Group Title: Journal of Autoimmune Diseases 2005, 2:8
Title: Lack of correlation between the levels of soluble cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and the CT-60 genotypes
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Title: Lack of correlation between the levels of soluble cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and the CT-60 genotypes
Series Title: Journal of Autoimmune Diseases 2005, 2:8
Physical Description: Archival
Creator: Purohit S
Podolsky R
Collins C
Zheng W
Schatz D
Muir A
Hopkins D
Huang YH
She JX
Publication Date: 38656
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Lack of correlation between the levels of soluble cytotoxic
T-lymphocyte associated antigen-4 (CTLA-4) and the CT-60
Sharad Purohitti, Robert Podolskytl, Christin Collins', Weipeng Zheng1,
Desmond Schatz2, Andy Muir', Diane Hopkins1, Yi-Hua Huang' and Jin-
Xiong She*l

Address: 'Center for Biotechnology and Genomic Medicine, Medical College of Georgia, CA4095 Augusta, GA 30912 and 2Department of
Pediatrics, University of Florida, Gainesville FL 32607, USA
Email: Sharad Purohit; Robert Podolsky; Christin Collins;
Weipeng Zheng; Desmond Schatz; Andy Muir;
Diane Hopkins; Yi-Hua Huang; Jin-Xiong She*
* Corresponding author tEqual contributors

Published: 31 October 2005
journal of Autoimmune Diseases 2005, 2:8 doi: 10.1 186/1740-2557-2-8

Received: 16 August 2005
Accepted: 31 October 2005

This article is available from:
2005 Purohit et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) plays a critical role in
downregulation of antigen-activated immune response and polymorphisms at the CTLA-4 gene have
been shown to be associated with several autoimmune diseases including type-I diabetes (TID).
The etiological mutation was mapped to the CT60-A/G single nucleotide polymorphism (SNP) that
is believed to control the processing and production of soluble CTLA-4 (sCTLA-4).
Methods: We therefore determined sCTLA-4 protein levels in the sera from 82 TI D patients and
19 autoantibody positive (AbP) subjects and I 17 autoantibody negative (AbN) controls using ELISA.
The CT-60 SNP was genotyped for these samples by using PCR and restriction enzyme digestion
of a 268 bp DNA segment containing the SNP. Genotyping of CT-60 SNP was confirmed by dye
terminating sequencing reaction.
Results: Higher levels of sCTLA-4 were observed in TI D (2.24 ng/ml) and AbP (mean = 2.17 ng/
ml) subjects compared to AbN controls (mean = 1.69 ng/ml) with the differences between these
subjects becoming significant with age (p = 0.02). However, we found no correlation between
sCTLA-4 levels and the CTLA-4 CT-60 SNP genotypes.
Conclusion: Consistent with the higher serum sCTLA-4 levels observed in other autoimmune
diseases, our results suggest that sCTLA-4 may be a risk factor for TI D. However, our results do
not support the conclusion that the CT-60 SNP controls the expression of sCTLA-4.

Effective T cell activation requires a 'costimulation' signal
that is mediated through CD28 interacting with B7 family

members on antigen presenting cells (APC) [1]. The cyto-
toxic T lymphocyte associated antigen-4 (CTLA-4) was ini-
tially described as a B7 binding protein and a receptor

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led Central


Journal of Autoimmune Diseases 2005, 2:8

expressed on the surface of activated T cells [2]. It belongs
to the immunoglobulin gene superfamily and shares
homology with CD28. CTLA-4 has been reported to be an
important negative regulator of autoimmune diseases
[3,4]. CTLA-4 blockade enhances T cell responses in vitro
and in vivo [5,6], augments antitumor immunity [7] and
exacerbates autoimmune diseases [8]. Several reports have
indicated that CTLA-4 deficient mice show a severe lym-
phoproliferative disorder and autoimmune disease with
early lethality [9,10]. Treatment with anti-CTLA-4 mAb of
BDC2.5/NOD mice provoked a rapid onset of diabetes,
indicating that a higher CTLA-4 presence was required for
suppression of autoimmune phenomenon in these mice
[11,12]. Recently, a soluble form of CTLA-4 (sCTLA-4)
was found to be expressed constitutively by unstimulated
human T cells [13]. Circulating sCTLA-4 protein was
found to be present in human serum and is shown to pos-
sess an inhibitory effect on mixed leucocyte response [14].

Several studies have demonstrated a genetic association
between polymorphisms within or near the CTLA-4 gene
and T1D [15-19] as well as other autoimmune diseases
[20-24]. This susceptibility locus has been recognized as
IDDM 12. Our previous studies indicated that CTLA-4 was
the only gene contained in the IDDM12 susceptibility
interval, suggesting that CTLA-4 is indeed the IDDM12
gene [16]. In a recent report by Ueda et al [25] the suscep-
tibility interval was further narrowed to a 6.1 kb region at
the 3' UTR of the CTLA-4 gene and the CT60-A/G single
nucleotide polymorphism (SNP) was suggested to be the
etiological mutation. The susceptible CT60-G allele was
reported to produce a lower amount of soluble CTLA-4
mRNA in the peripheral blood lymphocytes than the dis-
ease resistant CT60-A allele. These results suggested that
sCTLA-4 may confer protective effect against T1D. If this
effect is indeed true, one would predict lower sCTLA-4 in
the serum in T1D patients compared to controls. How-
ever, the prediction is in direct conflict with the observa-
tions in other autoimmune diseases including
autoimmune thyroid disease [26], systemic lupus ery-
thematosus [27] and myasthenia gravis [28], in which the
serum sCTLA-4 levels are increased in patients compared
to controls. The measurement of serum sCTLA-4 protein
in a larger sample set is vital in evaluating the potential
role of sCTLA-4 in T1D, and to better understand the
molecular and functional basis underlying the genetic
association between the CTLA-4 gene and T1D.

Patient sera
The study population consists of 218 subjects from the
South-eastern United States. All study subjects were geno-
typed for HLA-DQB1 and evaluated for three autoanti-
bodies (IA-2A, GADA and IAA) using established methods
[29,30] Subjects used in this study are participants in the

prospective assessment in newborns for diabetes autoim-
munity (PANDA) program. Briefly, PANDA screens new-
boms from the general population as well as children
with a first degree relative with T1D using HLA genotyp-
ing. Those subjects with high risk genes are monitored for
the appearance of islet autoantibodies and clinical diabe-
tes. Therefore, most of the autoantibody-negative (AbN)
subjects also have high risk HLA genes and the AbN group
is not randomly selected from the general population. The
autoantibody-positive (AbP) subjects have been tested
persistently positive for two or more islet autoantibodies.
Based on our results from PANDA and previous studies,
the AbP group has 70-80% of chance to progress to T1D
[31] and indeed represent a very high risk group. Since
autoantibody production is one of the hallmarks of
autoimmunity, the AbP and T1D group can be combined
to assess the impact of autoimmunity on the CTLA-4 lev-
els. Appropriate institutional review boards approved the
study design and informed consent was obtained from all

Assay of sCTLA-4
A sandwich ELISA assay as described by Oaks et al [26]
was used to measure the serum sCTLA-4 levels in a total of
218 subjects, including 117 autoantibody-negative
(AbN), 19 autoantibody-positive (AbP) and 84 patients
with T1D. The 96-well microtiter plates (Pierce Biotech-
nology, Rockford, IL) were coated with 1.0 ug/ml anti-
CTLA-4 monoclonal antibody (clone BNI3; Pharmingen,
San Deigo, CA). After blocking, 100 ul of 1:10 diluted
serum samples were added to each well and the plates
were incubated for 2 hr in a humid chamber at 37 C and
then washed to remove unbound material. After washing,
100 ul biotinylated anti-CTLA-4 mAb (1.0 ug/ml, clone
AS-33, Antibody Solutions, Palo Alto, CA) was added and
the reactions were incubated for another 1 hr at 37C in a
humid chamber. Reactions were developed using strepta-
vidin-peroxidase complex (Biorad, Hercules, CA) and
3,3',5,5'-tetramethy benzidine substrate (Sigma, St. Louis,
MO) for 10 min at room temperature, the reaction was
terminated with 2N H2SO4 and optical density was read at
450 nm and 630 nm. A standard curve was generated
using a dilution series of commercially available CTLA-4-
Ig fusion protein (0.125 ng/ml to 10 ng/ml Ancell, Bay-
port, MN). This assay has a linear range between 0.5 and
10 ng/ml and the vast majority of the samples fall under
this range. Each sample was analyzed in duplicate.

Genotyping of 3' untranslated region of CTLA-4 gene
A fragment of 268 bp encompassing the CTLA-4 C/T-60
single nucleotide polymorphism (SNP) in the 3' untrans-
lated region was amplified using the forward primer
5'GCTTCATGAGTCAGCTITGC3' and reverse primer
5'ATAGGACCACAGGT3'. The amplified PCR products
were digested using the 10 units of HpyCH4IV (New Eng-

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Journal of Autoimmune Diseases 2005, 2:8

Table I: Clinical and Demographic characteristics of subjects
involved in the study with respect to number of individuals, age
of diagnosis, duration of disease, genotype and antibody





subsequently conducted separate analyses adding other
factors. These analyses were as follows: (1) factorial
ANOVA with phenotypic group and CTLA-4 genotype; (2)
factorial ANOVA with phenotypic group and gender; (3)
ANOVA with phenotypic group and age as a covariate,
and the interaction between age and phenotypic group;
(4) ANOVA with phenotypic group and duration of T1D
as a covariate, and the interaction between duration of
T1 D and phenotypic group; and (5) factorial ANOVA with
phenotypic group and HLA genotype.

Age of Diagnosis (years)

Duration of T I D (years)

Age: n (percent)
<20 years
>20 years

Sex: n (percent)

Genotype: n (percent)*

86 (73.51) 14(73.68)
31 (26.49) 5 (26.32)

53 (45.30) 9 (47.37)
64 (54.70) 10(52.63)

8 (6.84)
31 (26.50)
21 (17.95)
23 (19.66)
18 (15.38)

0 (0.00)
5 (26.32)
3 (15.79)
2 (10.53)

8.8 (0.8-41.2) Results
Clinical and demographic information is presented in
6.2 (0-41.0) Table 1. A majority of the subjects in the study were below
the age of 20 years (73%) in all three groups. The subjects
60(73.17) were tested for IAA, GADA and IA-2A autoantibodies as
22(26.83) well as HLA-DQB1 genotypes. HLA-DQB1 genotyping
information was available for 96.58, 94.74 and 98.78 per-
cent of patients from the AbN, AbP and T1D groups,
50 (60.97) respectively. Eighty-eight percent of T1D subjects were
32 (39.03) diagnosed with T1D before the age of 20, with an average
age of 8.8 (range 0.9-41.2). Fifty-seven out of eighty-two
i1( 1 85R T1'1D subjects have a T1D duration of five years and less.

6 (7.32)
38 (46.34)
I 1(13.41)
3 (3.66)

Age, age of diagnosis and duration of disease is presented as means
*Genotyping information was not available on one AbP, one TI D and
4 AbN individualss.
Sex and genotype are presented as number of individuals of individuals
(n). Values presented in parenthesis are percentage of total.

land Biolabs) and separated on 3% agarose gels. The C-60
allele yielded two bands of 151 and 103 bp and the T-60
allele yields a band of 268 bp. The genotyping technique
for the C/T-60 SNP was further confirmed by DNA
sequencing of a subset of samples using a 300XL DNA
sequencer (ABI Sciex).

Statistical Analysis
Absorbance values obtained at 450 nm were normalized
with the absorbance values at 630 nm. sCTLA-4 levels
were log-transformed prior to analysis. Two of the T1D
subjects had very high sCTLA4 levels with serum CTLA4
levels were > 12 ng/ml, or 3.5 standard deviations from
the mean of 2.57 ng/ml. Further, initial analyses involving
analysis of variance (ANOVA) indicated these two sub-
jects' values were outliers. As such, the data for these two
subjects were removed from all subsequent analyses. We
used linear-mixed model ANOVA (Proc Mixed procedure
of SAS) in which plate was included as a random effect to
examine differences in sCTLA4 levels. Initially we ana-
lyzed phenotypic group (AbN, AbP, and TID) alone, but

We first compared the serum sCTLA-4 levels between the
three phenotypic groups (i.e., AbN, AbP and T1D). The
protein levels for T1D (mean = 2.24 ng/ml, range = 0-
10.1 ng/ml) and AbP (mean = 2.17 ng/ml, range = 0.2-
7.7 ng/ml) were slightly higher than that in AbN (mean =
1.69 ng/ml, range = 0.0-11.5 ng/ml) (Table 2), although
these differences were not statistically significant.

The serum CTLA-4 levels were analyzed after stratification
by phenotypic groups (T1D, AbP and AbN) and the CTLA-
4 CT-60 SNP genotypes (A/A, A/G and G/G) (Table 2). A
mixed model ANOVA using phenotypic group and CT-60
genotypes as factorial fixed effects revealed no differences
in sCTLA-4 levels between CTLA-4 genotypes (p = 0.46) or
genotype/phenotype interactions (p = 0.82). A similar
ANOVA using CT-60 genotypes alone as a fixed effect did
not reveal any significant differences in sCTLA-4 levels
between CTLA-4 genotypes (p = 0.64).

We then analyzed the data after conditioning on genetic,
phenotypic or demographic parameters. Neither gender
showed differences in serum CTLA-4 levels between the
three phenotypic groups (Table 2). The relationship
between sCTLA-4 levels and age differed between the
three phenotypic groups (p = 0.022). The sCTLA-4 levels
decreased with age in the controls (p = 0.048; Fig. 1). In
contrast, sCTLA-4 levels increased with age in both the
T1D and AbP groups (Fig. 1), although these relationships
were not significant (p > 0.1). This difference in the rela-
tionship with age will result in AbN controls having lower
sCTLA-4 levels at later ages compared with both AbP and

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Total number
Age (years)
Age range

Journal of Autoimmune Diseases 2005, 2:8

Table 2: sCTLA-4 levels in AbN, TI D and AbP individuals. Values presented are mean and 95% confidence interval in ng/ml.




All subjects (p = 0.58)
CTLA-4 genotype
Male (p = 0.53)
Female (p = 0.70)
DQB 1*0201 positive
DQB 1*0201 negative
DQB 1*0302 positive
DQB 1*0302 negative

1.7 (0.6-3.6) (n = I 17) 2.2 (0.5-5.5) (n = 19) 2.2 (0.9-4.6) (n = 82)

1.9 (0.7-4.1) (n = 12)
1.5 (0.5-4.0) (n= 21)
1.6 (0.7-3.0) (n = 49)
1.7 (0.9-2.8) (n = 53)
1.7 (0.9-2.8) (n = 64)
1.3 (0.6-2.4) (n = 60)
2.2 (1.2-3.8) (n = 53)
1.6 (0.8-2.9) (n = 66)
1.9 (0.9-3.3) (n = 47)

2.5 (n = I)
1.7 (0.4-4.2) (n = 7)
2.5 (0.9-5.4) (n = 9)
2.3 (0.9-4.5) (n = 9)
2.1 (0.8-4.2) (n= 10)
1.8 (0.6-4.0) (n = 8)
3.3 (1.4-6.8) (n = 10)
2.4 (1.0-4.6) (n= 13)
2.6 (0.9-5.9) (n = 5)

1.9 (0.8-4.9) (n = 19)
1.9 (0.7-3.8) (n = 16)
2.4 (1.1-4.4) (n =26)
2.3 (1.3-3.7) (n = 51)
2.2 (1.2-3.6) (n = 31)
2.2 (1.1-3.8) (n =62)
1.9 (0.8-3.8) (n = 19)
2.3 (1.2-3.9) (n = 56)
1.8 (0.7-3.6) (n =25)

T1D subjects. Serum sCTLA-4 levels in T1D subjects did
not show an association with duration of disease (p = 0.4)
nor with the age at disease onset (p = 0.6; data not

The serum CTLA-4 levels were also analyzed after condi-
tioning on the HLA-DQB 1 genotypes by using phenotypic
group, HLA-DQB1*201, and HLA-DQB1*302 as factorial
fixed effects in a mixed model ANOVA. No differences
were observed in sCTLA-4 levels between HLA-
DQB1*0302 genotypes (p = 0.96), and the three pheno-
typic groups stratified by HLA-DQB1 genotype did not
show any differences (Table 2, p = 0.51). AbN subjects
with the DQBI*201 allele tended to have lower sCTLA
levels (1.3 ng/ml vs 2.2 ng/ml), although the difference
was not significant, a similar trend was observed in AbP
group (1.8 ng/ml vs 3.3 ng/ml). The T1D group subjects
with and without DQB1*201 allele have a very similar
levels of sCTLA-4 (2.2 ng/ml vs 1.9 ng/ml). The differ-
ences between the three phenotypic groups for subjects
with the DQB1 201 allele were not significantly different
from the differences observed for those without the
DQBI*201 allele (Table 2; p = 0.13). The main effect of
the DQBI*0201 allele in the factorial mixed-model
ANOVA was marginally significant (p = 0.08) with serum
CTLA-4 levels being lower in individuals with a
DQB1*0201 allele (mean = 1.8 ng/ml) than individuals
without a DQBI*0201 allele (mean = 2.4 ng/ml). We
decided to redo this analysis by combining the AbP and
T1D groups for three reasons: (1) sample sizes were small
for some of the genotype/phenotype combinations; (2)
subjects that are positive for multiple antibodies and with
a high risk HLA genotype are much more likely to develop
T1D in future; and (3) autoimmunity is the common
denominator of the AbP and T1D groups. When the data
were analyzed with these two phenotypic groups consid-
ered as a single group (AbP + T1D vs. AbN), the main
effect of DQB1 *0201 allele became significant (p = 0.02),
with the remaining effects still not significant. T1D and
AbP subjects did show a trend towards having larger

sCTLA-4 levels (mean = 2.2 ng/ml) compared to the AbN
subjects (mean = 1.3 ng/ml) when only subjects with the
DQBI*0201 were considered, however, the difference
was not statistically significant (p = 0.15).

Type-1 diabetes is marked by the production of pancreatic
islet p cell-specific auotantibodies and destruction of the
insulin-producing p cells by autoreactive T cells. A role of
CTLA-4 in the pathogenesis in T1D and other autoim-
mune diseases has been well documented. In this study
we provide some suggestive evidence that high risk
autoantibody positive subjects and T1D patients both
have increased levels of sCTLA-4 in serum compared to
autoantibody-negative subjects. We observed that larger
differences in sCTLA4 levels between T1D/AbP and AbN
subjects occur in the older age group. Further, we observed
a difference between AbP/T1D subjects and AbN subjects
for those carrying the DQB1 0201 allele.

As a negative regulator of T cell activation, blockade of
CTLA-4 by monoclonal anti-CTLA-4 antibody provokes a
rapid onset of diabetes in BDC2.5/NOD mouse model
[11]. Treatment of animals with recombinant CTLA-4Ig
molecule delays the onset ofT1D and other autoimmune
diseases [ 11,12,32-36]. However, the function and poten-
tial role of sCTLA-4 have not been well studied. sCTLA-4
is generated by alternative splicing of CTLA-4 mRNA,
which induces a frame shift by deletion of a transmem-
brane region of CTLA-4 resulting in a native soluble pro-
tein [13]. sCTLA-4 is constitutively expressed on
nonstimulated T cells and its expression is downregulated
after T cell activation [14]. The soluble form of surface
proteins is believed, in most cases, to play an inhibitory
role due to competition for ligands with their surface
counterparts. The finding that the sCTLA-4 expression
level remains at sustained levels suggests that sCTLA-4
blocks the B7-mCTLA-4 interaction, thereby enhancing T-
cell activation and autoreactivity by inhibiting the induc-
tion of energy [37,38]. Alternatively, sustained sCTLA-4

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Journal of Autoimmune Diseases 2005, 2:8




Figure I
Relationship of sCTLA-4 with age and phenotypic groups.
The lines shown are the estimated line based on the mixed
linear model.

levels may play a protective role via inhibition of the B7-
CD28 interactions. Therefore, the role of sCTLA-4 in
autoimmunity may depend on the relative binding affin-
ity of sCTLA-4 to B7.1 and B7.2. This question was indi-
rectly addressed in several autoimmune diseases by
comparing the sCTLA-4 levels in the serum of patients and
controls. Elevated sCTLA-4 has been reported in organ
specific autoimmune thyroid disease [26], systemic lupus
erythematosus [27] and myasthenia gravis [28]. These
observations suggest that sCTLA-4 may contribute to the
development of autoimmune diseases, probably through
inhibiting the B7-mCTLA-4 interaction and down-regula-
tion ofT cell activation.

We are unaware of any study that has examined sCTLA-4
levels in the serum of T1D patients. A recent study by
Ueda et al. [25], suggested that a SNP (CT60-A/G) in the
3' UTR of the CTLA-4 gene may determine the efficiency
of the splicing and production of sCTLA-4 mRNA. Based
on a small number of subjects, the susceptible G allele was
suggested to produce lower amounts of sCTLA-4 mRNA.
Based on these observations, the authors concluded that
sCTLA-4 expression is the functional basis for the
observed genetic association between T1D and the CTLA-
4 gene. If this conclusion were correct, T1D patients
would be expected to have lower serum CTLA-4 levels.
Our results found no evidence to indicate that sCTLA-4
levels are decreased in patients compared to controls. In
contrast, our data suggested that the serum sCTLA-4 levels
were slightly higher in T1D patients. Our results also indi-
cated that the increased sCTLA-4 levels in T1D patients
were not due to the hyperglycemic conditions because the
autoantibody positive subjects also had increased serum
sCTLA-4 levels. We also directly tested the correlation
between sCTLA-4 levels and the CT-60 SNP in all three
phenotypic groups (AbN, AbP and T1D) and found no

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significant correlation in any of these groups. The discrep-
ancies between our study and the previous report [25]
may be explained by a number of factors. First, mRNA was
studied in the previous report, while serum protein was
analyzed in this study. As the biological function of
sCTLA-4 is carried out at the protein level, our data is
more applicable to the role of sCTLA-4 in T1D pathogen-
esis. Second, the sample size in the previous report [25]
was extremely small and random variation is quite likely.
Although the sample size in our study is not very large, it
is several times larger than the previous report and is suf-
ficiently powered to detect large differences. As there is no
indication of a correlation between sCTLA-4 levels and the
CT-60 genotype, it is unlikely that the C/T-60 SNP plays a
major role in controlling the expression of sCTLA-4.

Consistent with the observations in other autoimmune
diseases in humans as well as data in the NOD mice, our
data suggest that sCTLA-4 is potentially a risk factor for the
development of T1D. Our data also raises a serious doubt
about the conclusion that the expression of sCTLA-4 is
controlled by the CT60 SNP in the 3' end of the CTLA-4
gene. Therefore, the functional basis for the genetic asso-
ciation between CTLA-4 and autoimmune diseases as well
as the etiological mutation in the CTLA-4 region should
be re-considered.

Competing interests
The authors) declare that they have no competing inter-

Authors' contributions
SP and JXS designed the studies, helped with the interpre-
tation and the writing of the manuscript. SP, CC and WZ
were primarily involved in carrying out the clinical assess-
ments and the acquisition of data. RP performed the sta-
tistical analyses and was involved in preparing the
manuscript. AM and DS were responsible for collecting
clinical samples and patient evaluation. DH and YH were
involved in sample and data collection.

This work was supported by grants from the National Institute of Child
Health and Development (2RO I HD37800 and I R2 I HD50196) toJXS. SP
was supported by a JDRF postdoctoral fellowship (JDRF #3-2004-195).

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