Anti-IAPP Monoclonal Antibody Improves Clinical Symptoms in a Mouse Model of Type 2 Diabetes
, Monique Vogel 1,2 and Martin F. Bachmann 1,2,4,*Round 1
Reviewer 1 Report
I recommend to accept the revised manuscript for publication
Author Response
I recommend to accept the revised manuscript for publication
We thank the reviewer for his positive feedback and recommendation to publish our manuscript.
Reviewer 2 Report
It would be important to include the body weight changes over time for the treatment group compared to the control group;
It is surprisingly to see that m81 increased the AUC of glucose changes after insulin injection in mice, which counteracts with the results of GTT; probably it is because the number of mice is too low; therefore, it would be important to include more mice to repeat the experiment.
Author Response
t would be important to include the body weight changes over time for the treatment group compared to the control group;
We agree with the reviewer that it is an important parameter. This is why we compared the weight of the mice treated with the mAb m81 and that treated with the control isotype antibody. The results indicated that only the control mice show an increase in weight while the mice treated with the mAb maintain a similar weight and normal blood sugar levels. In the latest version of the paper that we recently submitted these results are visible in Fig. 4B. We hope that this figure answers the reviewer's question.
It is surprisingly to see that m81 increased the AUC of glucose changes after insulin injection in mice, which counteracts with the results of GTT; probably it is because the number of mice is too low; therefore, it would be important to include more mice to repeat the experiment.
We agree with the reviewer that this result is not entirely obvious. However, we do not think that it is a problem of mouse number because we repeated the experiment at least two times and found the same results. Our explanation for these results come from our observations that control mice after week 12 reduced insulin production (acquiring features of type 1 diabetes) and therefore probably respond to isolated insulin injection in an accelerated way, resulting in lower glucose levels than seen in m81 treated mice, where insulin levels and insulin-responses are more normal. This is discussed now in the text (page 8 lines 290-296).
Reviewer 3 Report
It is opinion of the reviewer that this paper before acceptance needs several corrections/modification. My individual comments are listed below.
Tittle – Capital first letters should be written.
- – It should be “… Type 2 Diabetes”
5-11 – e-mail addresses and authors’ initial should be completed.
27 – It should be “diabetes mellitus”.
- 29 – References should be cited like as [1,2].
- 62 – It should be “Moreover, in vitro fibril …”.
86 = “E coli” must be written with italic.
91 – Remove “Vaccines”.
112 – How the chromatography was monitored?
115 – It should be “hexafluoroisopropanol”.
119 – The volume of Thioflavin T?
132 – For “OD450” “450” should be subscripted.
141 – pH of this buffer?
161 – In NaN3 “3” subscripted.
177 - – It should be “high fay diet”.
186 – It should be “glucose”.
196 – It should be “The ELISA was …”.
197 – It should be “interleukin 1 beta”.
202 – It should be citrate buffer”.
202 – It should be “H2O2”. (“2” subscripted).
204 – It should be “H2SO4” (“2” and “4” subscripted).
208 – It should be “two-way ANOVA”.
208 – The Tukey’s test should be mentioned.
293 – It should be “type 2 to type 1”.
A part of the Discussion is really like as Summary. The author cited only 3 references – ref [24] is very general.
I suggest to ad “Conclusions”.
References should be with doi.
Author Response
Response to Reviewer 3 Comments
Comments and Suggestions for Authors
It is opinion of the reviewer that this paper before acceptance needs several corrections/modification. My individual comments are listed below.
We thank the reviewer for the comment and agree with the corrections and modifications that he suggested.
Please find below the answers to his comments.
Tittle – Capital first letters should be written.
The title has been modified accordingly.
2. – It should be “… Type 2 Diabetes”
It has been changed
5-11 – e-mail addresses and authors’ initial should be completed.
E-mail addresses and author’s initial have been completed.
29 – References should be cited like as [1,2].
The formatting of the references has been changed accordingly.
62 – It should be “Moreover, in vitro fibril …”.
The text has been modified as suggested by the reviewer.
86 = “E coli” must be written with italic.
It has been changed.
91 – Remove “Vaccines”.
It has been changed.
112 – How the chromatography was monitored?
Chromatography was monitored at 280 nm wavelength absorbance which allows to determine protein concentration. This information has been now added in paragraph 2.3.4.
115 – It should be “hexafluoroisopropanol”.
H has been added
119 – The volume of Thioflavin T?
The volume of ThioT for the assay was 50ul
132 – For “OD450” “450” should be subscripted.
It has been changed.
141 – pH of this buffer?
The pH of the 4.3mM Na2HPO4 1.4mM KH2PO4 buffer, used for Dot Plot analysis, was 7.2
161 – In NaN3 “3” subscripted.
It has been changed
177 – It should be “high fay diet”.
It has been changed
186 – It should be “glucose”.
The letter g was added
196 – It should be “The ELISA was …”.
The text was modified accordingly
197 – It should be “interleukin 1 beta”.
It has been changed
202 – It should be citrate buffer”.
The letter c was added
202 – It should be “H2O2”. (“2” subscripted).
204 – It should be “H2SO4” (“2” and “4” subscripted).
The numbers have been subscripted
208 – It should be “two-way ANOVA”.
Two has been added
208 – The Tukey’s test should be mentioned.
post hoc Tueky’s HSD has been added to the text
293 – It should be “type 2 to type 1”.
II and I have been changed into 2 and 1.
A part of the Discussion is really like as Summary. The author cited only 3 references – ref [24] is very general.
We have modified the first part of the Discussion and added more references.
I suggest to ad “Conclusions”.
A conclusion has been added.
References should be with doi.
References have been reformatted and doi have been added.
Reviewer 4 Report
In this manuscript, Roesti and coworkers demonstrate that a specific antibody is able to inhibit IAPP toxic amyloid aggregation in tube tests, in cell cultures and in vivo. This topic is of utmost importance, due to the high prevalence of Diabetes mellitus worldwide. Next, the manuscript is well written and clearly organized and experimental methods are adequately accurate and sound.
I have only a minor suggestion:
To ensure better coverage of the field, I suggest mentioning a recent work reviewing the role of an abnormal IAPP proteostasis in the development of diabetes (See https://dx.doi.org/10.1021/acs.chemrev.0c00981)
Author Response
Response to Reviewer 4 Comments
Comments and Suggestions for Authors
In this manuscript, Roesti and coworkers demonstrate that a specific antibody is able to inhibit IAPP toxic amyloid aggregation in tube tests, in cell cultures and in vivo. This topic is of utmost importance, due to the high prevalence of Diabetes mellitus worldwide. Next, the manuscript is well written and
clearly organized and experimental methods are adequately accurate and sound.
We thank the reviewer for his positive feedback and for his suggestion. Please find below the answer to his comment.
I have only a minor suggestion:
To ensure better coverage of the field, I suggest mentioning a recent work reviewing the role of an abnormal IAPP proteostasis in the development of diabetes (See https://dx.doi.org/10.1021/acs.chemrev.0c00981)
The reference to the review was added to the discussion.
Round 2
Reviewer 3 Report
The authors corrected this paper properly taken under considerations all my comments. Therefore, I can accept it now.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The manuscript by Roesti et al describes the characterization of anti-IAPP antibody and demonstrates that the administration of this antibody to transgenic mice prevents the formation of IAPP aggregates. Overall, the manuscript is descriptive and the conclusions are largely supported by the provided data and results. As outlined below, I do have several comments, including the much needed control data from healthy human sections and from naïve mice that share the same genetic background.
Major comments:
- Line 198 – it is not clear why m81 was chosen and not for example 171.
- Fig 2c – Please add parallel section taken from healthy donor in order to demonstrate the specificity of this staining by the novel antibody. Without this control data, one cannot be convinced that this is a specific staining of the aggregates.
- Line 260 – it is not clear whether the increase in insulin levels were expected and how they fit into the protective activity of the antibody.
- Fig 4d – please add parallel section from naïve mouse that share the same genetic background as the transgenic mice.
- Fig 4d,f – it seems that there are less insulin secreting cells following ab treatment. Is it normal?
- Line 292 – the overall results demonstrate that the antibody do recognize the monomer, although to a lesser extent. Yet, this recognition may have some impact on the mice (and probably human) physiological status. The follow-up on the animal weight is not sufficient to verify that there are no toxic effects of this antibody.
Minor comments:
- Section 3.1 – please elaborate on how the screening was made, how many hybridomas were positive and why these 8 were chosen.
- Line 190 – only 6 are shown in fig. 1.
- 2a – what are the error bars? Of how many replicates?
- Fig 2a – what is the role of RNase-Nterm in this experiment?
- Fig 3 – the dots should be joined by a line
- Fig 4 – please indicate the exact number of mice in each figure. What are the error bars?
- Fig 4 – I believe that the untreated mice is not a "control" mice. As mentioned above, the real control group is of naïve mice that should also be added to these experiments.
Reviewer 2 Report
The authors claimed that “The here described monoclonal antibody (mAb) m81, specific for oligomeric and fibrils but not for soluble free IAPP, is able to prevent oligomer growth and aggregate formation in vitro and blocks islet inflammation and disease progression in vivo.” The authors only showed that m81 reduced the fasting glucose levels, increased the insulin content in the islets. The in vivo data should be strengthened, the most common gold-standard assay is the glucose tolerance test and insulin tolerance test for testing the insulin sensitivity. These experiments should be performed to confirm the improvement of insulin sensitivity.
It would be important to measure the fasting insulin levels in these mice to see if the mAb affect the insulin levels in serum. So HOMA-IR values could be calculated in the experiment.
The in vivo experiment was done in male mice, does this mAb m81 work for female mice?
It seems the fasting glucose levels were reduced after 10 weeks later, is this because this mice model starts developing insulin resistance on a HFD for 10 weeks?
Presence of pro-inflammatory cytokine IL-1β were determined using immunofluorescence staining, it would be important to confirm if m81 mAb affect the serum IL-1b levels.
Please indicate if there is any significant differences between groups in Figure 1, Figure 2A, Figure 3.
Is there any significant differences between groups at 12 weeks and 13 weeks in Figure 4C?
Figure 4, 14h-fasting glucose level [mmol/L] of m81 mAb immunized (orange square) and control (black circle) mice with corresponding area under the curve (AUC). n=at least 3 mice per group. Please clarify how many mice were used for each group. 3 mice are kind of low to get the robust in vivo results.
Please include the SEM bar into Figure 4E, 4F.
