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Effect of injected human immunoglobulins on fetal rat development. Spinal neural and osseous changes

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Effect of Injected Human Immunoglobulins on Fetal Rat
Development. Spinal, Neural and Osseous Changes
BENJAMIN B. KAMRIN 1
Institute of Rehabilitation Medicine, New York University
Medical Center, New York
ABSTRACT
Rabbit anti-rat-brain serum immunoglobulins injected into pregnant rats on the ninth, tenth, eleventh or twelfth day of gestation resulted in a
fetal resorption rate 15 to 30 times higher than that found in normal untreated
pregnant rats. Human serum immunoglobulins obtained from normal postpartum mothers produced a similar percentage of fetal resorption when injected
by the same routes into pregnant rats of the same gestational age. In neither of
the above experiments were malformations detected among the delivered 20-day
fetuses. Injection of human serum immunoglobulins obtained from mothers of
children with spina bifida manifesta into pregnant rats along similar routes and
periods gave comparable fetal resorption rates. Injection of the above immunoglobulin into the lumen of the uterus adjacent to implantation sites gave a low
fetal resorption rate and produced varying degrees of skeletal and soft tissue
malformations among the viable survivors.
The only difference which could be discerned between normal postpartum
immunoglobulins and those obtained from mothers of spina bifida manifesta
children was characterized in the latter by a two-fold increase in the IgG levels
and the immunoelectrophoretic reactivity of its immunoglobulins with human
spinal cord antigens, The developmental defects observed were: 1. Cranialthinning and bleb formation of skull bones; widening of the foramen magrium;
descent of the obex closer to the foramen magnum. 2. Skeletal-delayed or inhibited calcification of the bodies and spinous processes of the thoracic and
lumbar vertebrae; widening of the vertebral canal and central canal of the
spinal cord.
Alterations in the growth patterns of developing embryos and fetuses have been
induced by exposing them to tissue antiserum. Brent et al. (’61) demonstrated that
rabbit anti-rat kidney serum injected intravenously into pregnant rats on the eighth
day of gestation resulted in severe skeletal
congenital malformations in 100% of the
fetuses. McCallion (’68) reported that rabbit anti-chicken brain serum placed on
33-hour incubated chick embryos and incubated for a further five to seven days resulteld in the formation of localized or
generalized spina bifida in 10% of the surviving embryos. Kamrin (’70) reported
that immunoglobulins from mothers of
children with spina bifida manifesta produced lesions suggestive of spina bifida
and the Arnold-Chiari syndrome when
injected into rats on the tenth day of gesANAT. REC., 173: 173-180.
tation. It is evident in all of these experiments that the exogenous immunoglobulins arrested or slowed the differentiation
of somite cells to cartilage and their subsequent calcification. These experiments
also appear to confirm the findings of
Holtzer et al. (’55, ’61, ’68) that ( a ) using
somite tissue taken from chick embryos
and grown in the absence of spinal cord
or notochord, the production of cartilage
will be prevented; (b) cartilage could be
induced to appear by the addition of embryonic intermediate somitic mesoderm
(from which the kidney forms) and ( c )
“there is no such thing as an undifferentiated, uninstructed cell” and the inducers
or inhibitors are therefore acting on cells
Received Nov. 15, ’71. Accepted Feb. 4, ‘72.
1 Present address : Biophysics Research Laboratory.
Department of Physics, N.Y.U.,
New York, N.Y. 10003.
173
174
BENJAMIN B. KAMRIN
which have received some genetic information.
The following experiments were devised
to test whether the immunoglobulins (predominantly IgG) obtained from mothers of
children with spina bifida manifesta would
produce similar malformations in the rat
fetus after exposure to the material during
gestation. Prior to performing such an
analysis, a series of experiments involving
the injection of rabbit anti-rat brain serum
and normal postpartum serum via various
routes into the pregnant rat was attempted.
METHODS
Animal eqerimentation. Adult and
20-day fetal Wistar rats were anesthetized
and the cerebrum, cerebellum and medulla
oblongata dissected out under aseptic conditions and pooled according to age and
anatomical entity. Each pooled specimen
was homogenized in small volumes of
physiological saline (pH 7.2), placed in
dialyzing sacs and dialyzed against repeated changes of cold distilled water to
remove contained sodium chloride. After
lyophilization, each specimen was stored
in a separate vial in the freezer (-5°C)
until used.
Rabbit antiserum was prepared from
each specimen by injection of the reconstituted material ( 1 ml physiological
saline = 50 mg of antigen) with complete
Freund’s adjuvant into the rabbit’s footpads over a 30-day period. The animals
were bled at one and two weeks after the
last injection. Each of the antiserum was
titrated against the original antigen and
only antibody titers of 128 or more used
in subsequent experiments. The antiserum
was also examined for its IgG, IgA and IgM
content by the use of Hyland Immunoplates
(Hyland Laboratories, California) against
standards of pooled non-pregnant rat
serum and the enclosed standards.
The immunoglobulins in the serum were
precipitated from the antiserum by the addition of an equal volume of saturated ammonium sulfate, mixed thoroughly and
centrifuged at 2000 rpm for 20 minutes at
5°C. The precipitate was dialyzed against
frequent changes of distilled water in a
refrigerator for approximately 72 hours.
The cleared immunoglobulins were lyophilized and stored in individual vials in
the freezer (-5°C) until reconstituted for
use by the addition of 1 ml of physiological
saline (pH 7.2) for each 25 mg of material.
Treatment schedule. On the ninth,
tenth, eleventh or twelfth day of gestation,
pairs of pregnant rats were laporotomized
under ether anesthesia and the exact site
of each implantation charted for each
horn. One of each control/experimental
pair received needle pricks into the uterus
between implantation sites or into the site
itself; or an injection of saline into the
same areas. Or one of the pair would receive an injection of the specific reconstituted immunoglobulins (0.015 to
0.04 ml) intranvenously, into the uterine
lumen or intrafetally so that all the fetuses
were involved on one side. Each experimental animal received only one type of
injection on a specific day with a specific
antiserum.
Recovery and examination of animals.
On the twentieth day of gestation (term =
21 days), each animal was again anesthetized and a laporotomy performed. The two
horns were exteriorized and the number
and position of each fetus compared with
the original findings on the animal. Each
fetus was removed and inspected for gross
malformations. Abnormalities such as alterations in the eye bulge, cranial defects,
cleft lip or cleft palate, position of limbs
and number of toes, defects along the
dorsal midline such as depressions or
bubble-like bulges, or constricted vascularized areas of the limbs and tail were noted
if present. Each animal was weighed, then
pinned fully extended to a small corkboard which carried the notations of animal number, uterine position and malformation data. Each animal was fixed in
10% formalin. The position of each resorption site was noted and related to the particular treatment; nodular masses when
found, were fixed for histological examination or clearing.
The protocol for selection and ultimate
type of examination was as follows: The
implantation site nearest the bifurcation
(right or left) was designated as A, the
next B, C , and so forth to the tip of the
horn. If unresorbed, the fetuses lettered “B’
on the experimental side and in the control
animal were cleared and stained with alizarin. The “C” animals from the experi-
I
I
-
~
-.
HUMAN IMMUNOGLOBULINS ON FETAL RAT DEVELOPMENT
mental and control mothers were sectioned
sagitally and stained alternately in sequence with hematoxylin and eosin, Masson’s trichrome, alizarin and Bodian’s silver
stain. Sectioned material was examined at
X 25 magnification and measurements
made with a calibrated ocular reticle.
The cleared animals were examined for
calcification defects in the skull and vertebrae and for malformations of ribs and
limbs under the stereomicroscope at 6 X,
12 X, and 25 X magnifications.
Human material. Blood serum from
mothers giving birth to normal off-spring
(NS-M) was obtained from 24 hours to
five days postpartum. Blood serum was obtained from mothers of children with spina
bifida manifesta and meningomyelocele
(SpBi-M) at times ranging from one week
to seven months postpartum. Blood serum
was obtained from three spina bifida
children ( SpBi-Inf) 72 hours after birth.
Fresh infant spinal cord and associated
meningomyelocele were obtained at the
death of a seven-month-old child. The
spinal cord was separated from the meningomyelocele and associated tissues, then
homogenized by sonic vibration and grinding with physiological saline and glass
beads in a high speed blender. The resulting mixture was filtered under suction in
the cold, dialyzed to remove the saline and
finally lyophilized. The meningomyelocele
was also homogenized, dialyzed and lyophilized. Both of these materials, when
reconstituted, served individually as antigen in double gel diffusion and immunoelectrophoresis studies against the human
serum.
Prior to the precipitation of the immunoglobulins from the human serum, each individual serum was tested for IgG, IgA and
IgM levels on Hyland Immunoplates
against the contained controls.
Yn double gel diffusion studies, the antigens derived from the spinal cord and from
the meningomyelocele were placed in central wells in agar and the antibodies in the
various immunoglobulins placed in the
surrounding peripheral wells. They were
allowed to diffuse toward each other in the
agar. Where interaction occurs between
the antigen and antibody, whitish bands
are formed indicating that lines of pre-
175
cipitation are formed where antigen and
antibody meet.
When the precipitation lines are indistinct or multiple, as often occurs when
one is dealing with a complex antigen mixture, immunoelectrophoresis is used. The
mixture of antigen is placed in a small well
in the center of a microscope slide on
which has been placed a 3 mm thick layer
of agar. The agar has been mixed with a
buffer solution of about pH 8.2 and is connected to reservoirs at either end containing a similar buffer and electrodes. Under
the influence of the current, the various
proteins in the antigen mixture spread out
toward the anode and cathode because of
varying electrophoretic mobilities. After
several hours, the current is stopped and
the slides removed from the machine. A
long horizontal trough is cut at one side of
the slide. Immunoglobulins derived from
specific individuals are placed in the trough
and double diffusion takes place. Each protein antigen forms an arc of precipitate as
it interacts with its corresponding antibody. In this study, human immunoglobulins from NS-M, SpBi-M and SpBi-Inf sera
were processed against the antigens contained in the spinal cord and meningomyelocele.
RESULTS
In the control group (1) of 256 fetuses,
subjected to no treatment and laporotomy,
the spontaneous fetal resorption rate was
0.7%. Group 2, subjected to pricking of a
needle into the various sites where injections were to be made plus laporotomy
gave the following fetal resorption rate:
intraperitoneal - 0.8% ; intravenous 1.8; intrauterine - 2.0% ;intrafetal - 2.5%.
Group 3, subjected to injection of experimental amounts of physiological saline and
laparotomy showed that the vehicle for the
lyophilized rabbit anti-rat brain antibodies
and human immunoglobulins had a deleterious effect on the fetal resorption rate:
intraperitoneal - 1.0; intravenous - 5.0% ;
intrauterine-6.0% and intrafetal-12.0%.
The injection of equivalent amounts of
rabbit anti-brain serum immunoglobulins,
as well as NS-M, SpBi-M, and SpBi-Inf immunoglobulins showed a marked increase
in the resorption rate of fetuses. Comparison of the lethal effects produced by the
176
BENJAMIN B. KAMRIN
injection of different immunoglobulins
into specific sites adjacent to the developing fetus demonstrated that the closer the
material is deposited to the fetus, the
greater the risk of resorption:
~
Antirat-brain
Intrauterine
(lumen)
Intr af e t a1
NS-M SpBi-M SpBi-Inf
%
%
%
%
15.0
10.2
3.0
12.5
30.0
33.0
29.0
26.5
testing and confidence limits were made
by the use of the “t-test” suggested by Mainland (’52). Using these criteria, it must be
noted that normal human serum also
caused increases in width except for the
first thoracic level where it was similar to
the controls. Measurement of sagittal sections confirmed the data obtained from the
cleared, alizarin-stained animals. In addition, sagittal sections showed widening at
the levels of the foramen magnum, tenth
thoracic and first lumbar vertebrae (table 2). The finding of a significantly enlarged foramen magnum suggested the
presence of an Arnold-Chiari-likesyndrome
and directed attention to an examination
of the position of the cerebellum in the
sagittal sections. Comparison between the
controls and SpBi-M injected experimental
animals showed a marked downward movement of the cerebellum and a thickening
of the cervical spinal cord (in a greatly
enlarged third cervical level of the vertebral
canal). In control fetuses, the level of the
obex in sagittal sections is approximately
3 mm (range 2.5 to 4.0 mm) above the
upper limits of the mineralized occipital
condyle. In SpBi-M treated fetuses, the
obex descends to the upper limits of the
occipital condyle. Such caudal movement
signifies a corresponding downward movement of the cerebellum. Measurement of
the spinal cord central canal failed to show
significant (P = 0.1) enlargement except
in the region of the fourth lumbar vertebra
(table 3 ) .
Precipitin reaction tests in agar of the
various immunoglobulins against antigens
derived from human spinal cord and the
meningomyelocele demonstrated interaction only between SpBi-M immunoglobulins
and spinal cord antigen. Since the precipitate obtained was diffuse and not clear-cut,
immunoelectrophoretic studies were made
of the immunoglobulins obtained from
each of the mothers of children with spinal
bifida manifesta. The dispersed spinal cord
antigen usually showed one definite arc
and occasionally one or two more fuzzy
arcs.
No explanation can be offered why the
SpBi-M immunoglobulins showed a low resorption rate to intrauterine injection, except that they proved effective in obtaining
viable malformed animals.
Intravenous , intrauterine and intr af etal
injections of SpBi-M immunoglobulins into
9, 10, or 11 day pregnant rats produced
little overt external malformation in the
delivered fetuses. Several of the fetuses exposed to intrauterine injection of SpBi-M
had irregularly shaped heads and some encircling constrictions (vascularized depressions) on their limbs associated with the
umbilical cord. There were no signs of
cleft palate or deformed limbs. Clearing
of these specimens in alizarin disclosed
that the frontal and parietal skull bones
were thinned and had transparent blisterlike swellings. Sagittal sections through
these areas demonstrated that there were
small (approximately 1.0 mm) outpocketings of the skull in which the membranous
bone was not mineralized (not stained
with alizarin).
Further evidence of malformation or delayed development was seen in those
cleared animals in which the vertebral
bodies and spinous processes appeared to
be missing (fig. 1). Examination of the
sagittal sections showed that these structures were present but not mineralized.
Some animals showed varying degrees of
vertebral body mineralization with kidneyshaped or paired small centers of ossification at the lateral borders. Measurement
of the interpedicular widths of the cleared,
alizarin-stained specimens at the levels of
the third cervical, first thoracic, and fourth
DISCUSSION
lumbar vertebrae demonstrated a signifiAside from the increased resorption rate
cant enlargement of the width in the
SpBi-M Immunoglobulin-injected animals of fetuses, no specific malformations could
over the controls (table 1 ) . Significance be induced by the injection of rabbit anti-
HUMAN IMMUNOGLOBULINS ON FETAL RAT DEVELOPMENT
177
Fig. 1 Cleared, alizarin-stained fetal rats delivered by laparotomy on twentieth day of
gestation. Left fetus, 3 in left horn, received a total intrauterine injection of 0.03 ml
(0.75 m g ) SpBi-M immunoglobulins on tenth day of gestation. Right fetus 3 in right horn,
received a total intrauterine injection of 0.03 m l physiological saline. Rib and skull distortions caused by manipulation during photography.
rat cerebrum, cerebellum or medulla oblongata antisera. The same results were obtained with human NS-M and SpBi-Inf
sera. Only with serum obtained from
mothers of children with spina bifida manifesta (SpBi-M) could malformations be induced in injected fetuses.
The SpBi-M serum differed from all of
the other sera in its high IgG level and the
ability to react (precipitate) with spinal
cord antigen. The average IgG level for
NS-M was 1200 350 mg/100 ml with a
range of 1200 to 1750 mg/100 ml. The
SpBi-M range was 2200 to 3100 mg/ml
or a two-fold increase. The IgA and IgM
levels were in the same range for NS-M
and SpBi-M sera. If confirmed by a study
of a much greater number of mothers of
children with spina bifida manifesta, these
high IgG levels may provide a clue in determining the etiology of this affliction. It is
well known that IgG readily crosses the
+_
178
BENJAMIN B. KAMRIN
TABLE 1
Measurements o f vertebral interpedicular width derived f r o m the examination o f cleared alizarinstained rat fetuses delivered by laparotomy o n the twentieth day o f gestation
Measurements ( m m )
Treatment
3rd Cervical
vertebra
12
1.09%0.04 1,4
1.09 f0.04
1.01 f0.04
rare 2
none
1.08 f0.05
1.06 f0.04
rare
none
1.3620.02 4,5
1.18f0.03
frequent
frequent
1. None
2. Normal
serum 3
8
1.24 % 0.02
3. SpBi-M
serum
9
1.42f0.10
i34
1st Thoracic
vertebra
Non-c alcific ation
Total
subiects
4th Lumbar
vertebra
Thorac.
bodies
Lumbar
bodies
1 Mean and standard error.
2 Infrequent kidney-shaped partially calcified bodies in untreated and i
n normal serum injected animals; particularly i n 10, 11, and 12 thoracic vertebrae.
3 Saline controls ( 5 ) gave measurements intermediate between untreated and normal serum.
4 Significant difference - P = 0.05 to 0.1.
5 Significant difference - P = 0.1.
TABLE 2
Measurements o f histological sagittal sections o f whole 20 day rat fetuses delivered by
laparotomy on twentieth day o f gestation
Dimension of bony canal ( m m )
Treatment
1. None
Number
exam.
8
2. Normal
serum
10
3. SpBi-M
serum
7
1
2
8
Foramen
magnum
3rd Cerv.
region
1st Thorac.
region
10th Thorac.
region
1st Lumb.
region
4th Lumb.
region
1.03f0.04
0.96f0.04
0.96 f0.06 s
1.14 f0.05
1.05 2 0.05
1.41-10.042 1.41f0.103
1.42e0.09
1.26k0.07
1 . 0 1 ~ 0 . 0 2 1 1.06%0.062
~2
1.01-10.042 1.00%0.02
1.25" 0.03 3
1.23 2 0.02 3
1 . 7 1 f 0 . 0 7 2 ~ 31.55f0.063
1.18 f0.05
Mean and standard error.
Significant difference P = 0.05 to 0.1.
Significant difference - P = 0.1.
-
placental barrier by active transport; if it
carried an antibody against neural tissues,
the IgG molecule could alter the development of these tissues. It must be noted,
however, that examination of the IgG levels
of three babies with spina bifida manifesta
showed readings of 150,575 and 1750 mg/
100 ml, comparable to the findings of
McKay et al. ('67). Therefore, assuming
that the IgG can transport anti-tissue molecules through the placenta, it is possible
that the SpBi-M immunoglobulins interact
directly with the antigens on the predetermined mesenchymal somite cell membrane, thereby altering its metabolic activity (Kamrin, '69). Or, in view of the
evidence of Holtzer ('61) and Lash et al.
( ' 6 2 ) evidence that spinal cord or notochord cells or their products are necessary
for the differentiation of somite cells to
cartilage, the IgG carrying anti-neural antibodies may in some manner inhibit or
neutralize the factors elaborated by the developing neural cells.
CONCLUSIONS
Pregnant rats injected with rabbit antirat cerebrum, cerebellum or medulla oblongata serum immunoglobulins via various routes on the ninth, tenth, eleventh or
twelfth day of gestation did not produce
offspring with gross or microscopic (as
studied at 25 X ) deformities. The injected
immunoglobulins did cause an increase in
the resorption rates of the implanted
fetuses; the major increase occurred from
intrafetal injections. Injections of anti-rat
brain, NS-M, and SpBi-Inf sera into the
lumen of the uterus between the implantation sites resulted in a reduction to onehalf of the fetal resorption rate. Similar
injections of SpBi-M serum into the same
site resulted in a very low fetal resorption
rate (3.0% versus 10 to 1 5 % ) . It was in
HUMAN IMMUNOGLOBULINS ON FETAL RAT DEVELOPMENT
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I wish to thank Doctors C. A. Swinyard
and C. Sansaricq at the Institute of Rehabilitation Medicine, N. Y. U. Medical
Center for their aid and support in making
this study. I appreciate Miss Patricia
Muck's fine technical assistance.
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this latter group that the spinal, neural and
osseous changes were found.
The major differences found in the
SpBi-M serum compared with the serum
of NS-M were: a two-fold increase in the
IgG levels of the SpBi-M serum and the
ability of the SpBi-M serum to form reactivity bands and arcs in double gel diffusion
tests and immunoelectrophoresis preparations.
The teratogenic tendency of the SpBi-M
immunoglobulins was seen in the widening of the foramen magnum, the vertebral
canal and the central canal of the spinal
cord. These findings are accompanied by
apparent delay in calcification of the vertebral bodies and spinous processes and
downward movement of the cerebellum
and obex toward the foramen magnum.
Whether these findings apply to the formation of human spina bifida manifesta
and the Arnold-Chian syndrome is unclear.
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LITERATURE CITED
Brent, R. L., E. Averich and V. A. Drapiewski
1961 Production of congenital malformations
using tissue antibodies. I. Kidney antisera. Proc.
SOC.Exptl. Biol. Med., 106: 523-526.
Grobstein, C., and H. Holtzer 1955 In vitro
studies of cartilage induction i n mouse somite
mesoderm. J. Exp. Zool., 128: 333-358.
Holtzer, H. 1961 Aspects of chondrogenesis and
myogenesis. In: Synthesis of Molecular and
Cellular Structure. D. Rudnick, ed. Ronald
Press, New York, pp. 35-87.
1968 Induction of chondrogenesis: A
concept in quest of mechanisms. I n : EpithelialMesenchymal Interactions. R. Fleischmajer and
R. E. Billingham, eds. Williams and Wilkins,
Baltimore, pp. 15fG164.
Kamrin, B. B. 1969 Role of alpha globulins in
immuno-suppression: Reactive site occlusion
hypothesis. Transpl. Proc., 1: 506-510.
1970 Possible influence of serum immunoglobulins on embryological malformations.
Abstract p. 192 in IXth International Congress
of Anatomists. Leningrad, Russia.
Lash, J. W., F. A. Hommes and F. Zilliken 1962
Induction of cell differentiation. I. The in vitro
induction of vertebral cartilage with a low-
180
BENJAMIN B. KAMRIN
molecular-weight tissue component. Biochem.
Biophys. Acta, 56: 313-319.
Mainland, D. 1952 Elementary medical statistics. W. B. Saunders Co., Philadelphia,
pp. 147-208.
McCallion, D. J. 1968 The production of spina
bifida in the chick embryo by brain antibodies.
Abstract 10 in The Teratology Society. 8th Annual Meeting. Buck Hills, Pa.
McKay, E., H. Thom and P. Gray 1967 Immunoglobulins in umbilical cord plasma. 11. Congenital deformities, other abnormalities and
multiple pregnancies. Arch. Dis. Child., 42:
264-274.
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