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112
 
© 2008 Blackwell Publishing •
 
Journal of Cosmetic Dermatology
 
, 
 
7,
 
 112–119
 
Original Contribution
 
Blackwell Publishing Inc
 
ORIGINAL CONTRIBUTION
 
Neuroimmunomodulatory compound for sensitive skin care: 
 
in vitro
 
 and clinical assessment
 
Gustavo de Campos Dieamant, MD,
 
1,2
 
 Maria Del Carmen Velazquez Pereda,
 
1,2
 
 Samara Eberlin, MD,
 
1,2
 
 
Cecília Nogueira,
 
1,2
 
 Rejane Maria Werka,
 
1,2
 
 & Mary Luci de Souza Queiroz, PhD
 
1
 
1
 
Department of Pharmacology/Hemocenter, Faculty of Medical Science, State University of Campinas, Campinas, São Paulo, Brazil
 
2
 
Research and Development Department, Chemyunion Química Ltda, Sorocaba, Brazil
 
Summary
 
Background
 
The pathophysiology of sensitive skin consists of an inflammatory reaction
resulting from the abnormal penetration in the skin of potentially irritating sub-
stances, which occurs due to skin barrier dysfunction and changes in the production of
local neuromediators.
 
Aims
 
The therapeutic potential of 
 
l
 
-carnosine and 
 
Rhodiola rosea
 
, as antioxidant and
neuromodulatory, respectively, leads us to investigate the effects of the 
 
R. rosea
 
 extract/
 
l
 
-
carnosine–associated compound (RCAC) on sensitive skin alterations.
 
Methods
 
A double-blind comparative study was conducted on 124 volunteers with
sensitive skin, who were selected by their reactivity to stinging test. Two randomized
groups of 62 each received either a formulation containing 1% of RCAC or placebo,
which was applied twice a day for 28 consecutive days. One perceptibility questionnaire
was applied at the onset and at the end of the treatment to evaluate the subjective
response to test product. Additionally, 
 
in vitro
 
 studies were performed to investigate
RCAC neuroimmunomodulatory mechanisms.
 
Results
 
RCAC treatment produced
 
 in vivo 
 
protective effects in skin barrier function and
a positive subjective response of sensitive skin volunteers. 
 
In vitro
 
 treatment promoted the
release of proopiomelanocortin peptides and restored to normal the increased levels of
neuropeptides and cytokines produced by keratinocytes exposed to ultraviolet radiation.
Clinical effectiveness was measured by reduction of transepidermal water loss, positive
perceptions of improvements in skin dryness and skin comfort sensation, and reduction
of discomfort sensation after stinging test.
 
Conclusions
 
The protective effect of RCAC in skin barrier function and the positive
response produced in human subjects with sensitive skin could be partially explained by
our 
 
in vitro
 
 results showing a significant increase in opioid peptides release, an inhibitory
effect on neuropeptides production, and modulation of cytokines production by
keratinocytes under ultraviolet stress.
 
Keywords
 
: cytokines, neuroimmunomodulation, proopiomelanocortin peptides, 
 
sensitive skin, stinging test, transepidermal water loss
 
Introduction
 
The term “sensitive skin” is largely used by individuals who
consider themselves to have a more reactive skin to applied
products or environmental factors than the general
population.
 
1,2
 
 There is a relatively high evidence of this
 
Correspondence: Mary L. S. Queiroz, PhD, Department of Pharmacology/
Hemocenter, State University of Campinas, PO Box 6111, Campinas, São 
Paulo, Brazil 13083-970. E-mail: mlsq@fcm.unicamp.br
 
Accepted for publication January 17, 2008
 
Neuroimmunomodulatory compound for sensitive skin care •
 
G C Dieamant 
 
et al.
 
© 2008 Blackwell Publishing •
 
Journal of Cosmetic Dermatology
 
, 
 
7,
 
 112–119
 
113
 
manifestation, because approximately 40% of the population
believes to possess the characteristics of sensitive skin.
 
3
 
Based on current understanding, several authors define
sensitive skin as a condition that exhibits a reduced
tolerance to frequent or prolonged use of cosmetics and
toiletries, with symptoms ranging from visible signs of
irritation such as erythema, scaling, and skin dryness to
more subjective neurosensory forms of discomfort such
as stinging, burning, itching, tightness, and smarting.
 
4–6
 
The physiopathology of sensitive skin consists of an
inflammatory process resulting from the abnormal
penetration in the skin of potentially irritating substances
because of skin barrier dysfunction.
 
7,8
 
 In addition, the
presence of a nonspecific reaction has been related to
cutaneous sensory innervation in the establishment of
skin sensitivity.
 
9,10
 
 Neuropeptides released from cutaneous
nerves and skin resident cells such as substance P, calcitonin-
gene–related peptide (CGRP), and proopiomelanocortin
peptides (POMC peptides) – such as 
 
β
 
-endorphin and
enkephalin – are mandatory for a fine-tuned regulation
of cutaneous immune responses and tissue maintenance
and repair.
 
11,12
 
In response to noxious stimuli, substance P and CGRP
lead to vasodilatation and mast cell degranulation, originating
a process called neurogenic inflammation.
 
10
 
 Classical
pathways are then activated causing a nonspecific
inflammation in consequence of released cytokines and
eicosanoids such as interleukin-1
 
α
 
 (IL-1
 
α
 
), tumor necrosis
factor-alpha (TNF-
 
α
 
), prostaglandin-E
 
2
 
, and prostaglandin-
F
 
2
 
.
 
13
 
 On the other hand, POMC activities include antagonism
and down-regulation of adhesion molecules and reduced
inflammation by modulation of IL-10 production, which
contributes to the amelioration of the subjective neuro-
sensory forms of discomfort.
 
13,14
 
The biological activity of natural compounds has been
screened for the development of nonsynthetic skin-care
products. In this work, we studied the effects of a 
 
Rhodiola
rosea
 
 extract/
 
l
 
-carnosine–associated compound (RCAC)
in the reactivity of individuals with sensitive skin, using
the lactic acid facial stinging test model. Skin barrier
function was measured by transepidermal water loss
(TEWL), which is considered a noninvasive bioengineering
technique frequently used to substantiate the efficacy of
cosmetic products.
 
15
 
 Additionally, 
 
in vitro 
 
studies were
performed in order to reveal the neuroimmunomodulatory
effects of RCAC in human keratinocyte cell culture. Our
 
in vitro
 
 findings demonstrated that RCAC significantly
reduced the levels of CGRP and substance P and
increased the release of POMC peptides, in a dose-dependent
manner. Importantly, increased POMC peptide production
was observed at basal and ultraviolet (UV) statement
condition. In relation to the immunomodulatory response,
the RCAC restored the levels of IL-1
 
α
 
, TNF-
 
α
 
, and IL-10 to
basal condition after UV radiation (UVR). These results
could be responsible, at least in part, for the clinical
evidence of reduced TEWL and skin responsiveness of
volunteers after treatment with RCAC.
 
Materials and methods
 
Associated compound
 
RCAC (Relievene-SK™) was manufactured and provided
by Chemyunion Química Ltda. (Sorocaba, Brazil). RCAC
is composed of 
 
R. rosea 
 
extract and 
 
l
 
-carnosine. The
quantitative high-performance liquid chromatography
analysis of RCAC discloses the presence of 2.8% to 3.5%
p/p of total rosavins and 45.0% to 48.0% p/p of 
 
l
 
-
carnosine. RCAC is recognized through the order of
patent PI0700701-9, deposited in the National Institute
of Industrial Property, Brazil, on February 16, 2007.
 
Cell culture and treatment protocol
 
Normal human epidermal keratinocytes were obtained
from a commercial supplier (Cell Applications, Inc., San
Diego, CA) and subcultured at 37 
 
°
 
C in a humidified incubator
with 5% CO
 
2
 
. After confluence, cells were seeded into 24-
well culture plates (1 
 
× 
 
10
 
5
 
 cells per well). Twenty-fourhours
after seeding, cells were irradiated with UVR (Multiport
Solar UV Simulator Model 600, Solar Light Co., Philadelphia,
PA) in a dose of 100 mJ/cm
 
2
 
/day during five consecutive
days and immediately incubated with concentrations of
1.0, 0.5, 0.25, 0.12, and 0.06 mg/mL of test product
dissolved in culture medium. After 48 h of incubation,
cell-free supernatants were collected, and assays were
performed using commercial kits. Each experiment was
conducted in triplicate of three independent experiments.
Selection of these doses was based on previous results of
cytotoxicity assays (data not shown).
 
Quantification of POMC peptides, neuropeptides, 
and cytokines
 
β
 
-Endorphin, enkephalin, CGRP, substance P, IL-1
 
α
 
, TNF-
 
α
 
,
and IL-10 were quantified by using a commercially available
enzymatic immunoassay kit (Phoenix Pharmaceuticals,
Belmont, CA; R & D Systems, Minneapolis, MN).
 
Evaluation of clinical efficacy
 
The clinical studies were carried out in agreement with
the Ethics Committee of Leonor Mendes de Barros Hospital,
São Paulo, Brazil.
 
Neuroimmunomodulatory compound for sensitive skin care •
 
G C Dieamant 
 
et al.
 
114
 
© 2008 Blackwell Publishing •
 
Journal of Cosmetic Dermatology
 
, 
 
7,
 
 112–119
 
Subjective studies
 
One hundred and twenty-four volunteers, after informed
consent, were enrolled in this double-blind, comparative,
board-approved investigative review. Inclusion criteria
were as follows: human subjects 40 to 55 years old,
Fitzpatrick phototype I, II, or III; and all classified as
sensitive skin type by stinging test. The volunteers were
separated into two groups with 62 individuals each. Each
group received facial fluid serum containing 1% of RCAC
or placebo, which was applied on the cleaned face by
circular movements twice a day, for 28 days. Stinging
was evaluated at the onset and at the end of the treatment
by the sensation reported by each volunteer 2 and 5 min
after application of the product. Stinging intensity was
assessed as described elsewhere.
 
16
 
 Each volunteer
answered an perceptability questionnaire of 5-point scale
evaluating the effect of the treatment by comparing day 1
vs. day 28, taking into account the following parameters:
sensation of comfort, and skin dryness sensation.
Sensation of comfort was classified as follows: (i) very
satisfactory, (ii) satisfactory, (iii) indifferent, (iv) little
satisfactory, and (v) not satisfactory. Skin dryness sensation
was classified as follows: (i) very dry, (ii) dry, (iii) indifferent,
(iv) little dry, and (v) not dry. In addition, the level of skin
discomfort was evaluated by comparing the intensity of
discomfort after stinging test at day 1 and day 28.
 
Evaluation of skin barrier function by TEWL assessment
 
TEWL was measured using a TM 300 Tewameter™
(Courage + Khazaka Electronic GMBH, Cologne, Germany)
and expressed in g/m
 
2
 
 h. Thirty-nine volunteers were
enrolled in this study following the above-mentioned
Parameters
Subjective Studies
Levels of Skin Comfort (% of answer)
Placebo RCAC
Day 1 Day 28 Day 1 Day 28*
Not satisfactory 11.7 1.56 5.62 0.89
Little satisfactory 27.66 5.64 16.99 3.31
Indifferent 22.51 34.04 19.58 5.67
Satisfactory 35.6 45.93 52.38 62.37
Very satisfactory 2.53 11.83 5.43 27.76
Parameters
Levels of Skin Dryness Sensation (% of answer)
Placebo RCAC
D1 D28 Day 1 Day 28†
Not dry 24.28 25.39 16.85 42.20
Little dry 31.06 31.42 27.07 31.63
Indifferent 25.57 25.01 28.89 16.78
Dry 17.86 17.04 25.26 8.85
Very dry 1.22 1.15 1.92 0.54
Sensation of Discomfort after ST (% of answers)
Placebo RCAC‡
Day 1 50 67.74
Day 28 50 32.26
Data are expressed in percentage of volunteers in accordance with these parameters 
comparing day 1 to day 28. *P < 0.05, in relation to RCAC day 1 and P < 0.001, in relation to 
placebo day 28; †P < 0.001, in relation to RCAC day 1 and P < 0.05, in relation to placebo day 
28; ‡P < 0.05, in relation to RCAC day 1 and P < 0.001, in relation to placebo day 28 (SAS 
PROC MIXED).
Table 1 Levels of skin comfort, skin 
dryness sensation, and sensation of 
discomfort after stinging test in sensitive 
skin subjects after application of a 
formulation containing 1% (w/w) of 
Rhodiola rosea extract/l-carnosine–
associated compound (RCAC) for 28 
consecutive days
 
Neuroimmunomodulatory compound for sensitive skin care •
 
G C Dieamant 
 
et al.
 
© 2008 Blackwell Publishing •
 
Journal of Cosmetic Dermatology
 
, 
 
7,
 
 112–119
 
115
 
inclusion criteria. The forearms of each volunteer were
treated during 56 days with a fluid serum containing 1% of
RCAC or placebo. The latter was applied in the corresponding
area of the forearm opposite to that receiving RCAC.
According to a standard procedure, before each test, the
subjects rested for 30 min in an air-conditioned area with
a constant 22 ± 1 
 
°
 
C room temperature and 50 ± 5%
relative humidity. The kinetics of TEWL was performed at
days 1, 28, and 56, before (T
 
0
 
) and after (T
 
30
 
, T
 
60
 
, and
T
 
120
 
 min) the application of both products (2 mg/cm
 
2
 
).
 
Statistical analysis
 
For all 
 
in vitro
 
 analyses, a parametric method, the one-way
analysis of variance (
 
anova
 
) followed by the Tukey test,
was used to compare data among all groups. For the
clinical trials, the Generalized Linear Models were used to
evaluate treatment and time effects. To consider repeated
measurements, Generalized Estimating Equations were
used. Analysis of results was performed using the procedure
MIXED of SAS 9.1 system (SAS PROC MIXED, SAS
Institute, Cary, NC). Statistical significance was considered
when 
 
P
 
 < 0.05.
 
Results and discussion
 
Sensitive skin is identified as being hypersensitive to
stimuli, and its manifestation seems to occur due to
increased permeability of the stratum corneum and
exacerbation of the nerve response in skin.
 
4
 
 Subjective
perceptions are derived from the observations of patients
regarding stinging, burning, pruritus, and tightness
following various environmental stimuli. Attempts to
identify more objective clinical parameters in the subjective
response showed that in these individuals, there is a
tendency of a less hydrated, less supple, more erythematous
and more telangiectatic skin, compared with normal
population.
 
9
 
 Classical and subjective methods of sensory
testing, such as stinging test and perceptibility of consumers,
have been increasingly utilized to provide more accurate
information on sensitive skin reactions.
 
10
 
In this work, we investigated the effects of the RCAC on
skin sensitivity in individuals exposed to stinging test. Clinical
results showed that after a 28-day treatment, the levels of
skin comfort reported in the group treated with RCAC were
significantly higher than those obtained in the placebo
group (
 
P
 
 < 0.05; Table 1). Additionally, the RCAC reduced
skin dryness sensation (
 
P
 
 < 0.001) when compared with
the percentages of answers at days 1 and 28, whereas no
significant effects were observed in the placebo group
(Table 1). Importantly, the results obtained in the binary
questionnaire concerning the intensity of skin discomfort
sensation corroborated these findings. As we can see in
Table 1, RCAC treatment significantly decreased the
sensation of skin discomfort when compared with the
placebo group (
 
P
 
 < 0.05). In addition, RCAC prevented
water loss, compared with placebo, after 28-day (
 
P
 
 < 0.001)
and 56-day (
 
P
 
 < 0.001) treatments. Concurrently, the
RCAC promoted a progressive effect during the period
of treatment (days 0, 28, and 56), thus indicating an
important improvement of skin barrier function and
stratum corneum homeostasis (Table 2).
The 
 
in vitro 
 
results using human keratinocyte cell
culture suggested that the mechanisms involved in the
protective effects ofRCAC are mainly related to modula-
tion of cytokines and growth factors released by activated
keratinocytes. In the last two decades, it has become clear
that keratinocytes, which represent 95% of epidermal cells,
play an important role in the initiation and perpetuation
Table 2 Transepidermal water loss in subjects with sensitive skin 
after application of a formulation containing 1% (w/w) of Rhodiola 
rosea extract/l-carnosine–associated compound (RCAC) for 28 
consecutive days.
TEWL (g/m2 h)
Day 1
Time (min)
T0 T30 T60 T120
Placebo 7.7 ± 1.6 7.7 ± 1.7 7.8 ± 1.7 8.4 ± 1.7
RCAC 7.8 ± 1.7 7.6 ± 1.7 7.6 ± 1.7 8.0 ± 1.6
Day 28
Time (min)
T0 T30 T60 T120
Placebo 7.3 ± 1.8 8.6 ± 1.7 8.6 ± 1.8 8.7 ± 1.8
RCAC 7.0 ± 1.8* 7.8 ± 1.7 8.0 ± 1.7 8.1 ± 1.8
Day 56
Time (min)
T0 T30 T60 T120
Placebo 6.5 ± 1.4 8.0 ± 1.4 8.1 ± 1.4 7.8 ± 1.4
RCAC 6.0 ± 1.4† 7.0 ± 1.4 7.2 ± 1.5 7.1 ± 1.4
Data are expressed as mean ± SD. †P < 0.001, in relation to RCAC 
day 1/T0, P < 0.01, in relation to RCAC day 28/T0 and P < 0.001, 
in relation to placebo day 56/T0; *P < 0.001, in relation to RCAC 
day 1/T0 and P < 0.001, in relation to day 28/T0 (SAS PROC MIXED).
Neuroimmunomodulatory compound for sensitive skin care • G C Dieamant et al.
116 © 2008 Blackwell Publishing • Journal of Cosmetic Dermatology, 7, 112–119
of skin inflammatory and immunological reactions.17
Whereas resting keratinocytes produce some cytokines
constitutively, a variety of environmental stimuli can
induce epidermal keratinocytes to release growth factors
and humoral cytokines.18 Among all the cytokines
produced by keratinocytes, IL-1α, IL-1β, and TNF-α
activate a sufficient number of effector mechanisms to
trigger cutaneous inflammation.19
The ability of cellular components of the skin immune
system to mount various types of immune responses is
largely dependent upon their ability to release and to
respond to different signals provided by immunoregulatory
mediators such as cytokines and neuropeptides.11,20
The endogenous peptides, especially POMC peptides
(such as β-endorphin and enkephalin) and neuropeptides
(such as substance P and CGRP), have an important
role in skin homeostasis.13,20,21 They are released from
sensory or autonomic nerve fibers, and certain neuropeptides
have also been reported to be produced by immuno-
competent, epidermal, and dermal cells.11 POMC
activities include antagonism and down-regulation of
adhesion molecules and proinflammatory cytokines,
modulation of immunosupressive cytokine IL-10 produc-
tion, and inhibition of the nuclear transcription factor
(NF-κB) by various inflammatory agents, including
TNF-α and IL-1.12,20,22 Conversely, substance P and CGRP
not only function as neurotransmitters for pain and itch but
also may act as mediators of immunity and inflammation.
The release of these neuropeptides in the skin after UVR
is of particular interest, because they alter a number of
biological functions of skin cells during cutaneous neurogenic
inflammation, including cytokine and chemokine produc-
tion, cell proliferation, adhesion molecule expression, and
antigen-presenting cell functions.23,24
In the present study, neuroimmunological evaluation
of the effects of the RCAC in the skin was performed
through the production of IL-1α, TNF-α, IL-10, β-endorphin,
enkephalin, substance P, and CGRP. The results showed
an immunomodulatory pattern of response to RCAC,
because it restored to normal values the increased levels
of proinflammatory IL-1α and TNF-α, and of the immuno-
suppressive cytokine IL-10, induced by the UVR (Fig. 1).
Figure 1 In vitro effects of Rhodiola rosea 
extract/l-carnosine–associated compound 
(RCAC) on the production of interleukin-
1alpha (IL-1α), tumor necrosis factor-
alpha (TNF-α), and interleukin-10 (IL-10) 
by human keratinocytes under basal and 
ultraviolet radiation (UVR) exposure. IL-
1α, TNF-α, and IL-10 were measured in the 
supernatant of cultures after 48 h of 
incubation. Data are presented as mean ± 
SD of three independent experiments 
(n = 18). *P < 0.05, in relation to basal 
control group; #P < 0.05, in relation to 
UVR control group; &P < 0.05, in relation 
to UVR/RCAC 1.0 and 0.05 mg/mL 
(anova, Tukey).
Neuroimmunomodulatory compound for sensitive skin care • G C Dieamant et al.
© 2008 Blackwell Publishing • Journal of Cosmetic Dermatology, 7, 112–119 117
Moreover, increased levels of β-endorphin and enkephalin
were produced by the treatment with RCAC, reaching levels
up to threefold higher than normal and UV-treated control
(Fig. 2). RCAC was also able to promote a significant decrease
of about 50% in the production of substance P and
CGRP after UVR (Fig. 3). It is important to mention
that, in all parameters evaluated, the compound pro-
moted a dose-dependent effect and the optimal biological
Figure 2 In vitro effects of Rhodiola rosea 
extract/l-carnosine–associated compound 
(RCAC) on the production of β-endorphin 
and enkephalin by human keratinocytes 
under basal and ultraviolet (UVR) 
exposure. β-Endorphin and enkephalin 
were measured in the supernatant of 
cultures after 48 h of incubation. Data are 
presented as mean ± SD of three 
independent experiments (n = 6). 
*P < 0.01, in relation to basal control (BC) 
group; #P < 0.001, in relation to BC; 
φP < 0.001, in relation to BC, to BC/RCAC 
1.0 mg/mL and to BC/RCAC 0.25 mg/mL 
groups; δP < 0.001, in relation to BC, to 
BC/RCAC 0.25 mg/mL and to BC/RCAC 
0.06 mg/mL groups; $P < 0.001, in 
relation to UVR control (UVRC) group; 
ωP < 0.05, in relation to UVRC group; 
θP < 0.001, in relation to UVRC, to UVR/
RCAC 1.0 mg/mL and to UVR/RCAC 
0.25 mg/mL groups; πP < 0.001, in 
relation to UVRC and to UVR/RCAC 
0.25 mg/mL groups (anova, Tukey).
Figure 3 In vitro effects of RCAC on the 
production of substance P and calcitonin 
gene–related peptide (CGRP) by human 
keratinocytes under basal and ultraviolet 
(UVR) exposure. Substance P and CGRP 
were measured in the supernatant of 
cultures after 48 h of incubation. Data are 
presented as mean ± SD of three 
independent experiments (n = 18). 
*P < 0.001, in relation to basal control (BC) 
group; #P < 0.001, in relation to UVR 
control (UVRC) group; $P < 0.001, in 
relation to UVRC, P < 0.01, in relation to 
UVR/RCAC 1.0 mg/mL groups; 
φP < 0.001, in relation to UVRC, to UVR/
RCAC 1.0 mg/mL and to UVR/RCAC 
0.5 mg/mL groups (anova, Tukey).
Neuroimmunomodulatory compound for sensitive skin care • G C Dieamant et al.
118 © 2008 Blackwell Publishing • Journal of Cosmetic Dermatology, 7, 112–119
response was observed at concentrations ranging from
1.0 to 0.25 mg/mL.
Additional in vitro studies in progress in our laboratory
show that the RCAC is able to promote an important
effect in other mechanisms involved in pathophysiology
of sensitive skin, such as inhibition of matrix metallopro-
teinases, increase of endogenous antioxidant enzymes
activity, and induction of tissue growth factors produc-
tion. These findings and the results presented here
indicate that RCAC could be considered as an important
tool for the prevention of neurosensory symptoms related
to sensitive skin. In addition, in view of the fact that
similar inflammatory mechanisms seem to be involved
in the physiopathology of skin disorders related to hyper-
sensitivity reactions, such as allergic and contact der-
matitis,25 the neuroimmunomodulating ability of RCAC
to restore the balance in the production of inflammatory/
anti-inflammatory mediators encourages its clinical use
as an adjuvant for the treatment of these dermatological
disorders.
Conclusion
Evidence suggests that various neuropeptides and
neurohormones play a significant immunoregulatory role,
and this complex cascade of interacting mediators is
involved in the physiopathology of sensitive skin. In this
work, we showed that RCAC exerts a protective effect in
skin barrier function and in the reactivity of human subjects
with sensitive skin. These clinical responses couldbe
partially explained by the in vitro findings showing
stimulating effects on POMC peptide production, inhibition
of neuropeptides release, and immunomodulation of
cytokines production by keratinocytes exposed to UV stress.
Acknowledgments
This work was supported by grants from the Chemyunion
Química Ltda. The authors also express many thanks to
the Instituto de Bioengenharia da Pele EVIC Brazil for
clinical assessment performed and to Sueli Cristina de
Oliveira, Raphael Marcos Pereira, and Jéssica E. P. S.
Silveira for their technical assistance.
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