Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 3195–3199, April 1997
Effects of a polymorphism in the human tumor necrosis factor ?
promoter on transcriptional activation
(genetics?major histocompatibility complex?cytokine?gene regulation?autoimmune diseases)
ANTHONY G. WILSON†, JULIAN A. SYMONS†, TARRA L. MCDOWELL†, HUGH O. MCDEVITT‡,
AND GORDON W. DUFF†§
†Section of Molecular Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, United Kingdom; and‡Departments of Microbiology and
Immunology, and Medicine, Stanford University, Stanford, CA 94305
Contributed by Hugh O. McDevitt, December 23, 1996
immunomodulator and proinflammatory cytokine that has
been implicated in the pathogenesis of autoimmune and
infectious diseases. For example, plasma levels of TNF? are
positively correlated with severity and mortality in malaria
and leishmaniasis. We have previously described a polymor-
phism at ?308 in the TNF? promoter and shown that the rare
allele, TNF2, lies on the extended haplotype HLA-A1-B8-DR3-
DQ2, which is associated with autoimmunity and high TNF?
production. Homozygosity for TNF2 carries a sevenfold in-
creased risk of death from cerebral malaria. Here we dem-
onstrate, with reporter genes under the control of the two
allelic TNF promoters, that TNF2 is a much stronger tran-
scriptional activator than the common allele (TNF1) in a
human B cell line. Footprint analysis using DNase I and B cell
nuclear extract showed the generation of a hypersensitive site
at ?308 and an adjacent area of protection. There was no
difference in affinity of the DNA-binding protein(s) between
the two alleles. These results show that this polymorphism has
direct effects on TNF?gene regulation and may be responsible
for the association of TNF2 with high TNF? phenotype and
more severe disease in infections such as malaria and leish-
Tumor necrosis factor ? (TNF?) is a potent
Tumor necrosis factor ? (TNF?) is a potent cytokine with a
wide range of proinflammatory activities (1). It is classically
produced by monocytes?macrophages, although other cell
types, such as T and B cells, also produce significant amounts.
The TNFA gene lies in the class III region of the major
histocompatibility complex (MHC), ?250 kilobases centro-
meric of the HLA-B locus and 850 kilobases telomeric of
HLA-DR. In view of its biological effects and gene location it
has been speculated that polymorphism within this locus might
contribute to MHC associations with autoimmune and infec-
tious diseases (2), particularly those in which TNF? has been
implicated in initiating or sustaining the inflammatory re-
sponse, such as rheumatoid arthritis (3), or where increasing
blood levels have been shown to be predictive of poorer
outcome, such as malaria (4). This has been supported by the
association of specific MHC haplotypes with different TNF?
phenotypes: DR3 and DR4 haplotypes produce higher levels
of TNF? (5, 6) while DR2 haplotypes are associated with low
production (5, 7), suggesting that functional polymorphism
might exist within regions that regulate the TNFA gene.
Studies in mice have implicated the TNF locus with disease
phenotype. Polymorphisms have been described in the pro-
of TNFA in different strains of mice. Further, variation in the
production of TNF? from macrophages has been shown to
vary between strains (10). Several of these polymorphisms
have been correlated with TNF? production and with suscep-
tibility to, or severity of, several diseases. The (NZB ?
NZW)F1mouse develops a severe autoimmune disease that is
very similar to systemic lupus erythematosus (SLE) in humans.
is correlated with low production of TNF? and with the
development of lupus nephritis (11), and replacement therapy
with recombinant TNF? delays the onset of nephritis with
increased survival rate (12). Correlation of TNF? polymor-
phism with production of TNF? mRNA and resistance to
development of murine Toxoplasma gondii encephalitis has
also been demonstrated in the BALB?c strain (13). A study of
Th2 cell-mediated local inflammatory responses has shown
that the TNF dependence of this phenomenon is related to
H2D haplotypes and corresponding TNF? production pheno-
types, suggesting, at least in mice, that differential expression
of TNF? from distinct alleles may influence the nature of an
immune response (14).
TNF has potent biological actions, and control of its pro-
duction is tightly regulated, occurring both at the transcrip-
tional and posttranscriptional levels (15). In response to lipo-
polysaccharide stimulation of macrophages, TNF transcription
increases 3-fold, TNF mRNA increases 50- to 100-fold, and
protein secretion increases by a factor of ?10,000-fold (16).
Sequences within the 1100-bp stretch of DNA between the 3?
end of the lymphotoxin alpha gene and the 5? end of TNFA
have been shown to be central to the control of transcription
Recently we and others have described two polymorphisms
in the human TNFA promoter at ?308 (19) and ?238 (20),
both involving the substitution of guanine by adenosine in the
uncommon alleles. We showed that the rare allele at ?308
(TNF2) is part of an extended MHC haplotype HLA-A1-B8-
DR3-DQ2 (21), which is associated with high TNF? produc-
tion (5, 6). Studies in large populations have indicated that
carriage of TNF2 is associated with a worse outcome in
cerebral malaria (22) and in leishmaniasis (23).
To test whether the ?308 polymorphism has a functional
significance, we have investigated its effects on transcription
using reporter gene assays. Our results show that the TNF2
allele is a much more powerful transcriptional activator than
the common allele. Although we can demonstrate specific
binding of a nuclear protein and DNase I hypersensitivity at
the polymorphic site, there was no obvious difference in
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Copyright ? 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA
PNAS is available online at http:??www.pnas.org.
Abbreviations: TNF?, tumor necrosis factor?; TNFA, gene for TNF?;
MHC, major histocompatibility complex; SLE, systemic lupus ery-
thematosus; CAT, chloramphenicol acetyltransferase; EMSA, elec-
trophoretic mobility-shift assay; IL, interleukin; PMA, phorbol 12-
§To whom reprint requests should be addressed.
affinity of the protein(s) for the two alleles. These results
indicate that this polymorphism may have a direct effect on
transcriptional activity and may underlie the association of the
HLA-A1-B8-DR3 haplotype with high TNF? production and
be directly responsible for the poorer outcome reported in
malaria and leishmaniasis with carriage of the TNF2 allele.
MATERIALS AND METHODS
Generation and Cloning of TNF? Promoter Fragments. A
fragment of 691 bp (?585 to ?106) of the TNFA gene was
amplified by PCR using primers 5?-GCTTGTCCCTGCTAC-
CCGC-3? and 5?-GTCAGGGGATGTGGCGTCT-3?, and cy-
cles as described (19). The fragments were cloned into the TA
vector and used to transform Escherichia coli (strain INVF??)
(Invitrogen, United States Biochemical). Following selection
and propagation, pure plasmid DNA was prepared by standard
methods (24). The TNF promoter alleles were removed from
the TA vector by restriction enzyme digestion with HindIII and
XbaI (Promega) to allow for directional cloning into the
pBLCAT3 expression vector (25). The sequences of the inserts
were checked by the dideoxy chain termination method using
Sequenase (United States Biochemical).
Transfection of Human B Cells. Experiments were per-
formed using the human Raji B cell line. Cells were cultured
in 1? RPMI 1640 medium adjusted to pH 7.4 with 1 M NaOH,
buffered with 7.5% (vol?vol) sodium bicarbonate, and supple-
mented with 5% (vol?vol) fetal calf serum (GIBCO?BRL),
penicillin (100 units per ml), streptomycin (100 ?g?ml), and
glutamine (2 mM) (Northumbria Biologicals, Northumber-
land, England). Cultures were incubated at 37?C in a humid-
ified 5% CO2?95% air atmosphere. Raji cells, which were
maintained in rapid growth phase by change of medium and
passaged 1:10 every 3 days, were centrifuged at 400 ? g for 5
min. The cells were washed once with medium, centrifuged,
and then resuspended at a concentration of 1 ? 107cells per
ml of medium; 800 ?l of this was used for each transfection. In
each experiment three different plasmids were transfected: (i)
pBLCAT3, (ii) TNF1-pBLCAT3, and (iii) TNF2-pBLCAT3.
Each time 80 ?g of DNA was used. Electroporation was
performed with a single pulse from a gene pulser apparatus
(Bio-Rad) with a capacitance extender unit at settings of 300
V and 960 ?Fd. Cells were incubated at room temperature for
10 min before and after electroporation. The cell suspension
was added to 19.2 ml of medium and incubated for 24 hr. Each
cell culture was then split in half and again resuspended in 20
ml of medium. Phorbol 12-myristate 13-acetate (PMA) (Sig-
ma) was added to one culture from each duplicate to a final
concentration of 50 ng?ml and the cultures incubated for a
further 48 hr. The cells were then harvested by centrifugation,
and the pellets were resuspended in 0.25 M Tris?HCl (pH 8.0)
and stored at ?70?C. The cell suspensions were subjected to
three episodes of rapid freezing?thawing to obtain lysates.
Quantification and Normalization of Chloramphenicol
Acetyltransferase (CAT) Expression. Measurement of CAT
protein production in transfected cells was performed using a
commercially available enzyme-linked immunosorbant assay
(Boehringer Mannheim). The lower detection limit was 100
pg?ml CAT. Quantification of protein concentrations in cel-
lular extracts was determined using a modification of the
micro-Lowry technique (protein assay kit; Sigma).
To determine transfection efficiencies, a dot-blotting pro-
cedure was followed (26) using a ?-counter (Tri-Carb 260-DU,
Packard). The probe used was isolated from pBLCAT3 by
digesting the plasmid with EcoRI. The fragment, ?1500 bp in
length, spanning the CAT cDNA, was randomly labeled with
[?-32P]dCTP [3000 Ci?mmol (1 Ci ? 37 GBq); Amersham]
using a T7 Quickprime kit according to the manufacturer’s
Extraction of DNA-Binding Proteins from Raji Cells. Nu-
clear proteins were extracted from Raji cells as described (27).
Prior to use, the protein concentration was determined.
Generation of Radiolabeled TNFA Promoter Fragment. A
119-bp fragment (?345 to ?226) of the TNF? promoter was
amplified by PCR from TNF1 and TNF2 homozygous indi-
viduals using primers 5?-CAAAAGAAATGGAGGCAAT-3?
and 5?-TCCTCCCTGCTCCGATTCCG-3?. The two allelic
fragments were then cloned into the TA vector, and the
sequences were confirmed as above. Probe was prepared by
restriction digestion using NsiI and HindIII, [?-32P]dCTP
end-labeled using the Klenow fragment (Promega), and then
purified with a Sephadex G-50 NICK column (Pharmacia).
The specific activity of the probe was measured with a
Electrophoretic Mobility Shift Assay (EMSA). Nuclear pro-
tein extract (10 ?g) was incubated with 2 ?g of poly(dI-dC)
(Pharmacia) in 25 ?l of buffer composed of 10 mM Tris?HCl
(pH 7.5), 75 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA,
12.5% glycerol, and 0.1% (vol?vol) Triton X-100 at room
temperature for 30 min. After the addition of 2.5 ng of labeled
probe and incubation for a further 30 min, 2.5 ?l of loading
buffer consisting of 250 mM Tris?HCl (pH 7.5), 0.2% bromo-
phenol blue, 0.2% xylene cyanol, and 40% glycerol was added
Tris?64.6 mM boric acid?2.5 mM EDTA, pH 8.3)?4% non-
denaturing polyacrylamide gel. Visualization of bands was by
autoradiography. Quantification of competitor DNA concen-
trations was by measurement of optical density at 260 nm. This
was checked by comparing band intensities following agarose
gel electrophoresis and ethidium bromide staining.
DNase I Footprint. Initially 80 ng of Raji nuclear extract was
incubated at 37?C for 20 min in 50 ?l of buffer containing 25
mM Hepes (pH 7.8), 50 mM KCl, 0.05 mM EDTA, 0.5 mM
DTT, 0.5 mM phenylmethylsulfonyl fluoride, 5% glycerol, and
100 ng poly(dI-dC). Labeled probe (40,000 cpm) was then
added and incubated for a further 20 min. Digestion was
performed at 0?C with 0.01 units of DNase I (Boehringer
Mannheim) for 1 min following the addition of 1 mM CaCl2
?l stop solution consisting of 0.375% (wt?vol) SDS, 15 mM
EDTA, 100 mM NaCl, and 100 mM Tris?HCl (pH 7.6). The
products were then incubated at 37?C for 15 min with 0.18 mg
proteinase K and 10 ?g of tRNA in a final volume of 163 ?l,
and phenol?chloroform extracted and ethanol precipitated. At
the same time a Maxam–Gilbert guanidine ladder was gener-
ated as described (28). The fragments were separated on a 6%
denaturing polyacrylamide gel. The gel was then dried and
visualized by autoradiography.
Induction of CAT Protein by TNFA Promoter Fragments.
Raji cells were transfected with three plasmids: a negative
control consisting of the pBLCAT3 vector alone and plasmids
containing either of the two TNF allelic promoter fragments.
The experiments were performed four times using DNA from
different plasmid preparations. Efficiency of transfection was
assessed by Southern blot analysis of the CAT gene and
quantified by ?-counting. Total protein was also measured in
cellular lysates. The CAT protein concentration was corrected
using these results to minimize differences due to transfection
efficiency and cell numbers and was expressed in picograms of
CAT protein per milligram of total protein. The results of this
experiment are shown in Fig. 1. As expected, the negative
control produced very low levels of CAT protein in both the
unstimulated and PMA-stimulated cells. There was a signifi-
cantly higher production of CAT from the TNF2-CAT plasmid
compared with TNF1-CAT in both the unstimulated and
stimulated cells. The TNF1-CAT plasmid showed no evidence
3196 Immunology: Wilson et al.Proc. Natl. Acad. Sci. USA 94 (1997)
of inducible production of CAT. Although there appeared to
be some inducibility of the TNF2-CAT plasmid by PMA, this
was not significant. This result was replicated four times with
different batches of Raji cells.
EMSA of TNF? Fragments. To investigate protein–DNA
interactions in the vicinity of the polymorphism we performed
EMSAs using extract from Raji cells and a 119-bp TNF
fragment. The results of this experiment are shown in Fig. 2.
Binding of at least one protein is demonstrated with both
allelic fragments (lanes 2 and 7). The specific nature of binding
is demonstrated by the disappearance of both DNA–protein
complexes using competition with a 100-fold excess of unla-
beled TNF1 probe (lanes 3 and 8) or TNF2 probe (lanes 5 and
10), but not when a similar-sized fragment of the interleukin
1A (IL-1A) promoter was used (lanes 4 and 9).
DNase I Footprint of TNF? Promoter Region Fragment.
The exact site of DNA–protein interaction around the poly-
morphic site was defined in a footprint assay. A hypersensitive
site was induced at ?308 with an adjacent protected region
(Fig. 3). No other evidence of interaction was seen.
Competitive EMSA. To examine whether the difference in
transcriptional activity was due to a difference in affinity of the
two alleles for the binding protein, a competition EMSA was
performed using labeled TNF1 and increasing excesses of cold
oligonucleotides. There was no evidence of a major difference
in affinity between the two alleles (Fig. 4).
Several features make it difficult to determine whether a
particular DNA variant within the MHC is directly responsible
for a disease association. This is the most polymorphic region
of the genome and contains many genes that encode proteins
that are involved in inflammatory and immune responses.
Another important feature is the strong linkage disequilibrium
between alleles across the MHC. Thus, for example, the
haplotype HLA-A1-B8-DR3-DQ2 occurs much more fre-
quently than the product of the individual allelic frequencies
would suggest. Therefore, the association of MHC haplotypes
with TNF? phenotypes might not be due to polymorphism
within the TNF gene itself, but rather to variation in a linked
gene that regulates expression of this cytokine. A possible
example is the recent cloning of an IkB-like gene within 90
kilobases of the TNF locus (29). There are at least three NFkB
consensus sequences within the TNF? promoter and these
have each been demonstrated specifically to bind a B cell
nuclear protein (17). Therefore, it is important to show a direct
functional effect of any polymorphism, because the association
with a disease may be entirely due to linkage disequilibrium
with the true etiological gene.
Our results demonstrate that the polymorphism at ?308 has
a significant effect on transcriptional activity in reporter gene
assays and that this could explain the association between the
high TNF? phenotype and the DR3 haplotype. The molecular
mechanism of this difference is not completely clear because
there was no evidence of a major difference in affinity of the
DNA-binding protein(s) to the two allelic forms of the TNFA
promoter, at least in Raji cells. This should have shown up in
the competition experiment if based on protein–DNA inter-
actions. Perhaps as a result of difference in the DNA?
chromatin structure at the polymorphic site, the interaction of
transcription factors is enhanced leading to stronger transac-
tivation of the TNF? gene. Our results demonstrating a lack of
B cells are in keeping with a previous study which demon-
strated that the minimum promoter fragment required for
PMA responsiveness in the 729–6 B cell line extended to
?1105 bp with a high basal activity and poor inducibility
compared with a T cell and monocytic cell line (30), and with
the finding of high constitutive levels of TNF? mRNA in Raji
cells (18). Although the polymorphic site lies in a consensus
sequence for AP2 we found no evidence, using recombinant
human protein in a gel retardation assay, of AP2 binding to the
polymorphic site (data not shown). Interestingly, a homolo-
transfected into Raji cells. Raji cells (0.8 ? 107) were transfected with
the pBLCAT3 vector containing either no insert, which served as a
negative control, or 691 bp of each allelic promoter. After 24 hr the
cells were split in two, and one flask of each duplicate was stimulated
with PMA (50 ng?ml). After a further 48 hr incubation the cells were
harvested. Results have been corrected for transfection efficiency by
Southern blot analysis of CAT DNA and also for total cell numbers by
measuring total protein. The experiments were performed four times
and means and standard errors are shown.
Induction of CAT protein from reporter gene constructs
nuclear protein. Nuclear extract (10 ?g) from Raji cells was incubated
with each of the two TNF promoter fragments with (lanes 3–5 and
8–10) or without (lanes 1, 2, 6, 7) 100-fold molar excess of unlabeled
probe. The gel retardation complex is indicated by “Protein?DNA
complex”. Lanes 4 and 9, competition with unlabeled IL-1 DNA
fragment did not disrupt the complex.
Specific binding of the TNF promoter fragment by a
Immunology: Wilson et al. Proc. Natl. Acad. Sci. USA 94 (1997)3197
gous sequence in the TNFA promoter (?254 to ?230) has
been shown to bind a transcriptional repressor and not AP2
(31). It may be, therefore, that a novel protein binds to the
polymorphic TNFA ?308 site. A number of other groups have
studied the effects of this polymorphism on gene expression.
Results similar to ours have been found in one study in both
Jurkat cells or U937 cells with evidence of binding of a protein
only to the TNF2 allele (32). Two other groups have been
unable to demonstrate a difference in transcriptional activity
between the promoter alleles. This may be because of differ-
ences in cell types, stimulants, and reporter gene constructs
used (33, 34). For example, we found that TNFA fragments
shorter than those described here were inactive in reporter
gene assays (data not shown). However, a recent study of
TNF? production from peripheral blood mononuclear cells
stimulated with anti-CD3 and anti-CD28 has shown a higher
TNF? production phenotype in TNF2 carriers, and of two
TNF haplotypes, differing only at ?308, the TNF2 ?ve
haplotype produced significantly more TNF? (35). There is,
therefore, evidence that the TNF2 genotype is associated with
increased TNF? productions in vitro.
An interesting observation is the low incidence of SLE in
areas of West Africa in which malaria is endemic (36), in
contrast to the high incidence in Afro-American populations
who are mostly of West African descent (37). Furthermore
infection of the (NZB ? NZW)F1mouse with Plasmodium
Berghei leads to protection from the spontaneous lupus-like
disease (38). In humans with malaria, the highest levels of TNF
are seen in fatal cases of cerebral malaria (4). A study of TNF
genotypes in West African malaria patients has shown that
homozygosity for TNF2 is associated with a 7-fold increased
risk of death or severe neurological complications due to
cerebral malaria (22). The TNF2 allele may therefore be
responsible for the lower incidence of SLE in Africa as a
consequence of endemic malaria causing higher levels of TNF
production, while the absence of this stimulant of TNF pro-
duction in the United States allows for the increased incidence
of lupus seen in Afro-Americans. Despite the adverse effects
of homozygosity in malaria, TNF2 is maintained at similar
levels in West African and Northern European populations,
suggesting that compensatory pressures in Africa exist to
maintain the allele. Perhaps it has beneficial effects in other
major infectious diseases, such as measles, meningococcal
disease, leprosy, or tuberculosis. There may also be heterozy-
The associations of HLA-DR4 and -DR2 with high and low
TNF? production, respectively, has not been explained. It
seems reasonable to speculate that polymorphism, which may
exist in other regulatory regions of the TNFA gene or in linked
genes, plays a role in TNF? production. The most important
region in the regulation of gene expression seems to be the 3?
untranslated region, which contains a tandem repeat of an
octameric sequence, TTATTTAT (39). The corresponding
UA-rich sequence in mRNA binds an inducible cytoplasmic
factor (40), resulting in mRNA instability and translational
blockade (41). It may be that DNA variants in this region are
in linkage disequilibrium with HLA-DR4 or -DR2. Microsat-
ellite alleles in the TNF locus can be used to subdivide DR4
haplotypes into high and low TNF phenotypes, and this has
suggested that functionally important DNA variants do indeed
exist within, or close to, this locus. TNF? is believed to be one
of the key mediators of the chronic inflammatory response
seen in rheumatoid arthritis (42). Severe forms of this disease
are associated with homozygosity of the DRB1*0401 allele
(43). It will therefore be interesting to see if this subtype of
DR4 is associated with a high TNF? phenotype.
A study of the ?238 TNFA promoter polymorphism has
demonstrated strong linkage disequilibrium of the rare allele
with two extended MHC haplotypes: B18-DR3 and B57-DR7.
However, no genotype–phenotype association could be seen,
and the high TNF? production of the B18-DR3 haplotype
could not be further differentiated by typing this variant (35),
suggesting that it does not have a direct effect on gene
expression. Polymorphisms may of course also exist in the
coding region of the gene, as has been found in the nearby
lymphotoxin alpha gene (44). With regard to ?308 TNFA2,
I footprint analysis of TNF1 allele (coding strand) is shown. The
unfilled arrowhead indicates an area of protection, and the filled
arrowhead a DNase I hypersensitive site. Lanes: 1, Maxam–Gilbert
guanidine ladder; 2, naked DNA control; 3, plus Raji cell nuclear
Detection of a hypersensitive site at ?308. In vitro DNase
and Raji nuclear extracts. Labeled TNF2 was used. Lanes: 1 and 7, no
competitor; lanes 2–6 and 7–12, increasing excess of unlabeled TNF2
and TNF1, respectively, as competitor. No difference in affinity for
nuclear proteins between TNF alleles was apparent.
Competitive EMSA using allelic TNF promoter fragments
3198 Immunology: Wilson et al. Proc. Natl. Acad. Sci. USA 94 (1997)
however,thereisevidencethatitisoverrepresentedindiseases Download full-text
where TNF? levels are associated with poor prognosis. TNF?
production from this allele has been reported to be higher in
vitro, and now we have shown an increased transcriptional rate
from the TNFA2 promoter in reporter gene assays. These
observations begin to make a strong case that the ?308
variation is of biological significance.
Polymorphisms within the human IL-1 gene cluster on
chromosome 2 have been characterized, and associations with
a number of autoimmune diseases have been described (45–
47). An allele of a TaqI restriction fragment-length polymor-
phism in the IL-1? gene is associated with a high IL-1?
production phenotype and psoriasis (F.S. di Giovine, personal
communication). Different cytokine genotypes may exist in
the population as a result of the selective pressure of infectious
diseases. It may be that specific cytokine genotypes are ben-
eficial in the eradication of infectious diseases, but by creating
a ‘‘proinflammatory’’ phenotype, they predispose to chronic
inflammatory diseases or to a more severe form of inflamma-
tory disease with a worse clinical outcome, irrespective of
whether the initial triggering event is an infectious agent,
autoimmunity, or, indeed, any cause sufficient to stimulate an
inflammatory response. This possibility is currently being
tested in a wide range of human diseases.
We thank Gerald Crabtree, Francesco di Giovine, James Lorens,
and Martin Nicklin for advice and discussion. This work was supported
by an Arthritis and Rheumatism Council Copeman Travelling Fel-
lowship (A.G.W.), a program grant from the Arthritis and Rheuma-
tism Council (G.W.D.), a grant from Rho ˆne-Poulenc Rorer, and a
grant from the National Institute of Diabetes, Digestive, and Kidney
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