subsets were analyzed: CD4, CD25, CD4 CD25, Foxp3, CD4 Foxp3, CD4 CD25 Foxp3, CD4 CD25 Foxp3, CD8, CD8 Foxp3, CD8 CD25, CD8 CD25 Foxp3, CD8 CD25 Foxp3, CD3, NK1.1, and CD3 NK1.1."

However, this is just a random statement. How to classify the different immune cells without using a backbone flow cytometry, LCA? First major classification need to be done, followed by subset analysis. Where are the raw files of flow cytometry? What about autofluorescence and compensation? How did u confirm the expression of these markers using experimental and instrumental controls?
Western blot need to be done to confirm the existence of the markers in the given population. It is missing.

Results:

No flow cytometry graph or quadrant analysis. Just a mere bar diagram does not prove the analysis. Western blot has to be shown for all the markers as confirmed by flow cytometer.

In view of these facts, I cant be more positive and have to opt for rejection, unless all these issues are addressed.

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Biomedical genetics and diganostics

CITE

Report a concern

Author Response 28 Sep 2023

Yaron Ilan, Hadassah University Hospital, Jerusalem, Jerusalem, Israel

28 Sep 2023

Author Response
1. The Introduction section was revised as proposed for better clarity. The relevant references were inserted.
2. A supplementary file is included with all the data of
... Continue reading
The Introduction section was revised as proposed for better clarity. The relevant references were inserted.

A supplementary file is included with all the data of all experiments. The relevant section in the Methods was revised for better clarity.

Western blots were not conducted in the present study and are subject to future studies.

The data of all studies is included in the supplementary file. The graphs were not included as multiple analyses were conducted in these experiments.
The Introduction section was revised as proposed for better clarity. The relevant references were inserted.

A supplementary file is included with all the data of all experiments. The relevant section in the Methods was revised for better clarity.

Western blots were not conducted in the present study and are subject to future studies.

The data of all studies is included in the supplementary file. The graphs were not included as multiple analyses were conducted in these experiments.
Competing Interests: NA Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 28 Sep 2023

Yaron Ilan, Hadassah University Hospital, Jerusalem, Jerusalem, Israel

28 Sep 2023

Author Response
1. The Introduction section was revised as proposed for better clarity. The relevant references were inserted.
2. A supplementary file is included with all the data of
... Continue reading
The Introduction section was revised as proposed for better clarity. The relevant references were inserted.

A supplementary file is included with all the data of all experiments. The relevant section in the Methods was revised for better clarity.

Western blots were not conducted in the present study and are subject to future studies.

The data of all studies is included in the supplementary file. The graphs were not included as multiple analyses were conducted in these experiments.
The Introduction section was revised as proposed for better clarity. The relevant references were inserted.

A supplementary file is included with all the data of all experiments. The relevant section in the Methods was revised for better clarity.

Western blots were not conducted in the present study and are subject to future studies.

The data of all studies is included in the supplementary file. The graphs were not included as multiple analyses were conducted in these experiments.
Competing Interests: NA Close
Report a concern

Views

Reviewer Report 02 Sep 2022

Suprabhat Mukherjee, Integrative Biochemistry & Immunology Laboratory, Department of Animal Science, Kazi Nazrul University, Asansol, West Bengal, India

Approved with Reservations

https://doi.org/10.5256/f1000research.57980.r144783

Approval/reference number of the meeting of the Institutional Animal Ethics Committee for the Mice related experiments is required.
Figures need to be revised.
Names of the cytokines

Approval/reference number of the meeting of the Institutional Animal Ethics Committee for the Mice related experiments is required.
Figures need to be revised.
Names of the cytokines should be formatted correctly.
Please perform ANOVA following a post-hoc test to test the difference amongst the cytokine level.
Immunofluorescence data may be added to strengthen the data of FACS and ELISA.

These changes are necessary to improve the manuscript.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Immunology of Inflammatory and Infectious diseases

I 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.

CITE

Report a concern

Author Response 28 Sep 2023

Yaron Ilan, Hadassah University Hospital, Jerusalem, Jerusalem, Israel

28 Sep 2023

Author Response
1. Approval details were added.
2. All figures were replaced with a more explicit version.
3. Corrected.
4. The Mann-Whitney SPSS test (software version 1.0)
... Continue reading
Approval details were added.

All figures were replaced with a more explicit version.

Corrected.

The Mann-Whitney SPSS test (software version 1.0) was used for revised statistical analysis.

These were not performed in the current study and are subject to future experiments.
Approval details were added.

All figures were replaced with a more explicit version.

Corrected.

The Mann-Whitney SPSS test (software version 1.0) was used for revised statistical analysis.

These were not performed in the current study and are subject to future experiments.
Competing Interests: NA Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 28 Sep 2023

Yaron Ilan, Hadassah University Hospital, Jerusalem, Jerusalem, Israel

28 Sep 2023

Author Response
1. Approval details were added.
2. All figures were replaced with a more explicit version.
3. Corrected.
4. The Mann-Whitney SPSS test (software version 1.0)
... Continue reading
Approval details were added.

All figures were replaced with a more explicit version.

Corrected.

The Mann-Whitney SPSS test (software version 1.0) was used for revised statistical analysis.

These were not performed in the current study and are subject to future experiments.
Approval details were added.

All figures were replaced with a more explicit version.

Corrected.

The Mann-Whitney SPSS test (software version 1.0) was used for revised statistical analysis.

These were not performed in the current study and are subject to future experiments.
Competing Interests: NA Close
Report a concern

Comments on this article Comments (0)

Version 3

VERSION 3 PUBLISHED 22 Nov 2021

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Version 3 (revision) 12 Mar 24			read
Version 2 (revision) 28 Sep 23	read		read
Version 1 22 Nov 21	read	read

Suprabhat Mukherjee, Kazi Nazrul University, Asansol, India
Biswadeep Das, KIIT Deemed to Be University, Bhubaneswar, India
Rohit Malik, Gurugram Global College of Pharmacy, SRM Modi Nagar College of Pharmacy, SRMIST, Delhi-NCR Campus, Ghaziabad, Gurugram, India

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Reviewer Report

11 Views

15 Apr 2024 | for Version 3

Rohit Malik, Department of Pharmacology, Gurugram Global College of Pharmacy, SRM Modi Nagar College of Pharmacy, SRMIST, Delhi-NCR Campus, Ghaziabad, Gurugram, Haryana, India

11 Views Cite this report Responses(0)

Approved

The Authors updated the manuscript as per the suggestions.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Ethnopharmacology and Natural Product Chemistry

I 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.

Respond to this report

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Reviewer Report

13 Views

28 Feb 2024 | for Version 2

Rohit Malik, Department of Pharmacology, Gurugram Global College of Pharmacy, SRM Modi Nagar College of Pharmacy, SRMIST, Delhi-NCR Campus, Ghaziabad, Gurugram, Haryana, India

13 Views Cite this report Responses(1)

Not Approved

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Ethnopharmacology and Natural Product Chemistry

Respond to this report

Responses (1)

Author Response

13 Apr 2024

Yaron Ilan, Hadassah University Hospital, Jerusalem, Jerusalem, Israel

Response to reviewers

The Abstract's Results section is unclear, as it merely presents outcomes without clearly articulating the determination process as stated.

Response: The paragraph was rewritten for improved clarity.

2) Reconstruct the Conclusion section of the Abstract to align with your findings.
Response: We accepted the comment, and the paragraph was revised.

3) Add the complete abbreviations as mentioned in the manuscript.
Response: Corrected

4) Major revisions are needed in the Introduction section to justify the aspects of your study adequately.
Response: The Introduction section was revised for improved clarity.

5) Why n=4 in animals groupings, instead of n=6 Justify.
Response: The study design determined the number of animals to enable significance. We have revised the relevant section for improved accuracy.

6) Insert a graphical representation of the research protocol with the grouping of animals and day-wise plan.
Response: We have revised Table 1 and added Table 2 with a graphical representation of the research protocol as suggested.

7) Cross-check the sequence of references in the manuscript.
Response: Corrected.

8) Why was SPSS used instead of GraphPad Prism?
Response: The method was recommended by one of the reviewers.

9) Regenerate the figures to include standard deviation (SD), standard error of the mean (SEM), degrees of freedom (Df) values, comparisons among all groups, and R-squared values for a clearer understanding of the results.
Response: A new set of figures is included with the SD. We have revised the relevant paragraphs in the results to include the statistical data.

10) In the discussion part, mention the findings with figure numbers when compared with previous studies.
Response: Corrected as suggested.

11) Add the conclusion part to the manuscript.
Response: A conclusion section was added as suggested.

View more View less

Competing Interests

None

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Reviewer Report

5 Views

06 Oct 2023 | for Version 2

Suprabhat Mukherjee, Integrative Biochemistry & Immunology Laboratory, Department of Animal Science, Kazi Nazrul University, Asansol, West Bengal, India

5 Views Cite this report Responses(0)

Approved

Authors have addressed all the queries.

Competing Interests

No competing interests were disclosed.

I 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.

Respond to this report

Responses (0)

Back to all reports

Reviewer Report

26 Views

11 Sep 2023 | for Version 1

Biswadeep Das, School of Biotechnology, Kalinga Institute of Industrial Technology, KIIT Deemed to Be University, Bhubaneswar, Odisha, India

26 Views Cite this report Responses(1)

Not Approved

The introduction is too small and does not involve the rationale behind selecting such a research work. Need to elaborate significantly.
Methods are the major limitation: The selection of mice need to be well defined. Flow cytometry is very subtly written. The authors say that, "The following subsets were analyzed: CD4, CD25, CD4 CD25, Foxp3, CD4 Foxp3, CD4 CD25 Foxp3, CD4 CD25 Foxp3, CD8, CD8 Foxp3, CD8 CD25, CD8 CD25 Foxp3, CD8 CD25 Foxp3, CD3, NK1.1, and CD3 NK1.1."

However, this is just a random statement. How to classify the different immune cells without using a backbone flow cytometry, LCA? First major classification need to be done, followed by subset analysis. Where are the raw files of flow cytometry? What about autofluorescence and compensation? How did u confirm the expression of these markers using experimental and instrumental controls?
Western blot need to be done to confirm the existence of the markers in the given population. It is missing.

Results:

No flow cytometry graph or quadrant analysis. Just a mere bar diagram does not prove the analysis. Western blot has to be shown for all the markers as confirmed by flow cytometer.

In view of these facts, I cant be more positive and have to opt for rejection, unless all these issues are addressed.

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Biomedical genetics and diganostics

Respond to this report

Responses (1)

Back to all reports

Reviewer Report

32 Views

02 Sep 2022 | for Version 1

Suprabhat Mukherjee, Integrative Biochemistry & Immunology Laboratory, Department of Animal Science, Kazi Nazrul University, Asansol, West Bengal, India

32 Views Cite this report Responses(1)

Approved With Reservations

Approval/reference number of the meeting of the Institutional Animal Ethics Committee for the Mice related experiments is required.
Figures need to be revised.
Names of the cytokines should be formatted correctly.
Please perform ANOVA following a post-hoc test to test the difference amongst the cytokine level.
Immunofluorescence data may be added to strengthen the data of FACS and ELISA.

These changes are necessary to improve the manuscript.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Immunology of Inflammatory and Infectious diseases

Respond to this report

Responses (1)

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

[1] 1. Brizhik L, Foletti A:Nonlinear quantum phenomena and biophysical aspects of complexity related to health and disease. J. Biol. Regul. Homeost. Agents. 2014; 28(3): 357–66. PubMed Abstract

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Correlations between components of the immune system

Abstract

Background

Methods

Results

Conclusions

Keywords

Revised Amendments from Version 2

Abbreviations

Introduction

Methods

Animals

Experimental groups

Table 1. Experimental groups (n = 4 for each group).

Table 2. A representation of the research protocol.

Study design

Flow cytometry

Cytokine measurement

Statistical analysis

Results

Figure 3. The effect of the presence of a mouse in a cage on altering the expression of membrane epitopes and cytokine secretion was determined by analyzing the differences between the tubes from all groups and the tubes kept on empty cages (G and H).

Discussion

Conclusions

Data availability

Underlying data

Reporting guidelines

References

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