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PHYTOTHERAPY RESEARCH
Phytother. Res. 18, 957-'-962 (2004)
Published online in Wiley InterScience (www.interscience.wiley.com). DOl: 10.1002/ptr.1542
Supplementation with Glladin-combined
Plant Superoxide Dismutase Extract Promotes
Antioxidant Defences and Protects Against
Oxidative Stress
Ioannis Veuldeukis-", Marc Contf; Pascal Krauss', Caroline Kamaté'r', Samantha Blazquez',
Maurel Tefif, Dominiqne Mazier", Alphonse Calenda! and Bernard Dngas
1,2*
'ISOCELL Nutra SAS. 53 bd du Général Martial Valin, 75015, Paris, France
2INSER\1 U511, Irnmunobiologie Cellulaire et Moléculaire des Infections Parasitaires, CHU-Pitié Salptérière Paris VI, 75013,
Paris, France .
'INSER"! U477, Hôpital Cochin, 75006 Paris, France
The potential benefits to health of antioxidant enzymes supplied either through dietary intake or supple-
mentation is still a matter of controversy, The development of dietary delivery systems using wheat gliadin
biopolymers as a natural carrier represents a new alternative. Combination of antioxidant enzymes with this
natural carrier not only delayed their degradation (i.e. the superoxide dismutase, SOD) during the gastrointestinal
digestive process, but also promoted,
in vivo,
the cellular defences by strengthening the antioxidant statns.
The effects of supplementation for 28 days with a standamized melon SOD extract either combined (Gllsodin"
or not with gliadin, were evaluated on various oxidarive-stress biomarkers, As already described there was no
change either in superoxide dismutase, catalase or glutathione peroxidase activities in blood circulation or in
the liver' following non-protected SOD supplementaticn, Bowever, aninIals supplemented with GlisodinllD
showed a significant elevation in circulated antioxidant enzymes activities, correlated with an increased resist-
ance of red blood cells to oxidative stress-induced hemolysis, ln the presence of Sin-I, a chemical donor of
peroxynitrltes, mitochondria from hepatocytes regularly underwent membrane depolarîzation as the primary
biological event of the apoptosis cascade. Hepatocytes isolated from animais supplemented with Glisodinll>
presented a delayed depolarîzation response and an enbanced resistance to oxidative stress-induced apoptosis.
It is concluded that supplementation with gliadin-combined standardized melon son extract (Glisodin"
promoted the cellular antioxidant statns and protected against oxidative stress-induced cell death. Copyright
© 2004 John Wiley
&
Sons, Ltd.
Keywords: antioxidant; plant superoxide dismutase; gliadin; oxidative stress.
INTRODUCTION
ln all aerobic organisms, the consumption of oxygen is
crucial for life. It also produces reactive oxygen species
involved in the regulation of many different biological
pro cesses (Forman and Torres, 2002) and survival from
invading pathogens. Under physiological conditions the
production of these pro-oxidant molecules is control-
led at different levels by the antioxidant defences that
normally !imit the excess of free radical species (Wei
and Lee, 2002). These natural defences are essentially
composed of specialized enzymes such as superoxide
dismutase (SOD), catalase (Cat) and glutathione-
peroxidase (Gpx) and also by non-enzymatic antioxi-
dant molecules such as vitamins, thiols and f3-carotene.
Inflammatory or aging processes (Wickens, 2001) are
*
Correspondence to: Professor A. B. Dugas, ISOCELL Nutra SAS, 53
blvd du Général Martial Valin, 75015, Paris, France.
E-mail: bdugasïêtibcrtysurf.ïr
Contract/grant Sponsor: CIFRE fellowship ISOCELL Pharma SAS.
Copyright
©
2004 John Wiley
&
Sons, Ltd.
associated with the disruption of the oxidant/antioxidant
(redox) balance resulting in cellular and tissue oxidative
stress and cell death by apoptosis (Lang et al., 2002;
Chandra et al., 2000). Indeed, the progressive and dis-
crete imbalance of the endogenous redox system can
lead to the development of chronic degenerative dis-
eases (Lavrovsky et al., 2000; Tak et al., 2000). Thus it
seemed evident that nutritional antioxidant supple-
mentation could have health-promoting effects if it could
control the endogenous redox system (Fang et al., 2002;
Kritharides and Stocker, 2002). It is already admitted
that dietary antioxidants are very useful in general
health either by preventing or by supplementing the
usual drug treatments in a variety of diseases (Stephens
et
al"
1996; Kritchevsky, 1999; Burk, 2002). This sug-
gests that the use of a nutritional antioxidant formula
will provide better prevention of oxidative stress-me di-
ated diseases.
Until now the development of these new functional
foods has been limited by their poor capacity to pro-
mote efficient oral delivery of antioxidant enzymes
and also by the definition of the correct health bio-
markers to follow (Branca et
al.,
2001). However, the
Received
14
May 2003
Accepteâ 4June 2004
958
1.VOULDOUKIS ET AL.
development of new drug delivery and food pack-
aging systems (Weber et al., 2002; Takata et al., 2002)
make this new functional antioxidant formula possible
(Mosca et al., 2002; Stella et al., 1995; Regnault et al.,
1996). Among various different delivery systems the
wheat gliadin biopolymers presented a dual interest:
(i) their capacity to -trap and to delay the release
of the active ingredient during the gastrointestinal
digestive process (Arangoa et al., 2001), and (ii) their
bioadhesive properties with the intestinal mucosa to
improve and/or promote the delivery of the active in-
gredient, thus defining an orally bioactive SOD (Dugas,
2002).
This study investigated the froperties of an effective
nutritional formula (Glisodin ) made from the combi-
nation of a melon (Cucumis melo Le.) standardized
superoxide dismutase extract as the active ingredient
and wheat (Triticum vulgare) gliadin biopolymers as
the carrier. The antioxidant properties of the melon
SOD contained in Glisodin" were evaluated on anti-
oxidant biomarkers currently used to assess the poten-
tial health benefits of nutrition al products.
MATERLUS AND METHODS
Reagents. Dulbecco's modified Eagle's medium
(DMEM), L-glutamine, glucose, streptomycin-penicil
lin, fetal calf serum (FCS) and most of the chemical
reagents were from Sigma Chemical Co (St Louis, MO).
Hepatocytes were cultured in DMEM medium con-
taining 10% FCS, 1% L-glutamine, 2% streptomycin-
penicillin, in 5%
CO
2
at 37 "C, The chemical donor
nitrogen peroxide (Sin-I) was the kind gift of Dr J.P.
Kolb (INSER.t\1 U311, Paris, France). 2,2'-azobis-(2-
aminopropane )-dihydrochloride (AAPH) was obtained
from Calbiochem (Meudon, France). Wheat gliadin
(Gliamine") was purchased from HITEX (Vannes,
France). The standardized melon superoxide dismutase
extract (Extramel") was obtained from the strain
Cucumis melo L.e., genetically selected for its higher
grade SOD activity (90 ill/mg of dry powder), BIONOV
(Avignon, France).
The gliadin-combined SOD preparation. Briefly,
Glisodin® is a water dispersible form of superoxide
dismutase lyophilized extract from melon (standardized
to 90 ill/mg) combined with a 40% hydro-alcoholic
soft gel of gliadin at 50 "C, It is spray-dried using
maltodextrin as a support and the various ratios were
adjusted to obtain a theoretical activity of 1 lU/mg of
final dry powder.
The superoxide dismutase activity of the Glisodin"
was certified using a specifie enzymatic assay
(Beauchamp and Fridovich, 1971; Oberley and Spitz,
1984) from 5 g of dry product sonicated into 7 mL of
water. The solution was then centrifuged at 10000 g
for 20 min and the first supernatant
(SI)
made up to
10 mL with ultra pure water. The pellet was suspended
again in 1 mL of ultra pure water, homogenized and
centrifuged at 10 000 x g for 20 min at 6°-8 "C, The sec-
ond supernatant (S2) was then adjusted to 1 mL. The
activity in both fractions
(SI
and S2) was determined
on a native polyacrylamide gel electrophoresis against
the SOD melon extract (90 lU/mg).
Copyright © 2004 John Wiley
&
Sons, Ltd.
Delayed release of loaded SOD from the gliadin com-
bination. The progressive release of the SOD activity
trapped by the gliadin polymers was compared with
the parallel degradation of the non-protected SOD
(melon extract) during a pro cess that mimicked the
digestive transit (0.1
M
hydrochloric acid at pH
1
in the
presence of 1 !lM of pepsin at
37
DC) as already de-
scribed by Stella et al. (1995).
Animal population and treatment. Balb/c mice were
purchased from IFFA-CREDO (Orleans, France), aged
6-8 weeks and weighing 25-30 g. Each group consisting
of 10 animals randomly selecte d, received either a
normal diet, or a supplementation with gliadin, or a
supplementation with non-protected SOD melon ex-
tract (10 ill/day for 28 days) or Glisodin" (0.1, 0.5, 1,
5 mg/day for 28 days) by force-feeding.
Redox status. Blood samples were collected on heparin
at different time-points along the supplementation pe-
riod (0,7,14,21 and 28 days). Plasma and erythrocytes
were immediately separated by centrifugation at 800
x
g
for 20 min at 4 "C, Superoxide dismutase (RANSOD
kit, Randox) glutathione peroxidase (RA.t~SEL kit,
Randox) and catalase activities (was assayed by a
method in which the disappearance of peroxide is
followed spectrophotometrically at 240 nm) were then
deterrnined. Red blood cell (RBC) hemolysis, induced by
the free radical generator 2,2'-azobis-(2-amidinopropane)-
dihydrochloride (AAPH), was deterrnined as previously
described (Miki et al., 1987).
Peroxynitrite-induced apoptosis in hepatocytes. Apo-
ptosis was quantified by using the ApoAlert DNA frag-
mentation detection kit (Clontech, Palo Alto, CA). At
days 0, 7 and 28, hepatic cells were isolated and incu-
bated for 48 h (5 x 105cells/mL) in complete DMEM
medium in the presence or in the absence of 100
ngl
mL of Sin-1 (3-morpholinosydnonimine hydrochloride),
a potent generator of nitrogen peroxide. Data are
presented as the percentage of apoptotic cells among
various areas of 200 cells.
Measurement of the mitochondrial depolarization,
D.
'fi
m'
The
il
'Pmof isolated hepatocytes was measured by flow
cytometry using the J-aggregate-forrning lipophilic
cation, 5,5',6,6'
-tetrachloro-Ll
,3,3'-tetraethylbenzimida-
zolocarbocyanine iodide (JC-1) (Beltran et al., 2000).
Briefiy, aliquots of the cell suspension (106cells) were
incubated with JC-1 at a final concentration of
31J.M
at 37°C in the dark for 30 min before analysis. Pre-
lirninary experiments demonstrated that under these
conditions the dye reached near equilibrium distribu-
tion and gave a maximal fluorescence response to a
fall in
il
'Pm induced by the mitochondrial uncoupler
carbonyl cyanide m-chlorophenylhydrazone (5
ILro.f).
Flow cytometry was performed on a FACScan instru-
ment (Becton Dickinson). Data were acquired and ana-
lysed by using CELLQUEST software. The results are
expressed as the mean aggregate fluorescence (red)
alone.
Statistical analysis. Mean comparisons between the vari-
ous groups (with or without supplemented diets) were
conducted using Student's t-test. Differences were
considered significant when
p
<0.05).
Pltytother.
Res.
18, 957-962 (2004)
ORAL DELfVERY OF SUPEROXIDE DISMUTASE
959
Supplementation SaD (U/g Hb)
Table 1. Effect of a supplementation with non-protected SOD on circulating antioxidants
Catalase (kU/g Hb)Gpx (U/g Hb)
Control
Non proteeted SaD extra et
1125 ±55
1220
±
40
798
±
32
810
±21 30 ±2
33 ± 6
Animais
(n
=
10) were fed every day with control diet supplemented or not with 10 mg/mouse/day
of the non-protected SOD for 28
days,
Blood sam pies were collected and SaD, Gpx and catalase
activities were evaluated in erythrocytes. Data represent the mean.
±
SD
of
ten animals/group
from
one representative experiment.
RESULTS
Wheat gliadin carrier delays the SOD release in
conditions mimicking the digestive pro cess
Many investigations (Zidenberg-Cherr et al., 1983; Giri
and Misra, 1984) now including ours, demonstrated
that oral treatment with non-protected SOD did not
induce significant changes in the circulating redox sta-
tus since the levels of erythrocyte SOD, catalase and
Gpx activities remained constant (Table 1). This is con-
sistent with the poor bioavailability or rapid degrada-
tion of proteins during the digestive process. As a matter
of fact, a rapid disappearance of the non-protected
SOD activity was observed in a medium mimicking the
digestive pro cess (Fig. 1) demonstrating that the anti-
oxidant enzyme was destroyed during gastrointestinal
transit. However, when the SOD activity was trapped
by gliadin biopolymers (Glisodin®) a significant and
progressive increase of SOD activity was observed
probably correlating with the concomitant proteoly-
sis of the gliadin biopolymers. This suggested that
gliadin might delay the release and consequently the
degradation of the. SOD activity during gastrointestinal
transit.
~
100
~
-Or-
Free SOD
'>
!
--- Glisodin
n
80
i
T
III
0
0
,
(J)
60
1
!
L
~
40
!
:~
l
.•.
0
,
tf?
20
-1
o
2
5
10 30 60
Time (min)
Figure
1. Gliadin polymers delay the release of the melon SOD
activity in a medium mimicking the digestive process. An iden-
tical amount (100 units) of melon-SOD extract was submitted
free or combined with gliadin (Glisodin·"') to conditions mimick-
ing the digestive process, for 1 h at 37°C. The medium was
periodically sampled to measure the residual SOD activity ac-
cording to the reduction of ferricytochrome C. The data repre-
sent the mean
±
SD of quadruplicate samples of one treatment
out of six different experiments.
Copyright © 2004 John Wiley
&
Sons, Ltd.
Table 2. Effect of a supplementation with SOD-gliadin combina-
tion on circulating antioxidants
Supplementation
Control Glisodin"
Antioxidant status (rnrnol/Ll
SOD (U/g Hb)
Gpx (U/g Hb)
Catalase (kU/g Hb)
1.39
±
0.03
1720
±
125
800
±
33
35
±
5
1.98
±
0.06
3250
±
255
1210
±
89
95
±
6
Animais were fed every day with control diet or with con-
trol diet supplemented with 1 mg/mouse/day of Glisodin" for
28 days. Blood samples were collected and SOD, Gpx and
catalase activities were evaluated in erythrocytes. Data repre-
sent the mean
±
SD of ten animals/group
from
one representa-
tiva
experiment.
Glisodin@ supplementation modulated the circulating
antioxidant status
Supplementation of normal mice with the gliadin-
combined standardized melon SOD extract (Glisodin®)
for 28 days was.found to promote the circulating anti-
oxidant enzymes SOD, catalase and Gpx (Table 2). This
effect was formula specifie (Glisodin"), because the non-
protected SOD extract or the gliadin alone was unable
to promote these antioxidants. This promoting effect
was time dependent (Fig. 2A) since the circulating SOD
activity began to Increase after 7 days of supplementa-
tion to reach a maximum after 28 days (SOD returned
to the baseline after an addition al 28 days, data not
shown), The promoting effect was dose dependent
(Fig. 2B) since significant effects appeared only for doses
equivalent to 0.5 mg/day or higher with a maximal
effect .obtained at 5 mg/day.
As already demonstrated for different antioxidant
dietary supplementation (Peng
et al.,
2000), the supple-
mentation with Glisodin" for 28 days increased the re-
sistance of RBC
(p
<
0.01) to oxidative stress-induced
hemolysis (Fig. 3) in response to a chemical donor of
free radicals (AAPH). After 3 h of incubation at 37°C
in the presence of 50 mMAAPH, about 48% vs 74% of
hemolysis was observed for RBC isolated, respectively,
from animaIs supplemented or not with Glisodin".
Hepatoprotective effect of Glisodln" supplementation
As previously described
in vitro
(Vouldoukis
et al.,
2000), the SOD-gliadin combination also induced
in vivo,
a time-dependent increase in SOD activity in
Phytother. Res. 18, 957-962 (2004)
-- -- ----------~.
960
1.VOULDOUKIS
ET AL.
5000
l
AI
(J) 5000]
>-
-{,.~Control
(1j
1
"0
i
..,.. Free gliadin
TT
co
~ 4000
1
--e-
Free SOD
f
11
N
L-
4000 .
-o-Glisodin
Q) 1
;::=
1
i
3000
1
(1j
:0-
:c
1
el
3000
1
:3
-
(1j
!
>-
~ 2000
J
t:;t;t:J
!
-
.:;
2000
1
:;:;
1
o
(1j
el
1000
1
0
1000 .
CI')
0
7
14 21 28 0 0.1 0.5 5
Time of treatment (days) Gllsodirr"
(mg/day)
Figure
2.
Effect of a supplementation with
Glisodin"
on circulating SOD activity. A. Mice were fed for
28
days, with either a control
diet or supplemented with (a) melon SOD extract
(10
lU of non protected SOD), (b) gliadin
(1
mg) or (e) Glisodin"
(1
mg for
1IU).
B.
Mice were fed with different doses of Glisodin"
(0.1, 0.5, 1,
or
5
mg of Glisodin"/mouse/day). Blood was periodically sampled in
the study (A) while only at day
28
for study (B). SOD activity was measured as deseribed in materials and methods. Data represent
the mean
±
SEM of the different groups.
90
80 -+-Control
T
1
70
I
-0- Glisodin-treated
T
:H<OO11
c
60
1
.!!1
!
Ul
50
.L
>-
40
1
n .
'0
1
E30
t
1
Q)
1
:x:
1
20
..L
1
10
1
1
·06/1
00,5
1
1,5 2 2,5 3
Time (Hours)
Figure
3.
Effect of a supplementation with Glisodin" on ervthro-
eyte resistanee to oxidative stress-induced hemolysis. After a
28
day peri ad of supplementation with Glisodin"
(1
mg/day),
RBC were eolleeted and exposed ta the free radical generator
AAPH
(50
mM). Hemolysis was evaluated as deseribed in mate-
rials and methods. Data represent the mean
±
SEM of the dif-
ferent groups.
hepatocytes (Fig. 4). This inducing effect appeared to be
significant
(p
<0.05) after 14 days of Glisodin" supple-
mentation and reached a maximal effect after 21-28
days
(p
<
0.001). Such stimulation was not restricted to
the SOD activity because catalase and Gpx activities
were also increased (Table 3). As shown in Fig. 5 the
improvement of the hepatocyte antioxidant defences
correlated with an increased resistance (p <0.01) to
oxidative stress-induced apoptosis (Estevez and Jordan,
2002). After 8 h in the presence of the peroxynitrites
chemical donor Sin-I, it was observed that 20% of the
hepatocytes isolated from animals supplemented with
Gliscdin" underwent apoptotsis, whereas this rate in-
creased ta 72% in hepatocytes fram
untreated
animals.
Copyright © 2004 John Wiley
&
Sons,
Ltd.
Table 3. Effect of a supplementation with SOD-gliadin combina-
tion on Iiver antioxidants
Supplementation
Activity (unit/mg of protein)
SOD Gpx Catalase
2.5
±
0.2
13.5
±
0.6 0.21 ±0.05
0.80 ±0.02 40
±
1
68
±
3
Control
Glisodin"
Animais received every day either a control diet with or with-
out supplementation with
1
mg/mouse/day of Glisodin" for
28
days. Livers were then collected and then the SOD, catalase
and Gpx activities were evaluated from the various tissue ex-
tracts. Data represent the mean
±
SD of ten animals/group.
Effect of Glisodin" supplementation on animal
hepatocytes mitchondriaI
L1
'fi
m
exposed ex vivo to
Sin-1
As the mitochondrion is a key compartment involved
in the control of oxidative stress-induced cell death
(Akao et al., 2003a) the mitochondrial functions of
hepatocytes isolated from animaIs receiving a Glisodin"
supplementation were evaluated. As aIready described
(Li et al., 2002; Kahlert and Reiser, 2002; Makani et al.,
2002) mitochondria from normal hepatocytes exposed
to Sin-l showed a gradual decrease in
LI.'Pm
as described
by the mean aggregate fluorescence of the cationic
lipophilic fluorochrome (Je-l) (Fig. 6). Analysis of
mitochondrial
LI.
'f'
ID
of Sin-l-stimulated hepatocytes from
Glisodin" supplemented animals demonstrated that the
mitochondrial depolarization was substantially delayed.
DISCUSSION
This study investigated the potential effects of a
supplement containing a gliadin-combined plant SOD
Phytother. Res. 18, 957-962 (2004)
ORAL DELIv'ERY OF SUPEROXIDE DISMUTASE
961
10
-o-Contro!
<:
--*-Gliadin
Oi
"0
8
-0-
SOO extract
...
0.
-
~Glisodin
0
01
6
.§
2-
~
4.
~
Ü
<Il
0
2
0
rn
0
0
7
14 21
28
Time of treatment (days)
Figure 4. Effect of a supplementation with Glisodln" on liver
SOO activity. Mice were fed with a control diet supplemented
or not with
1
mg/mouse/day of
Gllsodin".
Animais were killed
periodically each 7 days. Liver proteins were extracted and the
SOO activity was evaluated. The results are expressed as units
per mg of protein and data represent the mean
±
SEM of the
different grou ps.
100
f&!l
Control
i
80
l
B Glisodin
.g
60
1
.B
0-
o
g.
40
'5
><
1
20 .
l
p
«
0.001
~
T
Figure 5. Effect of a supplementation with Glisodin"' on the
resistance of hepatocytes to nitrogen peroxide-induced
apoptosis.
Balb/c
mice
(n
=
10 per group) were fed a control
diet supplemented or not with , mg/mouse/day of Glisodin"'
and killed after
28 davs,
After isolation Iiver ceIls were submit-
ted to Sin-t. The results are expressed as a percent of apoptotic
ceIls and data represent the mean
±
SEM of four different
experiments.
extract on several redox biomarkers. The results of this
animal study were dual: the Glisodin" dietary supple-
mentation not only promoted the circulating and
tissue antioxidant defences (increased SOD, Gpx and
catalase activities) but also improved cell resistance to
oxidative stress. ln the circulation, RBC from animals
receiving Glisodin" were less susceptible to oxidative-
stress-induced hemolysis. ln addition hepatocytes from
animaIs receiving Glisodin" dietary supplementation
Copyright
©
2004
John
Wiley
&
Sons, Ltd.
140
11
il
g
1 •••••••
Control
ii
t: '
Co
~?
120 ~
1-0-
Glisodin
î.
(I)~ I~'- ~
~~1001
T ~~
a
801 ~ ~~ 1
~~ i
= }~~
~ 60 T
<ll
œ
40
1
3
4
o
1
2
Time after serum withdrawal (hours)
Figure 6. Effect of a supplementation with Glisodin"' on
sln-t-tnduced mitochondrial membrane depolarization
Ll'!'
m
in
hepatocytes. Changes in
t.\Pm
of isolated hepatocytes from
normal or Glisodin"'
(n
=
10 per group) supplemented animais
were followed after exposure to the chemical peroxynitrite
donor, Sin-l
(100
ng/mL) over a period of 4 h as described in
materials and methods. Data represent the mean
±
SO of ail
animais.
were resistant to peroxynitrite-induced apoptosis and
mitochondrial depolarization.
The combination of the melon SOD extract with
gliadin biopolymers
is
mandatory for obtaining this
health promoting effect, confirrning that the wheat
gliadin is a helpful carrier for the oral delivery of active
food ingredients (Arangoa
et al., 2001) .
Many studies have reported that a long-lasting intake
of fruit and vegetable antioxidants reduced the Iikeli-
hood of cardiovascular and proinfiammatory diseases
as weil as certain cancers (Block
et
al., 1992; Diplock
et al.,
1987;
Madar and Stark,
2002;
O'Byrne
et al., 2002;
Akao
et al.,
2003b): Soit appears that the improvement
of antioxidant defences is a biological key event in the
health promoting effects of antioxidant nutrients. The
present work not only confums and extends these
scientific and clinical studies but also provides useful
information for the development of functionally active
food ingredients.
This new formula shows real benefits for health since
functional antioxidant enzyme supplementation (here
melon SaD) is now able to promote cellular resistance
to stress by strengthening the host antioxidant defences.
Nevertheless, the mechanism by which it exerts its bio-
logical effect remains to be clarified.
The present study does not only confirms the effi-
cacy of dietary antioxidant supplementation but also
describes an orally active plant superoxide dismutase
demonstrating that functional enzymes can be used in
dietary supplementation.
Acknowledgements
Caroline Kamaté is a PhD student in receipt of a CIFRE fellowship
from Isocell Pharma SAS.
Pliytother. Res.
18, 95ï-962 (2004)
962
I. VOULDOUKIS
ET AL.
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