Anticardiolipin Antibodies From Patients With the
Antiphospholipid Antibody Syndrome Recognize Epitopes
in Both ␤2-Glycoprotein 1 and Oxidized
Low-Density Lipoprotein
Sohvi Hörkkö, MD, PhD; Tsaiwei Olee, PhD; Lian Mo, PhD; D. Ware Branch, MD;
Virgil L. Woods, Jr, MD; Wulf Palinski, MD; Pojen P. Chen, PhD; Joseph L. Witztum, MD
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Background—We recently suggested that many anticardiolipin antibodies bind only to oxidized cardiolipin (OxCL) and/or
to OxCL–␤2-glycoprotein 1 (␤2GP1) adducts but not to a “reduced” cardiolipin that is unable to undergo oxidation. To
test this hypothesis, we investigated 24 sera, 4 protein A–purified IgG fractions, and 3 human monoclonal antibodies
that were all isolated from patients with antiphospholipid antibody syndrome (APS); testing was also performed in 7
controls. Two monoclonal antibodies (IS3 and IS4) were selected for binding to CL and one was selected for binding
to ␤2GP1 (LJB8).
Methods and Results—By chemiluminescent immunoassay, all APS sera samples bound only to OxCL and not to reduced
CL, and the binding was inhibited ⬎95% by OxCL but not reduced CL. All purified IgG fractions bound to ␤2GP1 but
only when the ␤2GP1 was plated on microtiter wells coated with OxCL. All 3 monoclonal antibodies bound only to
OxCL. On Western blots, IS4 and LJB8 bound to ␤2GP1 as well as to delipidated apoB of oxidized LDL but not to
native apoB. IS3 also bound to oxidized apoB on Western blot. Covalent modification of ␤2GP1 with oxidation products
of CL made it more antigenic for APS serum samples, for purified IgG fractions, and for the monoclonal antibodies.
Conclusions—These data support the hypothesis that oxidation of CL is needed to generate epitopes for many
anticardiolipin antibodies and that some of these epitopes are covalent adducts of OxCL with ␤2GP1 or apoB.
(Circulation. 2001;103:941-946.)
Key Words: antibodies, antiphospholipid 䡲 lipoproteins 䡲 autoimmunity
A
fatty acids in CLred are hydrogenated to saturated fatty
acids). We proposed that aCL bind to epitopes generated
when CL undergoes oxidation.8,9
␤2GP1 is a phospholipid-binding apolipoprotein (also
called apolipoprotein H) that seems to be necessary for the
binding of some aCL.2,3 It has been proposed that, as a result
of noncovalent protein-lipid interactions, novel, conformational epitopes are created on the plated CL, on ␤2GP1, or on
an admixture of these two, or that ␤2GP1 alone is the target
antigen.3–5,10,11 We demonstrated that some aPL bind to
proteins like ␤2GP1 only as a consequence of covalent adduct
formation between oxidized phospholipids (OxPL) and the
protein.9 The formation of neoepitopes between OxPL and
associated proteins would be analogous to LDL oxidation,
which generates immunogenic neoepitopes.8,12,13 Autoantibodies to OxLDL are present in the sera of animals and
humans and are increased in those with increased atherosclerosis.14,15 Indeed, aCL in patients with systemic lupus erythematosus cross-react with oxidized LDL (OxLDL).16
ntiphospholipid antibodies (aPL) are heterogeneous autoantibodies detected in vitro by solid-phase immunoassays as antibodies binding to phospholipids such as cardiolipin (CL) or to complexes of phospholipid and ␤2glycoprotein 1 (␤2GP1).1– 6 Patients with high levels of aPL
are prone to fetal loss, autoimmune thrombocytopenia, and
thrombotic events in either the venous or arterial circulation.4 –7 Elevated levels of aPL in combination with one or
more clinical features has been termed the antiphospholipid
antibody syndrome (APS).
The exact nature of the epitope(s) for anticardiolipin
antibodies (aCL) has been controversial.4 – 6 We recently
demonstrated that CL is rapidly oxidized when plated on
microtiter wells and exposed to air—as is done in conventional solid-phase aCL immunoassays.8 We also showed that
a few selected reference sera and affinity-purified aCL-IgG
from APS patients progressively bound to CL as it was
oxidized but not to a “reduced” CL (CLred) analogue that
was unable to undergo lipid peroxidation (all 4 unsaturated
Received July 12, 2000; revision received October 9, 2000; accepted October 16, 2000.
From the Department of Medicine, University of California, San Diego, and the Department of Obstetrics and Gynecology, University of Utah, Health
Sciences Center, Salt Lake City (D.W.B.).
Correspondence to Joseph L. Witztum, MD, or Sohvi Hörkkö, MD, PhD, Dept of Medicine, University of California, San Diego, 9500 Gilman Drive,
La Jolla, CA 92093-0682. E-mail [email protected] or [email protected]
© 2001 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
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Circulation
February 20, 2001
In the present article, we demonstrate that oxidized CL
(OxCL) and covalent adducts of OxCL with ␤2GP1 are
epitopes for many aCL. In addition, we show that “native”
␤2GP1 and OxLDL share common epitopes recognized by
monoclonal antibodies cloned from APS patients.
Methods
Materials
CL (diphosphatidylglycerol, bovine heart) containing 4 unsaturated
fatty acids and hydrogenated CL (CLred) containing 4 saturated fatty
acids were obtained from Avanti Polar Lipids. Fatty acid analysis
confirmed that linoleic acid accounted for ⬎92% of the fatty acids of
CL, whereas all fatty acids in CLred were saturated (18:0).8 CL was
oxidized by air exposure to generate various decomposition products, as previously descibed.8 Human ␤2GP1 was purified as previously described.17
Human Subjects
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Serum samples from 21 women and 1 man with APS and from 7
healthy controls were collected at the Department of Obstetrics and
Gynecology of the University of Utah Hospital. Two patients had
donated a serum sample at 2 different times, 7 and 8 years apart, for
a total of 24 serum samples from the APS patients. APS patients had
ⱖ1 of the following clinical features: (1) a history of either ⱖ1 fetal
deaths or ⱖ3 consecutive pregnancy losses, (2) venous or arterial
thrombosis, or (3) autoimmune thrombocytopenia. Six patients had
systemic lupus erythematosus. All patients had aCL IgG, as measured by a standardized assay18 in Dr Branch’s laboratory (19
samples were ⬎100 IgG phospholipid-binding units and 6 samples
were 30, 40, 44, 47, 62, and 75 IgG phospholipid-binding units,
respectively).
Figure 1. IgG binding of 24 APS serum samples (from 22
patients) and 7 control samples to OxCL and CLred. Samples
were diluted 1:50 with 10% adult bovine serum buffer. Bound
IgG was detected with alkaline phosphatase–labeled goat anti–
human-IgG antibody.
Generation of Monoclonal IgG aCL Antibodies
Human monoclonal IgG antibodies IS3 and IS4 were generated from
a patient with primary APS, as recently described,17,19 by selecting
for their ability to bind to CL. Antibody LJB8 (not previously
described) was cloned from another APS patient in a similar manner,
but it was selected for binding to ␤2GP1. The absence of bovine
␤2GP1 in the monoclonal antibody preparations was verified by
SDS-PAGE gel electrophoresis and silver staining.
Western Blot
Chemiluminescent Immunoassay for
Antibody Binding
CL or CLred in 100% ethanol was added at 25 ␮g/mL into white,
round-bottomed High Binding Microfluor (Dynex) microtitration
plates and exposed to air for the indicated time at room temperature
to induce oxidation. Absolute ethanol was added to blank wells. The
wells were washed with PBS buffer containing 0.27 mmol/L EDTA
and blocked with 10% fetal bovine serum, 1% bovine serum albumin
(BSA), or 0.25% gelatin in indicated experiments. The primary
antibodies were incubated for 1 hour, and the amount of antibody
bound was measured with alkaline phosphatase-labeled goat antihuman IgG (Sigma) using LumiPhos 530 (Lumigen) as the substrate.
Luminescence was measured in relative light units (RLU) with a
Dynex Luminometer (Dynex Technologies).8,9 Each point in each of
the figures is the mean of triplicate determinations.
Preparation of OxCL-␤2GP1
CL was dried and exposed to air for 3 hours. Purified human ␤2GP1
(in PBS and 20 ␮mol/L EDTA) and NaCNBH3 (10 mmol/L) were
added and incubated at 37°C for 6 hours. After incubation, 80
mmol/L octylglucoside was added and dialyzed against PBS to
remove the unbound CL.
Protein A Purification of IgG
Whole IgG fractions were purified using ImmunoPure Plus Immobilized Protein A IgG Purification Kit (Pierce). The absence of
␤2GP1 in the IgG fractions was verified by a capture assay. Samples
were incubated in wells coated with polyclonal goat anti-human
␤2GP1 antibody (10 ␮g/mL; Enzyme Research Laboratories) and by
detecting the amount of ␤2GP1 captured with the biotinylated
anti-human ␤2GP1 antibody and alkaline phosphatase–labeled avidin
(Pierce).
Proteins were electrophoresed on SDS 4% to 12% trisglycine
polyacrylamide gels and electrotransferred to nitrocellulose membranes. Transfer was confirmed with 0.1% Ponceau S (Sigma)
staining, and the membranes were blocked with Super Block (Pierce)
or 0.25% gelatin and immunostained with either the human monoclonal antibodies or goat anti-human ␤2GP1 antibody. Antibody
binding was detected by appropriate alkaline phosphatase–labeled
secondary antibodies and visualized with Alkaline Phosphatase
Conjugate Substrate Kit (Bio-Rad).
Results
Antibody Binding to OxCL and CLred
Figure 1 demonstrates that IgG binding was substantially
higher to OxCL than to CLred for each of the APS serum
samples. Control samples had low binding to both antigens.
When the assay was repeated in the absence of 10% bovine
serum, virtually identical results were observed (data not
shown).
Because the aCL binding has been suggested to depend
exclusively on the presence of ␤2GP1, we tested whether
␤2GP1 binds to both OxCL and CLred to the same extent.
Figure 2A shows an example in which there is increased IgG
binding from one APS serum sample to the OxCL but no
binding to CLred. In the same experiment, we used an
anti-␤2GP1 antibody to demonstrate that substantial amounts
of human ␤2GP1 (from the added serum) bound to both
OxCL and CLred when measured in parallel wells under
identical conditions (Figure 2A). This suggests that the
binding of aCL to phospholipids does not depend exclusively
on the presence of ␤2GP1.
Hörkkö et al
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Figure 2. A, IgG binding from 1 APS serum sample (1:50 dilution in 1% BSA-PBS) to OxCL (●) and CLred (Œ) plated on
microtiter wells. Amount of ␤2GP1 bound to OxCL (䡩) and CLred
(‚) in parallel wells using goat anti-human ␤2GP1 antibody is
also shown. B, Competition immunoassay of IgG binding from 1
APS serum sample. Serum sample (1:50 in 1% BSA-PBS) was
preincubated for 60 minutes with increasing amounts of OxCL
(●) or CLred (Œ), and then immune complexes were pelleted by
centrifugation (13 000 rpm for 30 minutes) and the supernatants
were tested for IgG binding to OxCL. Amount of ␤2GP1 remaining in supernatant after absorption with OxCL (䡩) or CLred (‚) is
also shown. Samples were incubated in microtiter wells coated
with anti-human ␤2GP1 antibody, and amount of ␤2GP1 captured was measured with biotinylated anti-human ␤2GP1 antibody. Results are expressed as percentage remaining after
absorption when compared with a control incubation performed
under same conditions in absence of any phospholipid.
Specificity of aCL IgG Binding
The serum samples were preincubated with OxCL or CLred,
and the supernatants were then tested for binding to OxCL.
Figure 2B shows that the preincubation of 1 APS serum
sample with OxCL but not CLred removed ⬎95% of the
original aCL binding. Using a capture assay (see Methods)
we found that ⬍20% of the total ␤2GP1 content was absorbed
from the 1:50 dilution of serum during the preincubation with
OxCL or CLred (Figure 2B).
Figure 3 demonstrates that preincubation of all APS serum
samples with OxCL removed ⱖ95% of the IgG binding to
OxCL. A control incubation for each sample without phospholipid did not remove any IgG binding to OxCL (data not
shown). To examine whether a population of antibodies
binding to OxCL could also bind to another OxPL epitope,
we tested the IgG binding to copper-oxidized LDL (CuOxLDL). Figure 3 also shows that preincubation with OxCL
absorbed ⬇40% of the binding to CuOx-LDL. Preincubation
with CLred did not remove any IgG binding to either OxCL
or CuOx-LDL. Using similar competition assays, we demonstrated that even the slight degree of binding to CLred
observed with a few of the APS samples (Figure 1) was
nonspecific (data not shown).
Binding of Human Monoclonal
IgG aCL Antibodies
Figure 4 shows that monoclonal antibodies IS4 (selected for
binding to CL) and LJB8 (selected for binding to ␤2GP1) had
high binding to OxCL but not to CLred. Both of these
monoclonals also bound to CuOx-LDL and to another model
epitope, malondialdehyde-modified LDL (MDA-LDL) but
not native LDL. Monoclonal antibody IS3 (selected for
aCL Recognize ␤2GP1 and OxLDL
943
Figure 3. Absorption immunoassay for IgG binding of 24 APS
serum samples. Each serum sample was diluted 1:50 with 2%
BSA–triethanolamine-buffered saline and preincubated with 25
␮g of OxCL or CLred. Immune complexes were pelleted by centrifugation, and supernatants were tested for IgG binding to
OxCL or CuOx-LDL. Results are expressed as percentage
remaining after absorption when compared with control incubations performed under same conditions in absence of
phospholipid.
binding to CL) also showed identical results (data not shown).
To test if these monoclonals specifically recognized lipidprotein adducts, we performed Western blot analyses. Figure
5A shows that IS4 bound to the protein of both MDA-LDL
(lane E) and CuOx-LDL (lane F), but not to native LDL (lane
D) or BSA (lane B). In addition, IS4 showed strong binding
to ␤2GP1 (lane C). Figure 5A also demonstrates the absence
of human ␤2GP1 on native-LDL (lane I) or CuOx-LDL (lane
J). Figure 5B shows that LJB8 bound to ␤2GP1 (lane B),
MDA-LDL (lane D), and CuOx-LDL (lane E) but not to
native-LDL (lane C) or BSA (lane F). IS3 (Figure 5B) also
showed binding to MDA-LDL (lane I) and CuOx-LDL (lane
Figure 4. Binding of human monoclonal antibodies from APS
patients to OxCL (‚), CLred (●), MDA-LDL (ƒ), CuOx-LDL (䡩), or
native LDL (䡲). IS4 was originally selected for binding to CL, and
LJB8 was selected for binding to ␤2GP1. Antigen-coated wells
were blocked with 0.25% gelatin, and monoclonal antibodies (5
␮g/mL) were diluted in 0.25% gelatin. Amount of bound IgG
was detected with alkaline phosphatase–labeled anti-human IgG
antibody.
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February 20, 2001
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Figure 6. Immunoassay of protein A–purified IgG fractions from
sera of 4 APS patients (APS-IgG #1 through #4) and 1 control
subject (human IgG). Wells were coated with OxCL or CLred (25
␮g/mL) or ethanol only. After 4 washes, wells were incubated
with ␤2GP1 (10 ␮g/mL in PBS buffer) or 1% BSA-PBS buffer for
1 hour at room temperature, and amount of IgG or ␤2GP1
bound to wells was measured with appropriate antibodies.
Figure 5. A, Western blots of binding of human monoclonal
antibody (IS4) originally selected for binding to CL (lanes A
through F) and binding of goat anti-human ␤2GP1 antibody
(lanes G through J). Lane A, standard; lane B, BSA; lane C,
␤2GP1; lane D, native LDL; lane E, MDA-LDL; lane F, CuOxLDL; lane G, BSA; lane H, human ␤2GP1; lane I, native LDL; and
lane J, CuOx-LDL. B, Western blots of binding of monoclonal
antibody (LJB8) originally selected for binding to ␤2GP1 (lanes A
through F) and monoclonal antibody (IS3) originally selected for
binding to CL (lanes G through J). Lane A, standard; lane B,
␤2GP1; lane C, native LDL; lane D, MDA-LDL; lane E, CuOxLDL; lane F, BSA; lane G, ␤2GP1; lane H, native LDL; lane I,
MDA-LDL; and lane J, CuOx-LDL.
aCL. To further test this hypothesis, we prepared covalent
adducts between OxCL and ␤2GP1 (OxCL-␤2GP1). Figure 7
demonstrates that although most of the APS serum samples
showed increased binding to the native unmodified ␤2GP1
compared with BSA (mean, 30 776 versus 6445 RLU/100
ms, respectively; P⬍0.001 when using Student’s paired t
test), the binding to OxCL-␤2GP1 (mean, 138 907 RLU/100
ms) increased 4-fold (P⬍0.001). The control samples showed
very little binding to either native ␤2GP1 or OxCL-␤2GP1
(Figure 7). There was a positive correlation between the
measurements of IgG binding to OxCL and to OxCL-␤2GP1
among the APS samples (r⫽0.84, P⬍0.0001, linear regression analysis). The protein A–purified IgG fractions IS4 and
LJB8 also had increased binding to OxCL-␤2GP1 compared
with the native ␤2GP1 (data not shown).
J) but not to native LDL (lane H) or to ␤2GP1 (lane G). These
data clearly demonstrate that IS4 and LJB8 recognize similar
oxidatively-modified protein moieties of CuOx-LDL, MDALDL, and ␤2GP1 but that IS3 seems to recognize a different
epitope.
Antibody Binding to Oxidatively Modified
Human ␤2GP1
To test whether OxCL on microtiter wells could modify
␤2GP1 and create epitopes for aCL antibodies, we first dried
OxCL, CLred, or solvent only (ethanol) in microtiter wells
before adding ␤2GP1. Figure 6 demonstrates that protein
A–purified IgG fractions isolated from different APS patients
bound to the ␤2GP1 added to OxCL-coated wells but not to
the ␤2GP1 added to CLred- or ethanol-coated wells. Note that
equal amounts of ␤2GP1 were present in the wells under
different conditions. Under these conditions, these purified
IgGs did not bind to OxCL alone (eg, BSA wells).
The data in Figure 6 strongly suggest that OxCL can
modify ␤2GP1 in such a way that it forms epitopes for some
Figure 7. Binding of IgG from 24 APS serum samples and 7
control samples to BSA, ␤2GP1, or ␤2GP1 covalently modified
with CL oxidation products (OxCL-␤2GP1). Serum samples were
diluted 1:50 with 2% BSA–triethanolamine-buffered saline, and
bound IgG was detected with alkaline phosphatase–labeled
anti-human IgG antibody.
Hörkkö et al
Discussion
On the basis of previous observations, we proposed that
some aCL bind to neoepitopes of OxPL or to neoepitopes
generated by adduct formation between reactive breakdown
products of OxPL and associated proteins. We now demonstrate that most aCL in the tested APS sera require CL
oxidation for the CL to be an antigen. Furthermore, our data
using purified IgG fractions indicate that CL oxidation is
required even in the presence of ␤2GP1 (ie, ␤2GP1 alone or
bound to CLred does not yield epitopes). Two monoclonal
antibodies from two APS patients (IS4 and LBJ8) bound to
both ␤2GP1 and to the apoB of OxLDL but not to native
apoB, implying similar oxidized lipid-protein epitopes on
these different proteins. Antibody IS3 bound to both OxCL
and oxidized apoB but not to ␤2GP1, implying variations in
oxidized lipid-protein epitopes. We postulate that a large
number of different lipid-protein and even lipid-lipid adducts
could form when phospholipids undergo oxidation. This
would be analogous to the adduct formation between oxidized lipids and apoB that occurs during LDL oxidation.20,21
Indeed, the recent observation that CL is found in the
circulation22 suggests that OxCL-apoB adducts form on
OxLDL.
␤2GP1 has been reported to be the primary serum “cofactor” or target antigen for many aCL.2–5,10,11 We and others
have reported that other proteins, such as polylysine, LDL,
and apoAI, also have cofactor activity.9,23 ␤2GP1 seems to be
an excellent cofactor because of its high avidity to phospholipids and its ability to form adducts with OxCL (and possibly
other phospholipids9). To test this idea, we demonstrated that
the binding of the purified IgG fractions to ␤2GP1 occurred
only when the ␤2GP1 was plated with OxCL (Figure 6). Also,
we demonstrated that APS sera, purified IgG fractions, and
human monoclonals all showed strong binding to the OxCL␤2GP1 adduct. Furthermore, among all APS sera, the binding
to OxCL-␤2GP1 correlated well with the binding to OxCL.
These data suggest that not only does ␤2GP1 readily form
adducts with OxPL in the microtiter wells, but it may already
contain some oxidized lipid-protein epitopes. In support of
this, we demonstrated that monoclonal antibodies cloned
from APS patients recognized epitopes not only on native
␤2GP1 but also on oxidatively modified apoB on Western
blots. These data strongly indicate that these epitopes are
covalently oxidized lipid-protein adducts on ␤2GP1 and
apoB. In the case of ␤2GP1, this may occur in plasma in vivo
or during the isolation procedure (␤2GP1 is often isolated
with a method involving perchloric acid precipitation that
generates a strong pro-oxidant condition24).
There has been considerable difficulty in generating reliable and reproducible clinical assays for measuring aCL,25,26
which we believe is partly because of the oxidation of CL. In
fact, there is variability not only between different preparations but even in the same CL preparation depending on its
“age” (even if stored at ⫺70°C under argon). Because the rate
of CL oxidation is extremely difficult to control or standardize, an alternative approach might be to use an adduct
between OxCL and ␤2GP1 (or other protein) as an antigen.
If many aPL are, in fact, directed against oxidationdependent epitopes and because many OxPL products can
8,9
aCL Recognize ␤2GP1 and OxLDL
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form, it is likely that some aPL are against unique oxidationspecific structures whereas other aPL are against more
common oxidation-dependent structures. These data imply
heterogeneity even among aPL to OxCL epitopes. Moreover,
our data do not address the observations that there are many
oxidation-independent antibodies that bind exclusively to
conformational changes or even primary sequences of ␤2GP1,
independent of any bound lipid or lipid- ␤ 2 GP1 adducts.3,4,10,11,27,28 Furthermore, there may also be antibodies
against conformational changes in either CL,2,29 ␤2GP1,30 or
prothrombin.31
There is controversy over which type of antibodies are best
associated with various aspects of clinical disease.4 – 6 Knowledge that many aPL can be oxidation-dependent may give
insight into some of the pathogenic events underlying the
clinical manifestations of APS. These data suggest that
inflammatory conditions and an attendant pro-oxidant state
are associated with the generation of epitopes to many aPL.
Recently, Iuliano and colleagues32 tested this hypothesis and
reported a strong correlation between aCL and lipid peroxidation (isoprostane excretion) in patients with systemic lupus
erythematosus; treatment with vitamin E led to a reduction in
isoprostane excretion.33 If antiphospholipid antibodies are
indeed pathogenic or even simply a marker of enhanced lipid
peroxidation, then therapies aimed at the underlying inflammation and, in particular, at ameliorating the pro-oxidant state
may be beneficial.
Acknowledgments
These studies were supported by NIH grants HL57505 (to J.L.W.),
HL56989 (La Jolla SCOR), and AR42506 (to P.P.C.) and by a
Postdoctoral Research Fellowship from the American Heart Association, California Affiliate (to S.H.).
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Anticardiolipin Antibodies From Patients With the Antiphospholipid Antibody Syndrome
Recognize Epitopes in Both β2-Glycoprotein 1 and Oxidized Low-Density Lipoprotein
Sohvi Hörkkö, Tsaiwei Olee, Lian Mo, D. Ware Branch, Virgil L. Woods, Jr, Wulf Palinski,
Pojen P. Chen and Joseph L. Witztum
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Circulation. 2001;103:941-946
doi: 10.1161/01.CIR.103.7.941
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