Note: Descriptions are shown in the official language in which they were submitted.
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DISCLOSURE
Field of the Invention
The invention teaches methods of modulating the immune system of a mammal in
order to prevent spontaneous abortion.
Background
Recurrent Spontaneous Abortion
Recurrent Spontaneous Abortion (RSA) affects 1-2% of women in North America
(1). RSA is believed to be caused by abnormal activation of Thl cells, these
cells
are generally suppressed in pregnancy. Lin et al demonstrated successful
murine
pregnancy is associated with upregulated production of the Th2 cytokines IL-4,
IL-5 and II,-10 in the fetoplacental unit (2). This was postulated to inhibit
development of Thl cells, cells associated with induction of inflammatory
responses. The Th2 biasing of immune response by pregnancy can be seen in
C57BL/6 mice infected with Leishmania. Normally C57BL/6 mice are resistant
to such infection because of a Thl bias. When these mice are pregnant, due to
the
Th2 biasing of pregnancy, infection with Leishmania is possible (3).
Administration of Thl cytokines to pregnant mice can increase the rate of
abortion (4), whereas administration of Th2 cytokine suppresses it (5).
Therapies
which prevent RSA, such as paternal lymphocyte infusion are associated with
augmentation of Th2 responses (6,7). Therefore it appears that successful
pregnancy requires suppression of Thl and promotion of Th2 immune responses.
Paternal lymphocyte infusion therapy was originally devised by Govallo based
on
the idea that RSA occurs due to improper recognition of paternal antigens by
maternal immune system cells (8). Through immunization with paternal
lymphocytes, the mother's immune system will be primed to recognize the
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paternal antigens on the placenta, thus immune response will occur. However,
Govallo believed that stimulation of antiplacental immune response by
injection
of paternal lymphocytes would not cause an inflammatory response, but an
immune response which would actually help the placenta to grow. A parallel
concept was introduced by Prehn in cancer immunology who should that host
antitumor immune response in some circumstance can promote tumor growth (9).
Although some clinical trials using paternal lymphocyte infusion have reported
a
decreased number of RSA (10,11), a recent double blind study reported no
difference between treated and control groups (12). Nevertheless this practice
is
still in use today.
Rational exists for the systemic administration of Th2 cytokines in women
prone
to RSA. Since cytokine like IL-10 can be safely administered, and prevent
activation of Thl immune responses (13). The problem with systemic cytokine
therapy is cost associated, as well as the possibility of global immune
suppression.
Thus there is a need for treatment of RSA in an economical, safe and effective
manner.
Apoptosis
Apoptosis, in contrast to necrosis, is a type of cell death in which membrane
integrity of the cell is kept, thus preventing leakage of intracellular
organelles to
the extracellular mileau (14). The process of apoptosis exists in order to rid
the
organism of unwanted cells without causing an inflammatory response. In
contrast, cells dying by necrosis release various intracellular components
causing
inflammation and tissue damage. The concept of "danger signals" was originally
proposed by Matzinger (15) with the idea that innate signals of danger exist,
which instruct the body to launch immune response or to ignore the antigen.
Under Matzinger's paradigm, danger is associated with tissue necrosis, whereas
lack of danger occurs when cells die by apoptosis. This is exemplified by
studies
showing that intracellular danger signals are released by cells undergoing
necrotic
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but not apoptotic cell death. Such danger signals include free-floating heat
shock
proteins which can activate dendritic cells and macrophages ( 16, 17).
Following
with the idea that cells undergoing necrosis cause immune activation through
the
danger pathway, apoptotic death should inhibit activation of immune response.
One method by which apoptotic cells inhibit immune response is through the
production of immunosuppressive cytokines such as II,-10. In a study by Gao et
al, T cells induced to undergo apoptosis secreted IL-10 protein, as well as
increased production of IL-10 transcripts. In the same study it was
demonstrated
that apoptotic T cells can program macrophages to inhibit activation of pro-
inflammatory Thl immune responses (18). In another study, Voll et al
demonstrated that apoptotic lymphocytes can suppress production of 1'NF-oc and
IL-12 from peripheral blood mononuclear cells activated with
lipopolysaccharide
(LPS) (19).
Conclusion
In light of observations that paternal lymphocytes infusions seem to diminish
spontaneous abortion, and that lymphocytes undergoing apoptosis inhibit
inflammatory responses, we investigated whether transfer of paternal and
maternal origin apoptotic lymphocytes into abortion-prone mice would protect
the
fetus from immunologically mediated destruction.
Summary of the Invention
The disclosed invention teaches a method of inhibiting abortogenic immune
responses through administration of treated blood cells into a pregnant
mammal.
More specifically, paternal or maternal leukocytes are treated with apoptotic
stimuli after which they are introduced into a pregnant female. This process,
we
postulate, results in the inhibition of inflammatory immune responses,
allowing
for completion of pregnancy in situations under which the immune response
would normally result in fetal destruction.
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The invention discloses methods of preparing paternal or maternal leukocytes,
in
a manner which endows the cells with anti-inflammatory properties. Disclosed
is
the utility of these cells in inhibiting anti-fetal immune responses. The
direct
effect of administration of the treated leukocytes on the pregnant female,
that is
the anti-abortogenic effect, is also disclosed.
Brief Description of the Drawings
Figure I illustrates time-dependent apoptotic effects of serum starvation on
murine
leukocytes.
Figure II illustrates the dose-dependent antiabortogenic effects of infusing
apoptotic paternal cells to a pregnant mother induced to undergo abortion by
LPS
administration.
Figure III illustrates the dose-dependent antiabortogenic effects of infusing
apoptotic maternal cells to a pregnant mother induced to undergo abortion by
LPS
administration.
Figure IV illustrates the dose-dependent antiabortogenic effects of infusing
paternal apoptotic cells into the CBA/J X DBAJ2 model of spontaneous abortion.
Figure V illustrates the dose-dependent antiabortogenic effects of infusing
maternal apoptotic cells into the CBA/J X DBA/2 model of spontaneous abortion.
Detailed Description of Preferred Embodiments
This invention teaches methods of preventing immunologically-mediated
spontaneous abortions through administration of apoptotic lymphocytes. This
effect, we believe is mediated through production of soluble and membrane
bound
components on apoptotic cells which suppress inflammatory immune responses.
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It is these inflammatory responses which bring about the demise of the
conceptus.
In the data displayed apoptosis was achieved through serum starvation of the
leukocytes. In other studies we performed similar antiabortigenic effects can
be
seen by injection of leukocytes in which apoptosis was induced through other
methods such as irradiation or treatment with protein synthesis inhibitors. In
agreement with previously published studies, the immunomodulatory effects of
apoptotic leukocytes are independent of the method by which apoptosis was
induced (18, 19).
One embodiment of the invention mends itself applicable to the current
practice
of immunizing pregnant women at risk of spontaneous abortion with paternal
lymphocytes. Paternal leukocytes can be purified from whole blood of the
father
through the FicollTM technique or other methods known to one skilled in the
art.
Apoptosis can be induced in these cells through serum starvation or by other
methods known such as irradiation, treatment with protein synthesis
inhibitors,
treatment with ozone gas, or treatment with hyperthermia. Quality of the
apoptotic cells can be assessed by Annexin-V staining as well as propidium
iodide
in order to ensure a high percentage of apoptotic but not necrotic cells are
administered. Injection of apoptotic cells into the mother can occur via
direct
intravenous administration using syringe and needle, or continuous drip
administration. The same procedure can be carried out using leukocytes derived
from the mother.
Detection of cytokines associated with spontaneous abortion such as interferon-
y
can serve as an immune marker to guide the number of apoptotic cells injected,
as
well as the frequency of injection.
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Examples
Example I
Induction of Apoptosis by Serum Starvation
BALB/c mice were euthanized by carbon dioxide asphyxiation and spleens were
collected under asceptic conditions. Spleens were disassociated with a wire
mesh
and erythrocytes were lysed with hypotonic buffer. Lysed erythrocytes were
removed by centrifizgation. Splenocytes were cultured at a concentration of
105
cells/well in serum-free RPMI media in 96 well plates in a total volume of 200
p,1.
At day 0. 1, 2, 3, and 4 apoptosis was assessed using ApoAlertT"' Annexin V-
FITC apoptosis kit (Clontech, Palo Alto, CA) according to the manufacturer's
instructions. As illustrated in Figure I a time dependent increase in
apoptotic cells
was observed.
Example II
Dose Dependent Inhibition of LPS-Induced Spontaneous Abortion by Infusion of
Apoptotic Paternal Leukocytes
Pregnancy was induced in 8 week old BALB/c females by crossing with C57B6
males. Day zero of pregnancy was established as the day copulatory plug was
observed. On day 5 of pregnancy a single injection of 4 p,g lipopolysaccharide
from Escherichia coli (LPS: Sigma Chemicals, St. Louis, MO), was administered
in 100 p1 of saline through the intraperitoneal route. On day 5 an injection
of
apoptotic paternal (C57B6) leukocytes was administered intravenously through
the tail vein. Apoptotic leukocytes were induced by serum starvation for 4
days
as described in Example I. Mice were sacrificed on day 12 of pregnancy. As
shown in Figure II administration of fresh splenocytes (Untreated leukocytes)
did
not inhibit LPS-induced abortions whereas injection of apoptotic leukocytes
induced a dose-dependent inhibition of abortions. Abortions were quantified as
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percent resorption identified by morphological appearance. Each bar represents
the average (~Standard Deviation) of 7 mice.
Example III
Dose Dependent Inhibition of LPS Induced Spontaneous Abortion by Infusion of
Apoptotic Maternal Leukocytes
Pregnancy was induced in 8 week old BALB/c females by crossing with C57/B6
males. Day zero of pregnancy was established as the day copulatory plug was
observed. On day 5 of pregnancy a single injection of 4 p,g lipopolysaccharide
from Escherichia coli (LPS: Sigma Chemicals, St. Louis, MO), was administered
in 100 p,1 of saline through the intraperitoneal route. On day 5 an injection
of
apoptotic maternal (BALB/c) leukocytes was administered intravenously through
the tail vein. Apoptotic leukocytes were induced by serum starvation for 4
days
as described in Example I. Mice were sacrificed on day 12 of pregnancy. As
shown in Figure III administration of fresh splenocytes (Untreated leukocytes)
did
not inhibit LPS-induced abortions whereas injection of apoptotic leukocytes
induced a dose-dependent inhibition of abortions. Abortions were quantified as
percent resorption identified by morphological appearance. Each bar represents
the average (~Standard Deviation) of 7 mice.
Example IV
Dose-Dependent Inhibition Spontaneous Abortion in The CBAl,I X DBAl2 Model
by Infusion of Apoptotic Paternal Lymphocytes.
CBA/J female mice were bred with DBA/2. Day zero of pregnancy was
established as the day copulatory plug was observed. On day 5 an injection of
apoptotic paternal (DBA/2) leukocytes was administered intravenously through
the tail vein. Apoptotic leukocytes were induced by serum starvation for 4
days
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as described in Example I. Mice were sacrificed on day 12 of pregnancy. As
shown in Figure IV administration of fresh splenocytes (Untreated leukocytes)
did
not inhibit LPS-induced abortions whereas injection of apoptotic leukocytes
induced a dose-dependent inhibition of abortions. Abortions were quantified as
percent resorption identified by morphological appearance. Each bar represents
the average (~Standard Deviation) of 7 mice.
Example V
Dose Dependent Inhibition Spontaneous Abortion in The CBAlJX DBAl2 Model
by Infusion of Apoptotic Maternal Lymphocytes.
CBA/J female mice were bred with DBA/2. Day zero of pregnancy was
established as the day copulatory plug was observed. On day 5 an injection of
apoptotic maternal (CBA/J~ leukocytes was administered intravenously through
the tail vein. Apoptotic leukocytes were induced by serum starvation for 4
days
as described in Example I. Mice were sacrificed on day 12 of pregnancy. As
shown in Figure V administration of fresh splenocytes (Untreated leukocytes)
did
not inhibit LPS-induced abortions whereas injection of apoptotic leukocytes
induced a dose-dependent inhibition of abortions. Abortions were quantified as
percent resorption identified by morphological appearance. Each bar represents
the average (~Standard Deviation) of 7 mice.
The invention discloses a method of inhibiting immunologically mediated
abortions in a mammal. It is to be understood that the above examples are
presented only for clarity and that the invention may take other embodiments
as
practiced by one skilled in the art.
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