1Wellcome Trust Research Laboratory, Department of Gastrointestinal Sciences, Christian Medical College and Hospital, Vellore 632004, India
Submitted 26 July 2002 ; accepted in final form 7 March 2003
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ABSTRACT |
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cytoprotection; barrier function
Heat shock response is a universal response to cell injury, which has been
also documented in inflammatory bowel diseases and other human colonic
diseases, suggesting a close link between heat shock protein (HSP)70
expression and disease etiology
(23,
37). The heat shock response
is mediated by a family of chaperone proteins of which HSP70 is an important
member. The heat shock cognate 70 is constitutively expressed in many cells,
whereas inducible forms of HSP70 and HSP72 are synthesized in response to
injury (5,
15). Butyrate has recently
been reported to increase cellular levels of HSP25 but not HSP72 or HSC73 in
rat IEC-18 cells in culture, and this was associated with partial protection
against oxidant-induced injury
(32). Although the induction
of HSPs is generally considered to be protective
(29,
46), it is now realized that
there is a complex interaction between this response and activation of
cytokines in the inflammatory process. In fact, recent articles by Cobb and
colleagues (12) and Malhotra
and Wong (25) discuss this
heat shock paradox, where induction of heat shock in cells primed by
inflammation can have damaging effects. These studies indicate that an
integrating feature of the heat shock pathway and inflammation is the
NF-B pathway. The transcription factor NF-
B plays a central role
in regulating the expression of a number of cytokines involved in immune and
inflammatory responses (40).
In both CD and UC, an increased mucosal synthesis of proinflammatory cytokines
such as IL-1
, -16, -8, and -6, and TNF-
characterize the
excessive local immune response
(27). Activation of
NF-
B has been demonstrated in mucosa of patients with UC
(16), and butyrate has been
shown to inhibit NF-
B activation in lamina propria macrophages of
patients with UC (24).
Epithelial cell expression of HSP70 is enhanced in active UC
(23). Heat shock response can
induce activation of proinflammatory cytokines such as IL-6, and evidence from
animal and human studies suggests that excessive production of IL-6 is
involved in the pathogenesis of inflammatory bowel disease
(20). The promoter regions of
human IL-6 have also been shown to contain consensus-binding motifs for
NF-B (20). These
observations led us to the hypothesis that the mechanism of protection by
butyrate could be through modulation of HSP and NF-
B. This was tested
in dextran sulfate sodium (DSS) colitis in rats, which is a recognized animal
model resembling UC in many respects
(11), including decreased
colonocyte energy production
(1) and impaired barrier
function (42).
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MATERIALS AND METHODS |
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Adult Wistar albino rats, weighing 200 g, were administered DSS
(
40,000 molecular mass, 19% sulfation, a kind gift from Professor S. N.
S. Murthy, Allegheny University, Philadelphia, PA). DSS was mixed with rat
chow in 4% concentration and fed to the experimental group, whereas control
rats were pair-fed with chow not containing DSS. We
(43) have earlier shown that
epithelial cell viability and barrier function in rat colon were affected
after 1 day of DSS administration, whereas ulceration developed after 5 days
and fibrosis after 7 days of DSS administration. In initial studies, rats were
fed DSS for 5 days and killed on the 6th day, and colonocytes were isolated
for examination of the HSP response. In the studies reported here, animals
were administered DSS only for 3 days because we were particularly interested
in the early epithelial changes before establishment of ulceration and
fibrosis (43). Early clinical
changes in DSS-fed rats also included the presence of occult blood in the
stool with no apparent weight loss or decreased appetite. Because butyrate is
normally present in the rat colon from bacterial fermentation of fiber, and
because we intended to test the effects of butyrate enemas in DSS colitis, the
chow fed to rats was modified to minimize fermentable fiber in the colon and
to reduce colonic butyrate concentrations. A purified fiber-free diet based on
the American Institute of Nutrition (AIN)-93M diet
(31) was fed for 3 days after
which 4% DSS was added to the same diet, whereas controls received the AIN-93M
diet without DSS. Animals received single, daily, rectal enemas of 3 ml Ringer
solution containing either 140 mM NaCl, pH 7.4 (control), or 115 mM NaCl with
25 mM butyrate, pH 7.4 (test group) for 3 days, and were killed 1 day after
the last enema. Colonocytes were isolated for viability assay and assessment
of HSP70 production. Sections of colon were fixed in 10% buffered formalin for
immunohistochemistry and for histological quantification of neutrophil
infiltration. Pieces of distal colon were also obtained for measurement of
barrier function in vitro. The institutional animal ethics committee of the
Christian Medical College approved the protocol.
Colonocyte Isolation and Cell Viability
Animals were killed by ether overdose, and the colon was flushed with saline and removed. Colonocytes were isolated by using oxygenated Ca2+-free Krebs-Henseleit (K-H) solution containing 0.01 M ethylene diamine tetra acetic acid (1). Purity of colonocytes by this method was found to be >80%. Cell viability was measured by quantitating release of lactate dehydrogenase into the medium (18).
Mucosal Barrier Function
The barrier function of the distal colonic mucosa was assessed in short-circuited Ussing chambers by measuring electrical resistance and permeability to 14C-mannitol as previously described (42). 14C was measured in the withdrawn samples by liquid scintillation spectrometry by using a Rack Beta counter (LKB Wallac). Fluxes of mannitol were calculated by using standard formulas (42).
Assessment of Inflammatory Cell Infiltration
Paraffin-embedded sections (4-µm thick) of distal colon were stained with hematoxylin and eosin and examined under the x40 high-power objective and the number of polymorphonuclear neutrophil (PMN) in 10 high-power fields was counted and expressed as PMN/10 high-power fields as described elsewhere (40). Nonoverlapping fields were used for counting.
Immunohistochemistry
Sections of colon were fixed in formalin and embedded in paraffin. Sections (5 µm) were cut from these blocks and floated onto poly-L-lysine-coated slides, dewaxed with xylene, and brought to water. The sections were covered with diluted (1:15,000) mouse anti-HSP70 monoclonal antibody (SPA-810; Stressgen, Victoria, BC, Canada) and incubated for 30 min at 23°C. This antibody is specific for the inducible form of HSP70. Initial studies were done by using another antibody to HSP70, H5147 (Sigma, St. Louis, MO), which reacts with both inducible and constitutive forms of HSP70, but were then repeated by using the more specific antibody for inducible HSP70. The sections were drained and covered with biotinylated rabbit anti-mouse antibody (DAKO) diluted 1:200. Endogenous peroxidase was blocked with 0.5% H2O2 in methanol. The sections were then covered with peroxidase-conjugated avidin (DAKO), developed with fresh diaminobenzidine solution containing H2O2, and counterstained with hematoxylin. The sections were dehydrated, cleared, mounted in Gum Dammar, and examined.
SDS-PAGE and Autoradiography
Isolated colonocytes were resuspended in K-H solution in the presence or
absence of different concentrations of butyrate. To 250 µl of suspension,
60 µCi/ml of [35S]methionine was added, and colonocytes were
incubated at 37°C for 45 min. The cells were then centrifuged at 3,000 rpm
for 3 min, and the cell pellet was resuspended in 150 µl K-H solution.
Resuspended colonocytes were lysed by sonication of 2-µ amplitude for 2 min
(MSE Soniprep 150). The protein concentration of the sonicated suspensions was
assayed by Lowry's method. Samples were initially boiled for 5 min in protein
dissociation buffer (9% SDS, 16% -mercaptoethanol, 15% glycerol, 1 M
Tris, pH 6.7, containing 3 mg bromophenol). Equal amounts of protein were then
loaded onto 10% SDS-PAGE gel. Ten percent separating gel contained 10%
acrylamide, 0.1% SDS, 0.075% ammonium persulfate, 0.05%
N,N,N',N'-tetramethylethylenediamine (TEMED),
and 0.373 M Tris, pH 8.8. Stacking gel contained 5% acrylamide, 0.1% bis
acrylamide, 0.1% SDS, 0.2% ammmonium persulfate, 0.069% TEMED, and 0.138 M
Tris, pH 6.8. Electrophoresis of the gels was carried out for 6 h at 120 V in
buffer containing 0.02 M Tris, 0.192 M glycine and 0.1% SDS, pH 8.6. They were
then stained overnight in Coomassie blue stain (methanol/acetic acid/water
50:10:40 vol/vol/vol containing 0.1% Coomassie brilliant blue R). Gels were
destained, dried, and exposed to X-ray film at 80°C for 14 days.
The films were developed and scanned by using an HP scanner, and density of
the protein bands was quantitated by using Scion Image for Windows (Scion
1998).
Western Blot Analysis
Western blot analysis was carried out as described by Towbin et al.
(41). After electrophoresis,
gels were immersed in transfer buffer (25 mm Tris, 192 mM glycine, 20%
methanol) and blotted onto 0.45-µm nitrocellulose membranes (Millipore) by
using a Bio-Rad power supply. The membranes were covered with mouse anti-HSP70
monoclonal antibody (SPA-810; Stressgen) diluted 1:5,000 with blocking
solution and incubated for 2 h at 23°C. After being washed three times,
the blot was incubated with goat anti-mouse antibody conjugated with alkaline
phosphatase (Genei, Bangalore, India) diluted 1:1,000 with blocking solution
for 2 h at 23°C. After further washing, alkaline phosphatase was developed
by using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Genei)
as substrate. All incubation procedures were performed under gentle agitation
at 23°C. Experiments performed with other short-chain fatty acids acetate
and propionate did not inhibit HSP or NF-B synthesis; thus butyrate was
used for further studies.
Gel Mobility Shift Assay
The gel or EMSA was carried out with two separate objectives: (1)
to detect the binding of activated heat shock factors (HSF) in the cytoplasm
of colonocytes from DSS colitis to heat shock element (HSE) DNA and
(2) to detect the binding of translocated NF-B in the nuclei
of colonocytes from DSS colitis to the appropriate binding site of DNA.
HSF-HSE binding. Colonocytes were isolated as described earlier
from control rats, colitic rats receiving saline enema, and colitic rats
receiving butyrate enema. Cells were suspended in protein dilution buffer (20
mM Tris, pH 7.9, 150 mM KCl, 1 mM dithiothreitol, 10% glycerol, and 50
µg/ml bovine serum albumin) and quick frozen at 70°C. Cells were
lysed with repeated freeze and thaw to get the protein extract. These extracts
were then incubated at room temperature for 40 min with synthetic
double-strand HSE oligonucleotide (containing bases 80 to 115 of
the human promoter of the gene encoding HSP70), end-labeled with
[-32P]ATP (LCP1; BRIT, Mumbai, India). The sequence of this
oligonucleotide (a kind gift from Dr. Honorine Ward, Tufts University, Boston,
MA) was 5'-GCG AAA CCC CTG GAA TAT TCC CGA CCT GGC GCC AGG TCG GGA ATA
TTC CAG GGG TTT CGC-'3
(21). The binding reaction
mixture (20 µl) contained 20% glycerol, 100 mM Tris·HCl (pH 8.0),
300 mM KCl, 25 mM MgCl2, 500 µg BSA, 0.1 M DTT, labeled DNA, and
20 µg protein extract. These were then subjected to electrophoresis on a 6%
nondenaturing polyacrylamide gel at 4°C by using 1 x Tris and
glycine buffer (0.25 M Tris base and 1.9 M glycine, pH 8.3) at 160 V for 90
min. The gel was then dried and autoradiographed. Additional experiments were
done after adding 0.5 and 1.0 mM butyrate to colonocytes isolated from colitic
rats that had received saline enema. The specificity of the binding was
checked by competition binding assay in which large excess (200x) of
unlabeled DNA was added along with labeled DNA.
NF-B. Nuclear extracts were prepared from
colonocytes of control rats and colitic rats receiving either saline enema or
butyrate enema (17). These
extracts were incubated for 40 min at room temperature with synthetic
double-strand oligonucleotide containing the consensus sequence of NF-
B
binding region, 5'-TGAGGGGACTTTCCCAGGC-'3 (GSN15; Genei), and
end-labeled with 32P, by using [
-32P]ATP (LCP1;
Brit) and a DNA end-labeling kit (model KT-6; Genei). These were then
subjected to 6% nondenaturing gel electrophoresis, dried, and autoradiographed
as described earlier. Experiments were done in parallel by using a mutated
motif of NF-
B binding oligonucleotide,
5'-TGAGGCGACTTTCCCAGGC-'3 (GSN15; Genei), to establish the
specificity of the interaction.
Statistics
A minimum of three animals was used for each experiment. All experiments were performed in triplicate. Colonocytes for the experiments were from individual animals. All numerical values were expressed as means ± SE. The two-tailed Mann-Whitney U-test was used to assess significance of differences between means of groups. P values <0.05 were considered statistically significant.
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RESULTS |
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Colonocytes isolated from rats with DSS colitis that had received saline enemas showed significantly reduced cell viability measured as percent release of lactate dehydrogenase (56.1 ± 2.1%), compared with colonocytes from normal rats (89.6 ± 0.9%) (P < 0.01). Colonocytes from colitic rats that had received butyrate enemas showed significantly higher colonocyte viability (77.6 ± 0.9%) compared with those that had received saline enemas (P < 0.01) (Fig. 1).
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Mucosal Barrier Function and Effect of Butyrate
Tissue electrical resistance, an indication of barrier function, was significantly lower in the colon of rats with DSS colitis receiving saline enemas (60.2 ± 0.7 µohm · cm2 · h1) compared with normal control rats (115.2 ± 2.0 µohm · cm2 · h1) (P < 0.01). Colons from rats administered butyrate enemas showed significantly higher tissue electrical resistance (102 ± 3.1 µohm · cm2 · h1) than those that received saline enemas (P < 0.01). Measured flux of [14C]mannitol across the colonic mucosa, reflecting passive permeation, was significantly increased in DSS colitis (26.3 ± 1.5 µmol · cm2 · h1) compared with normal control rats (8.3 ± 1.1 µmol · cm2 · h1) (P < 0.01). Passive flux of mannitol was significantly reduced in colitic rats that had received butyrate enemas (10.3 ± 2.4 µmol · cm2 · h1) compared with those that had received saline enemas (P < 0.005) (Fig. 1).
The lamina propria of the colon contained very few neutrophils in control animals (6.7 ± 1.1 per 10 hpf). Neutrophil infiltration in the lamina propria was significantly increased in rats with DSS colitis that had received saline enemas (36.4 ± 7.8 per 10 hpf) (P < 0.005), and this was markedly reduced in colitic rats that had received butyrate enemas (13.4 ± 2.5 per 10 hpf) (P < 0.05 vs. control and P < 0.01 vs. DSS saline) (Fig. 1).
HSP70 Synthesis in DSS Colitis and Effect of Butyrate
Colonic sections from normal rats did not stain for HSP70 by using a monoclonal antibody. On the other hand, colon from colitic rats showed intense staining for HSP70 in both surface and crypt epithelium. Mild focal staining was also observed in stromal cells (Fig. 2). The intensity of staining for HSP70 was less in rats treated with butyrate enemas compared with those that had received saline enemas (Fig. 2). Inhibition of HSP70 expression in DSS colitis by butyrate enemas was confirmed independently by Western blot analysis by using specific antibody to the inducible form of HSP70 (Fig. 2).
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We also examined the effect of adding butyrate in vitro on colonocyte synthesis of neoproteins in DSS colitis. Because DSS colitis colonocytes were expected to contain formed HSP70 at the time of isolation, immunohistochemistry and Western blot analysis would not be appropriate to examine the effect of adding butyrate after isolation on HSP70 synthesis. Hence, incubation with [35S]methionine followed by SDS-PAGE and autoradiography, which would detect only protein newly formed after colonocyte isolation, was used. Colonocytes isolated from normal control rats did not show significant incorporation of [35S]methionine into new proteins during the 45 min of incubation. On the other hand, colonocytes isolated from rats with DSS colitis showed significant [35S]methionine incorporation into new proteins, the most prominent band being at the 70-kDa position (Fig. 3). This band reacted with HSP70 antibody after immunoblotting. When colonocytes from colitic rats were incubated in vitro with butyrate, inhibition of neoprotein production was noted generally and of the 70-kDa band, specifically. Scanning densitometry of these protein bands revealed that butyrate inhibited HSP70 formation by 5868% compared with colonocytes not incubated with butyrate (P < 0.001) (Fig. 3).
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Gel Mobility Shift Assays
HSFs bind to the DNA HSE to induce HSP70 production. The EMSA to detect DNA-protein binding by using radiolabeled HSE DNA and colonocyte lysates from animals with DSS colitis was carried out to determine whether butyrate would inhibit this binding. Cell extracts of colonocytes from control animals did not show any protein bands that bound radiolabeled HSE (Fig. 4). Colonocytes isolated from rats with DSS colitis exhibited a large radioactive band, indicating activation and binding of HSF to radiolabeled HSE, thus producing an electrophoretic mobility shift (Fig. 4). This shift was not found in colitic rats that had received butyrate enemas in vivo. The addition of butyrate in vitro to colonocytes from colitic rats also inhibited the mobility shift, indicating that butyrate directly inhibited formation of the HSF/HSE complex.
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Further experiments were undertaken to exclude the possibility that the
anti-inflammatory effect of butyrate in DSS colitis was due to blocking of
NF-B activation. Nuclear extracts from the colonocytes of control rats
did not bind the labeled NF-
B binding oligonucleotide in the gel
mobility shift assay (Fig. 5), whereas colonocyte nuclear extracts from colitic rats (receiving saline
enemas) showed significant NF-
B DNA binding activity, indicating
colonocyte NF-
B activation in DSS colitis. The specificity of binding
was demonstrated by the fact that binding was inhibited when 200-fold molar
excess of unlabeled NF-
B binding oligonucleotide was added, or when a
mutant NF-
B binding probe with one nucleic acid substitution was used.
Binding was markedly inhibited by the administration of butyrate enemas to
colitic rats and was also inhibited by the in vitro addition of butyrate
(Fig. 5). The HSP70 inhibitor
quercetin also inhibited activation of NF-
B both alone and in
combination with butyrate.
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DISCUSSION |
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Inflammation is a major component in the etiology of UC, and HSPs can
activate the immune system in various ways to perpetuate inflammation and
tissue damage (38). HSP70 is
an inducible HSP produced in response to a variety of different cellular
stresses and increased colonic expression of HSP70 has been found in the
inflammatory bowel diseases
(23,
39). However, no study to date
has reported the effect of butyrate on HSP70 synthesis and expression in DSS
colitis. The present study demonstrates that colonocyte production of HSP70
was markedly increased in DSS colitis, with immunohistochemistry showing that
inducible HSP70 was predominantly expressed diffusely in colonocytes. Although
a link of HSP70 expression to regenerative activity has been suggested by a
study in ischemic bowel disease that found HSP70 immunoreactivity to be
highest in viable crypt and surface epithelial cells adjacent to foci of
necrosis (22), no such pattern
was discernible in DSS colitis in the present study. There are likely several
reasons for increased colonocyte expression of HSP70 in DSS colitis. Colitis
results in the release of specific cytokines, some of which, such as IL-6 and
TNF- in addition to oxygen free radicals released during inflammation,
are thought to induce HSPs. Cellular ATP depletion may lead to HSP70 remaining
complexed to proteins, and therefore not available for recycling, leading to
increased HSF activation (19).
In DSS colitis, colonocyte oxidation of butyrate is markedly suppressed
(1,
23,
39), presumably leading to
reduced cellular ATP levels, and this may be yet another mechanism explaining
increased colonocyte expression of HSP70.
Interestingly, treatment with butyrate resulted in an inhibition of this increased HSP70 synthesis. The immunohistochemical data and immunoblots using monoclonal antibodies from two different sources confirm that we were detecting HSP70 and that butyrate prevented the increase in HSP synthesis induced in colitis. Although most studies suggest that the heat shock response provides protection against epithelial and mucosal injury in the intestine and colon, the role of cellular HSP70 activation in colitis remains unclear. Induction of the heat shock response before any injury has a protective effect, whereas activation of the heat shock response subsequent to a proinflammatory stimulus has been shown to have cytotoxic effects (7, 9, 13, 14). In intestinal epithelial cell lines, heat shock before exposure to lipopolysaccharide increased cell survival, whereas heat shock after exposure to lipopolysaccharide reduced enterocyte viability leading to cellular dysfunction and increased apoptosis (48). Thus it is becoming evident that increased expression of HSP can also have cytotoxic effects and the protective effect of butyrate could then be mediated by inhibition of these proteins.
Butyrate and short-chain alcohols have been reported to suppress HSP synthesis in cultured Drosophila cells (28), possibly by suppressing the initiation of transcription of heat shock genes. Butyrate has also been shown to inhibit expression of the HSP Grp 94 in colorectal carcinoma cells (47). In our studies, there was binding of HSF to HSE as shown by the EMSA in colitic rats. Butyrate suppressed this binding of HSF to HSE both when added in vitro to colonocytes isolated from colitic rats and when given as an enema in vivo to colitic rats. Whether butyrate has a dual role, reducing the activation of HSF and inhibiting HSF/HSE binding, is not very apparent from our studies. Butyrate is also known to influence gene transcription through effects on histone acetylation, potentially providing another mechanism for suppression of HSP70 synthesis by butyrate.
A major link between the heat shock response and cellular inflammatory
responses has been found to be the transcription factor NF-B
(25), which is implicated in
the regulation of a variety of genes during immune and inflammatory responses
(4). NF-
B activation has
been noted in inflammatory bowel disease in humans
(30,
35) and in DSS colitis in mice
(26). The decrease in mucosal
PMN infiltration in colitic rats receiving butyrate enemas coupled with its
effect on HSP70 prompted us to examine the effect of butyrate on this key
transcription factor. Butyrate inhibited the activation of NF-
B both
when administered in vivo as enemas and when added in vitro after colonocyte
isolation. Butyrate has been shown to inhibit NF-
B activation in lamina
propria macrophages of patients with UC
(24). Interestingly,
quercetin, an inhibitor of HSP70, also decreased the activation of NF-
B
in colonocytes isolated from rats with DSS colitis, suggesting that inhibition
of HSP70 was linked with suppression of NF-
B activation. HSP70 has been
reported to induce cytokine production through a CD14-dependent pathway
(3). The expression of
proinflammatory cytokines such as TNF-
, IL-1
, and -6 was
upregulated, and NF-
B was activated, in monocytes exposed to exogenous
HSP70. Butyrate has been reported to reduce the release of IL-8 from HT-29
cells in response to TNF-
; the role of HSP was not evaluated
(2). Butyrate has also been
reported to inhibit NF-
B transcription in peripheral blood mononuclear
cells exposed to lipopolysaccharide
(36), and in HT-29 cells
exposed to TNF-
. In the latter studies, butyrate appeared to suppress
degradation of inhibitory factor-
B, resulting in maintenance of
NF-
B in the inactive state.
In conclusion, our studies suggest that butyrate reduces mucosal
inflammation and improves epithelial cell integrity and barrier function in
DSS colitis in rats and that these effects are associated with (and are likely
to be mediated by) inhibition of activation of inducible HSP70 leading to
reduced activation of NF-B. Further studies to examine this association
may prove to be useful in the development of therapies for the treatment of
inflammatory bowel disease and other diseases characterized by colonic mucosal
inflammation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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