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Original Studies |
Centre for Immunology (A.G.M., D.A.B., W.D.F., A.R.B., P.K.B., M.L.C.M., P.K.R., S.N.B.), St. Vincent’s Hospital and University of New South Wales, Sydney, New South Wales, Australia; Department of Obstetrics and Gynecology (E.M.W.), Monash University, Clayton, Victoria, Australia; and Prince Henry’s Institute of Medical Research (L.A.S.), Clayton, Victoria, Australia
Address all correspondence and requests for reprints to: Dr. Samuel N. Breit, Centre for Immunology, St. Vincent’s Hospital, Victoria Street, Sydney, New South Wales 2010, Australia. E-mail: s.breit{at}cfi.unsw.edu.au
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytokines in the TGF-ß superfamily are thought to have multiple functions during pregnancy. The ability of the TGF-ß isoforms to modulate cell-cell adhesion, cell migration, and tissue remodeling has led some authors to suggest that this molecule may control trophoblast invasion and implantation early in pregnancy. Other possible roles include regulation of fetal growth and suppression of the maternal immune responses.
Placental cells are a major source of TGF-ß superfamily cytokines and are regulated by them. For example, activin suppresses the production of inhibin and enhances secretion of progesterone, human CG (hCG), and GnRH by placental cells (13). Inhibin suppresses placental hCG, GnRH, and activin-induced progesterone release (13), whereas TGF-ß1 suppresses human placental lactogen (PL) production. Activin and TGF-ß3 have also been shown to have opposing effects in regulating extravillous trophoblast invasion in early pregnancy (11, 14). These findings suggest that TGF-ß1, TGF-ß3, activin, and inhibin regulate the growth and differentiation of the placenta in an autocrine manner. TGF-ß1, activin, and inhibin are also present in the embryo proper, where they have been demonstrated to regulate growth and differentiation. In particular, TGF-ß superfamily members are well known for their ability to promote mesoderm induction.
TGF-ß superfamily proteins may promote fetal survival. Experimental evidence suggests that the amniotic fluid concentration of the proinflammatory cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor rise during labor. Furthermore, proinflammatory cytokine production accompanying intrauterine infection has been associated with fetal rejection or preterm labor (15, 16, 17). TGF-ß1 and inhibin have been shown to suppress production of proinflammatory cytokines by macrophages and lymphocytes, respectively (18, 19), whereas activin has proinflammatory effects on macrophages and the amnion (20, 21). This has led to the suggestion that TGF-ß1 and inhibin promote fetal survival by suppressing the production of potentially harmful proinflammatory cytokines by the maternal immune system.
We have recently cloned and characterized a divergent member of the TGF-ß superfamily, MIC-1 (1). Its expression is associated with macrophage activation. Like several other members of this superfamily, MIC-1 messenger RNA (mRNA) was identified as being highly expressed in placenta (22, 23, 24) and may also be involved in pregnancy. To better understand the role of MIC-1 in pregnancy, we have developed a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for its quantitation. Using this assay, we investigated the temporal relationship between maternal serum MIC-1 concentrations and gestational age. We also measured its concentration in amniotic fluid and placental extracts. To further delineate the origins of this cytokine, we have also assessed the capacity of a placental trophoblastic cell line (BeWo) to synthesize MIC-1.
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Materials and Methods |
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Serum samples were obtained from 22 healthy pregnant women with normal singleton pregnancies and on no medications. Approval from The Human Research and Ethics Committee of Monash Medical Center was obtained for all human sample collection. In each case, gestational age was determined by an early pregnancy ultrasound scan. All women subsequently had a normal vaginal delivery, at term (37–41 weeks), of a healthy normally grown infant. Serum samples were collected from 6 women between 10–14 weeks of pregnancy, and 8 women between 26–30 weeks and 37–40 weeks of pregnancy. The time periods indicated correspond to the end of each trimester. Samples corresponding to each trimester were pooled before measurement of MIC-1 levels. Serial maternal serum samples were also taken, on a weekly basis, from another 4 women, from 30 weeks of gestation to delivery. Again, all 4 women were healthy, with a normal singleton pregnancy, and had a normal vaginal delivery (at term) of a normal healthy infant. In addition, amniotic fluid was obtained from 10 women undergoing amniocentesis, at 15–17 weeks of gestation, for fetal karyotyping. In all cases, the indication for karyotyping was advanced maternal age (>37 yr). Amniotic fluid samples were also pooled before measurement of MIC-1 levels. Normal nonpregnant serum samples were obtained from 6 healthy women with a regular menstrual cycle and not on hormonal contraception. These women were in the luteal phase of their cycle when sampled.
Placental extracts
Between 100–150 mg placental tissue, obtained from normal term deliveries, (rinsed 4–5 times in saline solution and frozen in liquid nitrogen and stored at -80 C) was homogenized in 1 mL PBS. Homogenates were centrifuged at 10,000 rpm for 30 sec, and the supernatant was transferred to tubes. Total protein was measured by the BCA total protein assay (Pierce Chemical Co.), following the manufacturer’s instructions. BSA solutions, ranging between 0–1000 µg/mL, were used as standard solutions.
BeWo cell culture
The human choriocarcinoma trophoblastic cell line (BeWo) was purchased from ATCC (Rockville, MD). Cells were seeded into 96-well tissue-culture plates, at 5000 cells per well, in 250 µL DMEM (containing 4.5 g/L D-glucose, 110 mg/L sodium pyruvate, 0.584 g/L L-glutamine, 4 mg/L pyridoxine hydrochloride) (Life Technologies, Inc.) and 1x Nutridoma-SR (Roche Molecular Biochemicals, Germany) and cultured at 37 C in the presence of 5% carbon dioxide for 1–5 days. The culture plates were then spun at 1000 rpm for 10 min, and the supernatant was removed and stored at -20 C until quantification of MIC-1.
RT-PCR analysis of MIC-1 mRNA synthesis
Total RNA was isolated from BeWo cell monolayers, in 96-well plates, using Tri-Pure Reagent (Roche Molecular Biochemicals), method as provided by the manufacturer. RT was carried out in a total reaction vol of 20 µL using 1 µg RNA, a poly(T)15 primer, and 50 U of Expand Reverse Transcriptase (Roche Molecular Biochemicals) using the manufacturer’s recommended conditions. A 5-µL aliquot of the RT reaction was amplified in a PCR reaction using Pfu polymerase (Promega Corp.) and primers MSB-1 (5'-AGGACCTGCTAACCAGGCTGCGGGCCAACCAGAGC-3') and MSB-5 (5'-GGCTAACAAGTCATCATAGGTCTGGAGCGACAC-3'), which flank the single intron of MIC-1. PCR conditions were as follows: an initial denaturation step at 95 C for 1 min, followed by 35 cycles at 95 C for 30 sec, 60 C for 30 sec, and 72 C for 2 min. An RT reaction in which the RNA was omitted was used as a negative control, and a plasmid carrying the MIC-1 pre-pro-MIC/FLAG coding sequence (1) was included as a positive control. PCR products were separated on 0.8% (wt/vol) agarose gels.
Recombinant human (rh)MIC-1
Recombinant MIC-1 was expressed in the yeast Pichia pastoris and purified to homogeneity, using a multistep procedure, culminating in reverse-phase high-performance liquid chromatography (25).
Generation of MIC-1 antibodies
The sheep anti-MIC-1 polyclonal antibody (PAb) 233B3 was generated by immunization with rhMIC-1 in Complete Freund’s Adjuvant. Additional boosts were given over a period of 6 months, and the sheep were bled 10 days after the final injection. An enriched IgG fraction of normal sheep serum and 233B3 were prepared by caprylic acid precipitation, followed by ammonium sulfate precipitation. The IgG-enriched 233B3 fraction was designated 233B3-P.
The mouse anti-MIC-1 monoclonal antibody (MAb) 13C4H3-secreting hybridoma was generated from mice immunized with rhMIC-1. Hybridomas were cultured in DMEM (Life Technologies, Inc.) containing 4.5 g/L D-glucose, 110 mg/L sodium pyruvate, 0.584 g/L L-glutamine, and 4 mg/L pyridoxine hydrochloride supplemented with 20% FCS (CSL Melbourne). For MAb collection, the hybridomas were transferred into fresh DMEM-hi glucose, supplemented with Nutridoma-SR (Roche Molecular Biochemicals), for 7 days. The culture supernatants were spun at 2000 rpm for 10 min, to remove cell debris, and frozen until used. The sensitivity of the PAb and MAb preparations were examined by direct ELISA.
Direct ELISA
Ninety-six-well Maxisorp ELISA plates (Nunc) were coated (100 µL/well) with either 18 ng/mL rhMIC-1 or 20 ng/mL rhTGF-ß1 (R&D Systems) in coating buffer (0.1 mol/L carbonate in distilled H20, pH 9.4–9.8) at 4 C for 24 h. Plates were washed three times with 300 µL/well wash buffer [PBS containing 0.05% (vol/vol) Tween-20 (Sigma)]. Nonspecific binding was blocked with addition of 250 µL/well of 1% (wt/vol) BSA (Roche Molecular Biochemicals), in PBS for 2 h at 37 C. Hybridoma-conditioned, serum-free media, containing the MAb 13C4H3; sheep PAb 233B3-P diluted 1:500,000 in antibody diluent (Ab dil) [PBS containing 1% (wt/vol) BSA and 0.05% (vol/vol) Tween-20]; culture media conditioned by the mouse myeloma cell line SP2/0; DMEM+Nutridoma; IgG-enriched normal sheep serum diluted 1:500,000 in Ab dil; 200 ng/mL mouse IgG1 (R&D Systems) in DMEM+Nutridoma, or Ab dil alone were then added to the plates (100 µL/well) and incubated for 1 h at 37 C. The plates were washed three times, followed by the addition of 100 µL/well biotinylated donkey antisheep IgG (Jackson ImmunoResearch Laboratories, Inc.) or biotinylated goat antimouse IgG (Jackson ImmunoResearch Laboratories, Inc.) diluted 1:10,000 in Ab dil and incubated for 1 h at 37 C. The plates were washed three times, and 100 µL/well of horseradish peroxidase-conjugated strepavidin (Genzyme), diluted 1:2000 in Ab dil, was added to the plates and incubated for 30 min at 37 C. Plates were washed four times, followed by the addition of 100 µL/well of peroxidase substrate (1 mg/mL o-phenylenediamine dihydrochloride (Sigma) in 0.05 mol/L phosphate-citrate buffer containing 0.014% H2O2, pH5.0 (Sigma). Color development was allowed to proceed for 5–15 min and was terminated by the addition of 100 µL/well of 4N H2SO4. The absorbance was measured at 490 nm in a microplate reader (Pasteur Diagnostics).
MIC-1 sandwich ELISA
A MIC-1 sandwich ELISA was established using the mouse MAb 13C4H3 for antigen capture; and the sheep PAb 233B3-P, for detection. The optimum concentration of both antibodies was determined, then used for all subsequent studies. Ninety-six-well Maxisorp ELISA plates were coated with MAb 13C4H3 supernatant diluted 1:5 (final Ig concentration was approximately 20 ng/mL) in coating buffer at 4 C for 24 h. ELISA plates were then washed three times with 300 µL/well of wash buffer. Nonspecific binding was blocked with 250 µL/well of 1% (wt/vol) BSA in PBS for 2 h at 37 C. rhMIC-1 standards, tissue culture supernatant, maternal serum, placental extracts, or amniotic fluid in Ab dil were then added to the plates (100 µL/well) and incubated for 1 h at 37 C. The plates were washed three times, followed by the addition of 100 µL/well of the sheep PAb 233B3-P diluted 1:5000 in Ab dil and incubated for 1 h at 37 C. ELISA plates were then washed three times, and 100 µL/well of biotinylated donkey antisheep IgG diluted to 1:5000 in Ab dil was added and incubated for 1 h at 37 C. The plates were then developed, as for the direct ELISA. The concentration of hMIC-1 in the samples was determined by comparison with the rhMIC-1 standard curve. The standard curve was constructed using standard curve-fitting software supplied with the microplate reader (Pasteur Diagnostics). The level of rhMIC-1 in the standard curve was determined on the basis of a comparison of this standard to a master standard of highly purified recombinant MIC-1. The master standard protein concentration was determined by an average of eight estimations of total amino acid composition. All samples were assayed in triplicate on at least two occasions. Results are presented as the mean ± SD.
Immunohistochemistry
Term placenta was fixed overnight in 10% formaldehyde (vol/vol)
in PBS and paraffin embedded. Four-micrometer sections were placed on
sialine-coated slides. Sections were brought to water and microwaved
for 20 min in 0.1 mol/L citrate (Univar) buffer, pH 6.0.
Nonspecific binding was blocked using 4% BSA (wt/vol) in PBS for
2 h at 21 C. Slides were incubated with the sheep PAb 233B3 or
IgG-enriched normal sheep serum, diluted 1:5000 in 4% BSA (wt/vol) in
PBS, overnight at 4 C. To block nonspecific peroxidase activity,
sections were incubated in 0.03% peroxide (Analar) (vol/vol) in
methanol (Analar) for 30 min at 21 C. Biotinylated donkey antisheep IgG
(Jackson ImmunoResearch Laboratories, Inc.) diluted
to 1:200 in 4% BSA (wt/vol) in PBS and was incubated with slides for
45 min at 21 C. Streptavidin-HRP (DAKO Corp., large volume
kit) was incubated with samples for 10 min at 21 C, and the slides were
developed using DAB (Pierce Chemical Co.), 1:10 (vol/vol),
in stable peroxide buffer (Pierce Chemical Co.). A light
counterstain was performed by incubating the slides in Mayer’s
hematoxylin solution (Sigma) for 2 min, followed by
Scott’s bluing solution for 30 sec. Three washes with wash buffer
[Tris-phosphate 0.1 mol
(pH 7.6) buffer 0.1% Tween-20] were
performed between all steps, after the primary antibody incubation,
until counter staining, where tap water was used.
Immunoprecipitation
MAb 13C4H3 was adsorbed to protein-A Sepharose beads. Serum and medium samples (1 mL) were immunoprecipitated by incubating with beads overnight at 4 C and then washing 5 times with PBS containing 1% (vol/vol) Triton X-100. Bound proteins were eluted, using nonreducing SDS sample buffer, and analyzed by SDS-PAGE (26), followed by immunoblot analysis (1) with the sheep PAb 233B3 or MAb 13C4H3, in the presence or absence of 10 g purified recombinant MIC-1, or sheep preimmune serum. The 233B3 and sheep preimmune serum mix was used at a dilution of 1:14,000. Donkey antisheep IgG-biotin, at a dilution of 1:2,000 was used as the secondary antibody. Immunoblot analysis with Mab 13C4H3 was undertaken using serum free hybridoma supernatant, diluted 1:1 with PBS containing 0.1% Tween 20. In this case, goat antimouse IgG-biotin (at a dilution of 1:2,000) was used as the secondary antibody.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of the sheep PAb 233B3-P and the mouse MAb 13C4H3 to
bind to rhMIC-1 was examined by direct ELISA. It was found that both
undiluted tissue culture supernatant (containing the MAb 13C4H3) and
the sheep PAb 233B3-P (at a dilution of 1:500,000 in Ab dil) bound
strongly to 1.8 ng immobilized rhMIC-1 (Fig. 1A
). No reaction was observed between
rhMIC-1 and culture media conditioned by the mouse myeloma cell line
SP2/0, unconditioned culture media, mouse IgG1, Ig-enriched normal
sheep serum, or Ab dil. Minimal background binding to uncoated wells
was observed for all samples examined. No reactivity was detected when
either 13C4H3 or 233B3-P were incubated with immobilized
rhTGF-ß1.
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). All the
MIC-1 antibodies specifically recognized the 25-kD dimeric MIC-1.
Additionally, blocking of the antibodies was performed by preincubating
the antibody with purified recombinant MIC-1, before Western Blot
analysis. This greatly reduced the interaction of the antibody with the
MIC-1 specific 25-kD band, confirming specificity of the antibodies Mab
13C4H3 (Fig. 1B2) and 233B3-P (Fig. 1B4). Furthermore, those antibodies
tested failed to recognize inhibin (Fig. 1B, 4d and 5d), another member
of the TGF-ß superfamily, elevated in pregnancy.
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A sandwich ELISA, employing MAb 13C4H3 and the PAb 233B3-P, was
established. This assay could accurately quantify rhMIC-1 in the range
of about 20–900 pg/mL (Fig. 2). Above
this range, there was saturation demonstrated with the assay (Fig. 3). To examine the effect of factors
present in human serum and culture media on estimation of this
cytokine, 900 pg/mL rhMIC-1 was added to Ab dil containing either 10%
(vol/vol) normal human serum or 10% (vol/vol) DMEM+FBS, and then
quantified. It was found that the sandwich ELISA was accurate to within
5% of the correct value. Run-to-run variation was less than 5%.
TGF-ß1 and inhibin, assayed in the sandwich ELISA at a concentration
of 500 pg/mL each, yielded optical density readings no different from
negative controls in the sandwich ELISA system, indicating the total
absence of cross-reactivity. Parallelism of the sandwich ELISA was
confirmed with serial dilutions of culture and serum samples diluted in
culture medium and Ab dil (Fig. 2B).
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Pooled serum samples were diluted between 1:5 and 1:20 in Ab dil
before MIC-1 quantitation by sandwich ELISA. It was determined that
pooled normal human sera contained approximately 0.36 (± 0.04) ng/mL
MIC-1 (Fig. 4A). In pooled maternal
serum, the MIC-1 concentration was found to increase dramatically
during pregnancy. Maternal serum samples, corresponding to the first
trimester, contained approximately 6.3 (± 0.02) ng/mL MIC-1,
which rose to 12.2 (± 0.5) ng/mL during the second trimester and
peaked at 15.3 (± 1.3) ng/mL during the third trimester. The rise in
serum MIC-1 during pregnancy was found to be statistically significant
(P < 0.001), using the Student’s t
test.
Immunoprecipitation was used to confirm the presence of MIC-1 in pooled
maternal serum samples during pregnancy. MIC-1 was visualized by
immunoprecipitation with MAb 13C4H3, followed by immunoblot analysis
with PAb 233B3-P. A band corresponding to the disulphide-linked mature
MIC-1 peptide (approximately 25 kDa) can be observed in the second and
third trimester serum samples (Fig. 4B, lanes 3–4). The highest level
of MIC-1 was found in the third trimester sample. No similar band was
observed in normal serum or the sample corresponding to the first
trimester, presumably because of the lower sensitivity of immunoblot
analysis (Fig. 4B, lanes 1–2).
Maternal serum MIC-1 concentrations were also examined in serial
samples from four pregnant women. At 30 weeks of gestation, serum from
all four women examined contained approximately 3.6 ng/mL MIC-1 (Fig. 5). Maternal serum MIC-1 levels were
found to increase from 30 weeks of gestation until birth. Subjects
designated MH and JB exhibited a slight decrease in MIC-1 maternal
serum levels over the last week of pregnancy.
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In additional to maternal serum, amniotic fluid collected from 10
women during the second trimester for karyotyping purposes was pooled
before quantification of MIC-1 levels by sandwich ELISA. It was
determined that the pooled amniotic fluid sample contained
approximately 13.68 (± 0.16) ng/mL MIC-1 (Fig. 4A).
Immunoprecipitation and Western blot analysis of pooled amniotic fluid
revealed a band of approximately 25 kDa, which corresponds to the
disulphide-linked mature MIC-1 peptide (Fig. 4B, lane 5).
Placental MIC-1 is detected by immunohistochemistry
Term placental immunohistochemistry reveals that MIC-1 is produced
predominantly by the trophoblastic cells. Although there is production
by stromal cells, the result shown (Fig. 6.) indicates that the trophoblast cells
are the major producers of MIC-1 in the placenta.
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Whole-tissue Northern blot analysis indicates that MIC-1
transcripts are expressed abundantly in the placenta
(22, 23, 24), suggesting that the placenta is a major source
of circulating MIC-1 in the serum of pregnant women. To test further
the presence of MIC-1 in placental material, we have examined five
human placental extracts, for the presence of MIC-1, by sandwich ELISA.
All five samples were found to be positive for MIC-1 (Fig. 7), ranging in concentration from 5–54
ng/mL.
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Because high levels of MIC-1 were detected in placental extracts
and in placental immunohistochemistry, it seemed likely that the
placental trophoblastic cell line, BeWo, may also produces this
cytokine. To confirm this, we examined tissue culture media,
conditioned by BeWo cells under resting conditions, for the presence of
secreted MIC-1, by sandwich ELISA. BeWo-conditioned media (24 h)
contained approximately 21.6 (±2.95) ng/mL MIC-1 (Fig. 8A). The concentration of MIC-1 in the
culture media, after a 5-day incubation, increased to approximately 117
(±7.2) ng/mL. The ability of unstimulated BeWo cells to secrete MIC-1
was also examined by immunoprecipitation and Western blot analysis.
High levels of secreted mature MIC-1, as indicated by a band at
approximately 25 kDa, were observed in media conditioned by BeWo cells
for 5 days (Fig. 8B). Additional bands, migrating at 55-kDa and
12.5-kDa bands, were observed, which most likely represent incompletely
processed MIC-1 hemidimer and monomer, respectively. Culture media,
which had not been exposed to BeWo cells, contained no detectable MIC-1
when examined by sandwich ELISA or by immunoprecipitation.
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was in fact MIC-1,
the detection antibodies 233B3 and Mab 13C4H4 were preincubated with
purified recombinant MIC-1, as previously described, and compared with
nonabsorbed detection antibodies. This resulted in markedly diminished
staining (Fig. 1B: 1a, 2a, 4a, and 5a), confirming that the band does
represent MIC-1.
RT-PCR was used to confirm the presence of the MIC-1 transcript in
unstimulated BeWo cells. Total RNA was extracted from BeWo cells
cultured for 24 h and subjected to RT-PCR as described. A single
product of 0.4 kbp was observed, indicating that the MIC-1 transcript
was present in BeWo cells (Fig. 8C). No product was detected in the
absence of BeWo or plasmid DNA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although elevated levels of MIC-1 in maternal serum do not necessarily indicate fetal exposure, detection in the amniotic fluid does. These findings indicate that the developing fetus is exposed to substantial concentrations of MIC-1. The level of MIC-1 in amniotic fluid was comparable with that present in second- and third-trimester maternal serum and was well in excess of the serum level of nonpregnant women. The fetus is bathed in amniotic fluid, and there is significant recirculation of this ingested fluid, which is excreted renally during the pregnancy. This provides multiple routes of fetal exposure to MIC-1, topically to the gut and epidermis, as well as systemically through ingested amniotic fluid.
It has previously been demonstrated, both in our laboratory (22) and by others (23, 24, 28, 29), that the MIC-1 transcript is expressed most abundantly in placenta. To determine the source of MIC-1 in pregnancy, we measured MIC-1 concentrations in human placental extracts and undertook immunohistochemical localization. Placental extracts contain substantial amounts of MIC-1, in excess of pooled serum or amniotic fluid levels. Using immunohistochemistry, we confirmed previous observations (29) that the major source of placental MIC-1 is the trophoblast. We have also shown that the BeWo human trophoblastic cell line synthesizes and secretes MIC-1 constitutively. Together, these findings suggest that the trophoblastic cells within the placenta are a major source of the MIC-1 present in maternal serum and amniotic fluid. However, the localization of the MIC-1 transcript and protein to the developing epidermis, in day-18 rat embryos (29), suggests that the embryo may also contribute to the MIC-1 levels observed.
The precise role of MIC-1 during pregnancy is unknown. Experimental evidence suggests that MIC-1 has immunomodulatory activity. For example, rhMIC-1 inhibits the release of proinflammatory cytokines from LPS-activated macrophages (1) and suppresses the formation of erythrocyte and granulocyte/macrophage cell lineages from normal human nonadherent T-cell-depleted marrow cells (28). These findings suggest that MIC-1 may have wider modulatory effects, possibly suppressing both the development of the monocyte/macrophage lineage and/or their ability to produce proinflammatory mediators. Intrauterine inflammation accompanying proinflammatory cytokine production has been associated with fetal rejection or preterm labor (15, 16, 17). In this context, it is possible that MIC-1, present in the placenta and amniotic fluid, acts to maintain pregnancy by suppressing the production of proinflammatory cytokines. The finding that a placental extract, derived from a premature labor, contained depressed concentrations of MIC-1, compared with normal pregnancies, supports this hypothesis. Intensive examination of MIC-1 levels in maternal serum and amniotic fluid during multiple and abnormal pregnancies should help clarify the precise role of MIC-1 during pregnancy.
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Footnotes |
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2 Contributed equally to this work.
Received December 9, 1999.
Revised May 12, 2000.
Revised August 11, 2000.
Accepted August 11, 2000.
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 J. A. Keelan, K. Wang, T. Chaiworapongsa, R. Romero, M. D. Mitchell, T. A. Sato, D. A. Brown, W. D. Fairlie, and S. N. Breit Macrophage inhibitory cytokine 1 in fetal membranes and amniotic fluid from pregnancies with and without preterm labour and premature rupture of membranes Mol. Hum. Reprod., September 1, 2003; 9(9): 535 - 540. [Abstract] [Full Text] [PDF]
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D. H. Lee, Y. Yang, S. J. Lee, K.-Y. Kim, T. H. Koo, S. M. Shin, K. S. Song, Y. H. Lee, Y.-J. Kim, J. J. Lee, et al. Macrophage Inhibitory Cytokine-1 Induces the Invasiveness of Gastric Cancer Cells by Up-Regulating the Urokinase-type Plasminogen Activator System Cancer Res., August 1, 2003; 63(15): 4648 - 4655. [Abstract] [Full Text] [PDF]
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 K. A. Iczkowski and C. G. Pantazis Overexpression of NSAID-Activated Gene Product in Prostate Cancer International Journal of Surgical Pathology, July 1, 2003; 11(3): 159 - 166. [Abstract] [PDF]
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D. A. Brown, R. L. Ward, P. Buckhaults, T. Liu, K. E. Romans, N. J. Hawkins, A. R. Bauskin, K. W. Kinzler, B. Vogelstein, and S. N. Breit MIC-1 Serum Level and Genotype: Associations with Progress and Prognosis of Colorectal Carcinoma Clin. Cancer Res., July 1, 2003; 9(7): 2642 - 2650. [Abstract] [Full Text] [PDF]
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 J. B. Welsh, L. M. Sapinoso, S. G. Kern, D. A. Brown, T. Liu, A. R. Bauskin, R. L. Ward, N. J. Hawkins, D. I. Quinn, P. J. Russell, et al. Large-scale delineation of secreted protein biomarkers overexpressed in cancer tissue and serum PNAS, March 18, 2003; 100(6): 3410 - 3415. [Abstract] [Full Text] [PDF]
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J. B. Welsh, L. M. Sapinoso, A. I. Su, S. G. Kern, J. Wang-Rodriguez, C. A. Moskaluk, H. F. Frierson Jr., and G. M. Hampton Analysis of Gene Expression Identifies Candidate Markers and Pharmacological Targets in Prostate Cancer Cancer Res., August 1, 2001; 61(16): 5974 - 5978. [Abstract] [Full Text] [PDF]
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 W. D. Fairlie, H.-P. Zhang, W. M. Wu, S. L. Pankhurst, A. R. Bauskin, P. K. Russell, P. K. Brown, and S. N. Breit The Propeptide of the Transforming Growth Factor-beta Superfamily Member, Macrophage Inhibitory Cytokine-1 (MIC-1), Is a Multifunctional Domain That Can Facilitate Protein Folding and Secretion J. Biol. Chem., May 11, 2001; 276(20): 16911 - 16918. [Abstract] [Full Text] [PDF]
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