Molecular and Cellular Endocrinology, 68 (1990) R31-R36 R31

Elsevier Scientific Publishers Ireland, Ltd.

MOLCEL 02230

Rapid Paper

Regulation of ACTH-induced steroidogenesis in human fetal adrenals

by rTNF-a

M. J&tte% I, 0. Carp& I, U.-H. Stenman 2 and E. Saksela

Department of Pathology, University of Helsinki, SF-00290 Helsinki, Finland, and Department of Gynecology and Obstetrics, Helsinki
University Central Hospital, SF-00290 Helsinki, Finland

(Received 22 November 1989; accepted 24 November 1989)

Key words: Lymphokine; Steroidogenic enzyme; Radioimmunoassay

Summary

The presence of tumor necrosis factor type alpha (TNF-a) in different fetal tissue and adult adrenal
extracts was investigated by radioimmunoassay (RIA). Measurable levels of TNF-a were found in 12/22
fetal adrenals, but in none of the seven adult adrenals studied. Since it is known that (i) steroidogenesis in
fetal adrenals differs greatly from that in adult glands by having higher androgen/corticosteroid ratio, (ii)
and that macrophage-derived factors may cause adrenocortical suppression, the effect of TNF-a on
corticotropin-induced steroidogenesis in primary cultures of human fetal adrenals was studied. Results
show that TNF-a effectively suppresses, the production of cortisol and shifts the steroid synthesis towards
androgen production. The effect was not accompanied by any change in cell viability and could be
neutralized by addition of polyclonal rabbit anti-TNF-cY antiserum to cell cultures. These results suggest
that TNF-(U may take part in the regulation of human fetal steroidogenesis within the network of the
fetoplacental unit via inhibition of the cortisol synthesis.

Introduction 1972). Products secreted by the placenta have

The regulation of steroidogenesis and the secre-
tory pattern in human fetal adrenal differs greatly
from that in the adult gland. The peculiar feature
of the fetal adrenal is the synthesis of high levels
of androgens, especially dehydropiandrosterone
sulphate (DHEAS) for placental estrogen synthe-
sis, and low levels of A4-keto-3 steroids like corti-
sol, which reflects the low activity of 3 /3-hydroxy-
steroid dehydrogenase in the fetal gland (Villee,
Address for correspondence: Maja J%Lttell, Department
of Pathology, University of Helsinki, Haartmaninkatu
00290 Helsinki, Finland.
been shown to suppress the activity of this enzyme
in fetal adrenals (Voutilainen and Kahri, 1980),
but the mechanism of the suppression is not fully
understood. A number of hormones and cytokines
in addition to corticotropin (ACTH), the most
potent stimulus for steroidogenesis, may modulate
the growth and function of adrenocortical cells
(Winters et al., 1975; Seron-Ferrt et al., 1978;
Pedersen and Brownie, 1980; Kudo and Baird,
1984; Esch et al., 1985; Hotta and Baird, 1986).
Furthermore, factors produced in response to en-
dotoxemia in vivo and factors produced by
activated macrophages in vitro have been shown
to cause an impaired adrenal response to ACTH
3, SF- (Berry and Smythe, 1961; Sibbald et al., 1977;
Keri et al., 1981; Mathison et al., 1983).

0303-7207/90/$03.50 0 1990 Elsevier Scientific Publishers Ireland, Ltd.

R32
Tumor necrosis factor alpha (TNF-a) was ini-
tially described as a tumoricidal protein produced
by activated macrophages, endotoxin being the
most potent stimulus for the production (Carswell
et al., 1975). In addition to the cytotoxic effects,
TNF-(U has been shown to have a wide variety of
biological effects on non-transformed cells as well
(for review see Beutler and Cerami, 1988). Based
on these effects it has been postulated that TNF-a
has a crucial role in mediation of endotoxin shock,
cachexia, and inflammation.
We have earlier demonstrated measurable levels
of TNF-a in human amniotic fluids and in super-
natants of placental and decidual tissues (Jgattell
et al., 1988). The purpose of the present work was
to investigate further the presence of TNF-(U in
different fetal tissues. Since TNF-(Y was found in
most of the fetal adrenals and placentas, it was of
interest to determine whether TNF-(U in vitro al-
tered adrenal steroidogenesis. The results pre-
sented in this paper show that this multifunctional
cytokine has direct effects on cultured adrenal
cells regulating ACTH-stimulated steroidogenesis.
Materials and methods
Materials. Recombinant human TNF-(Y
(rTNF-cu) with specific activity of 6 X lo7 U/mg,
recombinant human TNF-P with specific activity
of 1 X lo* U/mg, and mouse monoclonal anti-
body to TNF-a with neutralizing capacity of 6000
U rTNF-a/mg were kindly provided by Dr. G.R.
Adolf (Ernst-Boehringer-Institut, Vienna, Austria).
Polyclonal antiserum to TNF-a was raised by
immunizing a rabbit 3 times 12.5 pg, 60 pg, and
60 pg of rTNF-cu (the first 2 times in Freunds
complete adjuvant and the last time in Freunds
incomplete adjuvant) at 2-week intervals. Anti-
serum collected 2 weeks after the last immuniza-
tion had a neutralizing capacity of 120 U rTNF-
a/p1 and no neutralizing capacity against
lymphotoxin.
Human fetal tissues (gestational age ranging
from 9 to 35 weeks) were obtained under ap-
proved protocols from spontaneous abortions or
abortions induced for socio-medical or medical
reasons. Histologically normal adult adrenals were
obtained at nephrectomies.
Tissue extracts. Tissues were removed, washed
carefully with phosphate-buffered saline (PBS).
minced, washed and frozen in PBS (about 1 g of
wet tissue/ml). After freezing and thawing twice
the tubes were centrifuged and the supernatants
were collected. Total protein concentrations of the
supernatants were analyzed by spectrophotometry
(Pet&n-Elmer, Co., Norwald, CT, U.S.A.) at a
wavelength of 280 nm, and the concentration of
TNF-a by radioimmunoassay (RIA).
Adrenal tissue cultures. For tissue culture pur-
poses five fetal adrenals (gestational age ranging
from 13 to 22 weeks) were removed aseptically
within less than 6 h of delivery and processed
immediately. Adrenals were decapsulated, washed
carefully with PBS, minced, washed twice, and
then cultured on 6-well plastic plates or in tissue
culture flasks (Nunc, Roskilde, Denmark) in
RPMI-1640 medium (Gibco, Paisley, U.K.) sup-
plemented with 25% heat-inactivated fetal calf
serum (FCS; Gibco), 0.29 mg/ml glutamine, and
antibiotics. To study the steroidogenesis during
the first week of culture about 10 mg of wet tissue
in 3 ml of medium was explanted per well on
6-well plates, and stimulated with ACTH
(Organon, Oss, Netherlands; 0.2 pg/ml of tissue
culture medium/day). rTNF-cu and its antiserum
were added on the first, third and fifth days of
culture at indicated concentrations. The super-
natants were collected after 6 days incubation at
37 o C in 5 % humidified CO, and stored at - 20 o C
until analyzed. To study the steroidogenesis dur-
ing the third week of culture about 30 mg of wet
tissue in 10 ml of medium was explanted into a
tissue culture flask. The medium was replaced on
the fifth day and after 10 days cells were
trypsinized (0.25% trypsin; Gibco), and recultured
on 6-well plates 5 x lo4 cells/ml of culture
medium). After 5 days fresh medium and ACTH,
rTNF-a, and the antiserum were added and the
supernatants were collected after 6 days as de-
scribed above. At the end of the third week of the
adrenal tissue culture, cell viability was de-
termined by trypan blue exclusion.
Radioimmunoassays. RIA for TNF-a employ-
ing iodinated (sodium I-iodine, 100 mCi/ml;
New England Nuclear, Boston, MA, U.S.A.)
rTNF-cw, mouse monoclonal anti-TNF-cY antibod-
ies and anti-mouse IgG agarose (Sigma Chemical
Co., St. Louis, MO. U.S.A.) was carried out as
 R33

described earlier (Jaattela et al., 1988). The TABLE 1

minimal detectable quantity of rTFN-a was 0.3 THE PRESENCE OF TNF-ol IN THE EXTRACTS OF DIF-
ng/ml as defined by the concentration causing FERENT FETAL TISSUES AND OF ADULT ADRENALS
ASSAYED BY THE RIA
10% binding inhibition. As the total protein con-
centration of the tissue extracts was adjusted to 20
Tissue Positive samples TNF (pg/mg protein)
mg/ml, the sensitivity of the assay was 15 pg/mg in the RIA a in positive samples
of protein in the extract. The coefficient of vari-
Brain 2/9 135 (+25)b
ation in the assay was 5.4%. rTNF-/3 did not Thyroid gland n.a. c
o/7
inhibit the binding of iodinated rTNF-cu to the Lung I/9 30
antibody at concentrations up to 1 yg/ml. Heart o/9 n.a.
Cortisol was determined by a direct assay using Thymus 219 82(*15)
Liver I/9 15
251-labeled tracer and separation by polyethylene
Pancreas I/7 n.d. d
glycol precipitation using reagents from Farmos Spleen n.a.
O/8
Diagnostica (Oulunsalo, Finland). All other assays Adrenal 12/22 156 ( f 12)
used separation with dextran-coated charcoal, tri- Kidney o/9 n.a.
tiated labels and, with the exception of DHEAS, Skin o/4 n.a.
Placenta 4/7 65 (k14)
before assay the steroids were extracted with
Adult adrenal o/7 n.a.
organic solvent as suggested by the antiserum
manufacturer. Antisera for androstenedione and a The detection limit for TNF-a was 15 pg/mg of protein.
11-deoxycortisol were from Radioassay Systems b The mean value (+ SD)
n.a., not applicable.
Laboratories (Carsin, CA, U.S.A.) for 17-hydroxy-
d n.d., not determined.
progesterone and dehydropiandrosterone (DHEA)
from Bioclinical Services (Cardiff, U.K.) and for
DHEAS from Steranti (St. Albans, U.K.). Triti- Results and discussion
ated DHEAS was from New England Nuclear,
other tritiated steroids were from Amersham In- TNF-a in tissue extracts and in the supernatants
ternational (Amersham, U.K.). The assay for of adrenal cultures. First we studied the presence
cortisol had a crossreaction of 15% with ll-de- of TNF-a in different fetal tissue extracts by the
oxycortisol and that for DHEAS a crossreaction RIA. TNF-a was detected in over half of the
of 1% with DHEA. Other crossreactions were adrenal and placental extracts and in a few ex-
insignificant for the present study. tracts of cerebral, thymic, pancreatic, pulmonal,
Cytotoxicity assay. The supematants of adre- and hepatic tissues but in none of the other fetal
nal tissue cultures were tested for the lytic capac- tissues studied, nor in histologically normal adult
ity against highly sensitive WEHI 164 clone 13 adrenals (Table 1). When adjusted to the protein
target cells (10; cells were kindly provided by Dr. concentration of the extract the average level of
T. Espevik) in a 24-h chromium release assay as TNF-a in 12 positive adrenal supernatants was
described earlier (JIattela et al., 1988) except that 156 pg/mg of protein and the range was loo-210
RPM1 supplemented with 25% FCS served as a pg/mg. Cytotoxicity assay could not be used to
control. Minimal detectable concentration of study the biological activity of fetal adrenal tissue
rTNF-a was 10 pg/ml as defined by amount extracts because of the presence of non-TNF-cu-
causing 10% cytoxicity. Specificity of the reaction mediated lytic activity in the extracts.
was confirmed by adding an excess of anti-TNF-a We then studied the presence of TNF-(Y in
to the assay. Antibody had no neutralizing capac- supernatants of cultures of ACTH-stimulated fetal
ity against cytotoxicity caused by up to 1 pg/ml adrenals by a cytotoxicity assay employing WEHI
of lymphotoxin. The coefficient of variation of the 164 cells as targets, and by the RIA. After the first
assay was under 10%. The spontaneous release of week of culture 80% of the culture supernatants
chromium was under 20% in all assays. (ten samples from five different fetuses) contained
Statistical analysis. Two-tailed t-test was used endogenous lytic activity corresponding to from
to test the statistical significance of the results. 30 pg/ml up to 250 pg/ml of rTNF-a tested in
R34

200 ,
parallel experiments. 50% of the supernatants (six
samples from three different fetuses) were still
positive in the cytotoxicity assay after 3 weeks of
culture containing lytic activity corresponding to
from 15 pg/ml to 60 pg/ml of rTNF-a. Addition
of monoclonal anti-TNF-cu to the assay neutral-
ized the cytotoxicity. All the samples were nega-
tive in the RIA as could be expected due to the
detection limit of the assay (300 pg/ml).
The results presented here demonstrate the DUEA DHEAS 170H-P andro 11Deoxyc
presence of TNF-cu in fetal adrenal and placental cortisolisteroid indicated

tissue extracts. The following evidence suggests 200

that the binding inhibition caused by fetal adrenal

extracts in the RIA is due to TNF-(r: (i) RIA
dilution curves of fetal adrenal extracts were al-
most identical with dilution curves of rTNF-cw; (ii)
culture supernatants of fetal adrenal tissues con-
tained measurable levels of biologically active
TNF-a; (iii) adult adrenal extracts did not cause
binding ~bition in the RIA; and (iv) recombi-
nant TNF-/I did not inhibit the binding of
WEA DHEAS 17OH-P sndro 11Deoxyc
iodinated rTNF-cw and the monoclonal anti-TNF-cu cortlsollsteroid indicated
antibody used in the assay. 200
The shorter the time between the abortion and C

the processing of the adrenals was, the more often

the extracts were positive in the RIA. Only 36%
150 J TT III
(4/11) of extracts of adrenals homogenized more
than 48 h after the abortion contained measurable
levels of TNF-(U while 73% (8/11) of adrenals
processed sooner were positive in the RIA. The

100
80 I---
s
z 60
C antiserum added every 48 h, crossed bars) on ratios of cortisol
Fig. 1. Effect of rTNF-n on ACTH-induced cortisol produc-
tion in the third week cultures of human fetal adrenals. Values
are means (*SD) of four cultures with no treatment (white
bars), four cultures with 1 ng/mf of rTNF-n (hatched bars,
P = O.Ol),four cultures with 3 ng,/ml of rTNF-ru (black bars,
P = 0.003). and two cultures with 3 ng/ml of rTNF-LX and 2
ml/ml of polyclonai anti-TNF-a antiserum added every 48 h
of culture (crossed bars).
-4
DHEA DHEAS 1?0H-P andro
ll-deoxycortisol/steroid indicated
Fig. 2. Effect of rTNF-a (1 ng/ml, hatched bars; 3 ng/ml,
black bars; 3 ng/ml with 2 PI/ml of poiyclonal anti-TNF-a
to other adrenal steroids. i.e. dehydroepiandrosterone (DHEA),
dehydroepiandrosterone sulphate (DHEAS), 17-hydroxypro-
gesterone (170H-P), androstenedione (androf, and 7 l-de-
oxycortisol (ll-deoxyc) in ACTH-stimuIated cultures of hu-
man fetal adrenals during the first week (A) and during the
third week (B) of culture. The ratios of ll-deoxycortisol to
other steroids are shown in C. In panel C data from the first
and the third week cultures have been combined. Values are
percentages of controls (cultures with no rTNF-u; white bars)
and means ( f SD) of five (A), four (B), nine (C; treated with
rTNF-a), and five (C; treated with rTNF-o and anti-TNF-ol)
different experiments are indicated. A: P-c 0.05 in all cases
whether treated with 1 or 3 ng/ml of rTNF-ol except for the
ratio of cortisol to 17-hydroxyprogesterone with 1 ng/ml of
rTNF-a. B: P < 0.001 in all cases. C: No significance.

presence of TNF-(Y in fetal adrenals showed no
correlation to sex, diagnosis, not to the gestational
age of the fetus. These observations suggest that
postmortally activated proteases may be responsi-
ble for destruction of TNA-a: in negative samples.
The effect of TNF-a on steroidogenesis. In order
to find clues to the possible biological role of
TNF-cr in fetal adrenals we investigated the effect
of rTNF-cw on the steroidogenesis in primary cul-
tures of human fetal adrenals. The supernatants of
the first and the third week cultures of ACTH
stimulated fetal adrenals with no treatment and
those treated for 6 days with rTNF-ol alone or
with polyclonal anti-TNF-cY were assayed for
DHEA DHEAS, 17-hydroxyprogesterone, andros-
tenedione, 11-deoxycortisol, and cortisol by RIAs.
As is shown in Fig. 1, the addition of 1.0 ng/ml
and 3.0 ng/ml of rTNF-a: to the third week cul-
tures every 48 h suppressed cortisol synthesis by
68.7% (mean of four cultures) and by 86.7% (mean
of two cultures), respectively. Most of this effect
could be neutralized by polyclonal anti-TNF-cY
antiserum. The addition of rTNF-a to these cul-
tures decreased also the production of DHEA,
DHEAS, 17-hydroxyprogesterone and ll-de-
oxycortisol, but to a condiserably lesser extent
than the production of cortisol. Because of the
crossreaction of 1 l-deoxycortisol in the cortisol
assay (IS%), the concentration of cortisol was
overestimated by 25550% in the experiments with
rTNF-cu in the third week cultures. Thus the sup-
pressive effect of rTNF-a on cortisol production
was actually larger than that observed.
The levels of the steroids in different treat-
ments could not be compared as such in the first
week cultures since the amount of tissue per well
could not be properly controlled. Therefore, we
then compared the relative content of the end-
product, cortisol, to the content of its precursors,
to study at which step of the steroidogenesis the
suppression occurs. In both the first and the third
week cultures the addition of rTNF-a significantly
lowered the ratio of cortisol to other steroids. This
effect was neutralized by addition of polyclonal
anti-TNF-cy to the cultures (Fig. 2) while non-im-
mune rabbit serum had no effect (data not shown).
Ratios of androstenedione concentrations to con-
centrations of steroids earlier in the pathway, e.g.
17-hydroxyprogesterone and DHEA, tended to be
R35
higher in cultures treated with rTNF-a: than in
cultures with no treatment but the changes were
not statistically significant. RTNF-(U had no clear
efffect on the ratios of other steroids.
These data suggest that addition of rTNF-cw to
fetal adrenal cultures decreases the production of
cortisol mainly by inhibiting the conversion of
11-deoxycortisol to cortisol mediated by P-450,,,.
There seems to be no significant inhibition of
3/3-hydroxysteroid dehydrogenase nor P-450,,, ,
P-450,,,. The possible effect of rTNF-a on P-450,,,
cannot be judged by these data. The effect is dose
dependent at least with the two different con-
centrations of rTNF-(Y tested, and it can be neu-
tralized by addition of polyclonal anti-TNF-a an-
tiserum. The slight accumulation of androstene-
dione in cultures treated with rTNF-a also sup-
ports the hypothesis of suppressed activity of P-
4%~ since this enzyme has been shown to con-
vert androstenedione to llp-hydroxyandrostene-
dione in cultures of fetal adrenals (Kahri et al.,
1976).
TNF-a has been shown to be cytotoxic or
cytostatic to many cells and cell lines. To study
whether the suppression of cortisol production
was due to toxic effects of rTNF-cq we studied the
cell viability of every culture. Addition of rTNF-a
or rTNF-e and its antiserum to adrenal cultures
did not have any effect on cell viability as de-
termined by trypan blue exclusion. Cell viability
after the third week of cultures was 91.8% (f2.1
SD) in control wells, 90.5% (k2.0) in wells with
rTNF-cY and 89.4% (k3.1) in wells with rTNF-a
and its polyclonal antibody.
Complex feedback mechanisms regulate the
production of steroid hormones and cytokines.
Tracey et al. (1987) have demonstrated an increase
in plasma levels of cortisol after an intravenous
administration of TNF-(Y in dogs. According to a
recent report by Milenkovic et al. (1989) this
increase could be due to a TNF-a-induced in-
crease in anterior pituitary release of ACTH. In
concordance with our results. the direct effect of
monocyte-derived factors (Mathison et al., 1983;
Hotta and Baird, 1986) and endotoxic plasma
(Keri et al., 1981) on adrenal steroidogenesis has
been shown to be suppressive. Hotta and Baird
(1986) have suggested that this suppression is
mediated by transforming growth factor p. Data
R36
presented here lend support to the hypothesis that
also TNF-a plays a role in regulation of ACTH-
induced steroidogenesis in adrenals brought about
by monocyte-derived factors. In addition to regu-
lation of steroidogenesis in adrenal glands, cyto-
kines have recently been shown to have many
regulatory effects in other steroid-producing
organs as well. TNF-a has been shown to alter
progesterone and androstenedione production in
preovulatory follicles (Roby and Terranova, 1988),
and to inhibit the aromatization of androgenic
steroid precursors induced by follicle-stimulating
hormone in ovarian granulosa cells (Adashi et al.,
1989).
In adipocytes TNF-(U has been shown to sup-
press the activities of a number of different en-
zymes (Pekala et al., 1983) and Cornelius et al.
(1988) have suggested that this suppression occurs
at the transcriptional level. TNF-a has also been
shown to regulate oncogene expression by affect-
ing the levels of mRNA (KriSnke et al., 1987;
Seliger et al., 1988). Furthermore, interferon-y has
been shown to inhibit steroidogenesis in Leydig
cells by inhibiting the accumulation of mRNA of
steroidogenic enzymes P-450,,, and P-450,,,. One
may thus speculate that the suppression of the
activity of P-450,,, brought about by rTNF-a
could also be due to a transcriptional regulation
and suppressed levels of P-450,,, mRNA. This
may have a significant role in directing the
steroidogenesis of the proportionally highly hyper-
plastic fetal adrenals to the production of the
precursors of placental steroid synthesis necessary
for the optimal function of the fetoplacental unit.
Acknowledgements
The excellent technical assistance of Maija-Li-
isa M%ntyll, Riitta Sissonen and Bjiirn Lindroos
and the constructive criticism by Raimo Vouti-
lainen are gratefully acknowledged. This work was
supported by the Finnish Cultural Foundation,
the Research Foundation of Farmos, the Finnish
Medical Foundation, and the Finnish Cancer
Organization.
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