Surgery – Miscellaneous – Methods
Reexamination Certificate
1998-05-29
2001-02-06
Isabella, David J. (Department: 3738)
Surgery
Miscellaneous
Methods
C436S504000, C436S518000, C435S005000, C435S007900
Reexamination Certificate
active
06182665
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to a method for determining whether a pregnant woman will successfully complete gestation based upon her immunological responsiveness to recall antigens.
BACKGROUND OF THE INVENTION
Recurrent spontaneous abortion, defined as three or more unexplained pregnancy losses prior to 20 weeks of gestation, occurs in approximately 0.3% of couples who desire children (Stirrat,
Lancet
336:728-733 (1990)). Among the factors identified as causing this disorder are: genetic or chromosomal abnormalities (3-5% of cases); endocrine etiologies (17%); infections (5%); and mularian anomalies (10%). The etiology of the remaining 50-60% of miscarriages is uncertain, but there are reasons to suspect that immunological factors may play a role.
Pregnancy is accompanied by a decline in the reactivity of maternal T-cells to HLA antigens (Tafuri, et al.,
Science
270:630-633 (1995)) and by a diminished immunological responsiveness of the mother (see e.g., Brunham et al.,
J. Clin. Invest.
72:1629-1638 (1983); Muchmore et al.,
J. Immunol.
138:2547-2553 (1987); and Tartof,
Clin. Exp. Immunol.
57:502-510 (1984)). This suggests that maternal immunological changes may help provide an environment conducive to fetal development.
Additional evidence comes from observations made in patients with immunological disorders. Women with rheumatoid arthritis (RA), generally experience an improvement in their symptoms during pregnancy (Cecere, et al.,
Clin. Rheum. Dis.
7:747-768 (1981) This may be related to the down-regulation of tumor necrosis factor alpha, a cytokine suspected of contributing to the severity of RA (Stalimach, et al.,
Lab. Invest.
73:384-392 (1995)). In contrast, women with systemic lupus erythematosus (SLE) often experience a worsening of the disease when they become pregnant (Nelson,
Arthritis Rheum.
139:191-194 (1996)). Diminished interleukin-2 production in response to recall antigens correlates with SLE disease activity and disease flares (Bermas, et al.,
J. Clin. Immunol.
14:169-177 (1994)). Since a similar decrease is also seen during normal pregnancy, this may explain why pregnant women with SLE experience an exacerbation of their symptoms.
Although immunological changes have been associated with pregnancy, the extent to which these changes are necessary for a successful completion of gestation has not been established. A test which correlates pregnancy outcome with a readily measurable immunological parameter would represent a clear advance in reproductive biology and would be of benefit in clinical practice. Ideally, such a test would be simple to perform and effective for the majority of pregnant women.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery that the immunological responsiveness of a pregnant woman to recall antigens can be used to predict whether she will carry a fetus to the point of viability. Women that fail to undergo a normal suppression of immunoresponsiveness have an increased risk of having a spontaneous abortion. In contrast, even women with a history of recurrent spontaneous abortion will maintain gestation to fetal viability when their responsiveness to recall antigens is reduced to an extent comparable to that of normal women.
In its broadest aspect, the invention is directed to a method for predicting if a pregnant woman will carry a fetus to viability by assaying her immunological responsiveness to recall antigens and comparing the results to those of a control group made up of one or more pregnant women known to have carried a fetus to viability. In cases where the woman undergoing testing shows a responsiveness that is not significantly greater than the responsiveness of the control group, it may be predicted that she will carry the fetus to viability. As used herein, a fetus has reached the point of viability when it is capable of surviving outside of its mother's womb, either with or without medical intervention. A woman has a significantly different responsiveness than the control group when p<0.05 using Student's T test.
Preferred recall antigens are those that nearly all of the population has been exposed to and include influenza antigens, tetanus antigens and Candida antigens. In general, assays should be performed on women between 6 and 9 weeks of gestation and will be most beneficial for women that have a history of reproductive failure, e.g., a history of having had at least one spontaneous abortion.
A preferred method for determining the immunological responsiveness of women is by collecting peripheral blood leukocytes (PBLs) and measuring their proliferation both in the presence and absence of the antigen. Cultures not exposed to the antigen serve as a measure of background proliferation, i.e., proliferation not induced by antigen. Background proliferation is subtracted from proliferation in the presence of antigen to determine immunological responsiveness. A preferred method for determining the rate at which cells proliferate is by determining their uptake up tritiated thymidine.
An alternative method for measuring responsiveness to a recall antigen is to determine the degree to which the antigen induces the secretion of a cytokine or growth factor related to the immune response. The preferred cytokines and growth factors for measurements are any of the interleukins, tumor necrosis factors, interferons, colony stimulating factors, leukemia inhibitory factor, transforming growth factors, or epidermal growth factor.
The ability to evaluate the likelihood that a woman will carry a fetus to viability can be used as part of a therapeutic program designed to reduce the likelihood that a pregnant woman will have a spontaneous abortion. This can be accomplished by first evaluating the likely outcome of pregnancy by one of the methods discussed above. If the results do not indicate that it is probable that the woman being tested will carry the fetus to viability, then she may be administered an immunosuppressive agent in an amount and for a duration sufficient to decrease her responsiveness to recall antigens. Ideally, the reduction in responsiveness should be just sufficient so that it no longer differs from the control group to a statistically significant degree. The effectiveness of the treatment regimen can be monitored by periodically repeating assays of responsiveness to recall antigens.
In a somewhat more specific aspect, the present invention is directed to a method for predicting if a pregnant woman will carry a fetus to viability by: a) obtaining PBLs from the woman undergoing testing; b) measuring the proliferation of the PBLs in the presence of one or more recall antigens; c) measuring the proliferation of the PBLs in the absence of the recall antigens; d) determining a stimulation index for the woman by dividing the amount of proliferation in the presence of recall antigens by the amount in the absence of the antigens; and e) predicting that the woman will carry the fetus to viability if the stimulation index is less than three.
The preferred method for assaying the proliferation of PBLs is by measuring the extent to which they incorporate tritiated thymidine and the preferred antigens are those associated with influenza A, tetanus and Candida. Typically, the method should be performed on a woman between 6 and 9 weeks of gestation and it is expected that it will find its greatest use for women selected because of a history of reproductive failure, e.g., a history of having had at least one spontaneous abortion. As with the method described above, the present method can be used as part of a therapeutic regimen for reducing the likelihood that a pregnant woman will experience a spontaneous abortion. This can be accomplished by evaluating the likelihood of her carrying a fetus to viability using the present method and then, if this evaluation indicates that it is not probable that she will carry the fetus to viability, administering an immunosuppressive agent. The agent should be administered in an amount and for a duration sufficient to dec
Bermas Bonnie L.
Hill Joseph A.
Brigham and Women's Hospital
Isabella David J.
O'Hara Kelly
Pillsbury Madison & Sutro LLP
Sanzo Michael A.
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