WO1994028425A1 - Facteurs embryotoxiques - Google Patents

Facteurs embryotoxiques Download PDF

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Publication number
WO1994028425A1
WO1994028425A1 PCT/US1994/005692 US9405692W WO9428425A1 WO 1994028425 A1 WO1994028425 A1 WO 1994028425A1 US 9405692 W US9405692 W US 9405692W WO 9428425 A1 WO9428425 A1 WO 9428425A1
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Prior art keywords
embryotoxic
factor
concentration
reproductive
cytokine
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PCT/US1994/005692
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English (en)
Inventor
Joseph A. Hill
Deborah J. Anderson
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Brigham And Women's Hospital, Inc.
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Publication date
Application filed by Brigham And Women's Hospital, Inc. filed Critical Brigham And Women's Hospital, Inc.
Priority to AU69544/94A priority Critical patent/AU6954494A/en
Priority to EP94918062A priority patent/EP0700523A1/fr
Publication of WO1994028425A1 publication Critical patent/WO1994028425A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/52—Sperm; Prostate; Seminal fluid; Leydig cells of testes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics

Definitions

  • This invention relates to methods and compositions for diagnosing and treating immunologic reproductive failure.
  • the methods include the administration of an immunomodulating agent for suppressing a cellular immune response.
  • CD4 T helper/inducer lymphocytes This dichotomous nature of the immune system is at least in part determined by regulatory CD4 T helper/inducer lymphocytes. Following activation by antigen, CD4 T lymphocytes produce one of two distinctive cytokine profiles which has led to their classification as T helper 1 (T Targetl) and T helper 2 (T Target2) cells (Mosmann T. et al . ,
  • T H 1 cells primarily secrete interferon-gamma (IFN-gamma), but also interleukin-2 (IL-2) and tumor necrosis factor-beta (TNF-beta, Lymphotoxin) , and induce cellular immunity.
  • T reflex2 cells primarily secrete interleukin-4 (IL-4), but also IL-5 and IL-10, and downregulate cellular immunity while playing a major role in the induction of antibody responses mediated by plasma cells (Kurt-Jones, E. et al. , 1987, 166:1774; Cher, D. et al . ,
  • the cytokine TNF- can be produced during both T interceptl and T formulate2 immune responses, although levels are higher in T formulatel responses and this cytokine is known for cytolytic effects that contribute to the efficacy of cellular immunity (Mosmann, T. et al.,
  • the present invention provides methods and compositions for diagnosing and preventing immunologic reproductive failure.
  • a method for diagnosing a predisposition to immunologic reproductive failure involves contacting a sample containing a plurality of leukocytes derived from the mammal with a reproductive antigen under conditions to stimulate release by the plurality of leukocytes of an extracellular embryotoxic factor, determining the concentration of the embryotoxic factor in the sample and diagnosing a predisposition to immunologic reproductive failure in the mammal by comparing the sample embryotoxic factor concentration to the concentration of embryotoxic factor present in at least one standard.
  • the standard can be a "positive" standard (which contains a concentration of the embryotoxic factor indicative of a predisposition to immunologic reproductive failure) or a "negative” standard (which contains a concentration of the embryotoxic factor indicative of the absence of a predisposition to immunologic reproductive failure) .
  • a diagnosis of a predisposition to immunologic reproductive failure is made if the sample embryotoxic factor concentration is elevated compared to the negative standard or is substantially the same as the positive standard.
  • a method for diagnosing a predisposition to immunologic reproductive failure in a non-pregnant mammal involves obtaining from the mammal a leukocyte secretion-containing sample, determining the concentration of an embryotoxic factor in the sample and diagnosing a predisposition to immunologic reproductive failure by comparing the sample embryotoxic factor concentration to the concentration of embryotoxic factor present in at least one standard as described above.
  • the sample concentration of embryotoxic factor is determined by, for example, an immunoassay that specifically recognizes the embryotoxic factor, an embryo development bioassay, a trophoblast assay or by a lymphocyte proliferation assay.
  • a third method for diagnosing a predisposition to immunologic reproductive failure involves stimulating a sample containing a plurality of leukocytes as described above to release a cytokine, determining the concentration of the cytokine to obtain a sample cytokine concentration and comparing the sample cytokine concentration to the concentration of cytokine present in at least one standard.
  • the standard can be a positive standard (which contains a concentration of cytokine indicative of a predisposition to immunologic reproductive failure) or a negative standard (which contains a concentration of cytokine indicative of the absence of a predisposition to immunologic reproductive failure) .
  • a diagnosis of immunologic reproductive failure is made if the sample cytokine concentration is elevated compared to the negative cytokine standard or is substantially the same as the positive cytokine standard.
  • a diagnosis of immunologic reproductive failure is made if the sample cytokine concentration is reduced compared to the negative cytokine standard or is substantially the same as the positive cytokine standard.
  • a diagnosis of immunologic reproductive failure can be made by observing an increase in the concentration of Trassit-l cytokines, e.g., interferon-gamma, tumor necrosis factor-alpha, tumor necrosis factor-beta or by observing a reduction in the level of certain cytokines, e.g., T Tha-2 cytokines such as interleukin-4 and interleukin-10, in stimulated leukocyte- or leukocyte secretion-containing samples.
  • T Thall cytokines e.g., interferon-gamma
  • tumor necrosis factor-alpha e.g., tumor necrosis factor-alpha
  • tumor necrosis factor-beta e.g., T Thall cytokines
  • T Thall cytokines e.g., interleukin-4 and interleukin-10
  • a method for preventing immunologic reproductive failure includes administering a therapeutically effective dose of an immunomodulating agent to a mammal diagnosed as having a predisposition to immunologic reproductive failure.
  • the method further includes isolating from the mammal a leukocyte secretion-containing sample; determining the concentration of embryotoxic factor present in the sample; and administering one or more subsequent doses of the immunomodulating agent.
  • a leukocyte-containing sample can be isolated from the mammal following initial administration of the immunomodulating agent.
  • the leukocyte-containing sample then is "stimulated", i.e., contacted with a plurality of reproductive antigens to release embryotoxic factor(s) in vitro.
  • the amount of released factor then is determined using one or more of the above-mentioned assays.
  • the subsequent dose of the immunomodulating agent is selected to cause a reduction in the amount of the embryotoxic factor released in vivo by the leukocytes.
  • the immunomodulating agent can be an agent that downregulates a T shiny-l immune response (e.g., a T H ⁇ 2 cytokine or an antibody which specifically binds to or otherwise regulates a T Zhao-l cytokine, thereby abrogating the cytokine's biological activity) or an agent that upregulates a T u ⁇ -2 immune response or otherwise increases the concentration of a T H ⁇ 2 cytokine in vivo.
  • the immunomodulating agent specifically modulates a cellular immune response to a reproductive antigen.
  • the preferred immunomodulating agent is a vaccine containing a reproductive antigen (or an antigen structurally-related thereto) in an adjuvant that downregulates a T Zhao-l immune response or in an adjuvant that upregulates a Tr meshl-2, response.
  • Nonspecific immunomodulating agents also are useful for the purposes of the invention, e.g., progesterone, glucocorticoids, cyclosporins, nifidipine, pentoxiphylline transforming growth factor-beta, intravenous immunoglobulin, anticytokines, cytokine receptor blockers, and antibodies to cytokine producing cells.
  • Progesterone is a preferred nonspecific immunomodulating agent.
  • kits for determining the concentration of an extracellular embryotoxic factor in a sample include a first vial containing an antibody to the embryotoxic factor; a second vial containing a positive or negative standard; and instructions for using the antibody to determine the concentration of embryotoxic factor in the sample and for comparing the sample embryotoxic factor concentration to the standard embryotoxic factor concentration to determine whether the mammal has a predisposition to recurrent immunologic reproductive failure.
  • the first vial includes a plurality of antibodies, each antibody being directed against a different embryotoxic factor. In this manner, the kits can be used to detect a plurality of different embryotoxic factors.
  • the second vial contains a positive standard containing an amount of the embryotoxic factor indicative of a predisposition to immunologic reproductive failure and the kit further includes a third vial containing a negative standard.
  • the negative standard contains an amount of the embryotoxic factor indicative of the absence of a predisposition to immunologic reproductive failure.
  • Kits that are to be used to determine the amount of embryotoxic factor present in a leukocyte-containing sample further contain an additional vial including a plurality of reproductive antigens for stimulating the leukocytes to release the embryotoxic factor(s) in vitro.
  • a vial containing an amount of one or more isolated reproductive antigens capable of stimulating a plurality of leukocytes to release an embryotoxic factor also is provided.
  • reproductive antigens include sperm antigens, rophoblast antigens and choriocarcinoma cell line-derived antigens.
  • the vial containing the reproductive antigens targeted for administration to a mammalian recipient i.e., for stimulating leukocytes in vivo
  • reproductive antigens or structurally-related antigens
  • adjuvant e.g., an adjuvant that downregulates a T Zhao-l cytokine immune response or that upregulates a T Zhao-2 cytokine response to the reproductive antigen
  • Fig. 1 shows the results of an active immunization experiment with various control groups indicating reduced fertility and increased fetal resorptions in sperm-immunized mice.
  • Fig. 2 shows the effects of passive transfer of T lymphocytes from immunized mice (from experiment depicred in Fig. 1), on fertility of recipient mice.
  • Fig. 3 shows the mean numbers + S.D. of T lymphocyte subpopulations and macrophages in uterine sections of mice after immunizations (from experiment depicted in Fig. 1).
  • Fig. 4 shows the mean numbers + S.D. of T lymphocyte subpopulations and macrophages in uterine sections of mice after passive transfer of T lymphocytes from immunized mice (same experiment as depicted in Figs. 1-3).
  • Fig. 5 shows preimplantation embryo recovery in mice following allogenic trophoblast immunization.
  • Immunization groups f, saline; g, adjuvant; adjuvant plus trophoblast.
  • Fig. 6 shows the number of viable offspring (1) and fetal resorption (2) in mice following allogenic trophoblast immunization. The groups are as described in Fig. 4.
  • Fig. 7 shows the dose response curve of Jeg-3 antigen in a lymphocyte proliferation assay.
  • Fig. 8 shows the trophoblast antigen fractions responsible for lymphocyte proliferation (SI>3).
  • Fig. 9 shows the correlation between lymphocyte proliferation (SI) and embryotoxic factors (% blastocysts) in response to trophoblast antigen stimulation.
  • the prior art reveals the difficulties of diagnosing non-genetic causes of recurrent spontaneous abortion.
  • the present invention provides a solution to this problem by disclosing a method for diagnosing in a patient a predisposition to immunologic reproductive failure.
  • immunologic reproductive failure refers to those reproductive failures (abortions occurring at any time post-conception) that are attributable to a cell-mediated immune response. Accordingly, the accurate diagnosis of a predisposition to immunologic reproductive failure is essential for designing effective therapies for preventing or reducing the occurrence of such abortions.
  • a method for diagnosing in a mammal a predisposition to immunologic reproductive failure.
  • the method involves contacting a sample containing a plurality of leukocytes derived from the mammal with a reproductive antigen under conditions to stimulate release by the plurality of leukocytes of an extracellular embryotoxic factor, determining the concentration of the embryotoxic factor in the sample to obtain a sample embryotoxic factor concentration, and diagnosing a predisposition to immunologic reproductive failure in the mammal by comparing the sample embryotoxic factor concentration to the concentration of embryotoxic factor present in at least one standard (described below) .
  • leukocytes embraces lymphocytes (e.g., T-lymphocyte ⁇ and natural killer cells) and macrophages, i.e., the cellular mediators of the cellular immune response).
  • lymphocytes e.g., T-lymphocyte ⁇ and natural killer cells
  • macrophages i.e., the cellular mediators of the cellular immune response.
  • leukocyte secretion-containing sample refers to a preparation containing soluble factors that have been released by lymphocytes or macrophages, in vivo or in vitro. These extracellular factors are released by the leukocytes in response to stimulation by reproductive antigens.
  • Exemplary leukocyte and leukocyte secretion-containing samples include peripheral blood and serum, peritoneal fluid, endometrial tissue, vaginal secretions and saliva.
  • the preferred leukocyte (e.g., lymphocyte/macrophage) and leukocyte secretion-containing sample is peripheral blood.
  • the sample containing a plurality of leukocytes derived (i.e., obtained) from the mammal can be used in the methods of the invention with or without prior culturing.
  • the leukocyte-containing sample is isolated from the mammal, cultured and stimulated in vitro (by contacting the leukocytes with a plurality of reproductive antigens) to release one or more embryotoxic factor(s).
  • contacting it is meant that the leukocytes are exposed to the plurality of reproductive antigens, under conditions such that the leukocytes are stimulated to release soluble factors. Exemplary conditions are provided in the Examples.
  • reproductive antigens refers to sperm antigens, trophoblast antigens, and/or choriocarcinoma cell line-derived antigens.
  • the sperm antigens are isolated from mammalian (e.g., human) motile sperm from fertile donors. Antigen isolation from tissue or cells involves subjecting the tissue or cells to homogenization, followed by washing and dividing the preparation into aliquots containing about 300 ug/ml protein, (see also Examples 1, 3 and 5). Endometrial antigens may be isolated from eutopic (intrauterine) or ectopic (endometriosis) endometrium.
  • the preferred reproductive antigens are derived from a trophoblast cell line, such as choriocarcinoma cell lines BeWo or Jeg-3.
  • Example 1 Diagnosis of Immunologic Recurrent Spontaneous Abortion
  • both sperm and trophoblast antigens stimulate peripheral blood lymphocytes and macrophages from women diagnosed with recurrent reproductive failure to secrete embryotoxic factors in vitro.
  • Embryotoxicity was measured by bioassay, e.g., observing the adverse effect of the released factors on embryo development and trophoblast proliferation in vitro.
  • the trophoblast antigens used in Example 1 were derived from the choriocarcinoma cells lines BeWo and Jeg-3. These cell lines were chosen as the trophoblast antigen model for three reasons: (1) they are homogeneous trophoblast cell lines uncontaminated by other cellular constituents such as stromal mesenchymal cells or lymphoid and myeloid populations that are always associated with normal placental trophoblast; (2) like normal trophoblast, they are devoid of classic major histocompatability complex class I and II antigens; and (3) they are established antigenic models of early normal trophoblast because of their invasive capabilities and cell surface antigen profiles that are similar to those of early normal human trophoblast.
  • the reproductive antigen(s) derived from the Jeg-3 cell line have been fractionated to determine their cellular location (see Example 5). Further purification of the Jeg-3 antigen is within the ordinary skill in the art, using conventional purification and characterization techniques. Thus, for example, gel filtration chromatography is used initially to fractionate a preparation based upon differences in molecular size and various additional chromatographic separation techniques (e.g. , ion-exchange HPLC) are used to isolate reproductive antigenic proteins that are capable of stimulating leukocytes to release an embryotoxic factor in vitro.
  • the Jeg-3 reproductive antigen proteins are isolated and cloned using procedures known to the artisan of ordinary skill in the art.
  • the relative amount or absolute concentration of embryotoxic factor present in a reproductive antigen-stimulated leukocyte-containing sample or in a leukocyte secretion-containing sample is determined using one or more of the following exemplary assays: an immunoassay that specifically detects the embryotoxic factor, an embryo development assay, a trophoblast proliferation assay and/or a lymphocyte proliferation assay (see Examples 1, 4 and 5 for assay protocols).
  • an immunoassay that specifically detects the embryotoxic factor
  • an embryo development assay e., a trophoblast proliferation assay and/or a lymphocyte proliferation assay (see Examples 1, 4 and 5 for assay protocols).
  • a trophoblast proliferation assay i.e., the concentration of embryotoxic factor in the sample
  • lymphocyte proliferation assay see Examples 1, 4 and 5 for assay protocols.
  • a "positive standard” refers to a standard which contains a concentration of the embryotoxic factor that is indicative of a predisposition to immunologic reproductive failure.
  • a “negative standard” refers to a standard which contains a concentration of the embryotoxic factor that is indicative of the absence of a predisposition to immunologic reproductive failure.
  • the preferred assays are im unoassays which specifically determine the amount of an embryotoxic factor present in the sample (see e.g., Example 4) and the lymphocyte proliferation assay (see, e.g. , Example 5).
  • the preferred immunoassays for the purposes of the invention are those which specifically recognize the following antigens: gamma-interferon, tumor necrosis factor-alpha, interleukin-1, interleukin-2, interleukin- , inte leukin-6, interleukin 10 and transforming growth factor-beta.
  • cytokines e.g., T u rl-1 cytokines
  • reduced levels of some cytokines e.g., T-.-2 cytokines such as interleukins 4 and 10
  • T-.-2 cytokines such as interleukins 4 and 10
  • the phrase "elevated concentration" in reference to an embryotoxic factor or cytokine refers to a concentration which is elevated with respect to the concentration of the embryotoxic factor or cytokine in the negative standard.
  • diagnosis of a predisposition to immunologic reproductive failure is made by an immunoassay specific for the embryc oxic factor if the ratio of the sample embryotoxic factor concentration to the negative standard embryotoxic factor concentration is at least about 2:1, i.e., the sample embryotoxic factor concentration is elevated two-fold compared to the concentration of embryotoxic factor in the negative standard.
  • a diagnosis of a predisposition to immunologic reproductive failure is made if the sample embryotoxic factor concentration is substantially the same as the concentration of embryotoxic factor present in the positive standard.
  • substantially the same it is meant that the sample embryotoxic factor concentration falls within the margin of error of the concentration of embryotoxic factor in the positive standard for the particular assay performed.
  • IFN-gamma interferon-gamma
  • TNF-alpha tumor necrosis factor-alpha
  • TNF-beta tumor necrosis factor-beta
  • T Trypsin-2 cytokines play a role in reproductive failure by mediating a cellular immune response to reproductive antigens
  • Tr.i,-2 cytokines are associated with a successful normal pregnancy and presumably play a role in downregulating a cellular immune response to reproductive antigens in vivo.
  • T.,-1 cytokines refers to the cytokines that are released by leukocytes in vivo as part of a T Formula-l type immune response.
  • Exemplary T Zhao-l cytokines include interferon-gamma, TNF-beta, IL-2, TNF-alpha, and potentially IL-12.
  • T H -2 cytokines refers to the cytokines released by leukocytes in vivo as part of a T Zhao-2 type immune response.
  • Exemplary T Zhao-2 cytokines include IL-4, IL-5, IL-6, IL-10 and potentially TGF-beta and colony stimulating factors. Accordingly, the invention is not limited in scope to cytokines that are presently known to be Tmony-l or TNase-2 cytokines but rather, embraces cytokines that are identified in future to fall within these categories.
  • the diagnosis of a predisposition to immunologic reproductive failure can be made by performing a lymphocyte proliferation assay and observing lymphocyte proliferation in response to stimulating the lymphocytes with the above-described trophoblast antigen.
  • Example 5 demonstrates that antigen-stimulated lymphocyte proliferation was significantly higher in women with recurrent abortion of unknown etiology than in fertile controls and that lymphocyte proliferation significantly correlated with embryotoxic factor activity in stimulated culture supernatants.
  • a diagnosis of a predisposition to immunologic reproductive failure was made if the stimulated leukocyte supernatant had a Stimulation Index greater than about 3.0. Fertile control subjects were observed to have a stimulation index that was less than 3.0.
  • Stimulation Index refers to the ratio of counts per minute (cpm) observed for unstimulated cultures to the cpm observed for antigen-stimulated cultures.
  • embryotoxic factor refers to an extracellular factor that is released by leukocytes (i.e., lymphocytes and/or macrophages) in response to stimulation by a reproductive antigen.
  • the embryotoxic factor is further characterized as being toxic to developing mouse embryos and human placental cell lines. Toxicity is determined using the above-recited bioassays. Applicants disclose herein at least three cytokines that are embryotoxic factors.
  • a preparation containing one or more embryotoxic factors has been partially purified and characterized (see Example 1).
  • the preparation is heat-sensitive and, based upon molecular weight dialysis experiments, contains a factor(s) having a molecular weight between about 10 and about 30 kD.
  • heat-sensitive it is meant that the ability of the embryotoxic factor to inhibit mouse embryo development is abrogated following exposure of the factor to 56°C for 1 hour.
  • Immunoassays also have been used to detect an elevated concentration of two other Tbericht-l cytokines (TNF-alpha, TNF-beta) in stimulated leukocytes from women having a history of recurrent spontaneous abortion compared to stimulated leukocytes from fertile controls (Example 4). Accordingly, at least gamma-interferon (one form of which has a molecular weight of 17,000) or a soluble molecule which closely associates (or works in synergy) with gamma-interferon, TNF-alpha and TNF-beta have been identified as embryotoxic factors.
  • TNF-alpha TNF-beta
  • Soluble products of activated macrophages e.g., tumor necrosis factor (TNF)
  • TNF tumor necrosis factor
  • the term embryotoxic factors is not limited to gamma-interferon, but rather embraces any and all soluble factors including cytokines (e.g., T effet-l cytokines) that are involved in a cell mediated immune response to a reproductive antigen.
  • cytokines e.g., T effet-l cytokines
  • Exemplary cytokines that modulate a cellular immune response are shown in Table I .
  • Example 4 also demonstrates that trophoblast antigen-stimulated leukocytes from women with recurrent reproductive failure do not release T Recipe-2 cytokines such as IL-2, IL-4 and IL-10 but that trophoblast antigen-stimulated leukocytes obtained from fertile parous controls do release these T formulate-2 cytokines, as measured in immunoassays which specifically detect each of the above-mentioned cytokines.
  • yet another method for diagnosing a predisposition to immunologic reproductive failure involves stimulating a sample containing a plurality of leukocytes as described above to release at least one cytokine, determining the concentration of the cytokine to obtain a sample cytokine concentration and comparing the sample cytokine concentration TABLE 1
  • GM-CSF cells and intracellular organisms
  • Chemotactic Factors Selectively mobilize and attract onocytes, neutrophils, eosinophils, or basophils to inflammatory site
  • Interleukin-4 Activates resting B and T cells Inhibits antibody class switching from IgM
  • Interleukin-7 Induces proliferation, but not maturation, of early B cells Stimulates proliferation of early B cells
  • Colony-Stimulating Factors • Stimulates granulocyte and monocyte (G -CSF, Interleukin-3) differentiation
  • Interleukin-1 Aids in stimulation of T cell IL-2 production
  • the standard can be a positive standard (which contains a concentration of cytokine indicative of a predisposition to immunologic reproductive failure) or a negative standard (which contains a concentration of cytokine indicative of the absence of a predisposition to immunologic reproductive failure) .
  • a diagnosis of immunologic reproductive failure is made if the sample cytokine concentration is elevated compared to the negative cytokine standard or is substantially the same as the positive cytokine standard.
  • cytokine that is a T Zhao-2 cytokine
  • a diagnosis of immunologic reproductive failure is made if the sample cytokine concentration is reduced compared to the negative cytokine standard or is substantially the same as the positive cytokine standard.
  • a diagnosis of immunologic reproductive failure can be made by observing an increase in the concentration of T..-1 cytokines, e.g., interleukin-2, interferon-gamma, tumor necrosis factor-alpha, tumor necrosis factor-beta or by observing a reduction in the level of certain cytokines, e.g., T Thall-2 cytokines such as interleukin-4 and interleukin-10, in stimulated leukocyte- or leukocyte secretion containing samples.
  • T..-1 cytokines e.g., interleukin-2, interferon-gamma, tumor necrosis factor-alpha, tumor necrosis factor-beta
  • T..-1 cytokines e.g., interleukin-2, interferon-gamma, tumor necrosis factor-alpha, tumor necrosis factor-beta
  • the invention also provides a method for preventing immunologic reproductive failure (see, e.g., Example 2 "Immunosuppressive Therapy in Pregnant Women for Prevention of Immunologic Spontaneous Abortion").
  • the method involves selecting a mammal diagnosed as having a predisposition to immunologic reproductive failure and administering to the mammal a therapeutically effective dose of an immunomodulating agent to prevent an immunologic reproductive failure.
  • the method for preventing immunologic reproductive failure further includes performing one or more of the above-described methods for diagnosing the condition for a leukocyte- or leukocyte secretion-containing sample as described above and administering to me mammal a subsequent dose of the immunomodulating agent to cause a reduction in the amount of embryotoxic factor released in vitro or in vivo by the mammal's leukocytes.
  • the instant invention provides a method for preventing immunologic reproductive failure, which method includes administering at least one subsequent dose of the immunomodulating agent to the patient.
  • the instant invention is not limited to administration of a single immunomodulating agent, but rather embraces the administration of one or more agents, depending upon the particular immunologic response of the patient to immunomodulating agent drug therapy.
  • This iterative treatment process i.e., administration of an immunomodulating agent followed by determination cf the concentration of embryotoxic factor released by patient leukocytes in response to immunomodulating agent drug therapy, allows the tailoring of treatment to the cellular immune response for each patient.
  • immunomodulating agent refers to an agent capable of modulating a cellular immune response and includes agents which directly or indirectly modulate the effective embryotoxic factor concentration in vivo.
  • the immunomodulating agent can be a nonspecific immunomodulating agent (i.e., not targeted to modulating an immune response to a particular target antigen) that downregulates a T-,-1 immune response or that upregulates a T tile-2 immune response.
  • nonspecific immunomodulating agents include glucocorticoids, cyclosporins, nifidipine, pentoxiphylline and progesterone.
  • the immunomodulating agent can be a specific immunomudulating agent that modulates the cellular immune response to a specific reproductive antigen.
  • the specific immunomodulating agent is a vaccine including a reproductive antigen contained in an adjuvant.
  • An adjuvant is selected that downregulates a T Zhao-l type immune response or that upregulates a T Formula-2 type immune response to the reproductive antigen in vivo.
  • Oral vaccines for modulating a cellular immune response to a specific antigen have been described (see, e.g., PNAS, USA 91:437-438 (1994); Immunology Today 12:383-385 (1991); Cellular Immunology 131:302 (1990); PNAS, USA 89:421-425 (1992); Science 259:1321-1324 (1993); and Science 261:1727-1730 (1993), the contents of which references are incorporated herein by references).
  • a particularly preferred cellular vaccine of the invention is an oral vaccine prepared by placing the Jeg-3 antigen in adjuvants such as those described in the above-identified references.
  • Adjuvants which downregulate a T-,-1 type response and/or upregulate a T Formula-2 type response can be selected by determining the T Zhao-l and/or T Zhao-2 cytokine profile following immunization of, for example, an animal with a vaccine containing a test antigen (e.g., BSA, reproductive antigen) contained in the test adjuvant.
  • a test antigen e.g., BSA, reproductive antigen
  • Exemplary adjuvants that upregulate a T H -2 type response (and thereby downregulate a T-,-1 response) include alum and squalene in oil.
  • Additional adjuvants which can be screened for their ability to downregulate a T Formula-l type response and/or to upregulate a T Formula-2 type response include the ISCOMS (Morein, B., et al. , Nature (Lond.). 308:457-459 (1984)), cholera toxin adjuvants (Quiding, M. , et al. , J. Clin. Invest. 88:143-148 (1991)) and complete Freunds adjuvant.
  • the ISCOMS are prepared by removing detergent in a controlled fashion from a mixture of cholesterol, protein, phospholipid, detergent and Quil-A (e.g., by dialysis and cent ifugation on a Quil-A-containing sucrose gradient).
  • Quil-A is a saponin extracted from the bark of the tree Quillaja saponaria. Purification of these saponins and their use as adjuvants is described in
  • immunomodulating agents refers to the concentration of cytokine that is available for binding to cytokine receptors, i.e., the concentration of cytokine that is capable of triggering a cellular immune response.
  • immunomodulating agents embrace agents which function by (1) reducing T leveragerl-l cytokine release from leukocytes; (2) reducing the concentration of receptors capable of binding to the T Zhao-l cytokines; (3) binding directly to T hinder-l cytokines, thereby preventing cytokine binding to receptors; (4) competing with the T Zhao-l cytokines for binding to cytokine receptors; as well as (5) agents which modulate the concentration of any of the above (e.g., T-,-2 cytokines) .
  • immunomodulating agents include agents which are known in the art for their ability to suppress an immune response (e.g., progesterone), as well as TGF-beta and antibodies to the embryotoxic cytokines and/or antibodies to the embryotoxic cytokine receptors (e.g., antibodies to gamma-interferon, tumor necrosis factor-alpha, interleukin-2 and interleukin-6 or antibodies to cytokine producing cells such as CD-3 and CD56 cells or to their receptors).
  • the immunomodulating agent is capable of modulating the cellular immune response in a localized area, i.e., the area in fluid or tissue communication with fetal cells, as distinguished from a humoral immune response.
  • the immunomodulating agents are administered in therapeutically effect amounts.
  • a therapeutically effective amount is that amount which is sufficient to reduce the level of embryotoxic factor(s) to an amount(s) that is insufficient to cause a reproductive failure.
  • the effective amount of agent will depend upon the clinical condition of the subject being treated.
  • a therapeutically effective amount can be determined in a number of ways using medical tech.iiques customary to one of ordinary skill in the art. For example, different amounts of immunomodulating agent are administered to mammals predisposed to reproductive failure, followed by assaying leukocyte- and/or leukocyte secretion-containing samples obtained from the subject. Exemplary assays for determining the level of embryotoxic factor in such samples are provided in the Examples.
  • the therapeutically effective dose of immunomodulating agent is selected which reduces the level of extracellular embryotoxic factor to that level observed in the samples of mammals not having a predisposition to reproductive failure (i.e., a "normal" level). Such levels are considered non-toxic. Likewise, embryotoxic levels that are above normal levels, but which are insufficient to cause reproductive failure, also are considered non-toxic.
  • a therapeutically effective dose of the immunomodulating agent to prevent immunologic reproductive failure is made in accordance with standard procedures known to one of ordinary skill in the art, taking into consideration the patient's clinical condition. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, and individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe does according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
  • a mouse model (see Example 3) is used to screen various immunomodulating agents (and/or the method of administration) for the ability to prevent immunologic reproductive failure.
  • Such screening is performed, for example, by beginning administration of the immunomodulating agent to the mouse prior to, or shortly after conception, and determining whether (1) the incidence of reproductive failure is reduced and/or (2) the level of embryotoxic factor present in mouse samples (i.e., leukocyte and/or leukocyte secretion containing) is reduced.
  • Administration of the immunomodulating agent is performed in accordance with methods known to one of ordinary skill in the art. Accordingly, a variety of administration routes are available. The particular mode selected will depend, of course, upon the particular drug selected, the particular condition being treated and the dosage required for therapeutic efficacy.
  • the methods of this invention may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces therapeutic levels of the agents of the invention without causing clinically unacceptable adverse effects.
  • modes of administration include oral, rectal, vaginal, topical, transdermal or parenteral (e.g. subcutaneous, intramuscular and intravenous) routes.
  • Formulations for oral administration include discrete units such as capsules, tablets, suppositories, patches, lozenges and the like.
  • an immunomodulating agent progesterone
  • compositions may conveniently be presented in unit dosage form, including oral vaccine form, and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing the active agents into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the agents into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product
  • Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the agents.
  • Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the agents, which is preferably isotonic with the blood of the recipient.
  • This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in polyethylene glycol and lactic acid.
  • a non-toxic parenterally-acceptable diluent or solvent for example as a solution in polyethylene glycol and lactic acid.
  • the acceptable vehicles and solvents that may be employed are water. Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectibles .
  • sustained release delivery systems can include sustained release delivery systems.
  • Preferred sustained release delivery systems are those which can provide for release of the agents of the invention in sustained release pellets or capsules.
  • Many types of sustained release delivery systems are available. These include, but are not limited to: (a) erosional systems in which the agents is contained in a form within a matrix, found in U.S. Patent Nos. 4,452,775 (Kent), 4,667,014 (Nestor et al.); 4,748,024 and 5,239,660 (Leonard) and (b) diffusional systems in which an active component permeates at a controlled rate through a polymer, found in U.S. Patent Nos. 3,832,252 (Higuchi et al .
  • a pump-based hardware delivery system can be used, some of which are adapted for implantation.
  • daily oral doses of active compound will be from about 0.01 milligrams/kg per day to 1000 milligrams/kg per day. Small doses (0.01 - 1 mg) may be administered initially, followed by increasing doses up to about 1000 mg/kg per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
  • Example 2 An exemplary therapeutic dose for administration of progesterone in suppository form is provided in Example 2.
  • the therapeutic dose of progesterone (a 50 mg progesterone suppository) was administered twice a day (beginning three days after ovulation in a subsequent conception cycle) to patients having a history of recurrent reproductive failure).
  • the therapeutically effective dose of immunomodulating agent is initially administered in the luteal phase prior to implantation or within one week post-conception.
  • immunomodulating agent drug therapy is continued until the risk of immunologic reproductive failure is eliminated (about 20 weeks) .
  • kits for determining the presence of an elevated concentration of an extracellular embryotoxic factor in a sample contains a first vial including an antibody to the embrytoxic factor, a second vial containing a standard including a concentration of embryotoxic factor indicative of the absence of a predisposition to immunologic reproductive failure ("negative" standard) or including a concentration of embryotoxic factor indicative of a predisposition to immunologic reproductive failure ("positive" standard); and instructions for using the antibody to determine the concentration of the embryotoxic factor in the sample and to compare the sample embryotoxic factor concentration to the standard embryotoxic factor concentration to determine whether the mammal has a predisposition to immunologic reproductive failure.
  • the instructions delineate the assay protocol and the statistical analysis necessary for determining the relative amount or absolute concentration of embryotoxic factor present in the sample.
  • Such instructions are tailored to the specific type of immunoassay being performed, e.g., a competitive assay, a sandwich assay, according to the standard procedures.
  • the antibody to the embryotoxic factor is specifically reactive with the embryotoxic factor.
  • specifically reactive it is meant that the antibody recognizes and binds to an epitope on the embryotoxic factor and does not bind to extraneous molecules present in the sample preparation, which molecules do not include the embryotoxic factor epitope.
  • Tests for determining the specificity of an antibody for a particular protein are known to those of ordinary skill in the art. For example, to demonstrate antibody specificity in an immunoassay in which the signal measured in the immunoassay is generated by virtue of a label attached to the antigen (e.g., a radiolabel, fluorescent tag, or enzyme label), the immunoassay is performed in the presence of an increasing amount of unlabeled antigen.
  • a label attached to the antigen e.g., a radiolabel, fluorescent tag, or enzyme label
  • the second vial includes an amount of embryotoxic factor indicative of the absence of a predisposition to immunologic reproductive failure (negative standard) .
  • the amount of factor which satisfies this criterion is established by assaying (e.g. , bioassay and immunoassay) sample preparations (i.e., leukocyte - and/or leukocyte secretion- containing samples) obtained from fertile women who have no propensity to reproductive failure (see, e.g., the Examples).
  • the second vial serves as a standard to establish a reference value to which the amount of embryotoxic factor quantitated in the sample is compared.
  • the amount of embryotoxic factor present in the second vial is selected such that a ratio of sample embryotoxic factor concentration to standard embrytoxic factor greater than about 2:1 is indicative of a predisposition to immunologic reproductive failure.
  • kits further contain a third vial including a positive control, i.e., a vial including an amount of embryotoxic factor indicative of a predisposition to immunologic reproductive failure.
  • a positive control i.e., a vial including an amount of embryotoxic factor indicative of a predisposition to immunologic reproductive failure.
  • the amount of embryotoxic factor contained in the positive control vial is determined by assaying samples obtained from women diagnosed with immunologic reproductive failure as described above, (see also the Examples).
  • kits further contain a vial including a plurality of reproductive antigens for stimulating the leukocytes to release embryotoxic factors.
  • the reproductive antigens are sperm antigens, trophoblast antigens and/or antigens derived from a human choriocarcinoma cell line.
  • the reproductive antigens are purified from choriocarcinoma cell lines BeWo or Jeg-3.
  • kits for determining an elevated concentration of the embryotoxic factor can easily be adapted for determining the concentration of a particular T Sha ⁇ -l or a T ⁇ u -2 cytokine(s) in the sample (e.g., by substituting an antibody to the cytokine for the above-mentioned antibody to the embryotoxic factor) and including instructions which describe comparing the sample cytokine concentration to a positive or negative cytokine standard to determine whether the mammal has a predisposition to immunologic reproductive failure.
  • vials containing various isolated reproductive antigens and/or embryotoxic factors are also provided within the scope of the invention.
  • the antigen or factor has been removed from its natural matrix (e.g., blood, tissue) and exists in a substantially purified form (e.g., greater than about 80 % pure as assessed by gel electrophoresis) .
  • the vial can include one or more isolated antigens.
  • the isolated antigen has a purity exceeding 90%. A higher degree of purity is needed for the in vivo than is required for in vitro applications.
  • a vial which includes an amount of isolated reproductive antigen capable of stimulating a plurality of leukocytes to release an embryotoxic factor.
  • the vial containing the reproductive antigens for stimulating leukocytes in vivo further includes a pharmaceutically acceptable carrier.
  • Such carriers are known to one of ordinary skill in the art and include, for example, an isotonic saline solution.
  • a vaccine is prepared which includes the reproductive antigen in the above-described adjuvants for downregulating a T-ri,-1 type response or for upregulating a T culinary-2 type response.
  • Example 1 demonstrates that the preimplantation embryo is a vulnerable target of embryotoxic factors produced by activated leukocytes from many women with recurrent abortion.
  • Example 1 further demonstrates that determination of an elevated concentration of embryotoxic factor is indicative of a predisposition to immunologic reproductive failure.
  • Example 2 illustrates the use of immunomodulating agent drug therapy for the treatment of women previously diagnosed with im unologic reproductive failure.
  • Example 3 illustrates the anti-fertility effects of antisperm cell-mediated immunity in mice.
  • Example 4 demonstrates that lymphocytes from a majority of women with unexplained recurrent abortion make T ⁇ sustainl-type cytokines when exposed to trophoblast extracts, whereas lymphocytes from parous women with normal reproductive histories and men make T formulate2-type cytokines.
  • Example 5 demonstrates that lymphocyte proliferation in response to trophoblast antigen stimulation was significantly higher in women with recurrent abortion of unknown etiology than in fertile controls and significantly correlated with embryotoxic factor activity in culture supernatants.
  • the subjects in this study were 300 reproductive-aged, non-pregnant women undergoing evaluation for recurrent abortion at the Brigham and Women's Hospital Reproductive Endocrinology and Infertility Clinic between July 1, 1986 and June 31, 1989.
  • the women were between 22 and 42 years old and had a history of at least three prior spontaneous abortions with or without a stillbirth, ectopic pregnancy, or live birth. These women were grouped by clinical history as follows: women with primary abortion (no live births), women with secondary abortion (abortions subsequent to live births or stillbirths), women with previous ectopic pregnancy, or women with other criteria (abortions interspersed with live births).
  • Peripheral blood was also obtained from a comparison group of 30 non-pregnant, paid volunteer women who were randomly selected from the Obstetrical Nursing Service. Women in this control group were of reproductive age (23 to 41) with a history of at least three prior uncomplicated term deliveries and no history of either spontaneous abortion, ectopic pregnancy, or stillbirth. None of the women in the study were taking medication at the time of blood collection.
  • Trophoblast antigen extracts were prepared from the human gestational choriocarcinoma cell lines BeWo and Jeg-3 (American Tissue
  • the cells were diluted to 10 cells per milliliter in tissue culture media comprising Roswell Park Memorial
  • Semen was obtained from fertile donors by masturbation into sterile containers, and motile sperm were isolated by percoll density gradient centrifugation. Aliquots of 10 7 sperm per milliliter in tissue culture media were dounce homogenized. The suspension was then centrifuged at 400g for
  • tissue culture medium diluted in tissue culture medium to a concentration of 10 cells per milliliter.
  • Leukocyte suspensions were then divided into three 50 ml tissue culture flasks (Becton Dickinson, Lincoln Park, N.J.) containing
  • the cells were next washed once in RPMI medium and once in Whitten's medium and then resuspended in 1 ml of Whitten's medium supplemented with antibiotic-antimycotic solution (Gibco) that had been previously passed through a 0.45 mm filter (Corning Glass,
  • Trophoblasts were prepared from a choriocarcinoma cell line as described above (Example 1) .
  • Embryo culture was performed as previously described (Hill, J.A., et al., J. Immunol. 139:2250-2254 (1987)). Briefly, two-cell embryos were harvested from CD-I female mice (Charles River, Kingston, Ontario) 44 hours after human chorionic gonadotropin administration. Embryos were cultured in 20 ml drops containing a 1:1 dilution of study supernatant in Whitten's medium -0.3% bovine serum albumin under carbon dioxide - equilibrated paraffin oil in Falcon tissue culture dishes(Becton Dickinson) . Media and supernatants were equilibrated overnight in 5% carbon dioxide and 95% air before the addition of embryos.
  • Embryos cultured in Whitten's medium - 0.3% bovine serum albumin alone served as an additional control. At least 11 embryos were cultured in each 20 ml drop. Embryos were assessed for normal development after 4 days in culture (100 hours after human chorionic gonadotropin administration) by the criteria described by Ducibella (Dev. Biol. 79:356-66 (1980)). The experimental end point was the percentage of normal blastocysts in each culture after 4 days of development in vitro. The sensitivity and specificity of the assay were maximal when the median percentage of embryos advancing to the blastocysts stage of development was 50% of control values. The intraassay and interassay coefficients of variation were 8% and 12%, respectively, for the embryo-toxic bioassay.
  • Human gestational choriocarcinoma cells (10 cells per 0.1 ml tissue culture medium) were plated in triplicate into 96-well, flat-bottom microtiter trays (Becton Dickinson). Two hours after cell plating, 0.1 ml of leukocyte supernatant or media alone was added (four wells per test solution) . After a 2-day incubation at 37°C in 5% carbon dioxide and 95% air, 0.5 mCi of tritiated thymidine z(13.1 Ci/mmol; New England Nuclear, Boston) was added to each well. Cells were harvested at 72 hours after trypsinization onto glass fiber filter paper with a MASH II automatic sample harvester (Microbiological Assoc. Los Angeles).
  • the leukocyte proliferation assay is performed according to procedures known to one of ordinary skill in the art (see Example 5 and e.g., Immunol. Rev. 75:61-85 (1983) and J. Reprod. Immunol. 6:377-391 (1984), the contents of which references are incorporated herein by reference) . Briefly, the above-described preparation of reproductive antigen(s) is added to 2.5 x 10(5) leukocytes in culture, followed by assessment of tritiated thymidine incorporation to determine the extent of DNA proliferation. All assays are performed in triplicate. A diagnosis of a predisposition to immunologic reproductive failure is made if the lymphocyte secretion-containing sample has a Stimulation Index that is greater than about 3.0. Fertile control subjects have a stimulation index that is less than 3.0. As used herein, the term "Stimulation Index” refers to the ratio of counts per minute (cpm) for un ⁇ timulated cultures to the cpm for antigen-stimulated cultures.
  • Example 4 An exemplary IFN-gamma ELISA, as well as other cytokine immunoassays useful for the purposes of the invention are provided in Example 4.
  • a commercially-available enzyme-linked immunoassay e.g., Genzyme Corp., Cambridge, MA
  • Genzyme Corp. Cambridge, MA
  • assays also are useful for determining the amount of an embryotoxic factor in a leukocyte-secretion containing samples.
  • Trophoblast antigen-stimulated leukocyte supernatants from 15 patients that adversely affected embryo development were subjected to three different methods, to characterize the factor(s) responsible for embryo toxicity; molecular weight dialysis, heat inactivation, and affinity column purification. The supernatants were dialyzed for 12 hours against three changes of Whitten's medium - 0.3% bovine serum albumin in Spectrapor membrane tubing (Spectrum Medical Industries, Los Angeles) with molecular weight limits of 3500, 10,000 or 30,000 and then tested in the mouse embryo development assay.
  • Women with recurrent abortion were 80% white, 10% Hispanic, 8% black, 1% Native American, and 1% Asian.
  • the 300 women evaluated for recurrent abortion had experienced 1367 prior pregnancies, 1213 of which resulted in reproductive failure (median 4, range 3 to 25). Fetal loss occurred during the first trimester in 88%, whereas 12% of the abortions occurred during the second trimester (before 17 weeks of gestation) .
  • embryo development wa ⁇ more often and more ⁇ everely affected than trophobla ⁇ t proliferation in the presence of both sperm antigen-activated and trophoblast antigen-activated leukocyte ⁇ upernatants (effects of trophoblast and ⁇ perm antigen-activated leukocyte supernatants on embryo development versu ⁇ trophoblast proliferation p ⁇ 0.01).
  • Embryo development was more adversely affected by trophobla ⁇ t antigen-activated leukocyte ⁇ upernatant ⁇ than by sperm antigen-activated leukocyte supernatants, except in those women with a prior ectopic pregnancy.
  • Embryo development in the pre ⁇ ence of trophobla ⁇ t antigen-activated or ⁇ perm antigen-activated leukocyte ⁇ upernatant ⁇ from women with an unknown cause of abortion was significantly less than embryo development in comparable supernatant ⁇ from women with other cau ⁇ e ⁇ (p > 0.01).
  • Embryo development wa ⁇ also observed to be lower in trophobla ⁇ t antigen-activated and ⁇ perm antigen-activated leukocyte ⁇ upernatant ⁇ from women with recurrent abortion cau ⁇ ed by an endocrine or humoral immune abnormality (p ⁇ 0.001) but not in tho ⁇ e from the two groups of women with an anatomic or genetic etiology of recurrent abortions.
  • embryo development was unaffected by sperm antigen-activated leukocyte supernatants but was adversely affected by trophobla ⁇ t antigen-activated leukocyte ⁇ upernatants.
  • embryo-toxic factor ⁇ were produced in women with a diagnosis of endometriosi ⁇ a ⁇ their only abnormal finding in re ⁇ ponse to stimulation by both trophoblast and sperm antigens (p ⁇ 0.05).
  • trophoblast proliferation was also significantly inhibited in response to trophoblast antigen-activated leukocyte supernatant ⁇ from women with endometriosis (p ⁇ 0.05).
  • a woman with a pericentric inversion of chromosome 9 had low embryo development in response to both trophoblast antigen-activated and sperm antigen-activated leukocyte supernatants and low trophobla ⁇ t proliferation in re ⁇ pon ⁇ e to trophobla ⁇ t antigen-activated leukocyte supernatants. Otherwise, cytogenetic abnormalities were not as ⁇ ociated with either embryo or trophoblast toxicity.
  • recurrent abortion was defined as two or more pregnancy los ⁇ e ⁇ before 20 week's gestation.
  • lymphocytes and macrophages from the patients' blood were isolated and maintained in tissue culture in the presence of antigens obtained from a human trophoblast cell line and human sperm (described above) .
  • Supernatant ⁇ from the ⁇ e culture ⁇ were collected and added to two-cell mouse embryos in culture, with assessment of subsequent blastocyst development. A toxic effect was as ⁇ igned when blastocyst development was le ⁇ s than 50% of control values as previously described. All samples were coded and the assays performed by the same technician, who was not aware of the sample source or patient status.
  • the embryotoxic factor assay was predictive of pregnancy outcome (p ⁇ .01). Of 56 women still positive for embryotoxic factor ⁇ early in pregnancy, 40 (71%) mi ⁇ carried (37 in the fir ⁇ t 10 weeks of pregnancy and three between 12-16 weeks) and 16 (29%) delivered a viable infant. Of the 85 women who were found to be negative for embryotoxic factors on repeat testing, only 11 (13%) miscarried (all in the first trime ⁇ ter), whereas 74 (87%) delivered a viable infant. The positive and negative predictive values of the embryotoxic factor as ⁇ ay were 0.76 and 0.86, re ⁇ pectively.
  • Chromo ⁇ omal analy ⁇ e ⁇ of the aborted ge ⁇ tation ⁇ were requested, but not performed in every case. Abnormalities were found in at lea ⁇ t four of 11 karyotyped mi ⁇ carriages in women negative for embryotoxic factors early in pregnancy, including one complete mole, two triploidic partial mole ⁇ , and one auto ⁇ omal triso y 18. Chromosomal analysi ⁇ wa ⁇ available in nine ⁇ ubsequent abortions of women who were ⁇ till po ⁇ itive for embryotoxic factor ⁇ early in pregnancy. Abnormal karyotype ⁇ (two triploidic partial mole ⁇ and one autosomal trisomy 21) were found in three of these nine cases.
  • Leukocyte-containing and/or leukocyte ⁇ ecretion- containing ⁇ ample ⁇ are prepared as described above and are te ⁇ ted for the presence of an elevated concentration of Embryotoxic Factor(s) using the above-described as ⁇ ay ⁇ .
  • ⁇ ample ⁇ are te ⁇ ted u ⁇ ing an assay such as an immunoa ⁇ ay for the Embryotoxic Factor to allow for rapid a ⁇ e ⁇ ment of efficacy of the immunomodulating agent therapy. Accordingly, patient ⁇ ample ⁇ (containing leukocytes and/or leukocyte secretion products) are a ⁇ sayed for the presence of an elevated concentration of Embryotoxic Factor's) prior to and during the immunomodulating agent therapy regimen.
  • An immunomodulating agent such as progesterone, is administered to a patient previously diagnosed (as above) as having recurrent reproductive failure.
  • Leukocyte and/or leukocyte secretion-containing samples are prepared and as ⁇ ayed as described above to determine the amount of Embryotoxic factor present in the sample.
  • a sub ⁇ equent dose of the immunomodulating agent is administered to the patient, the sub ⁇ equent do ⁇ e being selected to cause a reduction in the amount of embryotoxic factor released in vivo by the leukocytes.
  • the immunomodulating agent therapy is precisely tailored to the cellular immune response of each patient.
  • Thi ⁇ iterative process is continued until the concentration of extracellular embryotoxic factor in vivo i ⁇ sufficiently low (as determined by assay and comparison with a standard concentration indicative of the absence of a predi ⁇ po ⁇ ition to reproductive failure) to preclude abortion of the fetu ⁇ .
  • the cell pellet containing motile ⁇ perm was washed 3 time ⁇ and re ⁇ u ⁇ pended at a final concentration of 1 X 10 7/ml m saline.
  • the cell suspen ⁇ ion wa ⁇ mixed 1:1 with CFA or IFA to form a homogenou ⁇ emul ⁇ ion, aliquoted and ⁇ tored at -70°C until use.
  • Spleen Mononuclear Leukocytes Splenic mononuclear leukocytes from DBA/2 retired breeders were isolated by centrifugation through Lympholyte-M
  • mice of the ⁇ ame age and ⁇ train were injected with: (1) ⁇ aline (0.9% NaCl in di ⁇ tilled H_0) a ⁇ a control for stres ⁇ , (2) ⁇ aline-plu ⁇ -CFA as a control for the effects of the CFA alone, and (3) human red blood cells and (4) paternal (DBA/2) mouse lymphocyte ⁇ in
  • Injections (0.1 ml) were admini ⁇ tered: (1) subcutaneously into the neck (s.c), (2) intraperitoneally (i.p.), or (3) intrauterine (i.u.) through the cervix which wa ⁇ dilated by injection 12 h earlier with 1 IU of Pregnant Mare' ⁇ Serum
  • mice were ane ⁇ thetized with 0.2 ml of Advertine ⁇ olution (2.5 ml tert-amyl alcohol) Fisher Labs, Medford,
  • mice in all immunization groups were primed with 1 IU of PMS to cycle the animals but not to induce hyperstimulation.
  • mice in all immunization groups were injected with 1 IU human chorionic gonadotropin (hCG; Sigma) and placed with breeder male ⁇ from our ⁇ tud colony; one female per male in a ⁇ ingle cage.
  • hCG human chorionic gonadotropin
  • Half of the mice were a ⁇ ses ⁇ ed for number of ova and 2-cell embryos 40 h later and the other half were killed at day 15 of pregnancy and the numbers of viable fetuses, non-viable fetuse ⁇ and re ⁇ orption ⁇ ite ⁇ were a ⁇ e ⁇ ed.
  • Blood wa ⁇ drawn by retroorbital puncture for a ⁇ e ⁇ ment of anti ⁇ perm antibodie ⁇ and uteri were snap frozen and stored at -70°C for subsequent immunohistologic examination.
  • Spleen ⁇ and lymph nodes were harvested from s.c./s.c/i.u. immunized C57BL/6 female mice one week after final immunization and from age and ⁇ train-matched non-immunized mice.
  • the lymphoid organs were minced with fine scissors and cells were passed through a fine nylon screen. Red blood cells were lysed according to Maruyama et al . (1985), J. Androl . 6, 127-135 and mononuclear leukocytes were separated by gradient centrifugation on Lympholyte-M at 500 X g for 30 min. and wa ⁇ hed three time ⁇ in RPMI-1640 medium (GIBCO, Grand Island, NY).
  • T lymphocytes were obtained by nylon wool separation following a standard procedure (Trizio and
  • Non-adherent cells (T cell enriched) were resu ⁇ pended in RPMI 1640 at 10 X
  • mice 7 10 /ml and p.l ml wa ⁇ injected intravenously into the tail vein of C57BL/6 female mice.
  • PMS (1 IU) wa ⁇ injected i.p. at the same time.
  • mice Forty-eight hours later, mice were given 1 IU hCG i.p. and mated.
  • mice were killed by cervical di ⁇ location and the numbers of viable fetuses, non-viable fetuse ⁇ and resorption sites were recorded.
  • Uteri were frozen in OCT gel (Baxter McGaw, Park, IL) on dry ice and stored at -70°C for sub ⁇ equent immunohistological a ⁇ e ⁇ ment.
  • Blood wa ⁇ collected by retroorbital puncture prior to termination and serum was ⁇ tored at -70°C for anti ⁇ perm antibody a ⁇ ay.
  • T lymphocytes used for the passive study were:
  • the ⁇ lides were counter ⁇ tained with hematoxylin and mounted with an aqueou ⁇ mounting solution (GVA, Zymed Laboratories, South San Francisco, CA) .
  • Spleen was u ⁇ ed a ⁇ a po ⁇ itive ti ⁇ ue control and PBS in ⁇ tead of the fir ⁇ t antibody as a negative control.
  • mice For each group of mice (5 uteri), three cross-sections from different areas of the block were evaluated. Localization of macrophages and lymphocyte subpopulations was noted de ⁇ criptively. For quantitation, po ⁇ itive cells were counted in five random fields from each ⁇ ection u ⁇ ing a light icro ⁇ cope (X 500 magnification) . The mean number ⁇ and ⁇ tandard deviation ⁇ of leukocyte
  • Fig. 1 shows the results of a single representative s.c. s.c./i.u. active immunization experiment with various control groups indicating reduced fertility and increased fetal re ⁇ orption ⁇ in ⁇ perm-immunized mice.
  • Immunization group ⁇ a, non-immunized; b, ⁇ aline + adjuvant; c, DBA/2 lymphocyte ⁇ + adjuvant; d, human RBC + adjuvant; e, DBA/2 ⁇ perm + adjuvant.
  • Fig. 2. ⁇ how ⁇ the effect ⁇ of pa ⁇ sive transfer of T lymphocytes from s.c./s.c./i.u. immunized mice (from experiment depicted in Fig.
  • T lymphocyte ⁇ were from the group ⁇ (a,b,c,d,e) identified in the de ⁇ cription of Fig. 1.
  • Fig. 3. ⁇ how ⁇ the mean number ⁇ + S.D. of T lymphocyte subpopulations and macrophages in uterine sections of mice after s.c./s.c./i.u. immunizations (from experiment depicted in Fig. 1). Immunization groups were as described above (Fig. 1).
  • Fig. 4. shows the mean numbers + S.D. of T lymphocyte subpopulation ⁇ and macrophage ⁇ in uterine sections of mice after pas ⁇ ive tran ⁇ fer of T lymphocytes from s.c./s.c./i.u.
  • Fig. 5 shows ⁇ prei plantation embryo recovery in mice following allogeneic trophoblast immunization. Immunization group ⁇ : f, ⁇ aline; g, adjuvant; adjuvant plus trophoblast.
  • Fig. 6 ⁇ how ⁇ the number of viable off ⁇ pring and fetal re ⁇ orption in mice following allogeneic trophoblast immunization. The groups are as described in Fig. 5.
  • mice were admini ⁇ tered T cell-enriched lymphoid cells from sperm-immunized, non-immunized and control-immunized syngeneic mice via the tail vein.
  • the transferred cells were primarily CD4+ lymphocytes; less than 15% of the cells were macrophage ⁇ or CD8+ T cell ⁇ . Numbers of viable fetuses and resorption sites were stored on day 15 of pregnancy.
  • the fertility rate wa ⁇ reduced in mice receiving lymphocytes from sperm-immunized animals (Table IV).
  • stage of e ⁇ tru ⁇ was not correlated with numbers of T cells in uterine horns of control mice or of sperm-immunized mice.
  • Both CD4+ and CD8+ subpopulation ⁇ were ob ⁇ erved in uterine ti ⁇ sue from sperm-immunized mice, but CD8+ cell ⁇ predominated.
  • Mo ⁇ t of the CD8+ cell ⁇ were located in the pe iglandular ⁇ pace, while CD4+ cell ⁇ were evenly ⁇ pread throughout the epithelium and periglandular region.
  • mice Titers of antisperm antibodies in pooled sera from s.c./s .c./s.c/ or i .u/i . ./i .u. ⁇ perm-immunized mice were 1/256 and 1/1014, respectively. Both groups showed a predominantly acro ⁇ omal ⁇ taining pattern. Pooled sera from s.c. s.c./i.u. ⁇ perm-immunized mice had a titer of 1/1014 and reactivity directed again ⁇ t both the sperm acroso e and tail region ⁇ . Sera from mice which were pa ⁇ ively immunized with lymphocyte ⁇ from actively sperm-immunized ( ⁇ .c./ ⁇ ,c .
  • mice were negative for anti ⁇ perm antibodie ⁇ .
  • Normal mou ⁇ e sera negative control
  • sera from mice immunized with ⁇ aline and from mice injected with lymphocyte ⁇ from non-immunized mice were also negative.
  • FIG. 5 The re ⁇ ults of the trophoblast antigen immunizations are shown in figures 5 and 6. Immunizations were conducted using the above-described protocols for the immunization of mice with sperm. Specifically, C57BL/6 female mice were immunized with allogeneic trophoblast in complete Freund's adjuvant given subcutaneously, intraperitoneally and transcervically (intrauterine). These experiments demonstrate that trophoblast immunization result ⁇ in a decreased number of recovered preimplantation embryos (Fig.
  • mice C57BL/6 female mice were immunized with allogeneic (DBA/2) sperm in Freund' ⁇ adjuvant either subcutaneously (s.c), transcervically into the uterine lumen (i.u.), or with a combination of s.c. and i.u. immunization approaches.
  • Control mice received DBA/2 lymphocytes, human erythrocytes or saline in adjuvant using the same immunization protocols.
  • Immunization with ⁇ perm or control cell ⁇ in adjuvant exclu ⁇ ively by ⁇ .c. or i.u. approache ⁇ did not affect subsequent fertility, although sperm-injected mice from both protocols had high titer ⁇ of circulating anti ⁇ perm antibodies.
  • Mice receiving sperm by the ⁇ .c./i.u. protocol had high titer ⁇ of anti ⁇ perm antibodies and a marked infiltration of T lymphocytes and macrophages into the uterine endometrium.
  • T lymphocytes from spleens and pelvic lymph nodes of s.c./i.u. sperm-immunized mice and non-immunized mice were passively transferred to naive syngeneic female recipients which were subsequently mated.
  • the total number of fetuses on day 15 of pregnancy was significantly reduced in mice receiving T-lymphocytes from sperm-immunized mice and a significant increa ⁇ e in fetal re ⁇ orption ⁇ ite ⁇ wa ⁇ al ⁇ o ob ⁇ erved.
  • mice did not have detectable titers of circulating antisperm antibodies, but had a significant infiltration of CD4+T lymphocytes and macrophages in the uterine epithelium and endometrium.
  • the ⁇ e data indicate that intrauterine anti ⁇ perm cell-mediated immunity can be induced in mice by a combination of ⁇ ystemic and intrauterine immunizations and provide evidence for the existence of reproductive tract mucosal antisperm cellular immune responses that adversely affect fertility and pregnancy.
  • Example 4 T Helper 1-Type Cellular Immunity to Trophobla ⁇ t Antigens in Women with Recurrent Spontaneous Abortion
  • Peripheral blood was obtained from 244 non-pregnant women with a history of unexplained recurrent reproductive failure who were referred for evaluation to the Recurrent Mi ⁇ carriage Center, Brigham & Women's Hospital.
  • the women were between 26 and 40 years of age and had a history of at least three prior first trimester spontaneous abortions with or without a prior ectopic gestation or live birth.
  • the etiology of prior pregnancy losses was unexplained by conventional criteria (normal parental chromo ⁇ ome ⁇ , hysterosalpingography, luteal pha ⁇ e endometrial biop ⁇ y, hormonal analy ⁇ is, cervical cultures and antiphospholipid antibodie ⁇ ) .
  • Peripheral blood was also obtained from a control group of 13 paid volunteer non-pregnant women between 27 and 41 years of age with a history of at least two uncomplicated term deliveries with their last birth occurring within one year of blood collection and no history of spontaneou ⁇ abort.on, ectopic pregnancy or ⁇ till-birth.
  • blood wa ⁇ obtained from 10 men between 26 and 47 year ⁇ of age. All of the individual ⁇ in this study were in excellent health, had no history of allergy or atopy, and were taking no medication at the time of blood collection.
  • trophoblast antigen extract was prepared from the human choriocarcinoma cell line Jeg-3 (American Tissue Type Collection, Bethesda, MD) as described above (see al ⁇ o Hill, J. et al., 1992, Am. J. Ob ⁇ tet. Gynecoi. 166:1044). Briefly, mycopla ⁇ ma-free Jeg-3 cell ⁇ were cultured in flasks until 80% confluence in Minimum Es ⁇ ential Medium (MEM; Gibco, Grand I ⁇ land, NY) ⁇ upplemented with 10% fetal bovine ⁇ erum.
  • MEM Minimum Es ⁇ ential Medium
  • the cell ⁇ were harve ⁇ ted with a rubber cell scraper (Coaster, Cambridge, MA) and wa ⁇ hed three times in Hank's Balanced Salt Solution (GIBCO) .
  • the cells were then disrupted by Dounce homogenization (100 strokes) and the supernatant ⁇ aved after centrifugation at 400g for 10 minute ⁇ .
  • Protein concentration wa ⁇ determined by BCA reagent kit (Pierce, Rockford, IL) and antigen extracts were adjusted to 333ug/mL.
  • Fetal calf serum (GTBCO) was added to a final concentration of 10%, and 1 mL aliquots containing approximately 300ug of trophobla ⁇ t extract each, were stored at -70°C until use.
  • Peripheral venou ⁇ blood wa ⁇ collected into tube ⁇ containing pyrogen-free sodium heparin (Riches, P. et al., 1992, J. Immunol. Method 153:125), and mononuclear cells were isolated by Ficoll-Hypaque centrifugation as de ⁇ cribed above (see also Hill, J. et al., 1992, Am. J. Obstet. Gynecol . 166:1044).
  • wa ⁇ hed cell ⁇ were re ⁇ uspended to a concentration of 10 8 cells/mL in Roswell Park Memorial In ⁇ titute RPMI medium Gibco ⁇ upplemented with 0.3 mmol/L glutamine, 100 IU/mL benzylpenicillin pota ⁇ ium and 100 ug/mL streptomycin sulfate (Sigma Chemical Co., St. Louis, MO) and 10% fetal bovine serum.
  • TNF- ⁇ , TNF- ⁇ , and IFN-gamma kits were obtained from Endogen (Boston, MA; lower limit of sen ⁇ itivity, 10 pg/mL for TNF- ⁇ and IFN-gamma and 31pg/mL for TNF- ⁇ ), IL-2 kits from R&D sy ⁇ tem ⁇ (Minneapoli ⁇ , MN; lowe ⁇ t limit of sensitivity, 31 pg/mL) , IL-4 kits, from Amer ⁇ ham (International Place, UK; lowest limit of sen ⁇ itivity, 31 pg/mL), and IL-10 kits from The Biosource CytoTM (Biosource International, Camarillo, CA; lowest limit of sen ⁇ itivity, 15 pg/mL) .
  • ELISA a ⁇ says were ⁇ olid pha ⁇ e ⁇ andwich ELISAs in which specific monoclonal antibodie ⁇ were attached to wells in 96 well plates, and a secondary enzyme-conjugated specific antibody was added for cytokine detection. Following incubation with substrate, the colored product was measured in each well on a Dupont microplate reader (Dupont, Dover, DE) using a wavelength appropriate for the ⁇ ub ⁇ trate used. Cytokine concentrations were calculated from a standard curve generated with specific cytokine ⁇ tandard ⁇ provided with each kit.
  • the T carefullyl cytokine IFN-gamma was found in 125 (51.2%) of 244 supernatants from women with unexplained recurrent reproductive failure and significantly correlated with embryotoxic factor activity (75.6%) of 160 supernatants with embryotoxic activity vs 4 (4.8%) of 84 ⁇ upernatants without embryotoxic activity contained IFN-gamma (p ⁇ 0.0001).
  • the range of IFN-gamma levels in these ⁇ upernatant ⁇ wa ⁇ 10.3 to 2295 pg/mL with a mean of 209 + 34 pg/mL.
  • T H l-associated cytokines Table V.
  • IFN-gamma range 10.95-2,295 pg/mL; mean 517+141 pg/mL
  • TNF-alpha range 11.75-5646 pg/mL; mean 1199+388 pg/mL
  • TNF- ⁇ wa ⁇ detected in 17 of the 20 supernatants (range 47.6-578.1 pg/mL; mean 287 + 46.1 pg/mL) .
  • IL-2 was initially detected (within the first 24 hour ⁇ ) but wa ⁇ not detected not detected after four days in any of the 20 supernatant ⁇ .
  • Supernatants from unstimulated cultures of women with recurrent abortion contained neither T deliberately nor T complicat2 type cytokines.
  • TNF-gamma (INF-gamma, IL-2, TNF- ⁇ , or TNF- ⁇ ); however, T deliberately ⁇ 2 cytokines were detected in all 13 supernatants from parous women (Table VI) and 9 of 10 ⁇ upernatants from men (Table VII).
  • IL-10 was detected in all 13 supernatant ⁇ from parou ⁇ control ⁇ (range 18.2-283 pg/mL; mean 65+23 pg/mL)
  • IL-4 wa ⁇ detected in three ⁇ upernatants (31.10, 40.58 and 81.65 pg/mL) .
  • IL-10 was identified in 9 of 10 trophoblast activated cell culture supernatants (range 22.4-10.90 pg/mL; mean 46 + 8.7 pg/mL), IL-4 was not demonstrable, while unstimulated cell culture supernatants from all parous women contained IL-10 (range 29.1-279.2 pg/mL; mean 76.8 + 19.2 pg/mL) . IL-10 was detected in 5 of 10 unstimulated cell culture supernatants from men (range 20.3-478.1 pg/mL; mean 260.2 + 75.4 pg/mL) . (4) Di ⁇ cu ⁇ sion
  • T lymphocytes from gravid women respond to trophoblast antigen ⁇ with a dichotomou ⁇ T helper immune response Lymphocytes from many women (51.2%) with a hi ⁇ tory of unexplained recurrent reproductive failure produced T H l-type cytokines following exposure to trophoblast cell extracts in vitro, wherea ⁇ lymphocyte ⁇ from women with a hi ⁇ tory of normal pregnancies and men when placed under identical conditions produced T confuse2-type cytokines.
  • peripheral blood mononuclear cells were obtained from 244 non-pregnant women with a history of unexplained recurrent reproductive failure by conventional testing criteria, 13 non-pregnant women with normal reproductive historie ⁇ , and 10 men.
  • IFN-gamma was detected in 51.2% of the trophoblast-activated mononuclear cell ⁇ upernatants from 244 women with otherwise unexplained recurrent reproductive failure and in none of the parou ⁇ control ⁇ or men.
  • Thi ⁇ data supports our hypothesi ⁇ for a new etiology for recurrent abortion based on an aberrant cellular immune response to trophoblast involving the T formulatel pathway.
  • TNF- ⁇ and TNF- ⁇ wa ⁇ detected in 20 and 17 ⁇ upernatant ⁇ re ⁇ pectively from the recurrent abortion group, and in none from the parou ⁇ control group or from men.
  • TNF- ⁇ i ⁇ a T culinaryl-type cytokine and becau ⁇ e TNF- ⁇ is produced in higher amounts in a T culinaryl response than in T confuse2 immunity (Romagnani, S. et al. , 1992, Int. Arch. Allergy Immunol. 4:279; Del Prete, G. et al . , 1993, J. Immunol. 150:353).
  • the cytotoxic effect exerted in T-,1 immunity involves IFN-gamma, TNF- ⁇ and TNF- ⁇ (Tite, J. et al.
  • Additional therapie ⁇ involving the admini ⁇ tration of an immunomodulating agent (to modulate the concentration the above-identified cytokine ⁇ that play a role in immunologic reproductive failure) , antibodie ⁇ to T reflexl cytokine ⁇ that abrogate the biological activity of the ⁇ e cytokine ⁇ , or admini ⁇ tration of intravenou ⁇ immunoglobulin through anti-idiotype binding to T-cell receptor idiotype ⁇ , ⁇ hould also prove beneficial in preventing reproductive failure due to T reflexl immunity.
  • Blood wa ⁇ obtained from 57 non-pregnant women with a history of recurrent abortion at the Recurrent Miscarriage Center, Brigham and Women' ⁇ Ho ⁇ pital between February and June, 1993 in accordance with Human Subject ⁇ ' Committee approval.
  • the women were between 29 and 41 years of age and had a history of at least three prior fir ⁇ t trime ⁇ ter ⁇ pontaneou ⁇ abortions, with or without a prior ectopic gestation or live birth. All women were evaluated according to a standard protocol by a single investigator (JAH) as previously described.
  • Trophobla ⁇ t antigen ⁇ were prepared from the human choriocarcinoma cell lines Jeg-3 and JAR (American Tis ⁇ ue Type Collection, Bethe ⁇ da, MD) .
  • the ⁇ e cell ⁇ were cultured in fla ⁇ k ⁇ until 80% confluence in Minimum E ⁇ sential Medium (MEM; GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) .
  • MEM Minimum E ⁇ sential Medium
  • FBS fetal bovine serum
  • the cells were harve ⁇ ted without tryp ⁇ inization with a rubber cell ⁇ craper (Coa ⁇ ter, Cambridge, MA) and wa ⁇ hed three times in RPMI 1640 media (GIBCO) .
  • the cells were then disrupted by dounce homogenization (greater than 100 ⁇ troke ⁇ ) and the supernatant was saved after centrifugation at 400g for 10 minute ⁇ .
  • Jeg-3 cell ⁇ were harve ⁇ ted without tryp ⁇ inization a ⁇ above and wa ⁇ hed three times in ice-cold phosphate buffered saline (PBS) .
  • the cell pellet was re ⁇ u ⁇ pended in ice-cold hypotonic buffer (lOmM Tris-HCl pH 7.6, 0.5 mM MgCl 2 ) for 10 minutes and the cell ⁇ u ⁇ pen ⁇ ion wa ⁇ added to a chilled Dounce homogenizer and delivered 35 ⁇ trokes.
  • Tonicity re ⁇ toration buffer (lOmM Tri ⁇ -HCl, pH 7.6, 0.5mM MgCl 2 , 0.15M NaCl), wa ⁇ then added to the homogenized cell ⁇ , and the mixture was centrifuged at 500g at 4°C for 5 minutes to obtain a crude nuclear pellet (designated a ⁇ Nuclear Fraction) . The ⁇ upernatant was then centrifuged at 20,000g, 4°C for 30 minutes to i ⁇ olate mitochondria, Golgi, microsomes and cell debris (designated the Organelle Fraction) .
  • Nuclear, Organelle and Membrane antigen pellet ⁇ were ⁇ olubilized by incubation at 60°C for 5 minute ⁇ in 20uL of 0.2% sodium dodecyl sulphate in 10 mM Tri ⁇ -HCl, pH 7.6 and protein concentration ⁇ were determined by BCA reagent kit (Pierce, Rockford, IL) . All trophobla ⁇ t antigen sources were sterilized by gamma-irradiation (150 Gy) and stored at -70°C until use.
  • % SI Percent stimulation index
  • Red blood cell ⁇ (RBC) were ⁇ eparated from donor ⁇ ' white blood cells after Ficoll-hypaque gradient centrifugation and removal of buffy coat containing neutrophils. RBC were incubated in hypotonic solution and wa ⁇ hed three time ⁇ at 500g, 4°C for 10 minutes to remove hemoglobin. RBC membrane control antigen was then prepared after dounce homogenization and centrifugation as described above. (b) Lymphocyte proliferation a ⁇ ay
  • Peripheral blood mononuclear cell ⁇ were i ⁇ olated from heparinized blood by Ficoll-hypague gradient centrifugation.
  • Washed cells (2 x 10 ) were cultured in 96-well round-bottom microtiter plate ⁇ (Corning, Corning, NY) containing RPMI 1640 media, 10% human ⁇ erum (type AB, GIBCO) and antibiotic ⁇ in a final volume of 200 uL. Twenty microtiter ⁇ of antigen extract or medium alone were added to triplicate wells. After a 6 day incubation at 37°C, 5%
  • peripheral blood mononuclear cell ⁇ were i ⁇ olated from heparinized blood by Ficoll-Hypaque gradient centrifugation a ⁇ de ⁇ cribed for the lymphocyte proliferation assay. Washed cells were re ⁇ u ⁇ pended in lOmL of RPMI medium ⁇ upplemented with 10% human ⁇ erum to a concentration of 10 cell ⁇ /mL. The cell ⁇ were cultured with or without Jeg-3 antigen (30ug/mL) for 96 hour ⁇ at 37°C in 5% C0 2 , 95% air.
  • the cell pellet wa ⁇ re ⁇ u ⁇ pended in 2mL of Whitten' ⁇ media ⁇ upplemented with 0.3% bovine ⁇ erum albumin. After an additional 24 hour incubation, cell ⁇ u ⁇ pen ⁇ ions were centrifuged and the supernatants were filter-sterilized and ⁇ tored at -70° until u ⁇ e.
  • Two-cell embryos were harvested from ⁇ uperovulated CF-1 female mice that had been bred to C57BL6 male mice. All media and supernatants were equilibrated overnight in 5% Co- before the addition of embryos.
  • Embryos were cultured in 20uL drops containing a 1:1 dilution of study supernatant in Whitten's medium ⁇ upplemented with 0.3% bovine ⁇ erum albumin under C0 2 -equilibrated paraffin oil in ti ⁇ ue culture di ⁇ he ⁇ (Falcon, Oxnard, CA) .
  • At lea ⁇ t 11 embryo ⁇ were cultured in each 20uL drop.
  • the embryo ⁇ were a ⁇ e ⁇ ed for normal development after 4 day ⁇ in culture by the criteria described by Ducibella (Ducibella T., 1980, Dev. Biol., 79:356).
  • Embryotoxic factors were considered present when the percentage of embryos advancing to the blastocyst stage of development was les ⁇ than 50% of control value ⁇ a ⁇ previou ⁇ ly de ⁇ cribed (Hill J. et al. , 1992, Am. J. Ob ⁇ tet. Gynecol. 166:1044; Ecker J. et al. , 1993, Ob ⁇ tet. Gynecol. 84).
  • the embryotoxic factor assay was performed by a single investigator without knowledge of sample source or results of the lymphocyte proliferation as ⁇ ay.
  • the mean SI of the 57 women with a history of recurrent abortion was significantly higher than that of the control group (3.99+2.83 vs. 1.64+0.34, p ⁇ 0.01).
  • the mean SI in response to RBC membrane antigen was not ⁇ ignificantly different between group ⁇ (1.33+0.63 v ⁇ 1.10+0.29).
  • lymphocytes from 27 women that had responded to the crude Jeg-3 antigen extract (30ug/mL) were te ⁇ ted again ⁇ t antigen extracts made from Jeg-3 derived Nuclear, Organelle, Cytosol and Membrane fractions.
  • the optimal concentration of each antigen extract was lOug/mL, as determined by do ⁇ e re ⁇ ponse studie ⁇ in the 6 positive women who had originally responded to Jeg-3 (data not shown). As shown in Fig.
  • trophoblast antigen derived from cytosol and membrane component ⁇ were responsible for lymphocyte proliferation in the majority of individuals (55.6% and 37.0%, re ⁇ pectively). Antigenicity wa ⁇ completely abrogated after trophobla ⁇ t antigen treatment with either trypsin or heat (data not shown) .
  • placental cell ⁇ derived from chorionic membranes or normal human placenta as antigenic source ⁇ with conflicting re ⁇ ult ⁇ reported (Goodfellow C. , 1983, Immunol. Rev. 75:61; Hunt J. et al. , 1984, J. Reprod. Immunol. 6:377). Stimulation may be due to contamination by nontrophobla ⁇ t cell ⁇ ⁇ uch a ⁇ fetal or maternal white blood cells expressing HLA.
  • Jeg-3 and JAR cell ⁇ were found to be ⁇ uitable antigenic ⁇ ource ⁇ , with Jeg-3 cell ⁇ being more antigenic than JAR in ⁇ timulating lymphocyte proliferation in women with unexplained recurrent abortion.
  • the trophobla ⁇ t antigen( ⁇ ) re ⁇ ponsible for activated lymphocyte respon ⁇ e ⁇ were abundant in both cyto ⁇ ol and membrane fractions. Whether cell-mediated immunity was due to a single antigen which located in cyto ⁇ ol and membrane ⁇ , or due to a variety of antigen ⁇ distributed in both fractions remains as yet unknown.
  • our data provide evidence that responsible antigenic epitopes contained a peptide/protein component since antigenicity was abrogated by tryp ⁇ inization or heat treatment.
  • Lymphocyte proliferation wa ⁇ not ob ⁇ erved in any of the samples following incubation with RBC membrane antigens.
  • RBC membranes were chosen for the control antigen becau ⁇ e, like villou ⁇ trophobla ⁇ t, RBC do not expre ⁇ s HLA.
  • lymphocyte proliferation as ⁇ ay i ⁇ an effective method to di ⁇ tingui ⁇ h women with activated cellular immunity to trophobla ⁇ t which may contribute to their recurrent abortion.
  • This technique in addition to the as ⁇ ay ⁇ de ⁇ cribed in Example 4, is useful in the diagnostic evaluation of women with immunologic reproductive failure and has applicability regarding the testing of potential therapeutics such as the above-described immunomodulating agent drug therapies.

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Abstract

L'invention concerne des procédés et des compositions de diagnostic et de traitement d'anomalies reproductives immunologiques chez le mammifère. Lesdits procédés consistent à déterminer la présence d'une concentration élevée d'un facteur embryotoxique extracellulaire dans un échantillon. Ledit échantillon contient des leucocytes ou des produits de sécrétion de leucocytes. L'invention porte également sur le matériel permettant de déterminer la concentration desdits facteurs embryotoxiques.
PCT/US1994/005692 1993-05-21 1994-05-20 Facteurs embryotoxiques WO1994028425A1 (fr)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
WO2002022877A2 (fr) * 2000-09-12 2002-03-21 The Brigham And Women's Hospital, Inc. Variants de gene il-1 chaine beta et de gene cd46 pour le diagnostic de perte de gestation recurrente non expliquee
WO2003045361A1 (fr) * 2001-11-30 2003-06-05 Solvay Pharmaceuticals Gmbh Prevention de fausse couche faisant appel a des composes gestagenes non endogenes d'immunomodulation
ITMI20130916A1 (it) * 2013-06-04 2014-12-05 Gexnano S R L Metodo di rilevazione di sostanze chimiche in campioni di materiale prelevabili da un soggetto, in particolare di rilevazione di fattori embriotossici
US10545156B2 (en) 2011-03-17 2020-01-28 RPI Consulting, LLC Diagnostic biomarker to predict women at risk for preterm delivery

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WO1987000183A1 (fr) * 1985-06-27 1987-01-15 Biogen N.V. Procedes de production d'uromoduline
WO1989008706A1 (fr) * 1988-03-18 1989-09-21 Institut National De La Recherche Agronomique (Inr Isoformes de la trophoblastine, nouveaux interferons constitues par lesdites isoformes, leurs procedes d'obtention et leurs applications
US4997646A (en) * 1989-02-23 1991-03-05 University Of Florida Research Foundation, Inc. Use of interferons of the alpha family to enhance fertility in mammals
WO1993009438A1 (fr) * 1991-11-04 1993-05-13 Adeza Biomedical Corporation Procede de depistage pour l'identification de femmes presentant un risque accru d'accouchement avant terme
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002022877A2 (fr) * 2000-09-12 2002-03-21 The Brigham And Women's Hospital, Inc. Variants de gene il-1 chaine beta et de gene cd46 pour le diagnostic de perte de gestation recurrente non expliquee
WO2002022877A3 (fr) * 2000-09-12 2003-07-10 Brigham & Womens Hospital Variants de gene il-1 chaine beta et de gene cd46 pour le diagnostic de perte de gestation recurrente non expliquee
WO2003045361A1 (fr) * 2001-11-30 2003-06-05 Solvay Pharmaceuticals Gmbh Prevention de fausse couche faisant appel a des composes gestagenes non endogenes d'immunomodulation
US10545156B2 (en) 2011-03-17 2020-01-28 RPI Consulting, LLC Diagnostic biomarker to predict women at risk for preterm delivery
ITMI20130916A1 (it) * 2013-06-04 2014-12-05 Gexnano S R L Metodo di rilevazione di sostanze chimiche in campioni di materiale prelevabili da un soggetto, in particolare di rilevazione di fattori embriotossici
WO2014195771A1 (fr) * 2013-06-04 2014-12-11 Gexnano S.R.L. Procédé de détection de substances chimiques dans des échantillons de matière qui peuvent être pris à partir d'un sujet, en particulier de détection de facteurs embryotoxiques
US11085919B2 (en) 2013-06-04 2021-08-10 Innovitas Vitae S.r.l. Method for detecting chemical substances in samples of material that can be taken from a subject, in particular for detecting embryotoxic factors

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