Reviewer's report
Title:An adjuvant free mouse model of oral allergenic sensitization to rice seeds
protein
Version: 1Date:28 July 2010
Reviewer: Gabriella Di Felice
Reviewer's report:
General
In this study, authors develop a mouse model of rice sensitization under
conditions mimicking the induction of human food allergy, such as the oral route
of immunization and the absence of adjuvants. Rice-specific IgE response,
hypersensitivity symptoms after oral challenge and histologic signs of jejunum
inflammation have been evaluated.
The contribution of robust in vivo model of food allergy to the understanding of
immunological mechanisms underlying the IgE-mediated response to allergenic
foods is well recognized, as well as their importance in the preclinical evaluation
of new therapeutic interventions. From this point of view, the information provided
by this study could be significant and original, mainly regarding the protocol of
oral immunization in the absence of adjuvants, that overcomes a common
paradox of food allergy model: the oral route is more similar to the human
situations, but in the model animals it results preferentially tolerogenic, thus
generally requiring an adjuvant for sensitization.
However, the study is mainly descriptive, and limited functional data are
provided. There is some basic and technical weakness that affect the robustness
of the results and as a consequence the overall quality and interest of the study
(see the following section).
-------------------------------------------------------------------------------
Major Compulsory Revisions
1. The Th2 cell response has not been studied at the systemic (spleen) or local
(mesenteric lymph nodes, jejunum tissue) level.
2. The anaphylactic response is evaluated by a classical symptom score ranging
from 0 to 5, but sensitized mice reach a maximum score of 1, with no statistical
evaluation in comparison with sham-sensitized mice. To be regarded as
significant, this low score should be at least accompanied by other quantitative
parameters (body temperature decrease, evaluation of mediators such as
histamine in serum or gut content, etc.).
3. The methods applied to the statistical analysis of results are not described at
all, and it is impossible to know which statistical tests have been used in Table 1
and Figure 6.
4. The study design should be explained in more details. The number of
experimental replicates is not reported (see also the minor essential revisions
below). It is not possible to evaluate the amount of rice protein contained in the
two doses of rice flour used for the immunization, to compare them with the dose
of rice proteins used in the challenge.
5. Several challenges with rice proteins have been repeated during the ongoing
sensitization with rice flour, but results for anaphylaxis symptom score and
vascular leakage have been produced in unique (different) days: are these the
only peak days for each parameter? Is a kinetic (time-response) study available
for both parameters? It should be related to the changes in specific IgE levels. It
should be specified case by case if the group “PBS” in each Figure refers to
sham-sensitized or sham-challenged mice.
6. Specific IgE response in the serum has been evaluated in terms of OD, that is
not suitable for comparison purposes. Quantitative evaluation independent from
the assay variability, at least with Arbitrary Units (calculated with an internal
standard serum), should be provided.
-------------------------------------------------------------------------------
Minor Essential Revisions
1. Please specify in each Figure legend how many experiment replicates have
been performed.
2. Methods, 5th paragraph (Detection of vascular leakage): “assess” instead of
“access”, and “sixth” instead of “six”.
3. Results, 1st paragraph: please report the protein yield of the extraction
procedure.
4. Figure 5. Black and white arrow are not reported on the Figure as indicated in
the legend.
5. Discussion, 2nd paragraph: jejunum inflammation has been evaluated at the
end of the experiment after mouse killing on day 32, and it must not be
associated with immediate anaphylactic symptoms recorded within 1 hour after
challenge on day 18.
6. Discussion, last paragraph: there are some references that are not correctly
cited. Regarding the immunization route, ref. 26 and 29 reports studies on
transdermal sensitization in the absence of adjuvants. As regards the use of
Cholera Toxin as adjuvant, it is widely known that it induces a mixed Th1/Th2
response with production of IFN-gamma and IgG2a. CT is used to overcome the
tolerogenic potential associated to the oral route of immunization with known
allergens, rather than to confer allergenic properties to “non-allergens”. Citation
of refs. 30 and 31 is not correct, since these studies does not demonstrate the
immunogenicity of rice proteins. On the contrary, they evaluate the tolerogenic
properties of transgenic rice (expressing pollen allergens) when orally
administered to pollen-sensitized mice.
-------------------------------------------------------------------------------
Discretionary Revisions
Not applicable.
Level of interest: An article of limited interest
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests
Reviewer's report
Title:An adjuvant free mouse model of oral allergenic sensitization to rice seeds
protein
Version: 1Date:28 July 2010
Reviewer: Gabriella Di Felice
Reviewer's report:
General
In this study, authors develop a mouse model of rice sensitization under
conditions mimicking the induction of human food allergy, such as the oral route
of immunization and the absence of adjuvants. Rice-specific IgE response,
hypersensitivity symptoms after oral challenge and histologic signs of jejunum
inflammation have been evaluated.
The contribution of robust in vivo model of food allergy to the understanding of
immunological mechanisms underlying the IgE-mediated response to allergenic
foods is well recognized, as well as their importance in the preclinical evaluation
of new therapeutic interventions. From this point of view, the information provided
by this study could be significant and original, mainly regarding the protocol of
oral immunization in the absence of adjuvants, that overcomes a common
paradox of food allergy model: the oral route is more similar to the human
situations, but in the model animals it results preferentially tolerogenic, thus
generally requiring an adjuvant for sensitization.
However, the study is mainly descriptive, and limited functional data are
provided. There is some basic and technical weakness that affect the robustness
of the results and as a consequence the overall quality and interest of the study
(see the following section).
-------------------------------------------------------------------------------
Major Compulsory Revisions
1. The Th2 cell response has not been studied at the systemic (spleen) or local
(mesenteric lymph nodes, jejunum tissue) level.
2. The anaphylactic response is evaluated by a classical symptom score ranging
from 0 to 5, but sensitized mice reach a maximum score of 1, with no statistical
evaluation in comparison with sham-sensitized mice. To be regarded as
significant, this low score should be at least accompanied by other quantitative
parameters (body temperature decrease, evaluation of mediators such as
histamine in serum or gut content, etc.).
3. The methods applied to the statistical analysis of results are not described at
all, and it is impossible to know which statistical tests have been used in Table 1
and Figure 6.
4. The study design should be explained in more details. The number of
experimental replicates is not reported (see also the minor essential revisions
below). It is not possible to evaluate the amount of rice protein contained in the
two doses of rice flour used for the immunization, to compare them with the dose
of rice proteins used in the challenge.
5. Several challenges with rice proteins have been repeated during the ongoing
sensitization with rice flour, but results for anaphylaxis symptom score and
vascular leakage have been produced in unique (different) days: are these the
only peak days for each parameter? Is a kinetic (time-response) study available
for both parameters? It should be related to the changes in specific IgE levels. It
should be specified case by case if the group “PBS” in each Figure refers to
sham-sensitized or sham-challenged mice.
6. Specific IgE response in the serum has been evaluated in terms of OD, that is
not suitable for comparison purposes. Quantitative evaluation independent from
the assay variability, at least with Arbitrary Units (calculated with an internal
standard serum), should be provided.
-------------------------------------------------------------------------------
Minor Essential Revisions
1. Please specify in each Figure legend how many experiment replicates have
been performed.
2. Methods, 5th paragraph (Detection of vascular leakage): “assess” instead of
“access”, and “sixth” instead of “six”.
3. Results, 1st paragraph: please report the protein yield of the extraction
procedure.
4. Figure 5. Black and white arrow are not reported on the Figure as indicated in
the legend.
5. Discussion, 2nd paragraph: jejunum inflammation has been evaluated at the
end of the experiment after mouse killing on day 32, and it must not be
associated with immediate anaphylactic symptoms recorded within 1 hour after
challenge on day 18.
6. Discussion, last paragraph: there are some references that are not correctly
cited. Regarding the immunization route, ref. 26 and 29 reports studies on
transdermal sensitization in the absence of adjuvants. As regards the use of
Cholera Toxin as adjuvant, it is widely known that it induces a mixed Th1/Th2
response with production of IFN-gamma and IgG2a. CT is used to overcome the
tolerogenic potential associated to the oral route of immunization with known
allergens, rather than to confer allergenic properties to “non-allergens”. Citation
of refs. 30 and 31 is not correct, since these studies does not demonstrate the
immunogenicity of rice proteins. On the contrary, they evaluate the tolerogenic
properties of transgenic rice (expressing pollen allergens) when orally
administered to pollen-sensitized mice.
-------------------------------------------------------------------------------
Discretionary Revisions
Not applicable.
Level of interest: An article of limited interest
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests
Please review this paper.
user
Reviewer's report
Title:An adjuvant free mouse model of oral allergenic sensitization to rice seeds
protein
Version: 1Date:28 July 2010
Reviewer: Gabriella Di Felice
Reviewer's report:
General
In this study, authors develop a mouse model of rice sensitization under
conditions mimicking the induction of human food allergy, such as the oral route
of immunization and the absence of adjuvants. Rice-specific IgE response,
hypersensitivity symptoms after oral challenge and histologic signs of jejunum
inflammation have been evaluated.
The contribution of robust in vivo model of food allergy to the understanding of
immunological mechanisms underlying the IgE-mediated response to allergenic
foods is well recognized, as well as their importance in the preclinical evaluation
of new therapeutic interventions. From this point of view, the information provided
by this study could be significant and original, mainly regarding the protocol of
oral immunization in the absence of adjuvants, that overcomes a common
paradox of food allergy model: the oral route is more similar to the human
situations, but in the model animals it results preferentially tolerogenic, thus
generally requiring an adjuvant for sensitization.
However, the study is mainly descriptive, and limited functional data are
provided. There is some basic and technical weakness that affect the robustness
of the results and as a consequence the overall quality and interest of the study
(see the following section).
-------------------------------------------------------------------------------
Major Compulsory Revisions
1. The Th2 cell response has not been studied at the systemic (spleen) or local
(mesenteric lymph nodes, jejunum tissue) level.
2. The anaphylactic response is evaluated by a classical symptom score ranging
from 0 to 5, but sensitized mice reach a maximum score of 1, with no statistical
evaluation in comparison with sham-sensitized mice. To be regarded as
significant, this low score should be at least accompanied by other quantitative
parameters (body temperature decrease, evaluation of mediators such as
histamine in serum or gut content, etc.).
3. The methods applied to the statistical analysis of results are not described at
all, and it is impossible to know which statistical tests have been used in Table 1
and Figure 6.
4. The study design should be explained in more details. The number of
experimental replicates is not reported (see also the minor essential revisions
below). It is not possible to evaluate the amount of rice protein contained in the
two doses of rice flour used for the immunization, to compare them with the dose
of rice proteins used in the challenge.
5. Several challenges with rice proteins have been repeated during the ongoing
sensitization with rice flour, but results for anaphylaxis symptom score and
vascular leakage have been produced in unique (different) days: are these the
only peak days for each parameter? Is a kinetic (time-response) study available
for both parameters? It should be related to the changes in specific IgE levels. It
should be specified case by case if the group “PBS” in each Figure refers to
sham-sensitized or sham-challenged mice.
6. Specific IgE response in the serum has been evaluated in terms of OD, that is
not suitable for comparison purposes. Quantitative evaluation independent from
the assay variability, at least with Arbitrary Units (calculated with an internal
standard serum), should be provided.
-------------------------------------------------------------------------------
Minor Essential Revisions
1. Please specify in each Figure legend how many experiment replicates have
been performed.
2. Methods, 5th paragraph (Detection of vascular leakage): “assess” instead of
“access”, and “sixth” instead of “six”.
3. Results, 1st paragraph: please report the protein yield of the extraction
procedure.
4. Figure 5. Black and white arrow are not reported on the Figure as indicated in
the legend.
5. Discussion, 2nd paragraph: jejunum inflammation has been evaluated at the
end of the experiment after mouse killing on day 32, and it must not be
associated with immediate anaphylactic symptoms recorded within 1 hour after
challenge on day 18.
6. Discussion, last paragraph: there are some references that are not correctly
cited. Regarding the immunization route, ref. 26 and 29 reports studies on
transdermal sensitization in the absence of adjuvants. As regards the use of
Cholera Toxin as adjuvant, it is widely known that it induces a mixed Th1/Th2
response with production of IFN-gamma and IgG2a. CT is used to overcome the
tolerogenic potential associated to the oral route of immunization with known
allergens, rather than to confer allergenic properties to “non-allergens”. Citation
of refs. 30 and 31 is not correct, since these studies does not demonstrate the
immunogenicity of rice proteins. On the contrary, they evaluate the tolerogenic
properties of transgenic rice (expressing pollen allergens) when orally
administered to pollen-sensitized mice.
-------------------------------------------------------------------------------
Discretionary Revisions
Not applicable.
Level of interest: An article of limited interest
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests
assistant