Sunteți pe pagina 1din 6

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


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

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

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


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

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
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
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.
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

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 -4
DHEA DHEAS 1?0H-P andro
ll-deoxycortisol/steroid indicated
80 I---
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
z 60
C antiserum added every 48 h, crossed bars) on ratios of cortisol
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
Fig. 1. Effect of rTNF-n on ACTH-induced cortisol produc- and the third week cultures have been combined. Values are
tion in the third week cultures of human fetal adrenals. Values percentages of controls (cultures with no rTNF-u; white bars)
are means (*SD) of four cultures with no treatment (white and means ( f SD) of five (A), four (B), nine (C; treated with
bars), four cultures with 1 ng/mf of rTNF-n (hatched bars, rTNF-a), and five (C; treated with rTNF-o and anti-TNF-ol)
P = O.Ol),four cultures with 3 ng,/ml of rTNF-ru (black bars, different experiments are indicated. A: P-c 0.05 in all cases
P = 0.003). and two cultures with 3 ng/ml of rTNF-LX and 2 whether treated with 1 or 3 ng/ml of rTNF-ol except for the
ml/ml of polyclonai anti-TNF-a antiserum added every 48 h ratio of cortisol to 17-hydroxyprogesterone with 1 ng/ml of
of culture (crossed bars). rTNF-a. B: P < 0.001 in all cases. C: No significance.

presence of TNF-(Y in fetal adrenals showed no higher in cultures treated with rTNF-a: than in
correlation to sex, diagnosis, not to the gestational cultures with no treatment but the changes were
age of the fetus. These observations suggest that not statistically significant. RTNF-(U had no clear
postmortally activated proteases may be responsi- efffect on the ratios of other steroids.
ble for destruction of TNA-a: in negative samples. These data suggest that addition of rTNF-cw to
The effect of TNF-a on steroidogenesis. In order fetal adrenal cultures decreases the production of
to find clues to the possible biological role of cortisol mainly by inhibiting the conversion of
TNF-cr in fetal adrenals we investigated the effect 11-deoxycortisol to cortisol mediated by P-450,,,.
of rTNF-cw on the steroidogenesis in primary cul- There seems to be no significant inhibition of
tures of human fetal adrenals. The supernatants of 3/3-hydroxysteroid dehydrogenase nor P-450,,, ,
the first and the third week cultures of ACTH P-450,,,. The possible effect of rTNF-a on P-450,,,
stimulated fetal adrenals with no treatment and cannot be judged by these data. The effect is dose
those treated for 6 days with rTNF-ol alone or dependent at least with the two different con-
with polyclonal anti-TNF-cY were assayed for centrations of rTNF-(Y tested, and it can be neu-
DHEA DHEAS, 17-hydroxyprogesterone, andros- tralized by addition of polyclonal anti-TNF-a an-
tenedione, 11-deoxycortisol, and cortisol by RIAs. tiserum. The slight accumulation of androstene-
As is shown in Fig. 1, the addition of 1.0 ng/ml dione in cultures treated with rTNF-a also sup-
and 3.0 ng/ml of rTNF-a: to the third week cul- ports the hypothesis of suppressed activity of P-
tures every 48 h suppressed cortisol synthesis by 4%~ since this enzyme has been shown to con-
68.7% (mean of four cultures) and by 86.7% (mean vert androstenedione to llp-hydroxyandrostene-
of two cultures), respectively. Most of this effect dione in cultures of fetal adrenals (Kahri et al.,
could be neutralized by polyclonal anti-TNF-cY 1976).
antiserum. The addition of rTNF-a to these cul- TNF-a has been shown to be cytotoxic or
tures decreased also the production of DHEA, cytostatic to many cells and cell lines. To study
DHEAS, 17-hydroxyprogesterone and ll-de- whether the suppression of cortisol production
oxycortisol, but to a condiserably lesser extent was due to toxic effects of rTNF-cq we studied the
than the production of cortisol. Because of the cell viability of every culture. Addition of rTNF-a
crossreaction of 1 l-deoxycortisol in the cortisol or rTNF-e and its antiserum to adrenal cultures
assay (IS%), the concentration of cortisol was did not have any effect on cell viability as de-
overestimated by 25550% in the experiments with termined by trypan blue exclusion. Cell viability
rTNF-cu in the third week cultures. Thus the sup- after the third week of cultures was 91.8% (f2.1
pressive effect of rTNF-a on cortisol production SD) in control wells, 90.5% (k2.0) in wells with
was actually larger than that observed. rTNF-cY and 89.4% (k3.1) in wells with rTNF-a
The levels of the steroids in different treat- and its polyclonal antibody.
ments could not be compared as such in the first Complex feedback mechanisms regulate the
week cultures since the amount of tissue per well production of steroid hormones and cytokines.
could not be properly controlled. Therefore, we Tracey et al. (1987) have demonstrated an increase
then compared the relative content of the end- in plasma levels of cortisol after an intravenous
product, cortisol, to the content of its precursors, administration of TNF-(Y in dogs. According to a
to study at which step of the steroidogenesis the recent report by Milenkovic et al. (1989) this
suppression occurs. In both the first and the third increase could be due to a TNF-a-induced in-
week cultures the addition of rTNF-a significantly crease in anterior pituitary release of ACTH. In
lowered the ratio of cortisol to other steroids. This concordance with our results. the direct effect of
effect was neutralized by addition of polyclonal monocyte-derived factors (Mathison et al., 1983;
anti-TNF-cy to the cultures (Fig. 2) while non-im- Hotta and Baird, 1986) and endotoxic plasma
mune rabbit serum had no effect (data not shown). (Keri et al., 1981) on adrenal steroidogenesis has
Ratios of androstenedione concentrations to con- been shown to be suppressive. Hotta and Baird
centrations of steroids earlier in the pathway, e.g. (1986) have suggested that this suppression is
17-hydroxyprogesterone and DHEA, tended to be mediated by transforming growth factor p. Data

presented here lend support to the hypothesis that Berry. L.J. and Smythe, D.S. (1961) J. Exp. Med. 114, 761-778.
also TNF-a plays a role in regulation of ACTH- Beutler. B. and Cerami, A. (1988) Annu. Rev. Biochem. 57,
induced steroidogenesis in adrenals brought about
Carswell, E.A., Olds, L.J.. Kassel, R.L., Green, S., Fiore, N.
by monocyte-derived factors. In addition to regu- and Williamson, B. (1975) Proc. Natl. Acad. Sci. U.S.A. 72,
lation of steroidogenesis in adrenal glands, cyto- 3666-3670.
kines have recently been shown to have many Cornelius, P., Enerback, S., Bjursell, G., Olivercrona. T. and
regulatory effects in other steroid-producing Pekala, P.H. (1988) Biochem. J. 249, 765-769.
Esch, F., Baird, A., Ling, N.. Ueno, N., Hill, F., Denoroy, L.,
organs as well. TNF-a has been shown to alter
Klepper, R., Gospodarowicz, D., Bijhlen, P. and Guillemin,
progesterone and androstenedione production in R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6507-6511.
preovulatory follicles (Roby and Terranova, 1988), Espevik, T. and Nissen-Meyer, J. (1986) J. Immunol. Methods
and to inhibit the aromatization of androgenic 95, 99-105.
steroid precursors induced by follicle-stimulating Hotta, M. and Baird, A. (1986) Proc. Natl. Acad. Sci. U.S.A.
83, 7795-7799.
hormone in ovarian granulosa cells (Adashi et al.,
Jalttell, M., Kuusela, P. and Saksela, E. (1988) Lab. Invest. 58,
1989). 48-52.
In adipocytes TNF-(U has been shown to sup- Kahri, AI., Huhtaniemi, I. and Salmenperl, M. (1976) Endo-
press the activities of a number of different en- crinology 98, 33-41.
zymes (Pekala et al., 1983) and Cornelius et al. Keri, G., Parameswaran, V., Trunkey, D.D. and Ramachan-
dran, J. (1981) Life Sci. 28, 1917-1923.
(1988) have suggested that this suppression occurs
Kriinke, M., Schliiter, C. and Pfizenmaier, K. (1987) Proc.
at the transcriptional level. TNF-a has also been Natl. Acad. Sci. U.S.A. 84, 469-473.
shown to regulate oncogene expression by affect- Kudo, T. and Baird, A. (1984) Nature 312. 756-757.
ing the levels of mRNA (KriSnke et al., 1987; Mathison, J., Schreiber, R.D., La Forest, A.C. and Ulevitch,
Seliger et al., 1988). Furthermore, interferon-y has R.J. (1983) J. Immunol. 130. 2757-2762.
Milenkovic, L.. Rettori, V., Snyder, G.D., Beutler, B. and
been shown to inhibit steroidogenesis in Leydig
McCann, SM. (1989) Proc. Natl. Acad. Sci. U.S.A. 86,
cells by inhibiting the accumulation of mRNA of 2418-2422.
steroidogenic enzymes P-450,,, and P-450,,,. One Orava. M., Voutilainen, R. and Vihko, R. (1989) Mol. Endo-
may thus speculate that the suppression of the crinol. 3, 887-894.
activity of P-450,,, brought about by rTNF-a Pedersen, R.C. and Brownie, A.C. (1980) Science 208,
could also be due to a transcriptional regulation
Pekala, P.H., Kawamaki, M., Angus, C.W., Lane, M.D. and
and suppressed levels of P-450,,, mRNA. This Cerami, A. (1983) Proc. Natl. Acad. Sci. U.S.A. 80,
may have a significant role in directing the 2743-2747.
steroidogenesis of the proportionally highly hyper- Roby, K.F. and Terranova. P.F. (1988) Endocrinology 123.
plastic fetal adrenals to the production of the
Seliger, B., Stark, G. and Pfizenmaier, K. (1988) J. Immunol.
precursors of placental steroid synthesis necessary 141, 2138-2144.
for the optimal function of the fetoplacental unit. Seron-FerrC, M., Lawrence, CC. and Jaffe. R.B. (1978) J. Clin.
Endocrinol. Metab. 46. 834-837.
Acknowledgements Sibbald. W.J., Short, A.. Cohen, M.P. and Wilson. R.F. (1977)
Ann. Surg. 186, 29-33.
Tracey, K.J., Lowry, S.F.. Fahey, T.J.. Albert. J.D., Fong. Y.,
The excellent technical assistance of Maija-Li- Hesse, D., Beutler, B., Manogue. K.R., Calvano, S.. Wei,
isa M%ntyll, Riitta Sissonen and Bjiirn Lindroos H., Cerami, A. and Shires, G.T. (1987) Surg. Gynecol.
and the constructive criticism by Raimo Vouti- Obstet. 164, 415-422.
lainen are gratefully acknowledged. This work was Villee, D. (1972) Am. J. Med. 53. 533-544.
Voutilainen, R. and Kahri. A.I. (1980) J. Steroid Biochem. 13.
supported by the Finnish Cultural Foundation,
the Research Foundation of Farmos, the Finnish Waage, A. and Bakke, 0. (1988) Immunology 63. 299-302.
Medical Foundation, and the Finnish Cancer Winters, A.J., Colston, C.. MacDonald, P.C. and Porter. J.C.
Organization. (1975) J. Clin. Endocrinol. Metab. 41, 626-629.


Adashi, E.Y., Resnick, C.E., Croft, C.S. and Payne, D.W.

(1989) J. Biol. Chem. 264. 11591-11597.