The Severity of Gastrointestinal Disorders and Autistic-Like Behaviors Could Be Associated with a Selective Humoral Response to Bovine Milk Caseins: A Case Series

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors set to investigate the IgA/IgG/IgE mediated immunoreactivity to bovine milk caseins in subjects with ASD and to relate it to the intensity of gastrointestinal symptoms and autism-like behaviors.

The study is well-planned with clearly set goals and well described procedures and informatively presented results, altogether bringing the reader to logically derived and documented conclusions.

The symptoms’ assessment and grading tools also are correctly selected and appropriate sources cited. I do not have any comments with regard to statistics, either.

However, there are 2 issues that I’d like to raise and that – in my opinion – require some clarification.

1)     The selection of subjects to the study is not clearly described: have they been consecutive patients; all patients taken care of  by the authors’ team; or randomly selected subjects form the larger group ? This may be necessary to comment on in view of the STROBE checklist for cross-sectional studies, that the authors have cited: #10 “how the study size was arrived at”

2)     In conjunction with the above raised question, the issue of ALL subjects being negatively tested with regard to specific IgE to cow’s milk caseins. It is not clearly explained, if this was purely accidental or have the IgE-sensitized subjects been a priori excluded form the study? This could be a source of bias (if intentional), therefore, should be addressed in the context of the same STROBE checklist #9.

Author Response

The authors appreciate all your comments and suggestions to improve the uniqueness and scientific soundness of our manuscript originally entitled “The severity of gastrointestinal disorders and autistic-like behaviors could be associated with a selective humoral response to bovine milk caseins: A case series” (gastroent-3051906). Suggested changes are highlighted in Red in the new version (v2) of our manuscript while a point-by-point response (R.) is described below. All authors wish to have answered adequately to each of your observations. 

• The authors set to investigate the IgA/IgG/IgE mediated immunoreactivity to bovine milk caseins in subjects with ASD and to relate it to the intensity of gastrointestinal symptoms and autism-like behaviors. The study is well-planned with clearly set goals and well described procedures and informatively presented results, altogether bringing the reader to logically derived and documented conclusions. The symptoms’ assessment and grading tools also are correctly selected and appropriate sources cited. I do not have any comments with regard to statistics, either. However, there are 2 issues that I’d like to raise and that – in my opinion – require some clarification.

R. We thank you in advance for your positive opinion, we hope having fulfilled your expectations with all manuscript´s changes (v2).

• 1)     The selection of subjects to the study is not clearly described: have they been consecutive patients; all patients taken care of  by the authors’ team; or randomly selected subjects form the larger group ?. This may be necessary to comment on in view of the STROBE checklist for cross-sectional studies, that the authors have cited: #10 “how the study size was arrived at”

R. Although the reference study is much larger and with a different purpose than that addressed in this new report (Herrera-Mejía et al. (2023) Diagnostics, 13, 2387; https://doi.org/10.3390/diagnostics13142387), the case study reported here only included those participants who agreed to provide serum samples, recruited on a case-by-case basis over a period of just over a year (Jan 2023-Apr 2024). For this reason, our study only partially complies with the STROBE standards, a fact that was commented within the methods section in the previous version (v1) and modified to satisfy your suggestion (v2; lines 97-99):

(v1) - The study reported here met most criteria of the Reporting of observational studies in epidemiology (STROBE, https://www.strobe-statement.org) checklist for cross-sectional studies [18].

(v2) - This case series partially complies with the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology; https://www.strobe-statement.org) standards [18].

• 2)     In conjunction with the above raised question, the issue of ALL subjects being negatively tested with regard to specific IgE to cow’s milk caseins. It is not clearly explained, if this was purely accidental or have the IgE-sensitized subjects been a priori excluded form the study? This could be a source of bias (if intentional), therefore, should be addressed in the context of the same STROBE checklist #9.

R. Thank you very much for this insightful observation. The fact that no subject had titers suggestive of type 1 hypersensitivity (IgE-mediated) was as surprising to us as it was for you, considering that no participant was intentionally excluded (see exclusion criteria section 2.2 lines, lines 90-92). several factors (alone or in combination) could be playing a role in this fact, including A) The natural switching to a long-term adaptative humoral response (mediated by IgA and IgG but not IgE), B) That immunoreactivity was tested passively (basal serum titers) and not actively (e.g. after protein oral challenge) or C) that true allergenic response to bovine milk proteins was probably to non-casein proteins. These arguments were included in the last paragraph of discussion section.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for sending me this work for consideration. I congratulate the authors, they have done a good work. I have some little advice.

1-At the beginning of the discussion section, all the conclusions of this study should be summarized in one paragraph. Then, each result should be examined separately in paragraphs, first comparing the results of other studies and then the results of this study.

2-Separate subheadings were made and examined in the discussion section. I think this is confusing, as if it has created a situation like an article within an article. I think the subheading in the discussion section is unnecessary.

3-Limitations should be added to the discussion section.

4-The conclusion section should be rewritten. There should be no limitations here. I think the most important conclusion that can be drawn from this study is that the defect that led to the severity of autism in these patients may also have caused humoral impairment and impaired immunoreactivity.

Author Response

The authors appreciate all your comments and suggestions to improve the uniqueness and scientific soundness of our manuscript originally entitled “The severity of gastrointestinal disorders and autistic-like behaviors could be associated with a selective humoral response to bovine milk caseins: A case series” (gastroent-3051906). Suggested changes are highlighted in Red in the new version (v2) of our manuscript while a point-by-point response (R.) is described below. All authors wish to have answered adequately to each of your observations.  

• Thank you for sending me this work for consideration. I congratulate the authors, they have done a good work. I have some little advice.

R. We thank you in advance for your positive opinion, we hope having fulfilled your expectations with all manuscript´s changes (v2).

 

• 1-At the beginning of the discussion section, all the conclusions of this study should be summarized in one paragraph. Then, each result should be examined separately in paragraphs, first comparing the results of other studies and then the results of this study.

R. Thank you very much for your suggestion. The following statement was added to comply with your request (lines 238-243):

 

(v2) - In this study, using a stepwise immunochemical assessment, we report that the basal immunogenic status (not assessed by oral challenge with dietary proteins) of most participants, resembles a mixed or type IV (IgG+/IgM+ > IgA+) rather than type I (IgE-mediated) food hypersensitivity tht was strongly associated with the severity of GIS and ASD-like behaviors in a progressive inmmunogenic manner [IgG-/IgA- (reference)< IgG+/IgA- (mono)< IgG-/IgA- (dual)].

.

• 2-Separate subheadings were made and examined in the discussion section. I think this is confusing, as if it has created a situation like an article within an article. I think the subheading in the discussion section is unnecessary.

R. Thank you so much for your opinion. The subtitles were removed accordingly.

• 3-Limitations should be added to the discussion section.

R. Thanks for your observation. The study limitations were removed form conclusion section and located in the last paragraph of discussion section (lines 339-345, “lastly…behaviors”).

• 4-The conclusion section should be rewritten. There should be no limitations here. I think the most important conclusion that can be drawn from this study is that the defect that led to the severity of autism in these patients may also have caused humoral impairment and impaired immunoreactivity

R. Thanks for your observation. Conclusion section was reduced as requested as follows (lines 346-350):

(v2)- IgA+ > IgG+ but not IgE+ response to BMC (mainly α-casein) seems to be related to the severity of GIS and ASD-like behaviors, although a larger number of ASD patients are needed to disentangle such the direction of causality of this phenomenon (worsening of ASD/GIS symptoms ↔ humoral response/immunoreactivity grading).

Author Response File: Author Response.pdf