Methods are provided for the diagnosis and treatment of patients with increased risk of preeclampsia. The methods involve measuring levels of TGF-.beta..sub.3, receptors of cytokines of the TG.beta. family, or HIF-1.alpha..

Description of the Invention

1. Diagnostic Methods 

As hereinbefore mentioned, the present invention provides a method for diagnosing in a subject a condition requiring regulation of trophoblast invasion comprising detecting TGF-.beta..sub.3, receptors of cytokines of the TGF.beta. family, or HIF-1.alpha. in a sample from the subject. In an embodiment of the diagnostic method of the invention, a method is provided for diagnosing increased risk of preeclampsia in a subject comprising detecting TGF-.beta..sub.3, its receptors, or HIF-1.alpha. in a sample from the subject. 

TGF-.beta..sub.3 is a cytokine of the TGF.beta. family and it has the structural characteristics of the members of the TGF.beta. family. TGF.beta. is produced as a precursor characterised by having an N-terminal hydrophobic signal sequence for translocation across the endoplasmic reticulum, a pro-region, and a C-terminal bioactive domain. Prior to release from the cell, the pro-region is cleaved at a site containing four basic amino acids immediately preceding the bioactive domain (Massague, 1990, Annu. Review. Cell Biol. 6:597). 

The precursor structure of TGF.beta. is shared by members of the TGF.beta. family, with the exception of the TGF.beta.4 precursor which lacks a distinguishable signal sequence. The degree of identity between family members in the C-terminal bioactive domain is from 25 to 90% (See Basler et al. Cell, 73:687, 1993, FIG. 2 (see Original Patent)). All nine cysteines are conserved in the bioactive domain in the TGF.beta. family. The bioactive domain is cleaved to generate a mature monomer. 

The TGF.beta. family includes five members, termed TGF.beta. 1 through TGF.beta. 5, all of which form homodimers of about 25 kd (reviewed in Massague, 1990). The family also includes TGF.beta. 1.2 which is a heterodimer containing a .beta.1 and a .beta.2 subunit linked by disulfide bonds. The five TGF.beta. genes are highly conserved. The mature TGF.beta. processed cytokines produced from the members of the gene family show almost 100% amino acid identity between species and the five peptides as a group show about 60-80% identity. The amino acid sequence and nucleic acid sequence of TGF-.beta..sub.3 are shown in FIG. 1 (see Original Patent) (See also sequences for GenBank Accession Nos. HSTGF31-HSTGF37 and HSTGFB3M). 

"Receptors of cytokines of the TGF.beta. family" or "TGF.beta. receptors" refers to the specific cell surface receptors which bind to cytokines of the TGF.beta. family, in particular TGF-.beta..sub.3, including the TGF-.beta. type I receptor (ALK-1 or ALK-5)) (R-I), TGF-.beta. type II receptor (R-II), betaglycan, endoglin and activin, and complexes of the receptors, in particular a RI-RII-endoglin complex. Endoglin binds TGF.beta..sub.1 and .beta..sub.3 with high affinity (K.sub.D=50 pM). Betaglycan has considerable sequence homology to endoglin (Chiefetz, S., et al J. Biol. Chem. 267: 19027, 1992; Lopez-Casillas, F., et al, Cell 67:785, 1991; Wang, X. F., et al, Cell 67:797, 1991), it can bind all three forms of TGF-.beta..sub.3, and it regulates access of the ligands to R-I and R-II which are serine/threonine kinases and unlike betaglycan, are necessary for signal transduction (Wrana, J. L. et al, Cell 71:1003, 1992, Lopez-Casillas et al, Cell 73:1435, 1993; Franzen, P., et al Cell 75:681, 1993; Laiho, M. et al, J. Biol. Chem. 266:9108; Massague, J. et al, Trends Cell Biol. 4:172, 1994). TGF.beta. R-II is an integral membrane protein which contains a short extracellular domain, a single transmembrane domain, and an intracellular serine/threonine kinase domain (Lin H. Y. et al., Cell 68:775, 1992). Serine/threonine kinases encoding type II receptors have been cloned which are structurally related to the type II receptors (Wrana, J. L. et al, Cell 71:1003, 1992, ten Dikje, P., et al, Oncogene 8:2879, 1993; Ebner, R., et al Science 260:1344, 1993; Ebner, R., et al Science 262:900, 1993). TGF.beta. R-I (human ALK-5), binds TGF.beta..sub.1 and .beta..sub.3 only in the presence of TGF.beta. R-II (Wrana, J. L. et al, Cell 71:1003, 1992). The human ALK-1 (TGF.beta. R-I) binds TGF.beta. when forming a heterodimeric complex with TGF.beta. R-II (Franzen, P., et al Cell 75:681, 1993). TGF.beta. R-II kinase, which is endogenously phosphorylated, phosphorylates and activates R-I which then initiates further downstream signals (Wrana, J. L. et al, Nature 370:341, 1994). 

Hypoxia-inducible factor-I (HIF-1) is present in nuclear extracts of many mammalian cells cultivated in a low oxygen atmosphere (Semenza, G. L. et al Mol. Cell. Biol. 12:5447, 1992; Wang, G. L. et al J. Biol. Chem. 268:21513, 1993). HIF-I binds as a phosphoprotein to a short DNA motif (BACGTSSK) identified in the 3-flanking regions of many hypoxia-induced genes (Semenza, G. L. et al. J. Biol Chem 269:23757, 1994; Liu, Y., et al Circulation Res. 77:638, 1995; Firth, J. D. et al Proc. Natl. Acad, Sci. USA 91:6496, 1994; Abe, M., et al, Anal. Biochem. 216:276, 1994). HIF-I binds DNA as a heterodimeric complex composed of two subunits of the inducible HIF-1.alpha. and the constitutively expressed HIF-I.beta.. 

TGF-.beta..sub.3, receptors of cytokines of the TGF.beta. family (e.g., TGF.beta. RI (ALK-1), TGF.beta. RII, or a complex of RI-RII-endoglin), or HIF-1.alpha. may be detected in a variety of samples from a patient. Examples of suitable samples include cells (e.g. fetal or maternal); and, fluids (fetal or maternal), including for example, serum, plasma, amniotic fluid, saliva, and conditioned medium from fetal or maternal cells. 

TGF-.beta..sub.3, receptors of cytokines of the TGF.beta. family, or HIF-1.alpha. may be detected using a substance which directly or indirectly interacts with the cytokine, TGF.beta. receptors, or HIF-1.alpha.. For example, antibodies specific for TGF-.beta..sub.3, the TGF.beta. receptors, or HIF-1.alpha. may be used to diagnose and monitor a condition requiring regulation of trophoblast invasion. A method of the invention using antibodies may utilize Countercurrent Immuno-Electrophoresis (CIEP), Radioimmunoassays, Radioimmunoprecipitations, and Enzyme-Linked Immuno-Sorbent Assays (ELISA), Dot Blot assays, Inhibition or Competition assays and sandwich assays (see U.S. Pat. Nos. 4,376,110 and 4,486,530; see also Antibodies: A Laboratory Manual, supra). 

Antibodies used in the methods of the invention include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab').sub.2 and recombinantly produced binding partners. Polyclonal antibodies may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, various fowl, rabbits, mice, or rats. Monoclonal antibodies may also be readily generated using conventional techniques (see U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993 which are incorporated herein by reference; see also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are also incorporated herein by reference). Binding partners may be constructed utilizing recombinant DNA techniques to incorporate the variable regions of a gene which encodes a specifically binding antibody (See Bird et al., Science 242:423-426, 1988). 

Antibodies may also be obtained from commercial sources. For example, antibodies to TGF-.beta..sub.3 may be obtained from American Diagnostics Inc., CT. USA, Oncogene Science, NY, USA, and Dimension Laboratories, Mississauga, Canada. 

The presence of TGF-.beta..sub.3 in a sample may also be determined by measuring the binding of the cytokine to compounds which are known to interact with TGF-.beta..sub.3 such as its receptors, or decorin, thrombospondin, the serum glycoprotein .alpha.2-macroglobulin, fetuin, or thyroglobulin (Y. Yamaguchi, D. M. Mann, E. Ruoslahti, Nature 346, 281 (1990); S. Scholtz-Cherry J. E. Murphy-Ullrich, J. Cell Biol. 122, 923 (1993); O'Conner-McCourt, L, M. Wakefield J. Biol. Chem. 262, 14090 (1987); and J. Massague Curr. Biol. 1, 117 (1991)). These compounds are referred to herein as "TGF.beta. Binding Compounds". 

The presence of receptors of cytokines of the TGF.beta. family may be determined by measuring the binding of the receptors to molecules (or parts thereof) which are known to interact with the receptors such as their ligands. In particular, peptides derived from sites on ligands which bind to the receptors may be used. A peptide derived from a specific site on a ligand may encompass the amino acid sequence of a naturally occurring binding site, any portion of that binding site, or other molecular entity that functions to bind an associated molecule. A peptide derived from such a site will interact directly or indirectly with an associated receptor molecule in such a way as to mimic the native binding site. Such peptides may include competitive inhibitors, enhancers, peptide mimetics, and the like as discussed below. 

The presence of HIF-1.alpha. may be determined by measuring the binding of HIF-.alpha.1 to DNA molecules which are known to interact with HIF-.alpha.1 such as hypoxia inducing genes. The TGF.beta. binding compounds and molecules that interact with the receptors and HIF-1.alpha. are referred to herein as "Binding Compounds". 

The antibodies specific for the TGF-.beta..sub.3, TGF.beta. receptors, or HIF-1.alpha., or the Binding Compounds may be labelled using conventional methods with various enzymes, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase, or acetylcholinesterase; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive materials include radioactive phosphorous .sup.32P, iodine I.sup.125, I.sup.131 or tritium. 

An antibody to TGF-.beta..sub.3, a TGF.beta. family receptor, or HIF-1.alpha., or a Binding Compound may also be indirectly labelled with a ligand binding partner. For example, the antibodies, or a TGF-.beta..sub.3 Binding Compound may be conjugated to one partner of a ligand binding pair, and the TGF-.beta..sub.3 may be coupled to the other partner of the ligand binding pair. Representative examples include avidin-biotin, and riboflavin-riboflavin binding protein. Preferably the antibodies are biotinylated. Methods for conjugating the antibodies discussed above with the ligand binding partner may be readily accomplished by one of ordinary skill in the art (see Wilchek and Bayer, "The Avidin-Biotin Complex in Bioanalytical Applications," Anal Biochem. 171:1-32, 1988). 

The antibodies or Binding Compounds used in the method of the invention may be insolubilized. For example, the antibodies or Binding Compounds may be bound to a suitable carrier. Examples of suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc. The insolubilized compound or antibodies may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling. 

Indirect methods may also be employed in which a primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against the cytokine. By way of example, if the antibody having specificity against TGF-.beta..sub.3 is a rabbit IgG antibody, the second antibody may be goat anti-rabbit gamma-globulin labelled with a detectable substance as described herein. 

TGF-.beta..sub.3, TGF.beta. receptors, or HIF-1.alpha. can also be assayed in a sample using nucleotide probes to detect nucleic acid molecules encoding a TGF-.beta..sub.3, the TGF.beta. receptors, or HIF-1.alpha.. Suitable probes include nucleic acid molecules based on nucleic acid sequences encoding TGF-.beta..sub.3, the TGF.beta. receptors, or HIF-1.alpha.. A nucleotide probe may be labelled with a detectable substance such as a radioactive label which provides for an adequate signal and has sufficient half-life such as .sup.32P, .sup.3H, .sup.14C or the like. Other detectable substances which may be used include antigens that are recognized by a specific labelled antibody, fluorescent compounds, enzymes, antibodies specific for a labelled antigen, and luminescent compounds. An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleotide to be detected and the amount of nucleotide available for hybridization. Labelled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.). 

A nucleic acid molecule encoding TGF-.beta..sub.3, TGF.beta. receptors, or HIF1.alpha. can also be detected by selective amplification of the nucleic acid molecules using polymerase chain reaction (PCR) methods. Synthetic oligonucleotide primers can be constructed from the sequences of the TGF-.beta..sub.3, TGF.beta. receptors, or HIF1.alpha. using conventional methods. A nucleic acid can be amplified in a sample using these oligonucleotide primers and standard PCR amplification techniques. 

In a preferred embodiment of the invention, a method is provided for diagnosing increased risk of preeclampsia in a subject comprising detecting TGF-.beta..sub.3, TGF.beta. R-I (ALK-1), TGF.beta. R-II, endoglin, HIF-1.alpha., or a complex of R-I (ALK-1)-R-II-endoglin in a sample, and in particular using antibodies specific for TGF-.beta..sub.3. Levels of TGF-.beta..sub.3, TGF.beta. receptors or complexes thereof, or HIF-1.alpha. may be measured during the first trimester of pregnancy (approximately 1 to 14 weeks). It is preferred that at least two measurements be taken during this period, preferably including a measurement at about 14 to 16 weeks. If the levels are significantly increased as compared to levels typical for women who do not suffer from preeclampsia, the patient is diagnosed as having an increased risk of suffering preeclampsia. Levels above those typical for women who do not suffer from preeclampsia may be suspect and further monitoring and measurement of TGF-.beta..sub.3, TGF.beta. receptors, or HIF-1.alpha. may be appropriate. The information from the diagnostic method may be used to identify subjects who may benefit from a course of treatment, such as treatment via administration of inhibitors of TGF-.beta..sub.3 as discussed herein. 

It will also be appreciated that the above methods may also be useful in the diagnosis or monitoring of choriocarcinoma or hydatiform mole which involves uncontrolled trophoblast invasion (i.e. may be associated with abnormally low levels of TGF-.beta..sub.3, TGF.beta. family receptors, or HIF1.alpha.). Further the above methods may be used to diagnose or monitor other pregnancy complications including intrauterine growth restriction, molar pregnancy, preterm labour, preterm birth, fetal anomalies, and placental abruption. The diagnostic and monitoring methods of the invention may also involve determining responsiveness of cells to oxygen. 

The invention also relates to kits for carrying out the methods of the invention. The kits comprise instructions, negative and positive controls, and means for direct or indirect measurement of TGF-.beta..sub.3, TGF.beta. receptors, or HIF1.alpha.. 

2. Regulation of Trophoblast Invasion in a Subject 

The invention also provides a method of regulating trophoblast invasion comprising directly or indirectly inhibiting or stimulating (a) TGF-.beta..sub.3 (b) receptors of cytokines of the TGF.beta. family, (c) HIF1.alpha.; and/or (d) O.sub.2 tension, preferably inhibiting or stimulating TGF-.beta..sub.3. Trophoblast invasion may also be regulated by optimizing oxygenation of tissues. 

In an embodiment of the invention, a method is provided for increasing trophoblast invasion in a subject comprising administering an effective amount of a substance which is an inhibitor of (a) TGF-.beta..sub.3, (b) receptors of cytokines of the TGF.beta. family, and/or (c) HIF-1.alpha.. In particular, methods are provided for treating a women suffering from or who may be susceptible to preeclampsia. 

In another embodiment of the invention, a method is providing for reducing trophoblast invasion in a subject comprising administering an effective amount of (a) TGF-.beta..sub.3; (b) receptors of cytokines of the TGF.beta. family; (c) HIF-.alpha.1; and/or (d) a stimulator of (a), (b) or (c). The method may be used to monitor or treat choriocarcinoma or hydatiform mole. 

The methods of the invention may also be used to monitor or treat other complications of pregnancy such as intrauterine growth restriction, molar pregnancy, preterm labour, preterm birth, fetal anomalies, or placental abruption. 

Substances that regulate trophoblast invasion can be selected by assaying for a substance that inhibits or stimulates the activity of TGF-.beta..sub.3, TGF.beta. receptors, or HIF-1.alpha.. A substance that regulates trophoblast invasion can also be identified based on its ability to specifically interfere or stimulate the interaction of (a) TGF-.beta..sub.3 and a receptor for the cytokine (e.g. the interaction of TGF-.beta..sub.3 and endoglin, or TGF-.beta..sub.3 and R-I, R-II, or a complex of R-I-R-II endoglin, or (b) TGF-.beta..sub.3 and HIF1.alpha.. 

Therefore, a method is provided for evaluating a compound for its ability to regulate trophoblast invasion comprising the steps of: 

(a) reacting TGF-.beta..sub.3 or a part thereof that binds to a receptor of a cytokine of the TGF.beta. family, with a receptor of a cytokine of the TGF.beta. family or a part thereof that binds to TGF-.beta..sub.3, and a test substance, wherein the TGF-.beta..sub.3 and receptor of a cytokine of the TGF.beta. family or parts thereof, are selected so that they bind to form a ligand-receptor complex; and 

(b) comparing to a control in the absence of the substance to determine the effect of the substance. 

In particular, a method is provided for identifying a substance which regulates trophoblast invasion comprising the steps of: 

(a) reacting TGF-.beta..sub.3 or a part thereof that binds to a receptor of a cytokine of the TGF.beta. family, and a receptor of a cytokine of the TGF.beta. family or a part thereof that binds to TGF-.beta..sub.3, and a test substance, wherein the TGF-.beta..sub.3 and receptor of a cytokine of the TGF.beta. family or parts thereof, are selected so that they bind to form a ligand-receptor complex, under conditions which permit the formation of ligand-receptor complexes, and 

(b) assaying for complexes, for free substance, for non-complexed TGF-.beta..sub.3 or receptor, or for activation of the receptor. 

The substance may stimulate or inhibit the interaction of TGF.beta. or a part thereof that binds the TGF.beta. receptor, and the TGF.beta. receptor. 

In an embodiment of the invention a receptor complex is employed comprising TGF.beta. R-I (ALK-1)-TGF.beta. RII-endoglin. 

Activation of the receptor may be assayed by measuring phosphorylation of the receptor, or by assaying for a biological affect on a cell, such measuring biochemical markers of trophoblast invasion such as cell proliferation, FN synthesis, integrin expression, up regulation of gelatinase and type IV collagenase expression and activity. 

The invention also provides a method for evaluating a substance for its ability to regulate trophoblast invasion comprising the steps of: 

(a) reacting TGF-.beta..sub.3 or a part of TGF-.beta..sub.3 that binds to HIF-1.alpha., HIF-1.alpha. or a part of the protein that binds to TGF-.beta..sub.3, and a test substance, wherein the TGF-.beta..sub.3 or part thereof, and HIF-1.alpha. or part thereof bind to form a TGF-.beta.3-HIF-1.alpha. complex; and 

(b) comparing to a control in the absence of the substance to determine the effect of the substance. 

The substance may stimulate or inhibit the interaction of TGF-.beta..sub.3 and HIF-1.alpha., or the activation of TGF.beta. by HIF-1.alpha. and thereby regulate trophoblast invasion. 

The substances identified using the methods of the invention include but are not limited to peptides such as soluble peptides including Ig-tailed fusion peptides, members of random peptide libraries and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), antibodies [e.g. polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, single chain antibodies, fragments, (e.g. Fab, F(ab).sub.2, and Fab expression library fragments, and epitope-binding fragments thereof)], and small organic or inorganic molecules. The substance may be an endogenous physiological compound or it may be a natural or synthetic compound. The substance may be a TGF.beta. R-I-TGF.beta. R-II-endoglin complex, which competitively inhibits the binding of TGF-.beta..sub.3 to its natural receptors. The invention contemplates isolated TGF.beta. R-I-TGF.beta. R-II-endoglin complexes and their use in regulating trophoblast invasion. 

The substances may be peptides derived from the binding sites of TGF-.beta..sub.3 and a receptor for the cytokine such as endoglin, R-I or R-II, or a complex of R-I-R-II-endoglin; or the binding sites of TGF-.beta..sub.3 and HIF1.alpha.. A peptide derived from a specific binding site may encompass the amino acid sequence of a naturally occurring binding site, any portion of that binding site, or other molecular entity that functions to bind an associated molecule. A peptide derived from such a binding site will interact directly or indirectly with an associated molecule in such a way as to mimic the native binding domain. Such peptides may include competitive inhibitors, enhancers, peptide mimetics, and the like. All of these peptides as well as molecules substantially homologous, complementary or otherwise functionally or structurally equivalent to these peptides may be used for purposes of the present invention. 

"Peptide mimetics" are structures which serve as substitutes for peptides in interactions between molecules (See Morgan et al (1989), Ann. Reports Med. Chem. 24:243-252 for a review). Peptide mimetics include synthetic structures which may or may not contain amino acids and/or peptide bonds but retain the structural and functional features of a peptide, or enhancer or inhibitor of the invention. Peptide mimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc. Natl. Acad, Sci USA 89:9367); and peptide libraries containing peptides of a designed length representing all possible sequences of amino acids corresponding to a peptide of the invention. 

Peptides may be synthesized by conventional techniques. For example, the peptides may be synthesized by chemical synthesis using solid phase peptide synthesis. These methods employ either solid or solution phase synthesis methods (see for example, J. M. Stewart, and J. D. Young, Solid Phase Peptide Synthesis, 2.sup.nd Ed., Pierce Chemical Co., Rockford III. (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254 for solid phase synthesis techniques; and M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, supra, Vol 1, for classical solution synthesis.) 

Peptide mimetics may be designed based on information obtained by systematic replacement of L-amino acids by D-amino acids, replacement of side chains with groups having different electronic properties, and by systematic replacement of peptide bonds with amide bond replacements. Local conformational constraints can also be introduced to determine conformational requirements for activity of a candidate peptide mimetic. The mimetics may include isosteric amide bonds, or D-amino acids to stabilize or promote reverse turn conformations and to help stabilize the molecule. Cyclic amino acid analogues may be used to constrain amino acid residues to particular conformational states. The mimetics can also include mimics of inhibitor peptide secondary structures. These structures can model the 3-dimensional orientation of amino acid residues into the known secondary conformations of proteins. Peptoids may also be used which are oligomers of N-substituted amino acids and can be used as motifs for the generation of chemically diverse libraries of novel molecules. 

A substance that regulates trophoblast invasion may be a molecule which interferes with the transcription and/or translation of TGF-.beta..sub.3, TGF.beta. receptors, or HIF-1.alpha.. For example, the sequence of a nucleic acid molecule encoding TGF-.beta..sub.3, TGF.beta. receptors (e.g. endoglin, R-I (ALK-1), R-II, or RI-RII-endoglin complex), or fragments thereof, may be inverted relative to its normal presentation for transcription to produce an antisense nucleic acid molecule. An antisense nucleic acid molecule may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. Examples of antisense molecules for TGF-.beta..sub.3 are 5'-CCTTTGCAAGTGCATC-3' and 5'-GATGCACTTGCAAAGG-3'. 

The treatment methods and compositions described herein may use substances that are known inhibitors of TGF-.beta..sub.3. For example, antibodies to TGF-.beta..sub.3, the TGF.beta. Binding Compounds including decorin, .alpha.2-macroglobulin, fetuin, and thyroglobulin, or peptides derived from the sites on these compounds that bind to TGF-.beta..sub.3, or chimeras of these molecules may be employed. 

Activin, another member of the TGF.beta. receptor family, triggers trophoblast invasion and therefore it may be used to enhance trophoblast invasion in a subject. 

The utility of a selected inhibitor or stimulator may be confirmed in experimental model systems. For example, the human villous explant culture system described by Genbacev et al. (21) can be used to confirm the utility of an inhibitor for treatment of preeclampsia. 

In a preferred embodiment of the invention a method is provided for treating a woman suffering from, or who may be susceptible to preeclampsia comprising administering therapeutically effective dosages of an inhibitor of TGF-.beta..sub.3 or TGF.beta. receptors, an inhibitor of HIF-1.alpha., or a substance identified in accordance with the methods of the invention. Preferably treatment with the inhibitor begins early in the first trimester, at about 10 to about 16 weeks, and may continue until measured TGF-.beta..sub.3 levels, TGF-.beta. receptor levels, or HIF-1.alpha. levels are within the normal range. Preferably, treatment with the inhibitor or substance is not continued beyond about 30 weeks of gestation. For the purposes of the present invention normal TGF-.beta..sub.3 levels, TGF.beta. receptor levels, or HIF-1.alpha. levels are defined as those levels typical for pregnant women who do not suffer from preeclampsia. Treatment with the inhibitor is discontinued after TGF-.beta..sub.3 levels, TGF-.beta. receptor levels, and/or HIF-1.alpha., levels are within normal range, and before any adverse effects of administration of the inhibitor are observed. 

One or more inhibitors or one or more stimulators of TGF-.beta..sub.3, TGF.beta. receptors, or HIF-1.alpha., or substances selected in accordance with the methods of the invention including Binding Compounds, may be incorporated into a composition adapted for regulating trophoblast invasion. In an embodiment of the invention, a composition is provided for treating a woman suffering from, or who may be susceptible to preeclampsia, comprising a therapeutically effective amount of an inhibitor of TGF-.beta..sub.3, TGF.beta. receptors, or HIF-1.alpha., or substance selected in accordance with the methods of the invention including TGF.beta. Binding Compounds, and a carrier, diluent, or excipient. 

The compositions of the invention contain at least one inhibitor or stimulator of TGF-.beta..sub.3, TGF.beta. receptors, or HIF-1.alpha., or substance identified in accordance with the methods of the invention, alone or together with other active substances. Such compositions can be for oral, parenteral, or local use. They are therefore in solid or semisolid form, for example pills, tablets, and capsules. 

The composition of the invention can be intended for administration to subjects such as humans or animals. The pharmaceutical compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle, carrier or diluent. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). 

The compositions of the invention may be administered together with or prior to administration of other biological factors that have been found to affect trophoblast proliferation. Examples of these factors include IL-11 (Ireland et al Blood 84:267a, 1994), G-CSF, GM-CSF and M-CSF (U.S. Pat. No. 5,580,554 to Keith). 

The compositions and other biological factors may be administered through any known means. Systemic administration, such as intravenous or subcutaneous administration is preferred. A therapeutically effective amount of an active ingredient e.g. inhibitor is an amount effective to elicit the desired therapeutic response but insufficient to cause a toxic reaction. The dosage for the compositions is determined by the attending physician taking into account factors such as the condition, body weight, diet of the subject, and the time of administration. 

For example, a therapeutically effective dose of an inhibitor, e.g. an amount sufficient to lower levels of TGF-.beta..sub.3 to normal levels, is about 1 to 200 .mu.g/kg/day. The method of the invention may involve a series of administrations of the composition. Such a series may take place over a period of 7 to about 21 days and one or more series may be administered. The composition may be administered initially at the low end of the dosage range and the dose will be increased incrementally over a preselected time course. 

An inhibitor or stimulator of TGF-.beta..sub.3, receptors of cytokines of the TGF.beta. family, or HIF-1.alpha., or substance identified in accordance with the methods of the invention may be administered by gene therapy techniques using genetically modified trophoblasts or by directly introducing genes encoding the inhibitors or stimulators of TGF-.beta..sub.3, or receptors of cytokines of the TGF.beta. family, or substances into trophoblasts in vivo. Trophoblasts may be transformed or transfected with a recombinant vector (e.g. retroviral vectors, adenoviral vectors and DNA virus vectors). Genes encoding inhibitors or stimulators, or substances may be introduced into cells of a subject in vivo using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of DNA into liposomes. Antisense molecules may also be introduced in vivo using these conventional methods. 

Claim 1 of 16 Claims

1. A method for diagnosing an increased risk of preeclampsia in a subject, the method comprising a) detecting a level of a protein selected from the group consisting of TGF-.beta..sub.3, TGF-.beta. type I receptor (ALK-I)(RI), TGF-.beta. type II receptor (R-II), and endoglin in a fluid sample from the subject; and b) comparing the level detected in the subject's fluid sample to a control level obtained from samples of the same fluid taken at the same time of pregnancy from women who did not develop preeclampsia; wherein an increased level of TGF-.beta..sub.3, TGF-.beta. type I receptor (ALK-I)(RI), TGF-.beta. type II receptor (R-II), or endoglin in the fluid sample from the subject over that of a control level obtained from samples of the same fluid taken at the same time of pregnancy from women who did not develop preeclampsia indicates an increased risk of preeclampsia. 

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