Legend to the figures
Figure 1: Development and
characterization of mouse model of cashew sensitization and
anaphylaxis. Mice were sensitized to cashew through skin (in red) or
oral (in green) routes. As a negative control, a group of naïve mice (in
white) was included. (A ) Cashew-specific IgE, IgG1 and IgG2a
antibody responses were evaluated by ELISA, from blood samples collected
one week after the end of the sensitization. Mice were orally challenged
to cashew one week after the end of the sensitization. (B ) Body
temperature was measured every 5 minutes following challenge for 60
minutes. (C ) Area under the curve was calculated for each
individual percentage of temperature variation curve using 100 % as a
baseline. mMCP-1 (D ) and mMCP-7 (E ) concentrations
were measured by ELISA from plasma collected immediately after the
challenge, (n = 8 per experimental group). Data are median with
interquartile range of individual values. P values were determined using
the Mann-Whitney unpaired t-test (**, p<0,01; ***,
p<0,001; n.s., non-significant). For B , C ,D and E panels, the level of significant measured
between each sensitized group and the negative control group is
indicated above each graph.
Figure 2: Validation of the oral route of sensitization as a
trigger of IgE-mediated anaphylaxis in mice. Mice were orally
sensitized to cashew. One week after the end of the sensitization, mice
received IgE blocking antibody (clone EM-95, in green), IgG blocking
antibody (anti-FcγRII/RIII clone 2.4G2, in blue) or relevant isotype
controls (in red). The day after, mice were challenged orally to cashew.
(A ) Body temperature was measured every 5 minutes following
challenge for 60 minutes and (B ) diarrhea occurrence and
severity was scored. mMCP-1 (C ) and mMCP-7 (D )
concentrations were measured by ELISA from plasma collected immediately
after the challenge, (n = 8 per experimental group). Data are median
with interquartile range of individual values. P values were determined
using the Mann-Whitney unpaired t-test (*, p<0,05; ***,
p<0,001; n.s., non-significant). For C and D panels, the level
of significant measured between each sensitized group and the negative
control group is indicated above each dot plot.
Figure 3: Cashew allergens are efficaciously delivered by
epicutaneous patches, leading to allergen capture by skin dendritic
cells . Mice were orally sensitized to cashew. One week after the end of
the sensitization, mice received a patch loaded with cashew protein
extract conjugated to Fluoroprobe-647 (F-647) for 48 hours. As negative
controls, mice received a patch containing excipient. Brachial draining
lymph nodes were collected, and cells were isolated and labelled for
FACS analysis. The absolute number of cashew-positive (F-647-positive)
cells was measured among each DC subsets, (n = 7-8 per experimental
group). Data are Median and interquartile ranges of individual values. P
values were determined according to the Mann-Whitney test (**,
p<0.01; ***, p<0.001).
Figure 4: Kinetic modulation of cashew-specific antibody
response following EPIT to cashew nut in mice . Mice were orally
sensitized to cashew. One week after the end of the sensitization, mice
were submitted to EPIT. To that end, mice received cashew patches
containing 50 µg of cashew protein extract, once a week for up to 16
weeks (in blue). Patches were applied for 48 hours. As negative
controls, mice received patches containing excipient (sham, in black) or
were kept untreated (naïves, in white). Blood samples were collected
before EPIT (B) or every two weeks during treatment to isolate plasma
(weeks 2, 4, 6, 8, 10, 12, 14, 16), as indicated on C panel.
Cashew-specific antibody titers were measured from plasma by indirect
ELISA (IgE, panel A ) or direct ELISA (IgG1 and IgG2a, panelB and panel C , respectively), (n = 8-24 per
experimental group – 8 mice of each group were challenged at weeks 8
and 12 and sacrificed). Data are Median and interquartile ranges of
individual values.
Figure 5: Evaluation of the level of protection afforded by EPIT
against anaphylaxis following 8, 12 or 16 weeks of treatment . Mice were
orally sensitized to cashew and treated as described in Figure
4 . Following 8 (A, B ), 12 (C, D ) or 16 (E,
F ) weeks of EPIT, 8 mice of each group were challenged orally to
cashew. (A, C, E ) Body temperature was measured every 5 minutes
following challenge for 60 minutes. Data are Median and interquartile
ranges of individual values. (B, D, F ) Clinical symptoms were
monitored every 5 minutes following challenge for 60 minutes. (n = 8 per
experimental group). Data are Mean with SEM of individual values. P
values were determined according to the Mann-Whitney test.
Figure 6: Evaluation of mast-cell activation induced by oral
challenge following 8, 12 or 16 weeks of EPIT . Mice were orally
sensitized to cashew, treated as described in Figure 4 and
finally challenged as described in Figure 5 . Blood samples were
collected 60 minutes after the challenge to isolate plasma. mMCP-1
(A, C, E ) and mMCP-7 (B, D, F ) concentrations were
measured from plasma by ELISA, (n = 8 per experimental group). Data are
median with interquartile range of individual values. P values were
determined using the Mann-Whitney unpaired t-test (**, p<0,01;
***, p<0,001).