Establishment of an Epicutaneously Sensitized Murine Model of Shellfish Allergy and Evaluation of Skin Condition by Raman Microscopy

Round 1

Reviewer 1 Report

The manuscript by Ichimura-Shimizu et al. describes the development of a shellfish allergy skin sensitisation model in mice and shows that shellfish allergy can be induce through tape-stripped skin which is also associated with some skin damage detected by Raman microscopy. The manuscript is well-written, and the topic of skin sensitisation is a very relevant and interesting area of research. However, the manuscript is very lightweight and does not provide much information.

 

This reviewer has some major concerns.

The authors describe their research as novel in the light of only OVA- and peanut-induced food allergy have been reported in animal models of skin sensitisation. It is truth that several models of skin sensitisation have been developed and applied for OVA and peanut allergy. However, many other models of skin sensitisation have been developed and applied for eg. hazelnut, sesame, cashew, cow’s milk, wheat, soy, green bean, and papaya using rats as well as mice. Hence, it is acknowledged that sensitisation can be induced through the skin in animal models.

The authors describe that the skin damage conferred by the tape-stripping led to the sensitisation, however, the authors have not included a control group with intact skin and hence cannot conclude that it is the damage that led to the sensitisation. It has previously been shown that allergens may also sensitise through an intact skin.

 

Specific comments:

 

Line 101-115: The description of the animal experimental procedure should be much more detailed. For example, how many times were the skin tape-stripped? How was the shrimp tropomyosin diluted? In PBS? For how long was the skin covered by the Finn chamber? On how large an area was the shrimp tropomyosin applied?

It is written in the result section and shown in Figure S1, that yet another animal experimental protocol was used. Please include a description of this as well.

 

Line 127-130: The authors describe the test of specific IgE in rMet-sensitised mice. Did you not test specific IgE in control mice?

 

Line 143: Why did the authors apply SDS on the skin?

 

Line 171-173: Please delete these sentences.

 

Line 186: Please provide more figure text.

 

Line 197: Please provide more figure text.

 

Line 212: Please delete text.

 

Line 252-255: References are missing.

 

Figure 1B: It would be nice to see the response of the individual mice instead of the mean.

 

Figure 2B: The pictures of the mice should be bigger.

 

Figure 3C: The histology pictures of the skin should be bigger.

Author Response

Thank you very much for your valuable comments. Your comments have been very helpful in allowing us to revise our manuscript. Now we revised our manuscript accordingly.

<Major points>

Point1: The fact that is already known to be a sensitization via the skin in rats and other animals.

Response 1: We had considered mice, which are smaller than rats, to be more useful as animal models in view of lower dosage in the development of therapeutic drugs, and therefore, only the mouse model had been discussed in our manuscript. However, as the reviewer pointed out, sensitization through the skin has been confirmed in many allergens and animals. Now we’ve omitted the sentence emphasizing novelty as an animal model of food allergy by epicutaneous sensitization (lines 70-71 and 236-237)

Point 2: Regarding the difficulty to conclude that damage by tape-stripping induced sensitization

Response 2: We totally agree to the reviewer’s comment that it cannot be concluded that it was skin damage that led to sensitization. Therefore, the skin damage caused by tape-stripping was considered only as a methodology for creating model mice and was removed from the conclusions (line 296, the sentence was omitted).

 

<Specific comments>

Point 3: The description of the animal experimental procedure should be much more detailed. For example, how many times were the skin tape-stripped? How was the shrimp tropomyosin diluted? In PBS? For how long was the skin covered by the Finn chamber? On how large an area was the shrimp tropomyosin applied?

It is written in the result section and shown in Figure S1, that yet another animal experimental protocol was used. Please include a description of this as well.

Response 3: Detailed procedures have been added in method section. In detail, three times tape-stripping was done before allergen exposure (line 94); Tropomyosin was diluted with PBS (lines 117-118); Allergens were soaked into 8 mm diameter filter paper and attached to the back skin with Finn chamber for 24h (lines 96-99).

Another animal protocol was added in the Supplemental Information.

Point 4: The authors describe the test of specific IgE in rMet-sensitised mice. Did you not test specific IgE in control mice?

Response 4: We measured specific IgE levels in both sensitized and control mice. Now we amended the text (line 133).

Point 5:Why did the authors apply SDS on the skin?

Response 5: Since the application of SDS solution is a typical technique used for epicutaneous sensitization as well as tape-stripping, we aimed to compare the skin condition affected by both procedures (lines 146-148).

Point 6: Please delete these sentences.

Response 6: We forgot to delete the template. Now unnecessary sentences were deleted.

Point 7:  Please provide more figure text.

Response 7: Legend in Figure 1 has been increased (lines 193-196).

Point 8: Please provide more figure text.

Response 8: Legend in Figure 2 has been increased (lines 208-213).

Point 9:  Please delete text.

Response 9: We forgot to delete the template. Unnecessary sentences were deleted.

Point 10: References are missing.

Response 10: We added a reference to the first sentence (line 267). The second sentence was a statement of our thoughts, so we modified the sentence (line 268).

Point 11: It would be nice to see the response of the individual mice instead of the mean.

Response 11: Individual values have been plotted in Figure1B.

Point 12: The pictures of the mice should be bigger.

Response 12: The picture size of the mice has been increased (Figure 2B).

Point 13: The histology pictures of the skin should be bigger.

Response 13: The picture size of the histology has been increased (Figure 3C).

Reviewer 2 Report

The article „ Establishment of an epicutaneously sensitized murine model of 2 shellfish allergy and evaluation of skin condition by Raman 3 microscopy” highlights the role of microscopy in diagnosis of food allergy, which is rare use in clinical practice. The authors also highlighted possible recommendations, still in experimental models. I have read the paper with interest and feel that it is relevant for area of Food Allergy diagnosis.

I suggest few minor revisions and comments are made below regarding the article.

1. Light punctuation correction is needed.
2. Please add “Limitations of the study” in discussion.
3. References should be modified according to the journal requests for publication:

 

Comments for author File: Comments.pdf

Author Response

Thank you very much for your valuable comments. Your comments have been very helpful in allowing us to revise our manuscript.

Point 1: Light punctuation correction is needed.

Response 1: Several punctuation were corrected.

Point 2:   Please add “Limitations of the study” in discussion.

Response 2: “Limitations of the study” was added in discussion (lines 284-289).

Point 3:   References should be modified according to the journal requests for publication:

Response 3: The citation style has been modified.

Reviewer 3 Report

Thank you for the opportunity to review the manuscript titled, “Establishment of an epicutaneously sensitized murine model of shellfish allergy and evaluation of skin condition by Raman microscopy.” On this manuscript, the authors used a “, a mouse model of shrimp allergy was generated by epicutaneous sensitization 27 and used to identify skin conditions associated with susceptibility to sensitization” (copied verbatim from manuscript).

 

1: Abstract: The results are reasonably well described, but offer no guidance on how to interpret “lower relative intensities.” This is a method that may be unfamiliar to many readers, and yet is seemingly central to the study given that the method is listed in the title. Please add some words of guidance.

2: Shellfish allergy is not one of the most common food allergies in all parts of the world, and indeed appears to vary by ethnicity and geography. Please revise the sentence in Line 42.

3: Shellfish allergy tends to present in early childhood and persist throughout life. Please revise Lines 46-47.

4: When speaking about food allergy, the word “cure” is rarely used. Rather, “clinical tolerance” is preferred. Please revise Lines 48-49.

5: Anaphylaxis may be life-threatening. Please revise Line 51.

6: Food allergy, not only anaphylaxis, is associated with poorer quality of life, and health-related quality of life, for both the individual with the allergy, as well as their family. Please revise Lines 51-52.

7: To what food crisis are the authors referring in Line 59?

8: The mention of “immature digestive function” in Lines 60-61 is not entirely correct. Please revise.

9: Methods: This section is poorly organized and lacks detail. How many mice were in this study? Respectfully, the authors are encouraged to consider describing the animal model first, then the preparation of recombinant shrimp tropomyosin. Also, it is unclear how long after exposure serum was collected for IgE analysis.  

10: Methods: Why were symptoms of anaphylaxis evaluated only 30-40 min after oral challenge? In human, anaphylaxis occurs in minutes. A lengthy delay would suggest a non-Type 1 hypersenstivity reaction (i.e. not IgE mediated), and therefore not anaphylaxis.

11: Methods: Please confirm the methods for the Raman microscopy are per manufacturer instructions.

12: Results, Lines 171-173, Line 212: Presumably these lines should be deleted.

13: Results: When noting that “significantly higher” levels were noted, please provide numeric results and p-values in the main text.

14: Discussion: Typically, the first sentence of a discussion is a summary of the major results, and does not include data or references to other studies.

15: Discussion: There are no remarks about knowledge translation or potential impact of the study.

16: The manuscript contains some linguistic and grammatical errors. Although these errors do not impede comprehension, they are nonetheless distracting and do need to be corrected.

Author Response

Thank you very much for your valuable comments. Your comments have been very helpful in allowing us to revise our manuscript. Now we revised our manuscript accordingly. 

Point 1: Abstract: The results are reasonably well described, but offer no guidance on how to interpret “lower relative intensities.” This is a method that may be unfamiliar to many readers, and yet is seemingly central to the study given that the method is listed in the title. Please add some words of guidance.

Response 1: The differences in Raman band intensity between sensitized mice and controls suggests that there was a change in the molecular species and structure that constitute the skin. The main focus of this study is that differences were detected. Future studies are needed to clarify the specific molecules which were observed to change. In this context, that sentence has been modified in abstract (lines 35-36).

Point 2: Shellfish allergy is not one of the most common food allergies in all parts of the world, and indeed appears to vary by ethnicity and geography. Please revise the sentence in Line 42.

Response 2: We amended that the frequency of shrimp allergy is higher in the Asia-Pacific region, not worldwide in the revise manuscript as the reviewer’s suggestion (line 43).

Point 3: Shellfish allergy tends to present in early childhood and persist throughout life. Please revise Lines 46-47.

Response 3: As the referee’s comment, we amended the text (lines 46-47).

Point 4: When speaking about food allergy, the word “cure” is rarely used. Rather, “clinical tolerance” is preferred. Please revise Lines 48-49.

Response 4: Thank you for your suggestion. Now we amended the text (line 48)

Point 5: Anaphylaxis may be life-threatening. Please revise Line 51.

Response 5: As the referee’s comment, we amended the text (line 50).

Point 6: Food allergy, not only anaphylaxis, is associated with poorer quality of life, and health-related quality of life, for both the individual with the allergy, as well as their family. Please revise Lines 51-52.

Response 6: As the referee’s comment, we amended the text (lines 51-52).

Point 7: To what food crisis are the authors referring in Line 59?

Response 7: Due to food shortages crisis, the use of insect diets such as crickets is being explored. The text indicates that shellfish allergic patients should be careful with insect diets that contain tropomyosin in the near future (line 59).

Point 8: The mention of “immature digestive function” in Lines 60-61 is not entirely correct. Please revise.

Response 8: We revised that “immature digestive function” may play a part in the pathogenesis of food allergy (line 60).

Point 9: Methods: This section is poorly organized and lacks detail. How many mice were in this study? Respectfully, the authors are encouraged to consider describing the animal model first, then the preparation of recombinant shrimp tropomyosin. Also, it is unclear how long after exposure serum was collected for IgE analysis.  

Response 9: The number of mice was added (lines 96 and 99). The order of the sections was rearranged, with Sensitization and Challenge of Mice written first and Preparation of Recombinant Shrimp Tropomyosin written after. Blood samples for IgE measurement were taken 24 hours after sensitization (lines 139-140).

Point 10: Methods: Why were symptoms of anaphylaxis evaluated only 30-40 min after oral challenge? In human, anaphylaxis occurs in minutes. A lengthy delay would suggest a non-Type 1 hypersenstivity reaction (i.e. not IgE mediated), and therefore not anaphylaxis.

Response 10: We evaluated the response after 30-40 minutes of challenge according to the references (Leung, P.S et al.), but actually received a response within at least 15 minutes. After reviewing the data of responses evaluated every 15 minutes after the challenge, the results were shown as data within 15 minutes (line 209), and the method was also amended (line 121).

Point 11: Methods: Please confirm the methods for the Raman microscopy are per manufacturer instructions.

Response 11: The instrument we used was a home-built laser-scanning confocal Raman microscope and there are no manufacturer instructions. We’re sorry that the sentences were written inaccurately. Now we amended the text (lines 157-159).

Point 12: Results, Lines 171-173, Line 212: Presumably these lines should be deleted.

Response 12: We forgot to delete the template. Unnecessary sentences were deleted.

Point 13: Results: When noting that “significantly higher” levels were noted, please provide numeric results and p-values in the main text.

Response 13: Numeric results and P-values were added to the main text (lines 180-182, 185-187, 202-204, supplemental figure legend. Also, we apologize but we’ve amended Figure 2C because it incorrectly showed a significant difference in some data.

Point 14: Discussion: Typically, the first sentence of a discussion is a summary of the major results, and does not include data or references to other studies.

Response 14: A summary of the results has been added to the first paragraph of the discussion (lines 233-236).

Point 15: Discussion: There are no remarks about knowledge translation or potential impact of the study.

Response 15: We added the possibility that skin diagnosis using Raman microscopy, based on this study, could be utilized for early detection of individuals at high risk for epicutaneous sensitization (lines 289-294).

Point 16: The manuscript contains some linguistic and grammatical errors. Although these errors do not impede comprehension, they are nonetheless distracting and do need to be corrected.

Response 16: Some linguistic and grammatical errors were corrected.

Round 2

Reviewer 3 Report

The authors are to be thanked for their attention to, and consideration of, earlier reviewer comments. Although many previous comments are satisfactorily addressed, some concerns remain, and new ones have been identified.

1. Lines 51-2 read as if being out of place. Consider if this text could be better placed elsewhere.
2. Figure 3: Th legends, particularly for Panel B, are difficult to read.

Author Response

We would like to thank the referees for the careful and constructive reviews. Based on the comments, we have revised our manuscript, which is detailed below. 

Point 1: Lines 51-2 read as if being out of place. Consider if this text could be better placed elsewhere.

Response 1: As reviewer commented, this sentence seems to be out of place. Now we removed it because we had stated at the beginning of the introduction that anaphylaxis is life-threatening (lines 45-46).

Point 2: Figure 3: The legends, particularly for Panel B, are difficult to read.

Response 2: Detailed explanation has been added to the legends of Figure 3B (lines 228-231).