Journal of General Virology (1991), 72, 1871-1876.
1871
Printed in Great Britain
Characterization of Sendai virus-induced human placental trophoblast
interferons
George Aboagye-Mathiesen, 1 Ferenc D. Tbth, 1,2 Claus Juhl, 1 Niels Norskov-Lauritsen, 1
Peter M . Petersen, 1 Vladimir Zachar, 1,3 and Peter Ebbesen 1.
1The Danish Cancer Society, Department of Virus and Cancer, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark,
2Institute of Microbiology, Medical University, H-4012 Debrecen, Hungary and 3Institute of Virology, Slovak Academy
of Sciences, Bratislava, Czechoslovakia
Human placental trophoblast cultures produce a
mixture of interferons (IFNs) when challenged with
Sendai virus. High-performance dye-ligand and immunoaffinity chromatography of a trophoblast IFN
(tro-IFN) preparation enabled the isolation of three
antigenically distinct IFNs, ~l, sill and/~, with MrS of
16K, 22K and 24K respectively, by reducing and nonreducing SDS-PAGE. The major IFN, responsible for
75 % of the total antiviral activity, was tro-IFN-/], with
the remaining activity being due to tro-IFN-aq and
tro-IFN-~ul, as determined by an antiviral neutralization test using specific anti-human IFN antibodies.
The antiviral activities of the tro-IFNs were stable at
pH 2.0 for 24 h and tro-IFN-~tli1 and -p were shown to
be glycoproteins. The three tro-IFNs showed different
antiviral activities when assayed on human and bovine
cell species; tro-IFN-~,i and -agti1 protected both human
and bovine (MDBK) cells from virus infection, whereas
tro-IFN-fl showed a high degree of species specificity,
protecting only the human cell types tested.
Introduction
placentae, however the cellular origins of these IFNs
have not been identified. Besides IFN-~ and -fl, Chin et
al. (1986) have reported that IFN-~ is produced by
placental lymphocytes, monocytes and granulocytes
after phytohaemagglutinin and interleukin 2 stimulation. Recently, T6th et al. (1990a) demonstrated that
IFN-fl is produced by human placental trophoblasts in
vitro after stimulation with poly(rI), poly(rC). Further
studies by T6th et al. (1990b) showed that challenge of
trophoblast cultures with Sendai virus resulted in the
production of a mixture of IFN-~ and -ft. In this paper we
describe the isolation and characterization of the Sendai
virus-induced human placental tro-IFN components.
Interferons (IFNs) are a family of proteins produced by
vertebrate cells after exposure to certain inducers, e.g.
viruses, synthetic dsRNA or mitogens. They exert a
broad spectrum of biological activities, the most prominent being the ability to impair virus replication. In
humans, three functionally related but antigenicaUy
distinct IFNs (~, fl and ~) have been identified and are
recognized by the Committee on the Nomenclature of
Interferons. However, other types of human IFN have
been reported by other workers (Wilkinson & Morris,
1983; Dianzani et al., 1986), but these have not been
characterized fully. In addition, Adolf (1987) and
Hauptmann & Swetly (1985) have reported that a new
type of IFN, IFN-og, is released from cells challenged
with Sendai virus. This unique IFN (designated IFN-o~I
by Adolf, 1987) has 60% sequence identity with human
IFN-~ and 26% with IFN-fl, and is different antigenically from human IFN-~, -fl and -% IFN<ol was therefore
considered to belong to a subfamily of IFN-~ (Capon et
al., 1985) and was termed IFN-~ subclass II (tro-IFN~ii1), in contrast to the classical IFN-~ in IFN-ct subclass
I (IFN-~0.
Several workers (Chard et al., 1986; Duc-Goiran et al.,
1985; Bocci et al., 1985) have reported the presence of
IFN<t and -fl in amniotic fluids and specimens from
0001-0071 © 1991 SGM
Methods
Cell culture and IFN induction. Human placental trophoblast cells
were isolated using immunomagnetic microspheres as described by
Douglas & King (1989). IFN was produced by challenge of trophoblast
cultures with Sendai virus as described by Tbth et al. (1990b). The
supernatants containing IFN were centrifuged at 10000g for 1 h,
adjusted to pH 2-0 by addition of concentrated HC1 and stored at 4 °C
for 24 h to inactivate the virus. The crude IFN preparation was
neutralized with concentrated NaOH and precipitated by adding 1 g
ammonium sulphate per 2 ml IFN preparation. The precipitate was
redissolved in 0.02 M-sodium phosphate buffer pH 7.2 and dialysed
against two changes of a 500-fold excess of the same buffer for 10 h at
4°C.
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1872
G. Aboagye-Mathiesen and others
1FN bioassay. The IFN antiviral assay was performed as described
by T6th et al. (1990a). Results were standardized in international units
(IU) by comparison with the National Institute of Health standards for
human IFN-fl (G-023-902-527) and IFN-ct (GA-023-902-530). IFN
titres, using human trisomic 21 fibroblast cell lines GM 2504 and GM
2767 (Cell Repository, Camden, N.J., U.S.A.), and bovine (MDBK)
cells, were determined similarly using vesicular stomatitis virus (VSV)
as the challenge; results were expressed as the highest dilution giving
50~ protection.
Antigenic characterization of lFN components. The antigenic specificities of the crude and isolated tro-IFN components were determined by
antibody neutralization tests. Polyclonal anti-human IFN-ct, antihuman IFN-fl, anti-IFN-y and anti-recombinant human IFN-~qll
antibodies were used for the assay and preparation of HPLC sorbents.
Polyclonal anti-IFN<t antibodies obtained from Boehringer Mannhelm (produced in BALB/c mice using human IFN-ct purified from
Sendal virus-stimulated human lymphoblastoid cells) neutralize human
IFN-ct, and recombinant human IFN<t I and IFN-cql 1, but do not react
with human IFN-/~ or -y. Polyclonal human anti-IFN-fl antibodies,
produced in horses immunized with human IFN-fl (fibroblast), were
obtained from Boehringer Mannheim; they do not react with human
IFN-ct, IFN-~qll or IFN-),. Anti-recombinant human IFN-ctnl antibodies were supplied by Giinther R. Adolf (Department of Biotechnology, Dr Boehringer-Gasse, A-1121 Vienna, Austria) and do not react
with IFN-~q, -fl or -?. Serial dilutions of IFN and a fixed dilution of
antibody in 10-fold excess were incubated for 1 h at 37 °C and residual
antiviral activity was determined as described by T6th et al. (1990a).
Preparation of HPLC sorbents. HEMA-BIO 1000 VS 3GA was
prepared by covalently attaching Cibacron Blue 3GA by a spacer arm,
1,4-diaminobutane, to vinyl sulphonate-activated HEMA-BIO 1000
(10 ilm particle size) as described previously (Aboagye-Mathiesen et al.,
1991). The prepared gel was then packed into a biocompatible PEEK
(Poly Ether Ether Ketone) column (250 mm x 7.5 mm internal
diameter; Tessek A/S).
Immunoadsorbents (anti-IFN-~t and anti-IFN-fl antibodies) and
concanavalin A (Con A) columns were prepared by adsorptionpromoted enhanced covalent-immobilization of polyclonal anti-IFN-ct
and anti-IFN-fl antibodies, and Con A on maeroporous HEMA-BIO
1000 VS. Briefly, 0.5 g dry HEMA-BIO 1000 VS was swollen with
immobilization buffer (0.1 M-sodium borate, 0.75 M-ammonium
sulphate buffer pH 8.0) and 5 mg antibodies or 10 mg of Con A
dissolved in 4 ml or 15 ml (for Con A) of immobilization buffer was
added to the wetted gels. Immobilization was allowed to proceed under
gentle rotation for 16 to 20 h at 4 °C. The gels were then settled by
centrifugation, the supernatants were removed, the gels were washed
eight times with distilled water and residual reactive groups were
blocked by incubation with 3 ml 0.1 M-ethanolamine in 0.1 u-sodium
borate buffer pH 9.0 for 6 h. The gels were then packed in
biocompatible PEEK columns (50 mm x 4.6 mm internal diameter).
Chromatographic procedures
(i) High performancedye-ligand affinity chromatography (HP-DLAC).
Concentrated crude tro-IFN (2.05 x 106 IU) was filtered through a 0-22
~tm Millipore filter and applied to a HEMA-BIO 1000 VS 3GA column
equilibrated with column buffer (0.02 M-sodium phosphate buffer pH
7.2). After the column had been washed with the same buffer
containing 0.2 M-NaC1, proteins were eluted with a linear gradient of
0.2 to 1.0 M-NaC1 for 35 min at a flow rate of 1.5 ml/min; elution from
the column was monitored continuously by measuring the absorbance
at 280 nm. The column was washed further with 1.0 M-NaC1 in column
buffer for 20 min until the absorbance was almost zero. Protein was
then eluted from the column with increasing concentrations of ethylene
glycol in 0.02 M-sodium phosphate buffer pH 7.2 containing 1.0 M-
NaCI; elution was with 0 to 50~ ethylene glycol for t0 min and 50~ for
35 min at a flow rate of 1.5 ml/min. Fractions of 1.5 ml were collected
and assayed for IFN antiviral activity.
(ii) High performance immunoaffinity chromatography (HP-IAC).
Crude tro-IFN preparations and the fractions containing IFN antiviral
activity from the HEMA-BIO 1000 VS 3GA column were applied
separately to HEMA-BIO 1000 VS-anti-IFN<t (polyclonal) antibody
or -anti-IFN-fl antibody columns. In each case, the columns were
washed for 10 min with PBS pH 7-4 and proteins were eluted with 0.1 Mglycine-HC1 pH 2.4. Fractions of 0.5 ml were collected and dialysed
against PBS for 16 h at 4 °C to restore the pH to neutral, and then
assayed for IFN antiviral activity.
PAGE. Slab SDS-PAGE was carried out according to Laemmli
(1970). Freeze-dried IFN samples were prepared with or without 5 ~ 2mercaptoethanol. After electrophoresis, the gels were silver-stained
using a Bio-Rad silver staining kit and scanned on a Biomed SLR
1D/2D scanner to determine the Mr by comparison with Bio-Rad
protein standards. To determine the IFN antiviral activity on
unstained gels, 2 mm slides of the gel were extracted at 4 °C for 24 h into
0-5 ml volumes of PBS pH 7.4 containing 0-1 ~ SDS and 0-02~ NaN 3.
Aliquots (100 ~tl) were then taken and assayed for IFN antiviral
activity.
Protein determination. The concentration of protein in crude tro-IFN
preparations was determined by the dye binding assay (Bradford, 1976)
using bovine serum albumin (BSA) as a standard. The concentration of
purified IFN was measured by absorbance at 280 nm, or derivatization
with fluorescamine and injecting the samples into a fluorescence
HPLC monitor, using known concentrations of BSA as a standard.
Glycoprotein analysis. The presence of sugar residues in the tro-IFNs
was determined by their binding to a Con A affinity column as
described previously (Aboagye-Mathiesen et al., 1990).
Results
Purification o f tro-IFNs
Fig. 1 i l l u s t r a t e s t h e H P - D L A C o f c r u d e t r o - I F N o n
HEMA-BIO
1000 V S 3 G A . T h e t r o - I F N s b o u n d
c o m p l e t e l y w h e n a p p l i e d in 0-02 M - s o d i u m p h o s p h a t e
b u f f e r p H 7-2 at l o w i o n i c s t r e n g t h (0.2 M-NaC1).
Development of the column with a linear concentration
g r a d i e n t o f N a C I s e p a r a t e d 20 ~ o f t h e t o t a l I F N a c t i v i t y
a p p l i e d i n t o five p e a k s ( f r a c t i o n s 21 to 40 e l u t e d b e t w e e n
0.6 a n d 0.8 M-NaC1). F u r t h e r d e v e l o p m e n t o f t h e c o l u m n
with a linear concentration gradient of the hydrophobic
solute e t h y l e n e g l y c o l p r o d u c e d t w o I F N p e a k s ( f r a c t i o n s
50 t o 53 a n d 54 to 70), e l u t e d f r o m t h e c o l u m n at e t h y l e n e
glycol c o n c e n t r a t i o n s o f 40 to 5 0 ~ a n d 5 0 ~ . A s u m m a r y
o f t h e H P - D L A C o f t h e t r o - I F N s is p r e s e n t e d in T a b l e 1.
Characterization o f tro-lFNs
The tro-IFN components were identified by antiviral
n e u t r a l i z a t i o n tests u s i n g p o l y c l o n a l a n t i s e r a to h u m a n
I F N - ~ , -fl a n d -~ a n d a n t i - I F N - c t n l a n t i b o d i e s . P o l y c l o n a l a n t i s e r u m to h u m a n IFN-~6 n e u t r a l i z e d 75 ~ o f t h e
a n t i v i r a l a c t i v i t y o f c r u d e t r o - I F N s a m p l e s ( i n i t i a l l y 800
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Sendai virus-induced trophoblast IFNs
I
I
I
I
I
I
I
I
I
I
I
I
1873
5
Q
-4
20 - 4 ~
f"_~
-
.~1.5-~
....
°"""t-
1.0-
f Ih
. . . . .
1-0
1
lO.,
s~ SS
'
s~Si
50
; /
sss,,
. . . . . .
02
0
l i
..--
5
10
15
20
25
30
"I0"4
--
3
40
Fraction number
Z
'
"6
S
×
.~;~
~
~o-
, s
0-5 -
3
till
"I"
"~
1
70
0
"~.
"
45
50
55
60
65
Fig. 1. H P - D L A C of crude tro-IFN on HEMA-BIO 1000 VS 3GA. I F N (2.05 x 106 IU) was applied to a column and I F N activity (11)
was eluted with a 60 min linear gradient of 0-2 M- to 1-0 M-NaCI (---) for 60 min. The column was further eluted with increasing
concentrations of ethylene glycol (---) from 0 to 5 0 ~ for 10 min and 5 0 ~ for 35 min. The A28o is shown by the continuous line.
Table 1. Purification of placental tro-IFN components by
HP-DLAC
Crude tro-IFN
Fractions 21-40
Fractions 50-53
Fractions 54-70
Total
activity
(IU
x 10-4)
Total
protein
(mg)
Specific
activity
(IU/mg
x 10-3)
Purification
(-fold)
Recovery
(~)
205
41
742
t38
401.9
0.328
0.070
0.076
5.10
1250
1060
18200
1
245.0
207.8
3568-6
100
20
3-6
67.32
Table 2. Antibody neutralization of tro-IFN components
isolated by HP-DLAC*
Residual antiviral
activity after incubation with antisera (~)
Anti-human
I F N sera
a
fl
O¢ll1
~,
+ fl
anl + 17
+ anl
Fractions
21-40
Fractions
50-53
Fractions
54-70
0
100
1O0
100
40
60
40
100
100
0
1O0
100
ND~
0
ND
100
0
0
40
ND
100
* Neutralization tests were performed with a 10-fold excess of the
respective antiserum.
t reD, Not determined.
IU/ml, 200 IU/ml after treatment), whereas the antiIFN-~ antibodies neutralized 25~ of the activity
(initially 800 IU/ml, 600 IU/ml after treatment) (Table
2). Fractions 21 to 40 (see Fig. 1), eluted from the column
with NaCI (0.6 to 0-8 M), were completely neutralized by
polyclonal anti-IFN-~ antibodies, but not with anti-IFN~XlI1, anti-IFN-fl or anti-IFN-y antibodies. This showed
that the five IFN peaks consisted of IFN-al. However,
the IFN peak fractions (50 to 53) that were eluted from
the column using ethylene glycol concentrations between
40 and 50 ~ were partially neutralized by polyclonal antiIFN-fl antibodies, and to the same extent by polyclonal
anti-IFN-a and anti-IFN-~nl antibodies. Surprisingly,
these IFN fractions were completely neutralized by a
combination of anti-IFN-anl and anti-IFN-/7 antibodies, and polyclonal anti-IFN-~ and anti-IFN-fl antibodies, but not polyclonal anti-IFN-~ and anti-IFN-~H1
antibodies. This suggested that fractions 50 to 53 were a
mixture of tro-IFN-aii1 and tro-IFN-/7.
Fractions 50 to 53 (see Fig. 1) were further characterized by passage through a HEMA-BIO 1000 VS-antiIFN-fl (polyclonal) antibody column. The antiviral
activity of the flowthrough fractions and the bound IFN
eluted from the column at pH 2.4 was characterized
further using antiserum to human IFN-cq IFN-fl and
recombinant human IFN-aII1 (Table 3). These assays
showed that the antiviral activity of the flowthrough
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1874
G. Aboagye-Mathiesen and others
(a)
(b)
1
94K-~,,
2
~:
3
4
67K-~:~
4 3 K - t l I iii'I
1
:
Table 4. Protection of cell lines from different species
against VSV infection
Antiviral activity*
67K--:~ ~
ii i
14.4K-:,,~llp~Wlllb
3
94K--: ,;~ ~rrz!' :~z,>
i '
20.1K--~:~:
2
43K--::~
q'eIll
t 1 ~ ~-ax
20.1K--:~ :
:. : !i
Species
Cell line
tro-IFN%
tro-IFN-cqnl
tro-IFN-fl
:::! i~ ::
Human
Human
Human
Bovine
WISH
GM 2504
GM 2767
MDBK
64
192
192
128
64
224
224
256
256
768
768
2
14-4K- " . :
Fig. 2. Silver-stained SDS-polyacrylamide gel containing purified troIFNs. (a) Lane 1, standard protein markers; lane 2, tro-IFN-cq and troIFN-cqll purified from a crude tro-IFN preparation on a HEMA-BIO
1000 VS-anti-IFN-ct antibody column; lanes 3 and 4, HP-IAC-purified
tro-IFN-au under reducing and non-reducing conditions respectively.
(b) Lane 1, standard protein markers; lanes 2 and 3, HP-DLACpurified tro-IFN-fl (from fractions 56 to 58 in Fig. 1) under reducing
and non-reducing conditions respectively.
Table 3. Neutralization of the antiviral activity of fractions
50 to 53 after passage through an anti-IFN-fl antibody
column
Residual antiviral activity (~)
Anti-human IFN serum*
Flowthrough
Bound
None
Ctlll
a
fl
100
0
0
100
100
100
100
0
* The anti-IFN immune sera were used in 10-fold excess.
fractions was not neutralized by antiserum to IFN-fl but
was completely neutralized by anti-IFN-~nl and polyclonal anti-IFN-~ antisera. However, the bound IFN
was completely neutralized by human anti-IFN-fl
antiserum.
Fig. 2 shows the patterns obtained by SDS-PAGE of
fractions from HP-IAC of crude tro-IFN preparations
and the HP-DLAC-purified tro-IFN-~i component (fractions 21 to 40 from Fig. 1) on a HEMA-BIO 1000 VSanti-IFN-~ (polyclonal) antibody column. As shown in
Fig. 2 (a) and by antiviral activity neutralization assays of
IFNs extracted from unstained gels [similar to the gel in
Fig. 2 (a) under reducing and non-reducing conditions],
tro-IFN-~ I and tro-IFN-~nl activities corresponded to
the protein bands with MrS of 16K and 22K respectively;
no IFN activity was associated with the 67K protein
band. Fig. 2 (b) shows an SDS-polyacrylamide gel of troIFN-fl (from fractions 56 to 58 in Fig. 1); tro-IFN-fl
migrated as a 24K protein on reducing and non-reducing
SDS-polyacrylamide gels (Fig. 2b, lanes 2 and 3
respectively).
* Antiviral activity is expressed as the highest dilution giving 50%
protection against VSV.
The antiviral activity of the crude tro-IFN preparation
and the purified IFN components were stable after 24 h
incubation at pH 2. Tro-IFN-~nl and tro-IFN-fl bound
to the Con A affinity column (data not shown), whereas
tro-IFN-ar did not bind. This suggested that tro-IFN-~n 1
and tro-IFN-fl are glycoproteins.
Antiviral specificity of tro-IFNs
Table 4 shows the antiviral activities of the tro-IFN
components on different human and bovine cell lines.
They exhibited a broad spectrum of antiviral activities
on the human cells (WISH, GM 2504 and GM 2767)
tested, but tro-IFN-ai and tro-IFN-~ii1 protected bovine
MDBK cells better than human cells (twofold and
fourfold, respectively, relative to human WISH cells).
More interestingly, the protection of MDBK cells by troIFN-0qI1 was twice that conferred by tro-IFN-0q. In
contrast, tro-IFN-fl did not protect bovine MDBK cells
but did protect all the human cell lines tested.
Discussion
Previous work (T6th et al., 1990a; Aboagye-Mathiesen
et al., 1990) in our laboratory has shown that human
placental trophoblast cultures stimulated with poly(rI).poly(rC) produce IFN-fl exclusively. The results
presented here show that trophoblast cultures infected
with Sendai virus produce a mixture of IFN-~, IFN-~nl
and IFN-fl; IFN production increased after differentiation of the cytotrophoblast to syncytiotrophoblast in
vitro (T6th et al., 1990a). Similarly, Burke et al. (1978)
have reported that the production of IFN and IFN
sensitivity change during differentiation of mouse
embryonal carcinoma ceils in vitro.
The tro-IFNs (cti, ~ii1 and fl) are antigenically distinct
and this may suggest that they are structurally different.
Antiserum to recombinant human IFN-anl could not
neutralize the tro-IFN-cq component (fractions 21 to 40
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Sendai virus-induced trophoblast I F N s
in Fig. 1) or tro-IFN-fl, but completely neutralized troIFN-~.I purified by HP-IAC. The ability of polyclonal
anti-human IFN-0t (lymphoblastoid) antiserum to neutralize both the antiviral activities of tro-IFN-0q and troIFN-cq[1 can be explained by the fact that IFN-Ctli1 is a
component of natural mixtures of lymphoblastoid and
leukocyte IFN preparations (Adolf, 1987) used to
prepare polyclonal antibodies. The neutralization results
support the suggestion (Adolf, 1987) that human type 1
IFN is made up of three antigenically distinct proteins,
IFN-cq, IFN-0qi1 and IFN-fl.
The response of different cell lines to the antiviral
effects of tro-IFNs when tested by inhibition of plaque
formation varied. The results presented in Table 4 show
that the tro-IFNs differ from one another in their ability
to confer protection against VSV infection of different
human and bovine cell species. Tro-IFN-fl shows a
degree of species specificity, protecting human cells but
not bovine (MDBK) cells. However, tro-IFN-ul and -ctnl
protect both human and bovine cells. The variations in
the relative antiviral activities of tro-IFNs in the
different cells may indicate that they differentially affect
biochemical pathways induced by IFNs, such as phosphorylation of the eukaryotic initiation factor 2 by the
dsRNA-dependent protein kinase or activation of the 2'5'-oligoadenylate system (Mari6 et al., 1990; Baglioni,
1979). That IFNs differentially induce biochemical
pathways which correlate with particular specific antiviral activities in various lines of cells is indicated by
studies with different IFN preparations (Rosenblum et
al., 1990). Differences between distinct IFNs or between
distinct cell types may be of importance for the
therapeutic application of IFNs, particularly when such
differences occur within the same organism.
The trophoblast layer of the human placenta constitutes the maternal-foetal interface and acts as a barrier
to the transmission of infection from mother to foetus.
The ability of trophoblast cells to produce IFNs may
represent a system for the protection of the foetus from
viral infection. This is shown by the fact that, in some
cases, maternal infection may spread to the placenta but
fail to progress to the foetus (Yamauchi et aL, 1974;
Klein et al., 1976; Remington & Desmonts, 1976). In
addition to the antiviral activity of IFNs, there is
increasing evidence that IFNs are also involved in
normal physiological and regulatory processes such as
cell proliferation and differentiation (Zullo et al., 1985;
Fisher & Grant, 1985). Cells of the cytotrophoblast have
been shown (Bulmer et al., 1988) to be highly proliferative, as measured by the proliferative marker Ki67 or the
presence of mitoses, but lose their proliferative activity
and begin to differentiate as they migrate into decidua
during implantation. Although the factors that control
such aspects of trophoblast behaviour are not known,
1875
IFNs may play a major role in the development of these
differentiated states. Furthermore, IFN-0t has been
shown to be involved in prolongation of allograft
survival (Hirsch et al., 1974; Mobraaten et al., 1973) and
suppression of graft-versus-host disease (Corettini et al.,
1973). Such modulation of the immune response by IFN
may be important in preventing the rejection of the
allogenic foetus.
The results presented in this paper have raised several
questions concerning the trophoblast IFN system. Will
different viruses (or non-viral inducers) induce different
levels of the tro-IFNs already identified ?; how many troIFNs can be expected to exist ?; is there a molecular basis
for the observed biological dissimilarities of the troIFNs?; and do the biological differences between the
tro-IFNs reflect structural dissimilarities of the molecules? Our present work is directed towards answering
these questions and we believe that the results will be
biochemically and virologically important for understanding the mechanism of action and involvement of
tro-IFNs in foetal development, as suggested by the
presence of apparently constitutive IFNs in human
placental blood (Duc-Goiran et al., 1985) and the proven
role of IFNs in maternal recognition of pregnancy in
sheep (Cross & Roberts, 1989).
We thank Dr Giinther R. Adolf for providing antiserum to
recombinant human IFN-o71. We also thank the Danish Society
against AIDS, Snedkermester Sophus Jacobsen og Hustru Astrid
Jacobsens Fond and The Danish Lions Club for supporting the project.
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'(Received 7 December 1990; Accepted 2 April 1991)
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