Center for Surgical Research and Department of Surgery, University of Alabama School of Medicine, Birmingham, Alabama 35294
Submitted 11 February 2003 ; accepted in final form 17 March 2003
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ABSTRACT |
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inflammation; immune suppression; steroid synthesis; T lymphocytes; cytokines
Steroid hormones regulate immune functions in vivo, and the mechanisms
involve not only the control of cytokine gene transcription by the classic
steroid hormone-receptor complex but also the tissue-specific metabolism of
sex steroids (11,
21,
24,
29,
33,
36,
47). Among the sex steroids,
estrogen is demonstrated to protect immune functions after TH because
proestrus females are not immunodepressed compared with male and OVX mice.
Furthermore, the depressed immune functions in males and OVX females after TH
can be normalized by parenteral E2 administration
(16,
17,
18). The ovary is the primary
site of estrogen synthesis in females, and despite that, the enzymes involved
in estrogen metabolism are also present in peripheral tissues, including
spleen and the T lymphocytes
(21,
37). The presence of
steroidogenic enzymes, especially in the T lymphocytes, suggests a role for
local synthesis of E2 for interaction with the estrogen receptor (ER) present
in the cells (39) and the
production of cytokines as needed. E2 is a highly potent regulatory sex
steroid involved in a variety of metabolic functions. Because of its
regulatory role, E2 seldom accumulates in the cells, and its synthesis is
dependent on the tissue requirement. Thus understanding the E2 metabolism in
the T lymphocytes is desirable for determining the basis for change in the
cytokine releases by these cells after TH. In this regard, the expression and
analysis of enzymes involved in steroid metabolism is meaningful compared with
active steroid quantification, because regulatory steroids have a short
half-life and quantification of steroid at subpicomole levels in the tissues
or cells is ambivalent. We therefore measured the activity and expression of
the enzymes involved in E2 metabolism in splenic T lymphocytes by using
relevant substrates. Because the promoter regions of the cytokine genes have
response elements for ER binding
(24,
32,
33,
35), E2 synthesis in T
lymphocytes was evaluated in conjunction with ER- and ER-
expression and IL-2 and IL-6 release (the cytokines whose release are altered
after TH) in proestrus and OVX mice after TH in the same cell preparations.
The results indicate that continued synthesis of E2 in splenic T lymphocytes
of proestrus females appears to be responsible for the maintenance of IL-2 and
IL-6 release in those cells and is probably one of the reasons why proestrus
females are not immunodepressed after TH.
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MATERIAL AND METHODS |
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Mice. Inbred C3H/HeN female mice, 68 wk old weighing 2025 g, were obtained from Charles River Laboratories (Wilmington, MA). The animal experiments were conducted according to the guidelines established by the National Institutes of Health and the protocols approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee.
Experimental groups. Proestrus female mice that showed a large number of nucleated epithelial cells and few cornified cells in the vaginal smear were used in the experiments. The procedure described by Waynforth et al. (52) was followed for ovariectomy, and 2 wk after ovariectomy the animals were used in experiments. Animals were assigned to the following four groups (n = 8 mice per group): female shams, females undergoing TH, OVX female shams, and OVX females undergoing TH.
Trauma-hemorrhage. The procedure for inducing trauma (i.e., midline laparotomy)-hemorrhage has been described in detail in our earlier publications (56, 57). Briefly, after overnight fast, soft-tissue trauma was induced in mice by performing a 2-cm ventral midline laparotomy, which was closed in two layers. Both femoral arteries were then catheterized, and the animals were allowed to awaken. The animals were then bled rapidly to a mean arterial pressure of 30 mmHg, maintained at that pressure for 90 min, and resuscitated with four times the volume of shed blood with Ringer lactate solution. Sham-operated mice underwent the same anesthetic and surgical procedures, but neither hemorrhage nor resuscitation was carried out. The animals were killed at 2 h after resuscitation, and the spleens were removed for analysis.
Preparation of T lymphocytes. The procedures for the preparation of splenocytes and enrichment of T lymphocytes have been described in an earlier publication (36, 38). The purity of enriched lymphocytes was >95% and consisted of both CD4+ and CD8+ subsets. All analyses were carried out in the same population of T lymphocytes prepared from one mouse in each group or from a pooled population of lymphocytes prepared from two mice in each group. Approximately 109 lymphocytes were used for preparation of homogenate in enzyme assays, 106 lymphocytes for mRNA expression by PCR analysis, and 5 x 106 for bioassays.
Enzyme assays. The modified assay procedures for
5-reductase and for 17
-hydroxysteroid dehydrogenase
(17
-HSD) oxidative and reductive activities have been described in
detail previously (1,
37,
48). The assay mixtures after
the enzyme reaction were extracted five times with methylene chloride, and the
steroids in the organic phase were analyzed by thin-layer chromatography (TLC)
using the mobile phase of chloroform-ethyl acetate (3:1, vol/vol). The
radioactivity of the separated steroids in the chromatographic plates was
measured by using InstantImager (Packard, Downers Grove, IL), and steroids
were identified by comparison with the Rf values of standards.
The aromatase activity was assayed by the procedure of Thompson and Siiteri (45). [3H]androstenedione and [14C]-testosterone were used as substrates in these assays. For estimation of 3H20 release, 1 ml of 10% activated charcoal with 1% dextran-T70 was added to the assay mixture. After centrifugation at 10,000 g for 10 min, the radioactivity in 500 µl of supernatant was measured after the addition of 5 ml of liquid scintillation cocktail in the scintillation counter (Wallac, Gaithersburg, MD). For estimating [14C]E2 conversion from [14C]testosterone, the reaction mixture was extracted twice with two volumes of dichloromethane. After removal of the organic solvent, the residue was dissolved in 100 µl of methanol and subjected to TLC on silica gel plates with chloroform-ethyl acetate (3:1, vol/vol) as the mobile phase. The separated steroids in the chromatographic plates were measured for radioactivity with InstantImager.
RT-PCR analysis. The RNA was prepared from T lymphocytes using the
Atlas total RNA kit (Clontech, Palo Alto, CA) and purified by DNase treatment
(1 U/µl) for 30 min at 37°C. Poly(A)+ mRNA preparation and
RT-PCR reactions were carried out using the Access RT-PCR kit (Promega,
Madison, WI). The primers used in PCR analysis
(Table 1) were chosen from the
cDNA sequences of the GenBank, and Primer3 software
(www.genome.wi.mit.edu/genome_software/other/primer3.html)
was used for the selection of primers. The PCR reactions were carried out in a
gradient Mastercycler (Eppendorf, Westbury, NY). The first cycle of the RT
reaction was carried out at 48°C for 45 min. The PCR cycle for
amplification consisted of 30 s of denaturation at 94°C, followed by 1 min
of annealing at 60°C and 2 min of extension at 68°C. The final
products were extended for 7 min at 68°C. Each enzyme was analyzed for
amplification between 5 and 38 cycles. The number of amplification cycles for
measuring expression differed considerably for each enzyme. Comparison of
expression between the sham and TH for each enzyme was made at the cycle where
expression was nearly 50%. -Actin expression was used as the internal
control. The PCR products were analyzed by electrophoresis on 1.5% agarose
gels in 1x TAE (Tris-acetate-EDTA) buffer and visualized by ethidium
bromide staining under UV illumination. The intensity of cDNA bands was
measured in the 500 Fluorescence Chemilimager (San Leandro, CA).
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Cytokine assays. The CTLL-2 cell line (TIB-214) for IL-2 assay and the hybrid cell line 7TD1 (CRL-1851) for IL-6 assay were obtained from the American Type Culture Collection (Rockville, MD). The bioassay procedures for IL-2 and IL-6 release in T cell culture supernatants have been described previously (57). The cells were stimulated with 10 µg/ml anti-CD3 (BD Biosciences, San Jose, CA) in complete Click's medium at 37°C for 36 h before the culture supernatants were assayed for the cytokine release. IL-2 activity in the T lymphocyte culture supernatants was determined by making serial dilutions of the supernatant (in 500 µl) to which CTLL-2 cells (1 x 105 cells/ml) were added. The cultures were incubated for 48 h at 37°C with 5% CO2. At the end of this time, 1 µCi of [3H]thymidine (specific activity 6.7 Ci/mmol; NEN) was added to each well and cultures were further incubated for 16 h. The cultures were then harvested with a multiple automated sample harvester (Skatron AS, Trombay, Norway) onto a glass fiber-filter mat and processed for liquid scintillation counting on a Betaplate (model 1205; Pharmacia/LKB Nuclear, Gaithersburg, MD). For the IL-6 assay, 100 µl of 7TD1 cells (5 x 105 cells/ml) were added to the serial dilutions of the lymphocyte culture supernatant and incubated for 72 h at 37°C in 5% CO2. At the end, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 5 mg/ml in RPMI 1640 medium) were added to each well, and the plate was incubated for an additional 4 h. The MTT crystals that incorporated into the viable cells were dissolved by aspiration of the supernatant from each well and addition of 100 µl of isopropanol containing 0.04 M HCl. The absorbance of fluids in each well was measured at 620 nm using an automated microplate reader (Bio-Tek Instruments, Winooski, VT). Relative units of cytokine activity were computed by comparison of the curves produced from dilution of the experimental samples to that generated by dilution of recombinant mouse IL-2 or IL-6 standards (R&D Systems, Minneapolis, MN).
Protein content. The protein content was determined by the micro Bradford method (Bio-Rad, Hercules, CA) with BSA as standard.
Enzyme kinetics. Kinetic constants for steroid substrates were determined by Lineweaver-Burk analysis. Assays were carried out in triplicate using microsomal preparations of tissue homogenates. Ten concentrations of substrates between 1 and 200 µM were used for each steroid. SigmaPlot software (vers. 2.0; Jandel Scientific, San Rafael, CA) was used to generate hyperbolic functions and nonlinear regression plots.
Statistical analysis. SigmaStat (vers. 2.0; Jandel Scientific) was
used in nonlinear regression analysis. Data were analyzed by separate one-way
ANOVA. When a significant F value was obtained, the effects were
differentiated using Tukey's test. Tests between effects were performed with
Student's t-test. Significance was achieved when P
0.05.
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RESULTS |
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E2 is also synthesized from androstenedione with estrone (E1) as the
intermediate in the reaction. Two enzymes participate in this catalysis:
aromatase for conversion of androstenedione to E1 and 17-HSD for
reduction of E1 to E2. The aromatase activity associated with androstenedione
conversion to E1 in T lymphocytes of proestrus and OVX animals after TH is
shown in Fig. 1, C and
D. Aromatase activity in all the tissues was low with
androstenedione as a substrate, compared with testosterone as the substrate,
suggesting low conversion to E1 in all the tissues. The reduction of E1 to E2
is catalyzed by 17
-HSD, and the activity of this enzyme in T lymphocytes
is shown in Fig. 2, A and
B. The 17
-HSD activities for conversion of E1 to E2
did not alter in the tissues and in T lymphocytes, after TH, in both the
proestrus and OVX animals.
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Enzyme kinetics in T lymphocytes. The activity of enzymes involved
in the metabolism of testosterone and E2 in the T lymphocytes of proestrus
females, with pertinent substrates, is given in
Table 2. The catalytic
efficiency of the enzymes (Vmax/Km)
indicates that the production of E2 is from androstenedione through
testosterone, and not E1. Moreover, the analysis showed the presence of
relatively low 5-reductase activity, indicating less
5
-dihydrotestosterone (DHT) synthesis in the T lymphocytes of proestrus
female mice.
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Testosterone metabolism. Testosterone is synthesized from
androstenedione by reductive catalysis. The activity of 17-HSD
associated in this catalysis in tissues of proestrus and OVX mice, after TH,
is shown in Fig. 2, C and
D. In proestrus females, significant increase in the
activity was observed only in the ovary, spleen, and T lymphocytes after TH
(Fig. 2C). The enzyme
activity in adipose tissue was lower in OVX mice compared with proestrus
females, and the enzyme activity did not change in this tissue in either group
after TH (Fig.
2D).
5-Reductase converts testosterone into DHT, which is a highly active
androgen. There was no change in 5
-reductase activity of T lymphocytes
or other tissues of both proestrus and OVX mice after TH
(Fig. 3, A and
B).
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Conversion to E1. E1 is an inactive estrogen because of low
binding affinity to ER. The 17-HSD converts E2 into E1 by oxidative
catalysis. Similar to reductase, the oxidation of E2 by this enzyme was low in
the adrenal gland, ovary, adipose tissue, spleen, and T lymphocytes from
proestrus females (Fig. 3, C and
D). TH did not alter the oxidative activity of the enzyme
in any of the tissues, including T lymphocytes from the proestrus or OVX
animals.
Enzyme expression in T lymphocytes. The expression of
5-reductase, aromatase, and oxidative isomers II, IV, and V of
17
-HSD in T lymphocytes of sham and TH female mice is shown in
Fig. 4. The expression of
aromatase did not change significantly after TH in proestrus females or in OVX
mice. Likewise, the expression of the 17
-HSD isomers was similar in both
sham and TH proestrus females, but expression was reduced after TH in OVX
females. 5
-Reductase expression was not different after TH in proestrus
or OVX mice.
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ER- and ER-
expressions. The
expression of ER-
and ER-
, in the splenic T lymphocytes from
proestrus and OVX female mice, after TH, is shown in
Fig. 5. The ER-
expression was low in OVX animals compared with ER-
expression. There
was no change in the ER-
expression in the T lymphocytes of proestrus
and OVX animals after TH. In contrast, in proestrus females ER-
expression decreased significantly after TH, whereas its expression
significantly increased in OVX females under those same conditions.
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Cytokine expression and release. The expression of IL-2 and IL-6 in T lymphocytes of proestrus and OVX mice, after TH, is shown in Fig. 6. The IL-2 expression was low in OVX females compared with proestrus females, and TH did not alter IL-2 expression in either group. Stimulation of T lymphocytes with anti-CD3, however, resulted in a significant reduction in the IL-2 release in OVX animals but not in proestrus females after TH. In contrast, IL-6 expression and release were different. Significant increase in IL-6 expression was observed in proestrus mice after TH; however, the expression decreased significantly in OVX animals. Moreover, anti-CD3 stimulation of T lymphocytes did not alter the release of IL-6 in T lymphocytes from proestrus females after TH, whereas a threefold decrease in the IL-6 release was observed in OVX females under such conditions.
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DISCUSSION |
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The major consequence of TH, besides impairment of the cardiovascular system, is severe depression of immune functions (51, 56, 57). The depression is profound in males but is not observed in proestrus females, indicating sexual dimorphism in the immune response after TH. The divergent immune responses after TH in males and proestrus females are also manifested by the altered release of cytokines IL-2 and IL-6 by the splenic T lymphocytes (2, 4, 18, 54, 55). Because T lymphocytes express receptors for E2 and are also capable of synthesizing E2 locally, the assessment of local active steroid synthesis in the release of cytokines by T lymphocytes becomes significant (37, 39). Hence, the metabolism of E2 and its effect on the release of IL-2 and IL-6 was assessed in T lymphocytes of proestrus and OVX mice after TH.
Enzyme kinetics show that synthesis of E2 from androstenedione is via the
formation of testosterone and not via E1. This study indicates a correlation
among increased endogenous synthesis of E2, low conversion to E1
(Fig. 7), and the persistent
release of IL-2 and IL-6 in the lymphocytes of proestrus female after TH. This
is substantiated by 1) the enhancement of aromatase activity, which
leads to E2 synthesis in T lymphocytes after TH, unlike reduction in the
enzyme activity in OVX females under the same conditions; 2)
increased production of testosterone from catalytic reduction of
androstenedione by 17-HSD in proestrus animals after TH, whereas this
enzyme activity was unchanged in OVX animals, indicating sustained
availability of testosterone for conversion to E2 by aromatase in proestrus
animals; and 3) the comparatively low oxidative catalysis by
17
-HSD in both proestrus and OVX animals, suggesting little or no
conversion of E2 into E1. The expression of 17
-HSD isomers was analyzed
by routine RT-PCR analysis, which is not quantitative. Our aim was to
determine whether different forms of the 17
-HSD isomers are expressed in
T lymphocytes and, if they are expressed, whether their expression is altered
after TH. The enzyme expressions were therefore evaluated in the same T
lymphocyte preparation that was used for enzyme assays and ER expression as
well as IL-2 and IL-6 expression and release. The results show changes in the
expression of 17
-HSD isomers after ovariectomy and after TH. It is,
however, necessary to quantify the 17
-HSD isomer expressions by a
quantitative PCR procedure for meaningful association of the different isomers
in the E2 metabolism in T lymphocytes. Testosterone is also the precursor of
DHT. No change in the 5
-reductase activity, either after ovariectomy or
after TH, in proestrus and OVX females was evident, indicating little change
in the production of DHT in T lymphocytes. Because DHT is considered to be an
inhibitor of aromatase activity
(6,
41), an increase in its
activity would have lowered E2 production.
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A significant observation of our study is the lack of correlation of aromatase expression in T lymphocytes with the enzyme activity in both proestrus and OVX animals. However, this is not surprising because E2 formation from testosterone is the result of a coupled reaction involving aromatase P-450 and a flavoprotein NADPH-cytochrome P-450 reductase (42), and the expression of aromatase alone was assessed in this study. Furthermore, this enzyme reaction requires NADPH as a cofactor. In this regard, our previous studies have indicated decreased splenocyte ATP levels and NAD-to-NADH ratios in tissues after hemorrhagic shock (25). This suggests that not only the expression but also the cofactor requirements are important for the assessment of aromatase activity.
The predominant biological effects of E2 are mediated through two
intracellular receptors, ER- and ER-
, and our study shows that
both these subtypes are present in the splenic T lymphocytes of female mice.
However, their expression in response to TH is different. ER-
expression did not change after ovariectomy or after TH, whereas ER-
expression decreased significantly after ovariectomy. Moreover, expression of
ER-
was significantly different in the proestrus and OVX animals after
TH; its expression was decreased in proestrus and increased in OVX animals.
The increased production of E2-attenuated expression of ER-
in the
proestrus and the opposite effects in the OVX females after TH suggest that
down-regulation of ER-
may be a factor associated with the change in the
cytokine releases by T lymphocytes. Because the T lymphocyte populations used
in the experiments consisted of both CD4+ and CD8+
phenotypes (38), analysis of
each phenotype for receptor expressions is needed for correlating the changes
in receptor subtype expression to lymphocyte differentiation or functional
changes, such as a particular cytokine release.
The present study compared E2 synthesis with the in vitro stimulated release of IL-2 and IL-6 in the same cell preparations obtained from different groups. We selected the release of IL-2 and IL-6 in these studies because 1) the alterations in the release of these cytokines are indications of the proinflammatory condition, i.e., Th1 to Th2 shift, and 2) our earlier studies demonstrated marked alterations in the release of these cytokines in OVX females but not in proestrus females after TH (18, 55). Furthermore, the release of these cytokines can be restored by administration of E2 in OVX females during or immediately resuscitation (18). In the present study, we observed that the expression and release of the proinflammatory cytokines IL-2 and IL-6 in T lymphocytes are also different in the proestrus and OVX animals and in response to TH. IL-2 expression, although low in OVX compared with proestrus animals, did not change after TH in either group. In contrast, IL-6 expression was similar in both proestrus and OVX mice, but it was augmented in proestrus and markedly decreased in OVX mice after TH. The release of the cytokines, determined by bioassay in response to anti-CD3 stimulation of T lymphocytes, was also different. The use of anti-CD3 as a stimulant for T lymphocytes cytokine releases is evocative because Con A is primarily a mitogen associated with cell proliferation, whereas anti-CD3 is associated with the T lymphocyte functions. The release of IL-2 and IL-6 was similar in proestrus and OVX females in sham controls, but significantly decreased release of both cytokines was observed only in the OVX animals after TH. A distinct association between E2 synthesis and cytokine release in different groups is evident in this study. Increased E2 synthesis in T lymphocytes proestrus females after TH appears to be associated with sustained release of IL-2 and IL-6 in those animals, because loss in E2 production is reflected by decreased release of these cytokines in the OVX females after TH. The cytokine releases in this study were determined in T lymphocyte preparation that contained CD4+ and CD8+ subsets. Analysis of cytokine expression and release in each T lymphocyte subset is important for any meaningful correlation.
Substantial emphasis has been focused recently on the regulation of
extragonadal biosynthesis of sex steroids. The local synthesis of active
steroids in T lymphocytes is essential for carrying out their specific
functions, especially the release of cytokines. The rate of formation of each
steroid depends on the level of expression of the specific androgen- and
estrogen-synthesizing enzymes in the tissue. Moreover, local synthesis of
active steroids is meaningful compared with availability in circulation,
because the steroid can be synthesized as needed and catabolized immediately
after fulfillment of tissue function. This is especially true for any
regulatory molecule, of which E2 is one. Our recent studies have shown
augmented synthesis and decreased catabolism of DHT as the likely cause for
loss of T lymphocyte functions in males after TH as reflected in the decreased
release of IL-2 and IL-6 by T lymphocytes
(58). In this study, we have
demonstrated enhanced synthesis of E2, which promotes the maintenance of IL-2
and IL-6 release by T lymphocytes in proestrus mice after TH. Thus both of our
studies suggest an important role for steroid-metabolizing enzymes in the
release of cytokines by T lymphocytes after TH. Among the sex
steroid-metabolizing enzymes, the activities of 17-HSD isomers appear to
be critical because they catalyze both the oxidative and reductive reactions
that are required for the synthesis of testosterone, DHT, and E2 as well as
their catabolism into inactive steroids
(21,
31). This enzyme is also
involved in the formation of 5-androstene-3
,17
-diol from
dehydroepiandrosterone (DHEA), which has been shown to bind to the ER
(21,
27,
40). In this regard, our
previous studies have demonstrated that DHEA administration after TH restores
immune functions in male mice, and the effects appear to be mediated via the
ER because tamoxifen blocked the salutary effects of this adrenal steroid
(4,
5). Thus, being at the final
steps of the formation and inactivation of active estrogens and androgens,
17
-HSD isomers play a unique role in the sex steroid-sensitive
physiological functions.
Clinical trauma is a pathological condition that produces an inflammatory response, and our recent retrospective study reveals female patients in premenopausal age range tolerating blunt trauma far better than the males (10). Our experimental results point to sex hormones significantly influencing the immune system in males and females after TH. Thus gender and the hormonal status of the host appear to be critical in the outcome of TH, and estrogen appears to be beneficial in the favorable outcome. Estrogen functions in different tissues and cells by distinct mechanisms, either by regulation of gene activity or by regulation of signal transduction processes (13, 19, 47, 50). Our studies show that the local synthesis of the active steroid appears to be important at least for the T lymphocyte cytokine releases. Thus a thorough understanding of the mechanisms of action of estrogen in different tissues as well as in different immune cells is important. Such studies are expected to lead to further understanding of the basis of the pathophysiology of TH and to help in the development of improved therapy to prevent/decrease morbidity and mortality after TH.
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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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