1Cincinnati Children's Hospital Medical Center, Division of Pulmonary Biology, Cincinnati, Ohio 45229; and 2School of Women's and Infants' Health, The University of Western Australia, Perth, 6009 Australia
Submitted 18 November 2002 ; accepted in final form 24 February 2003
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ABSTRACT |
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inflammation; fetal maturation; cytokines; interleukin-1; chorioamnionitis; endotoxin
Cytokines such as TNF- and IL-1 are being evaluated as causes or as
predictors of preterm labor, preterm delivery, and fetal inflammation/injury
(8,
28). However, the effects of
TNF-
on the fetus or premature newborn have not been evaluated. A
mediator that correlates with a clinical outcome is not necessarily the
primary effector molecule because each cytokine can induce a cascade of
secondary mediators. To begin to characterize the fetal effects of selected
cytokines, we evaluated the responses of fetal sheep to ovine recombinant
TNF-
given by intra-amniotic, intravascular, or intratracheal
injection. We compared TNF-
with IL-1
, which is known to cause
chorioamnionitis, lung inflammation, induced lung maturation, and systemic
effects (4,
32).
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METHODS |
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Bioactivities of recombinant cytokines. We evaluated the in vitro
bioactivity of recombinant TNF- and IL-1
by assaying for MAPK
phosphorylation and cytokine mRNA expression. We isolated alveolar macrophages
from bronchoalveolar lavage fluid (BALF) of healthy adult sheep using Percoll
gradients. Macrophages were cultured overnight at 37°C in 5%
CO2 in Ham's F-12 medium (Invitrogen, Carlsbad, CA) supplemented
with 1% fetal bovine serum. More than 95% of macrophages remained viable after
overnight culture as assayed by the trypan blue dye exclusion test. Cells (2
x 106 cells/ml) were incubated for 10 min with 10 or 100
ng/ml TNF-
or IL-1
. We evaluated phosphorylation of p44/42 MAPK
induced by the recombinant cytokines by Western blot using phosphospecific
antiphospho-ERK antibody (1:100) and anti-total p44/42 MAPK antibody (New
England Biolabs, Beverly, MA). IL-1
mRNA and IL-8 mRNA expression in
cultured macrophages was also determined by ribonuclease protection assays as
described in assessments of inflammation after 2-h incubation with 1 or 10
ng/ml TNF-
.
Inflammatory responses of adult mouse lungs to TNF- were evaluated
to assess bioactivity in vivo. The amino acid sequences of mouse TNF-
and ovine TNF-
are 75% identical. Recombinant ovine TNF-
was
given by intratracheal injection to 7- to 8-wk-old FVBN mice (Charles River,
Wilmington, MA). Mice were anesthetized with isoflurane (23%) and
orally intubated with a 25-gauge animal-feeding needle. Each mouse received
intratracheal saline (80 µl) as control or saline containing 50 µg of
recombinant ovine TNF-
or 50 µg of E. coli
lipopolysaccharide (serotype 055: B5; Sigma Chemical, St. Louis, MO)
(n = 5/group). Other groups of mice (n = 5/group) were
injected intravascularly with 50 µg of TNF-
or endotoxin. Mice were
deeply anesthetized with pentobarbital sodium (100 mg/kg ip) and were killed
by exsanguination 16 h after intratracheal or intravenous injection. The
trachea was cannulated, and five 1-ml aliquots of saline were flushed into the
lungs and withdrawn by syringe three times for each aliquot
(12). Total and differential
cell counts were performed on BALF from the mice. We used the supernatants of
the lung homogenates for assay of IL-1
using an ELISA kit (R&D
Systems, Minneapolis, MN).
Responses of fetal sheep lung to TNF- and
IL-1
. The animal protocols were approved by the Animal Use
Committees at the Cincinnati Children's Hospital Medical Center and the
Western Australian Department of Agriculture. Chorioamnionitis, lung
inflammation, and lung maturation were assessed in fetal sheep that received
140 µg of TNF-
by intra-amniotic injection 5 h, 2 days (d), or 7 d
before preterm delivery at 123 d of gestation
(14). To compare with
TNF-
, responses to 100 µg of IL-1
were evaluated 24 h and 15
d after intra-amniotic injection. We previously reported that IL-1
induced both lung inflammation and maturation 7 d after intra-amniotic
injection (32). Date-bred
Merino ewes were randomized to cytokine or saline injections, each given in a
volume of 2 ml of saline by ultrasound-guided intra-amniotic injection
(14). To verify intra-amniotic
rather than allantoic injection, Na+ and Cl-
concentrations were determined on samples of fluid aspirated immediately
before injection (13). The
methods for animal protocols used for the present study were the same as used
previously for the 7-d IL-1
group
(32).
Lambs evaluated for inflammation 5 h, 24 h, or 2 d after the intra-amniotic
injections were delivered and not ventilated
(20). Lambs evaluated for both
inflammation and lung maturation 7 d after intra-amniotic TNF- and
IL-1
or 15 d after the intra-amniotic IL-1
were ventilated for
40 min after the preterm delivery
(32). Because 40 min of
ventilation causes modest increases in indicators of inflammation
(20), separate saline-injected
control groups were used for the unventilated and the ventilated groups.
Each ewe was sedated with ketamine (1 g im) and xylazine (25 mg im) followed by spinal anesthesia (2% lidocaine, 3 ml). The fetal head was exposed through maternal midline abdominal and uterine incisions, and amniotic fluid was collected. The fetus was sedated (10 mg/kg ketamine im), and after administering local anesthetic (2% lidocaine sc), we performed a tracheotomy and secured a 4.5-mm endotracheal tube in place. Lung fluid was aspirated by syringe, the animals were delivered, and the umbilical cord was cut. The animals that were not ventilated received a lethal dose of pentobarbital by intravenous injection. After delivery, lambs were weighed.
Premature newborn lambs with antenatal exposure of the intra-amniotic cytokines 7 or 15 d before delivery were ventilated for 40 min to evaluate lung function as described previously (13). Temperature was maintained at 39°C with an overhead warmer and plastic wrap. An arterial catheter was advanced to the level of the descending aorta via an umbilical artery, and lambs were anesthetized with pentobarbital sodium (15 mg/kg). Animals were placed on pressure-limited infant ventilators set to deliver 100% oxygen at a rate of 40 breaths per min, inspiratory time of 0.75 s, and positive end expiratory pressure (PEEP) of 3 cmH2O pressure. Peak inspiratory pressure (PIP) was initially set at 35 cmH2O. Tidal volume was monitored continuously with a neonatal respiration monitor (Acutronic, Baar, Switzerland). Arterial carbon dioxide partial pressure (PaCO2) was measured every 10 min, and PIP was adjusted to maintain adequate ventilation. Other ventilator settings were not altered during the study. The target PaCO2 was 4550 mmHg; however, animals were permitted to become hypercarbic when the target PaCO2 was not achieved with the maximum PIP of 40 cmH2O and/or maximum tidal volume of 10 ml/kg. Compliance was calculated by dividing tidal volume by ventilatory pressure (PIP-PEEP) and then normalized to body wt in kg (13). Ventilation efficiency index (VEI), an index that integrates ventilation with respiratory support, was calculated according to the formula VEI = 3,800/(PxFxPaCO2), where 3,800 is a carbon dioxide production constant, P is ventilatory pressure, and F is the ventilation rate (26). At 40 min, postdelivery animals were deeply anesthetized with pentobarbital sodium. We degassed the lungs by clamping the endotracheal tube for 5 min. The chest was opened, and the lung was inflated to 40 cmH2O for 1 min and lung volume (V40) was determined (13).
Ventilated premature newborn lamb lung responses to TNF-.
To evaluate the response of prematurely delivered newborn lung to TNF-
,
we delivered premature lambs at 130-d gestation age by Cesarean section as
described above. Before the initiation of ventilation, lambs were given 100
mg/kg surfactant (Survanta; Abbott Laboratories, Columbus, OH) mixed with 50
µg of TNF-
intratracheally or an equal volume of saline
(11). Lambs were ventilated
for 6 h as described above, except FIO2 was
changed to maintain the target PaO2 of 100200
mmHg (18).
Systemic effects of TNF- and IL-1
on
fetal sheep. Fetal sheep had intravascular catheters placed at 110-d
gestation for subsequent evaluation of the systemic effects of TNF-
and
IL-1
. At 117 d of gestation, a 50-µg bolus intravenous injection of
TNF-
(n = 3) or IL-1
(n = 3) was infused with
continuous monitoring of blood pressure and heart rate and collection of fetal
blood for pH, blood gas, and lactate measurements for 8 h after the infusion.
The results were compared with animals monitored similarly for 8 h after a
saline injection (n = 4).
Processing of lungs. We removed the lungs from the chest, weighed each lung, and used the left lung for bronchoalveolar lavage (BAL) by infusing and withdrawing a sufficient volume of saline at 4°C to fully distend the lungs three times with five separate saline volumes (13). The five BALF were pooled. Tissue from the right middle lobe was used for a dry-wet wt ratio measurement. Tissue from the right lower lobe was frozen in liquid nitrogen for later analysis.
Assessments of inflammation. Amniotic fluid was incubated for 30 min at 37°C with 20 mg/ml N-acetyl-L-cysteine, 1 U/ml neuraminidase, and 20 U/ml hyaluronidase (Sigma) to reduce the high viscosity. Cells were isolated from aliquots of amniotic fluid and BALF by centrifugation at 500 g for 10 min, and the pellets were resuspended in PBS. After total cell counts by trypan blue exclusion to identify live cells, differential cell counts were performed on cytospin preparations stained with Diff-Quick (Dade Behring, Düdingen, Switzerland). Without liquefying the amniotic fluid, we could not collect cells by centrifugation for cell counts.
A capture ELISA assay was used to measure IL-1 concentration in
amniotic fluid. An IgG fraction prepared from rabbit anti-sheep IL-1
antiserum was used for the primary antibody, and guinea pig anti-sheep
IL-1
was the secondary antibody. Standard curves were constructed from
the absorbance of known amounts of sheep IL-1
with and without amniotic
fluid from the control groups. Addition of the amniotic fluid did not alter
the values of IL-1
. The ELISA assay was specific for sheep IL-1
and did not detect recombinant sheep IL-1
. Standard curves were
sensitive from 0.1 to 80 ng/ml with a correlation coefficient of 0.99 for all
assays.
Total RNA was isolated from the right lower lobe of the lung and from cell
pellets of BALF by guanidinium thiocyanate-phenol-chloroform extraction
(1). Ribonuclease protection
assays were performed with total RNA from lung tissue and cell pellets as
described previously (17). In
brief, RNA transcripts of ovine interleukins (IL-1, IL-6, IL-8) and
ovine ribosomal protein L32 as a reference RNA were synthesized with
[32P]UTP (Life Sciences Products, Boston, MA) using SP6 or T7
polymerase (Promega, Madison, WI). Aliquots of 10 µg of RNA were hybridized
with excess radiolabeled probes for cytokines and L32 at 55°C for 18 h.
Single-stranded RNA was digested with RNase A/RNase T1. Protected fragments
were electrophoresed on a 6% polyacrylamide-urea sequencing gel and visualized
by autoradiography. The protected bands were quantified on a PhosphorImager
using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Saturated phosphatidylcholine. Lipids were extracted from aliquots of the BALF with chloroform-methanol. Saturated phosphatidylcholine (Sat PC) was isolated from lipid extracts by neutral alumina column chromatography after exposure to osmium tetroxide (23). Sat PC was quantified by phosphorus assay (3).
Surfactant protein mRNA. The mRNA for surfactant protein (SP)-A, SP-B, and SP-C were measured using S1 nuclease protection assays as previously described (2). In brief, an excess of linearized probes for the ovine surfactant proteins and L32 were end labeled with [32P]ATP and hybridized at 55°C with 3 µg of total RNA from lung tissue. After digestion with S1 nuclease, the protected fragments were resolved on 6% polyacrylamide 8-mol urea sequencing gels, visualized by autoradiography, and quantified.
Statistical analyses. Unless otherwise stated, values are given as means ± SE. Normally distributed data were compared between control and treated groups by one-way ANOVA, and post hoc pairwise comparisons were made using Dunnett's procedure. For data not normally distributed, global comparisons were made by Kruskal-Wallis ANOVA on ranks, and post hoc pairwise comparisons were made using Dunn's procedure. Statistical significance was accepted for P < 0.05.
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RESULTS |
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Lungs from adult mice responded to intratracheal instillation or
intravascular injections of TNF- or endotoxin with increased total cell
counts in BALF, which were primarily monocytes
(Fig. 2). The cell responses to
TNF-
and endotoxin were similar. In contrast to the intratracheal
endotoxin, TNF-
increased the IL-1
in the mouse lungs eightfold
above control levels (P < 0.05). Thus recombinant sheep
TNF-
was biologically active when tested in adult mice.
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Responses of fetal sheep to intra-amniotic TNF- and
IL-1
. Control animals had very few inflammatory cells in amniotic
fluid, and intra-amniotic TNF-
did not increase cell numbers
(Fig. 3A). In
contrast, intra-amniotic IL-1
induced a striking increase in monocytes,
lymphocytes, and neutrophils within 24 h
(Fig. 3B). The
neutrophils decreased at 7 d and returned to control values by 15 d. However,
the monocytes and lymphocyte numbers remained elevated in amniotic fluid 15 d
after the intra-amniotic IL-1
exposure.
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Intra-amniotic TNF- did not increase IL-1
in amniotic fluid
at 5 h, 2 d, or 7 d as measured by ELISA. In contrast, after intra-amniotic
injection of 100 µg of IL-1
, the amniotic fluid levels of
IL-1
were 12 ± 2 ng/ml at 24 h, 0.4 ± 0.2 ng/ml at 7 d,
and not detectable at 15 d.
Intra-amniotic TNF- increased monocytes in BALF within 5 h, but
there were no significant increases in neutrophils at 5 h or 2 d
(Fig. 3C). Compared
with the ventilated controls, intra-amniotic TNF-
significantly
increased lymphocytes and neutrophils at 7 d, although these increases were
small. In contrast, intra-amniotic IL-1
resulted in large increases in
monocytes, lymphocytes, and neutrophils in BALF within 24 h, and these cells
remained elevated relative to the ventilated control values 15 d after the
intra-amniotic IL-1
(Fig.
3D). Cell numbers in BALF from ventilated lambs 15 d
after intra-amniotic IL-1
were lower than after the 7-d exposure
interval. The fetal lung had a detectable but minimal inflammatory cell
response to TNF-
but a large response to IL-1
.
Proinflammatory cytokine mRNA levels for IL-1, IL-6, and IL-8 in the
lung were not increased 5 h or 2 d after intra-amniotic TNF-
compared
with the saline controls (Fig.
4). Ventilation for 40 min increased these cytokine mRNAs
similarly for both the saline control and the 7-d TNF-
groups.
Intra-amniotic TNF-
had no effect on IL-1
, IL-6, or IL-8 mRNA
expression in the fetal lung.
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TNF- had no effect on the arterial pH and blood gas values,
compliance, VEI, V40, or the amount of Sat PC in BALF for the lambs
used for the assessments of lung maturation after 40 min of ventilation
(Table 1,
Fig. 5). In contrast, blood gas
values after 40 min of ventilation for the animals treated with intra-amniotic
IL-1
were significantly better than for the control lambs. The
IL-1
was associated with the need for less ventilatory pressure,
improved respiratory system compliance and VEI, and large increases in
V40. Intra-amniotic IL-1
also increased the amount of Sat PC
recovered by BAL. Cord plasma cortisol and dry-wet lung wt ratios were not
affected by any of the antenatal treatments. Intra-amniotic TNF-
did
not increase surfactant protein mRNAs for SP-A, SP-B, or SP-C, and SP-B mRNA
was decreased 7 d after the intra-amniotic TNF-
(Fig. 6). Expression of SP-A
mRNA and SP-B mRNA was increased eight- and sixfold, respectively, 24 h after
intra-amniotic IL-1
injection and returned to control levels at
15d.
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Systemic effects of cytokines in fetal sheep. Chronically
catheterized fetal sheep were given bolus intravenous doses of 50 µg of
TNF- or IL-1
. Intravenous TNF-
had no effect on fetal
blood gas pH values or blood lactate levels
(Fig. 7). Mean blood pressures,
heart rates, and WBC also did not change. In contrast, the 50-µg dose of
IL-1
decreased PO2 and pH and increased blood
lactate. Heart rate increased from a control value of 184 ± 19
beats/min to 235 ± 17 beats/min at 1 h (P < 0.05). The WBC
count fell from 5.0 ± 1.1 x 106 to 0.46 ± 0.03
x 106 cells/ml (P < 0.05) at 4 h. IL-1
given by intravascular infusion caused a shock-like syndrome in the fetus.
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Responses of ventilated premature newborns to intratracheal
TNF-. Prematurely delivered newborns at 130-d gestation were given
TNF-
mixed with surfactant in the airways before first breath, and lung
responses to TNF-
were measured after 6-h ventilation
(Table 2). Intratracheal
TNF-
did not affect pH, blood gas, ventilatory pressure, compliance,
VEI, or V40 values measured from the pressure-volume curves. There
were significant increases in neutrophil numbers in BALF
(Fig. 8). IL-1
and IL-8
mRNA in lung tissue and in BALF cells were significantly increased by
intratracheal TNF-
after 6-h ventilation
(Fig. 9). TNF-
mRNA was
also increased in lung tissue in the TNF-
-exposed group. The mRNA for
SP-C was decreased by the 6-h TNF-
exposure with no changes detected in
SP-A or SP-B mRNA (Table 2). In
contrast to the lack of fetal response to intra-amniotic TNF-
,
intratracheal TNF-
increased several indicators of lung inflammation in
ventilated premature newborns.
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DISCUSSION |
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Median values reported for IL-1 in women with chorioamnionitis and
women colonized with Ureaplasma urealyticum were
3.5 ng/ml in
amniotic fluid with some values as high as 80 ng/ml
(35). If one assumes an
amniotic fluid volume of
1 l for sheep at 120-d gestation, the 100 µg
of IL-1
given into the amniotic fluid in our study would yield a
concentration of
100 ng/ml or
30 times the median values reported
for clinical samples. IL-1
is usually associated on the cell surface
and not detectable in the fluid
(6). The chorioamnionitis
caused by 10 mg of endotoxin resulted in IL-1
levels in amniotic fluid
of 0.14 ± 0.05 ng/ml at 2 d and 1.3 ± 0.4 ng/ml at 5 d, with
IL-1
not detectable at 24 h and 15 d in the fetal sheep model. Median
TNF-
levels in amniotic fluid of women with chorioamnionitis were
reported to be
0.1 ng/ml
(34), and we gave an average
dose of 140 µg, resulting in an initial concentration in the amniotic fluid
in excess of 1,000 times the median concentration measured clinically.
Although these estimates are imprecise, we gave a large dose of TNF-
relative to the concentrations measured in clinical samples.
TNF- is an early response cytokine that is a potent inducer of a
generalized inflammatory response. Adult sheep mount a strong inflammatory
response to intravenous injections of recombinant human or ovine TNF-
(15,
21). In the adult sheep,
TNF-
and endotoxin induce lung inflammation, alter lung mechanics,
increase lung vascular permeability, and alter systemic hemodynamics. We
anticipated that TNF-
would be as potent as IL-1
as a
proinflammatory mediator in the fetal or newborn lamb and would induce effects
similar to endotoxin (19).
Bioactivity of the recombinant ovine TNF-
was confirmed in vitro and in
vivo. The cytokine induced the phosphorylation of MAPK and proinflammatory
cytokines in macrophages from adult sheep. We did not test the responses of
monocytes/macrophages from immature sheep because these animals have very few
cells that can be recovered by BAL. The TNF-
also induced lung
inflammation in mice similarly to endotoxin and increased IL-1
in the
mouse lung, demonstrating in vivo bioactivity.
We expanded our studies of IL-1 and demonstrated that it was a
potent inducer of chorioamnionitis and lung inflammation within 24 h and that
indicators of inflammation and lung maturation persisted for 15 d. IL-1
also caused a systemic inflammatory response when administered intravenously
to fetal sheep. Therefore, the animals were clearly capable of developing the
anticipated inflammatory and systemic responses to this potent proinflammatory
cytokine.
Relative to the responses that we previously demonstrated for IL-1
and endotoxin, fetal sheep at 125-d gestation are quite unresponsive to
TNF-
. Intra-amniotic TNF-
did not cause chorioamnionitis, but it
did induce small increases in inflammatory cells in the fetal lung. The
assessments of inflammation in response to TNF-
at 5 h and 2 d
bracketed the time of severe inflammation induced by IL-1
. Because
inflammatory responses to intra-amniotic endotoxin and IL-1
persist for
weeks, we do not think that we missed an inflammatory response of the fetal
lung to TNF-
. More striking increases in neutrophils in BALF were
measured 6 h after intratracheal TNF-
in 130-d gestation ventilated
preterm lambs, and these increases were accompanied by increases in cytokine
mRNA expression in the lungs. These small responses demonstrate that the
preterm sheep is not completely unresponsive to TNF-
. Nevertheless, the
very modest responses demonstrate that TNF-
is not likely to be a major
contributor to chorioamnionitis and inflammation of the fetal or premature
newborn sheep lung. No similar information is available for the human,
although TNF-
is increased in preterm infants and ventilated baboons
that subsequently develop BPD
(5,
16). Our study demonstrates
that mediators of biological effects in the preterm cannot be extrapolated
from information obtained from adult animals.
There is no specific information about how TNF- affects the fetus
outside of its presence as a component of inflammatory responses. Chronic
overexpression of TNF-
in transgenic mice with the lung-specific SP-C
promotor resulted in progressive fibrosing alveolitis
(24). The mice that died at
birth seemed to respond to TNF-
with lung injury during late gestation.
Our results with sheep suggest a late gestational development of the receptors
and/or cellular elements responsible for TNF-
signaling because we
found no responses to intravascular infusion of fetuses with TNF-
at
118-d gestation, very minimal responses to fetal exposures at preterm delivery
at 125-d gestation, and a cytokine and inflammatory cell response at 130-d
gestation.
We were particularly interested in the possible effects of TNF- on
the surfactant system. In the mature lung, TNF-
decreases the mRNA for
both SP-B and SP-C (1,
27). The effects of
IL-1
on the surfactant proteins depend on the developmental stage of
the lung. In the preterm fetal sheep in vivo, IL-1
induces a persistent
increase in the surfactant protein mRNAs as it does in preterm rabbits and
explants of rabbit lungs (4,
7,
32). However, at later
gestation and after term, IL-1
decreases surfactant protein mRNA
levels. In the fetal sheep, we found a significant decrease in SP-B mRNA 7 d
after intra-amniotic TNF-
. In the ventilated preterm lamb, the
TNF-
decreased SP-C mRNA. These effects are consistent with the effects
of TNF-
on the mature lung.
These studies were done on sheep without exposure to other factors that
might mature the TNF- response pathway, which is an important qualifier
to the conclusion that fetal sheep and newborn preterm lambs respond only
minimally to TNF-
. For example, chronic indolent chorioamnionitis may
persist for weeks without the onset of preterm labor, and it is associated
with increased fetal cortisol levels in the human
(31). Women at risk of preterm
delivery routinely receive antenatal glucocorticoid treatments that not only
induce lung maturation but may alter fetal immune responses as well
(9). A productive research area
will likely be characterizing how antenatal inflammatory exposures and
maturational treatments alter subsequent immune and inflammatory responses in
the fetus. For example, we recently reported that intra-amniotic endotoxin
given to sheep 30 d before preterm delivery altered the inflammatory responses
to mechanical ventilation
(10). Although
anti-TNF-
therapies have been explored for a variety of inflammatory
diseases, this study suggests that TNF-
may not be an important
mediator in the preterm.
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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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