WO2008056114A1 - Arginase in pregnancy - Google Patents

Abstract

Arginase activity is highly up-regulated in the placenta in pregnant females, but the up-regulation is not seen in their peripheral tissues. Moreover, the up-regulation is not seen in age-matched non-pregnant controls,. Thus arginase in the placenta is believed to induce reversible and local T cell hyporesponsiveness, thereby playing a crucial role in the maintenance of normal pregnancy.

Description

ARGINASE IN PREGNANCY

All documents cited herein are incorporated by reference in their entirety.

TECHNICAL FIELD

This invention is in the field of pregnancy and, in particular, the mechanism involved in maternal tolerance to the fetus.

BACKGROUND ART

A major paradox in immunology is the ability of the maternal immune system to exhibit immunological tolerance towards the semi-allogeneic fetus while maintaining responsiveness to exogenous pathogens. Over 50 years ago, Medawar proposed [1] three potential mechanisms that may assist maternal unresponsiveness: anatomic separation of the mother and fetus; antigenic immaturity of the fetus; and suppression or modification of the maternal immune system during pregnancy. It is now clear that the maternal immune system is aware of fetal alloantigens during gestation, but is functionally tolerant to them until shortly after parturition [2]. Thus transient suppression of the maternal immune response appears to be vital for fetal survival, and it is likely that multiple, interconnected mechanisms have evolved to prevent rejection of the fetus.

Failure of the correct immunosuppressive mechanisms during pregnancy is a possible cause of miscarriage and/or other pregnancy-related complications, and in particular of recurrent miscarriage and pre-eclampsia (reviewed in reference 3).

It is an object of the invention to provide methods and reagents for providing and maintaining the normal immunosuppressive environment associated with pregnancy.

DISCLOSURE OF THE INVENTION

The inventors have found that arginase activity is highly up-regulated in the placenta in pregnant females, but that the up-regulation is less pronounced in their peripheral blood mononuclear cells. Moreover, the up-regulation is not seen in age-matched non-pregnant controls,. Thus arginase in the placenta is believed to induce reversible and local T cell hyporesponsiveness, thereby playing a crucial role in the maintenance of normal pregnancy. This type of reversible and local immunosuppression has not previously been reported.

Arginase' s role in maintaining T cell hyporesponsiveness in pregnancy is unexpected, but it fits other observations. For example, the control of amino acid metabolism is emerging as a mechanism of regulation of lymphocyte responses, it is known that indoleamine 2,3-dioxygenase (IDO) plays a role in regulating maternal T cell immunity during murine pregnancy [4,5]. IDO catalyses the first and rate-limiting step in the oxidative degradation of tryptophan, an essential amino acid. Another amino acid that is important in regulating immune responses is L-arginine. This semi-essential amino acid, can be synthesised by the adult human, but must be supplemented by diet at times of physiological or pathological stress, including pregnancy and neonatal development [6]. In addition to its essential role as a building block for tissue proteins, and ammonia detoxification, L-arginine depletion has been shown to down-regulate the expression of CD3ζ [7], the signalling chain of the T cell receptor, through decreased CD3ζ mRNA stability, and impair lymphocyte function in a reversible manner.

The identification of arginase's role offers several opportunities in therapy and diagnosis, including:

• Maintaining increased arginase activity or providing increased arginase activity during pregnancy to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication. In particular, increase of arginase activity that is localised to the uterus, endometrium or placenta can be used.

• Reduction of arginine levels to reduce the risk of a miscarriage and/or other pregnancy-related complication or to improve the chance of becoming pregnant.

• Intervention in metabolic pathways, upstream or downstream of arginase, to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication or to improve the chance of becoming pregnant.

• Maintaining or providing increased arginase activity or reduction of arginine levels during pregnancy to reduce the likelihood of premature birth.

• Increasing arginase activity when attempting to become pregnant to improve the success rate. This treatment can be targeted to specific groups of patients, including those who have previously had a spontaneous miscarriage and/or other pregnancy-related complication.

• Assays of arginase levels, and/or of arginase genotype, both before and during pregnancy as an indicator of miscarriage and/or other pregnancy-related complication potential.

• Assays of arginase genotype, both in males and females, as an indicator of the risk of miscarriage and/or other pregnancy-related complication in future pregnancies.

• Optimisation of nutritional supplements suitable for use before or during pregnancy, with the aim of reducing arginine levels.

• Assays for identifying a compound that can be used for maintaining or providing increased arginase activity or reduction of arginine levels or suppression of the immune system during pregnancy.

• Reducing arginase activity, or over-supplying arginine, to inhibit fertility or to promote termination.

Therapeutic benefit is achieved by decreasing the anti-fetal immune response of a patient. Hyporesponsiveness can be achieved in a patient by increasing the arginase activity either systemically or more preferably in targeted organs, tissues and cells. Induction of targeted hyporesponsiveness can have a therapeutic benefit where there is a history of recurrent spontaneous miscarriage or infertility by interfering with localised immune response without impairing the systemic immune response of a patient. Induction of hyporesponsiveness is also useful in patients to prevent rejection of the embryo or fetus, such as during conception and pregnancy. Beneficial results are preferably achieved by targeting arginase or the modulators of arginase production or activity specifically to the cells or tissues in which induction of hyporesponsiveness is desired. Thus a decreased immune response can be localised to specific organs or tissues, such as the uterus, endometrium or placenta, or to specific cells in those organs or tissues, such as macrophages, neutrophils, dendritic cells or lymphocytes, to achieve beneficial results without compromising the systemic immune response. The present invention provides therapeutic benefit through diminishing immune responses.

Arginase

Two arginase isoenyzmes exist - arginase I and II, which differ in subcellular localization, regulation and possibly function. Arginase is produced by a number of cells such as tumor cells, transplanted cells, cells undergoing autoimmune reactions and macrophages. Arginase I, which is inducible in macrophages and dendritic cells, is primarily responsible for the depletion of arginine in the cells and surrounding environment, resulting in suppression of an immune response. The invention is aimed primarily at Arginase I.

These enzymes play a role in a number of metabolic and signalling pathways that result in modulation of the immune response amongst other things. For example, arginase activity results in (a) the production or ornithine, a precursor of proline, which favours cellular regeneration, wound healing and repair [8], (b) the production of polyamines, also from ornithine, which can regulate macrophage function [9] and decrease nitric oxide production [10] and (c) reduction of arginine availability and, therefore, decreasing nitric oxide production and other arginine-mediated processes such as normal T lymphocyte proliferation [11-13].

The human arginase polypeptide sequences have been given accession numbers P05089 and P78540 in the Entrez protein database and their mRNA sequences are given in NM_000045.2 and NMJ)01172.3.

SNPs of arginase are known. For ARGl, the following SNPs are known in the dbSNP database: rs2608898, rs2608897, rs2781664, rs2781666, rs2781665, rsl7788484, rs3756780, rsl0457573, rs2781658, rslO63493, rs2781659, rsl803151, rs2781667, rs2246012, rs2781668, rs2297637, rs3850245, rs2608937, rs7769790, rs2749935, rs9321303, rs2781621 and rs9493030. For ARG2, the following SNPs are known: rsl7104534, rsl885042, rs742869, rs8017597, rs7140310, rs4899215, rs2295643, rsl2884807, rs7144186 and rs4902505.

Arginine and the immune system

The bioavailability of L-arginine is determined by two inducible intracellular enzymes, arginase and nitric oxide synthase (NOS), which share L-arginine as a common substrate [14]. These enzymes are induced by T cell derived cytokines and regulated by complex intracellular biochemical pathways and negative feedback mechanisms. The balance of L-arginine metabolism via arginase or NOS is an important determinant of the inflammatory response of murine macrophages and dendritic cells. ThI cytokines such as interferon-γ induce NOS, which catalyses the metabolism of L-arginine to nitric oxide, while Th2 cytokines drive the alternative activation of macrophages including the induction of arginase, which hydrolyzes L-arginine into urea and L-ornithine. The importance of arginase in regulating T cell responses has been convincingly shown in vitro and in animal models, and arginase induction as a method of tumour evasion has been demonstrated in human cancers.

Arginine is catabolised by several enzymes including arginine decarboxylase, arginase, nitric oxide synthase and arginine:glycine amidinotransferase. Arginase and NOS compete for arginine and also inhibit each other [15]. NOS generates hydroxy-arginine which is an inhibitor of arginase and polyamines generated by the arginase pathway are inhibitors of NOS. Feedback between the pathways regulates the level of available arginine and therefore the level of suppression of the immune system.

Arginine regulates p70 S6 kinase activity and phosphorylation of 4EBPI through the mTOR signalling pathway, which involves y⁺ cationic amino acid transporters (such as Cat-1, Cat-2a, Cat- 2b and Cat-3) [16]. Activation of the p70 S6 kinase results in activation of the immune system.

Increasing arginase activity

Arginase activity may be increased either by increasing the specific activity of the enzyme, or by increasing the level of arginase.

Any one of a number of methods to maintain or provide increased arginase activity during pregnancy in order to reduce the risk of an immunologically-mediated miscarriage or other pregnancy-related complications. In one method, compounds that increase the specific activity of the enzyme may be provided. In a second method, the enzyme itself may be provided to a subject, or expression of endogenous arginase may be increased using a stimulator of arginase expression. Increasing the expression of endogenous arginase is referred to as upregulating arginase expression. As an alternative method, gene therapy may be used to upregulate arginase expression, where a gene encoding arginase may be provided.

In another method to maintain or provide increased arginase activity during pregnancy to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication, the production of arginase inhibitors may be inhibited. Arginase inhibitors and stimulators are further discussed in reference 17. The arginase stimulator may increase the arginase expression levels or increase the activity of the arginase.

Preferably the arginase level and/or specific activity is increased by 10% or more (e.g. 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200% or more).

Arginase inhibitors which may be targeted for blocking to prevent their inhibitory action on arginase include amino acids such as NG-hydroxy-L-arginine (NOHA), N(omega)-hydroxy-nor-L-arginine (nor-NOHA), 2 (S)-amino-6-borohexanoic acid (ABH), ornithine, lysine, and norvaline. Other inhibitors that downregulate arginase activity include IFNγ and IL- 12 [18].

Arginase stimulators which may be used in the method described above to increase arginase activity include beta-adrenergic agents such as catecholamines or catecholamine analogues, epinephrine, norepinephrine, isoproterenol, prostaglandines, dopamine and salbutamol; Th2 cytokines such as IL- 4, IL-IO, IL-13 and TGFβ; and arginase substrates. Alternatively the stimulator may be 8-bromo- cAMP [19], 8-bromo-cAMP plus lipopolysaccharide 8-bromo-cAMP and interferon-gamma or combinations thereof. An arginase enzyme obtained from a natural or synthetic source may also be provided.

Mn²⁺ has been identified as a co-factor of arginase and so is required for arginase activity. Ni²⁺ and Co²⁺ are also known to be activators of arginase while Hg²⁺, Ag²⁺ and Zn²⁺ are known to be inhibitors of arginase. Therefore, in one method to maintain or provide increased arginase activity during pregnancy to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication, specific arginase activity can be upregulated in a situation where Mn²⁺, Ni²⁺ and/or Co²⁺ is limiting, by providing a source of Mn²⁺, Ni²⁺ and/or Co²⁺.

Decrease ofarginine levels

Arginine levels in the body may be decreased by any one of a number of methods described below in order to reduce the risk of a miscarriage and/or other pregnancy-related complication or to improve the chance of becoming pregnant.

Arginine biosynthetic enzymes

The invention provides a number of methods that can be used to intervene in metabolic pathways upstream of arginase to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication or to improve the chance of becoming pregnant. Arginine levels may be decreased by reducing the specific activity of an arginine biosynthetic enzyme (inhibiting the enzyme) or by inhibiting the expression of the enzyme (downregulating the enzyme). For example, synthesis ofarginine from citrulline or other upstream precursors may be decreased. Other precursors of arginine biosynthesis that may be targeted for reduction include carbamyl phosphate, ornithine, citrulline, aspartic acid and argininosuccinic acid (ASA). Enzymes that control the metabolism of these compounds may also be targeted for inhibition or downregulation. Such enzymes include ornithine transcarbamylase, ASA synthetase and ASA lyase. Inhibitors may be competitive or noncompetitive. For example, antibodies may be used to bind to and sequester arginine biosynthetic enzymes. Preferably the arginine levels are reduced by 10% or more (e.g. 20, 30, 40, 50, 60, 70, 80, 90, 95, 97, 99% or more). Preferably the arginine concentration in the placenta is 2000μM or less (e.g. 1000, 750, 500, 250, 150, 75, 50, 25, 15, 10 μM or less). General physiological levels of L-arginine are between 50-15OmM. Other targets

Arginine is transported into mammalian cells primarily through system y⁺ cationic amino acid transporters [20]. If the expression of these transporters is upregulated or their activity is increased, then the immune response is suppressed as less arginine is transported into cells such as macrophages. Four such transporter genes have been identified which encode system y⁺-like activity (Cat-1, Cat-2a, Cat-2b and Cat-3). The invention therefore provides a method of reducing the risk of miscarriage and/or other pregnancy-related complications or improving the chance of becoming pregnant comprising upregulating the expression of y⁺ cationic amino acid transporters or increasing the activity of y⁺ cationic amino acid transporters. The Cat-1 protein has been shown to have a higher affinity for arginine than the other transporters [21]. Therefore, preferably the activity of Cat-1 is targeted.

Rapamycin is a known suppressant of the immune system. Therefore its receptor, the molecular target of rapamycin (MTOR) is a possible target for intervention in metabolic pathways downstream of arginase, to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy- related complication or to improve the chance of becoming pregnant. Rapamycin and wortmanin inhibit the effect of arginine on the p70 S6 kinase, resulting in immune suppression. Therefore, rapamycin and wortmanin, which activate mTOR and induce immune suppression, are attractive for use in reducing the risk of miscarriage and/or other pregnancy-related complications.

GCN2 kinase influences mRNA translation. Amino acid depletion can lead to the accumulation of uncharged tRNAs that bind to and activate GCN2 kinase. This kinase phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2a), which inhibits eIF2a Guanylate Exchange Factor, and the generation of eIF2a-GTP-Met-tRNAi complexes. These complexes are necessary for the initiation of translation and, in their absence, cellular protein synthesis is suppressed. One of the mechanisms that is responsible for the temporary downregulation of T cell responses during pregnancy might be dependent on the activation of GCN2 in T cells. The level of tRNA that is uncharged with amino acids following the local depletion of amino acids by arginase could increase and activate GCN2. Activation of GCN2 might then repress protein synthesis in T cells, therefore rendering them unresponsive. CD3ζ is expressed by T cells and forms part of the T cell receptor. It is essential for signal transduction by the T cell receptor. Increasing of arginase levels in the placental region during pregnancy leads to a decrease in CD3ζ expression, resulting in local hyporesponsivenessand a reduced risk of miscarriage and/or other pregnancy-related complications. Therefore, the invention provides a method of intervening in a metabolic pathway downstream of arginase, to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication or to improve the chance of becoming pregnant by decreasing CD3ζ expression.

The polyamines are organic compounds, such as putrescine, spermidine, and spermine, that are growth factors in both eukaryotic and prokaryotic cells. Polyamines are synthesised by amino acid decarboxylation reactions. Arginine is broken down by arginase into ornithine, which is then converted to putrescine (a polyamine) by ornithine decarboxylase. It is believed that these polyamines play a role in the suppression of the immune system by downregulating the activity of antigen presenting cells. Thus polyamines are attractive targets for inducing suppression of the immune system. Polyamines derived from proline, but not arginine, are synthesised in the porcine placenta during early pregnancy [22] and in pregnant guinea pigs polyamines are thought to be important in cell growth and development [23]. Therefore, an increase in polyamine level is likely to be beneficial during pregnancy.

In one embodiment the present invention provides a method involving intervention in metabolic pathways downstream of arginase to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication or to improve the chance of becoming pregnant, comprising increasing the activity of polyamine biosynthesis or polyamine availability. This can be done by increasing the activity of polyamine biosynthetic enzymes or by delivery of polyamines.

Arginine is broken down into citrulline and nitric oxide by inducible Nitric Oxide Synthase (NOS). Citrulline can then be recycled into arginine by ASS and ASL. Arginine levels can be reduced by increasing the activity of NOS, either by inducing overproduction of the enzyme or by increasing the activity of the enzyme already present. A further a method involving intervention in metabolic pathways downstream of arginase to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication or to improve the chance of becoming pregnant comprises increasing NOS activity.

Inhibiting arginine synthesis would also lead to lower arginine levels. Arginine is synthesised from L- citrulline via the intermediate L-argininosuccinate by the enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL). The complete arginine biosynthetic pathway is described in reference 24.

Any compound that reduces the level of arginine could be useful in the methods described above. Alternatively, arginine levels may be decreased by increasing the arginase activity.

The methods described above can be used from conception to parturition as a method of maintaining or providing increased arginase activity or reduction of arginine levels during pregnancy to reduce the likelihood of premature birth, or before conception as a method of increasing of arginase activity or decreasing arginine levels when attempting to become pregnant to improve the success rate.

Localised targeting

Any of the methods described above may be used to increase arginase activity or decrease arginine levels systemically or may be targeted to a specific organ, tissue or cell type.

To avoid systemic immunosuppression, it is preferred that a composition of the invention is localised to a specific organ, tissue or cell type in order to achieve localised immunosuppression. Preferably, arginase activity is increased or arginine levels are decreased by altering the activity or expression of the target in the uterus. More preferably, arginase activity is increased or arginine levels are decreased by altering the activity or expression of the target in the endometrium, even more preferably arginase activity is increased or arginine levels are decreased by altering the activity or expression of the target in the region of the placenta or in the placenta itself. Preferably macrophages, dendritic cells and/or neutrophils in the uterus, endometrium or placenta are targeted in order to modulate targets described above.

A targeted increase of arginase activity or decrease of arginine levels may be achieved by using localised delivery methods rather than systemic delivery. As an alternative, systemic delivery may be used if the delivered material is then targeted to the desired area e.g. using immunoliposomes, etc.

Localised delivery may be achieved by any one of a number of methods such as hydrogels, pastes, vaginal tablets, pessaries/suppositories, particulate systems, local injection, intravaginal tampon devices, sponges and rings. Intravaginal devices such as rings, tampons and sponges are either of the type where a medicament is impregnated into the device, or of the type that carries an encapsulated medicament. For example, reference 25 discloses a moist, medicated vaginal tampon that is impregnated with a contraceptive agent and a medicament for the control of venereal disease. Reference 26 discloses a tampon which has a capsule of disintegratable material partially embedded in one end. Such a tampon is inserted into the vagina and serves both to deliver and to retain the encapsulated medicament in the vaginal cavity. Reference 26 also discloses a means for pre-wetting the tampon in order to activate the capsule. Reference 27 discloses a sponge, impregnated with a liquid containing an effective amount of an active pharmaceutical agent, for insertion into the vaginal cavity. Reference 28 discloses a tampon assembly that is adapted for carrying a medicament within a longitudinal bore formed within the tampon. The medicament can be selectively expelled into the vaginal cavity from the bore using a tubular inserter. Further devices designed for prolonged or improved delivery are disclosed in references 29, 30 and 31.

The vaginal drug delivery system preferably provides a sustained delivery of the composition to the vaginal epithelium. If a paste or hydrogel is used, it should be of sufficient thickness/viscosity to ensure prolonged vaginal epithelium contact.

Muco-adhesive agents are preferably used to bring the released composition in solution into prolonged, close contact with the mucosal surface. The muco-adhesive agent is preferably a polymer such as an alginate, pectin or cellulose derivative.

Assays

The present invention also provides assays of arginase activity, both before and during pregnancy as an indicator of miscarriage and/or other pregnancy-related complication potential. Similarly, assays to determine Cat activity and/or levels of Cat expression can be used as an indicator, as can assays of CD3ζ or polyamine levels. Clinical tests for evaluating the state of immune competence in a patient, and measuring the efficacy of a treatment on a condition are also provided by the present invention.

The present invention provides an assay comprising the step of measuring the level of arginase activity, CD3ζ, Cat or polyamines in specific fluids, such as amniotic fluid or blood, in cells, such as uterine or placental cells, the peripheral blood cells or monocyte/macrophages or lymphocytes or in serum from a pregnant individual or an individual who is attempting to become pregnant. The level of arginase activity has a direct correlation on T-cell function in the immune system. This aspect of the invention can be performed by, for example, isolating the fluid or cells of interest from the patient and measuring the arginase, CD3ζ, Cat or polyamine levels and/or activity through known assays. Preferably the sample in which the arginase activity, CD3ζ levels, Cat activity or polyamine levels is measured is obtained from the placenta or in the region of the placenta, for example by chorionic villi sampling (CVS). Arginase activity can be measured by the conversion of L-arginine to L-ornithine or urea. CD3ζ levels can be measured by immunochemical techniques. Cat activity can be measured by determining the levels of L-arginine taken up by cells as described in [32] Arginase, CD3ζ and Cat protein levels can be directly determined by western blot or by ELISA. The expression of the arginase, CD3ζ or Cat genes can be detected by PCR, by PCR ELISA and by Northern blot. These assays may include directly measuring the protein level or measuring a relevant activity e.g. the ability of the fluid or cells to break down arginine. Polyamine levels can be measured by various techniques, including HPLC and fluorometry [33].

The assays described above can be used to give a single measurement of activity /level in a subject. A single measurement may be sufficient to determine whether an individual is immunosuppresed, for example when arginase activity in an individual that is at least 10% (e.g. 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200% or more) greater than expected, the individual is considered to be immunosuppressed. The assay can also be used to compare activity/level in an individual to a known normal baseline to determine whether activity/levels are greater than expected. For example in humans, an individual is considered to be immunosuppressed if the arginase activity in leukocytes, such as mononuclear cells (including monocytes and lymphocytes) and/or neutrophils is at least 100 mU/mg protein (e.g. >250mU/mg).

The assay can also be used to compare the activity/level in a pregnant individual to a known normal baseline in non-pregnant individuals to determine whether the pregnant individual is immunosuppressed. For example, pregnant individuals in whom arginase activity is at least 10% (e.g. 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200% or more) greater than in a non-pregnant individual are considered to be immunosuppressed.

The assay can also be used to compare activity/levels in a single subject before and after conception or throughout the pregnancy to determine whether the individual is immunosuppressed during pregnancy. The assay can be performed in either: (a) at least one sample taken from before conception and at least one sample taken after conception (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or more samples); or (b) at least two samples (e.g. 2, 3, 4, 5, 6, 7, 8, 9 or more samples) taken after conception.

The samples taken during pregnancy may be taken at different times spread throughout the entire pregnancy, the first trimester and/or the second trimester. For example, an individual is considered to be immunosuppressed if the arginase activity is at least 10% (e.g. 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200% or more) greater than at the pre-conception time point or the normal level for non-pregnant individuals.

In all the above embodiments where arginase levels are measured, if a pregnant individual or a nonpregnant individual who is attempting to conceive is found to have a low level of arginase activity, i.e. less than 95% (e.g. 90, 80, 70, 60, 50, 40, 30, 20, 10% or less) of normal arginase activity, then fertility problems may be indicated and treatment may be used to increase arginase levels and decrease arginine levels to improve the chances of conceiving and decrease the chance of a spontaneous miscarriage and/or other pregnancy-related complication. For example, in humans low arginase activity levels are defined as less than 100 mU/mg protein {e.g. less than 10 mU/mg) in leukocytes, such as mononuclear cells (including monocytes and lymphocytes) and/or neutrophils . The assay provides not only a measurement of arginase activity, but also provides a solution to the problem of low arginase activity.

The efficacy of a treatment of a condition, such as spontaneous miscarriage and/or other pregnancy- related complications, can be measured by determining the ability of said treatment to increase the level of arginase activity in the targeted organ, tissue or cells. This assay is similar to the assay discussed immediately above, however in this assay the cells are isolated prior to and after any treatment has been administered and the level of arginase activity is measure and compared. Accordingly, a determination can then be made whether the treatment has been effective.

New and existing immunostimulants can be tested for their ability to prevent spontaneous miscarriage and/or other pregnancy-related complications by determining the ability of these medications to increase the level of arginase activity in the uterus, endometrium or placenta. This embodiment of the invention is similar to the ones previously mentioned, the cells are isolated prior to, and after, any treatment has been administered and the level of arginase activity of the cells is measure and compared.

Determining arginase level systemically or in specific cells may also indicate if a patient is likely to respond to treatment. For example, some patients prone to recurrent spontaneous miscarriage and/or other pregnancy-related complications having low starting levels of arginase activity in the placenta may respond to treatment better than patients with normal starting levels of arginase activity. Low arginase levels are defined as less than 95% (e.g. 90, 80, 70, 60, 50, 40, 30, 20, 10% or less) of normal arginase activity.

The pregnancy complications that may be indicated by low arginase activity measured by the assays mentioned above include, but are not limited to, spontaneous miscarriage, recurrent miscarriage, partic serial miscarriage, intrauterine growth retardation, preterm labour, eclampsia or preeclampsia.

Genetic testing

The invention also provides a method of testing arginase genotype, before or during pregnancy as an indicator of miscarriage and/or other pregnancy-related complication potential by testing the arginase genotype of the patient for alleles of arginase I and/or arginase II, comprising the steps of detecting the presence or absence of known SNPs and/or unknown alleles of arginase and correlating the genotype to the frequency of spontaneous miscarriage and/or other pregnancy-related complication.

Assays of arginase genotype, both in males and females, can be used as an indicator of the risk of miscarriage and/or other pregnancy-related complications of future offspring. The method described above may be used to correlate the maternal, paternal and/or fetal arginase genotype to the risk of spontaneous miscarriage and/or other pregnancy-related complications.

Data gathered using the methods described above can be used to develop a genetic test that can predict the probability of having a miscarriage and/or other pregnancy-related complications by genotyping the individual and their partner for arginase.

In addition, or as an alternative, testing arginase genotype, polymorphism in Cat and/or the manganese transporter can be assayed. Thus the presence/absence of known SNPs and/or unknown alleles of Cat (or the manganese transporter) can be tested, and the genotype can be correlated to the frequency of spontaneous miscarriage and/or other pregnancy-related complication.

Patients

Preferably a patient being treated is human. However, the invention is not limited to humans, but includes other animals where it is desirable to maintain a successful pregnancy (such as horses, cows, sheep, pigs, dogs and cats), by providing high arginase levels. Conversely, compositions of the invention may be used to help prevent pregnancy or cause miscarriage in mammalian pests {e.g. rodents, marsupials, etc.) such as mice and rats, by providing low arginase levels.

The patient will typically be female.

The patient may have an arginase deficiency. Known arginase deficiencies include argininemia, familial argininemia, and hyperargininemia.

Preferably the patient is a primigravida, or a multigravida but not a primiparta. Preferably the patient is pregnant, wanting to get pregnant, suffers from or has suffered from preeclampsia, has previously had a spontaneous miscarriage or recurrent miscarriages, or is undergoing IVF treatment. Preferably the patient is post-pubescent and pre-menopausal, i.e. is aged between 10-65 years, preferably 14-55, more preferably 16-45 e.g. 20-40.

It is also possible to test males, as sperm may contribute to low arginase activity in a fetus. For example, a sperm sample could be tested for arginase and/or spermine levels.

Screening assays

Compounds that affect arginase can be identified by screening assays, as described below.

The assay methods may be for use in the identification of compounds for maintaining or providing increased arginase activity or reducing the level of arginine before or during pregnancy to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication. In all the assays described, the in vivo steps may be carried out in a non-human animal, for example a non-human mammal, preferably a non-human primate.

The various steps of the methods may be carried out at the same or different times, in the same or different geographical locations, e.g. countries, and by the same or different people or entities.

Screening for compounds that affect arginase activity

The invention provides a method for identifying a compound that increases arginase activity, said method comprising a step of assessing arginase activity in the presence and absence of a candidate compound and determining whether arginase in the presence of the compound shows increased activity. A compound that increases arginase activity is one that increases the specific arginase activity and/or increases the level of expression of arginase.

Optionally, the method may also include one or more of the steps of: testing a candidate compound for its ability to reduce T-cell activation in vitro; and/or testing a candidate compound for its ability to reduce T-cell activation in vivo, wherein the T-cells may be obtained from the placenta; and/or testing a candidate compound for downregulation of alloantigen responses in vivo, for example the downregulation of CD3ζ expression; and/or testing a candidate compound in vivo for unwanted side effects in pregnancy, such as teratogenic effects, premature birth and interuterine growth restriction (IUGR), and selecting compounds that do not posses any of these qualities.

For example, one screening method could comprise adding arginase to arginine in the presence or absence of the candidate compound and detecting the level or ornithine and/or urea produced.

Various assays of arginase activity have been previously described, [34, 35]. A further method involves mixing arginase with arginine and 2,3-butanedione and then measuring absorbance at 490nm against a blank containing no enzyme. One unit of enzyme releases one micromole of urea per minute at 37°C and pH 9.5.

The concentration of arginase in a sample can be detected using standard techniques such as western blot or ELISA. The expression of the arginase gene can be detected by PCR, PCR ELISA and by Northern blot.

Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding arginase in cells. For example, an ELISA may be constructed that measures secreted or cell-associated levels of arginase using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of arginase from suitably manipulated cells or tissues. The formation of binding complexes between arginase and the compound being tested may then be measured. Screening methods for compounds that regulate other targets

The method described above may also be used to identify compounds that increase the activity of other polyamine biosynthetic enzymes that act downstream of L-arginine, such as L-ornithine decarboxylase.

Various assays for as L-ornithine decarboxylase activity are known in the art, such as described in reference [36]. The activity of polyamine synthetic enzymes can also be measured by measuring the concentration of polyamines. The concentration of polyamines in a sample can be detected by various methods including HPLC and GC-MS.

The method described above can also be used for identifying compounds that upregulate enzymes that are themselves upregulated by increased arginase activity. The activity of arginine catabolic enzymes can be measured by any of the methods known in the art, for example the methods described in reference 37.

Arginine levels can also be altered by modulating the activity of the arginine transporters. A decrease in the specific activity or expression level of any arginine transporter would lead to a decrease in the intracellular level of arginine. The invention provides a method for identifying a compound that modulates the activity of any one of the arginine transporters, said method comprising the step of assessing the activity of the transporter in the presence and absence of a candidate compound and determining whether the transporter shows decreased activity in the presence of the compound. Preferably, the transporter is CAT-I.

The effect of arginase on the immune system is to decrease CD3ζ expression in TCR+ cells and to suppress their biological activities (e.g. proliferation, chemokine and cytokine secretion, and cytotoxicity). The invention therefore provides a further method of identifying a compound for use in suppressing the immune system during pregnancy that decrease the level of CD3ζ expression in TCR+ cells, said method comprising the step of assessing the level of CD3ζ expression in the presence and absence of a candidate compound and determining whether the level of expression is decreased in the presence of the compound.

Methods suitable for measuring the level of CD3ζ expression are known in the art and include, but are not limited to, RT-PCR, qRT-PCR, northern blotting, western blotting, and ELISA.

Several suppressors of the immune system that act through the same pathway as arginase are already known but are currently not recommended for use during pregnancy. One aspect of the present invention is the determination that compounds such as rapamycin, which act to suppress the immune system through the same pathway as arginase, are compatible with pregnancy and can prevent spontaneous miscarriage and/or other pregnancy-related complications. The present invention therefore provides a method for identifying a compound for use in suppressing the immune system during pregnancy that interact with mTOR to prevent the induction of the p70 S6 kinase by arginine, and which therefore suppress the immune system via a pathway that may be compatible with pregnancy. Optionally, the methods described above also include the one or more of the steps of: testing a candidate compound for its ability to reduce T-cell activation in vitro; and/or testing a candidate compound for its ability to reduce T-cell activation in vivo, wherein the T-cells may be obtained from the placenta; and/or testing a candidate compound for downregulation of alloantigen responses in vivo, for example the downregulation of CD3ζ expression ; and/or testing a candidate compound in vivo for unwanted side effects in pregnancy, such as teratogenic effects, premature birth and interuterine growth restriction (IUGR), and selecting compounds that do not posses any of these qualities.

Systems for carrying out screening methods

The in vivo steps of the screening methods of the invention may be carried out in cell-free systems or in cells or tissues. The cell-free system must contain all the necessary components for transcription of the reporter gene where the level of expression is detected by measuring mRNA levels, and all the necessary components for transcription and translation of the reporter gene where the level of expression is assessed by measuring protein levels.

It is preferred that the methods of screening of the invention be conducted in cell-free systems since this facilitates high-throughput screening of candidate compounds.

Indirect screening methods of the invention are preferably carried out in eukaryotic cells, such as mammalian {e.g. human) or yeast cells. They may also be performed in mammalian {e.g human) tissues. A typical cell is a macrophage.

Reference standards

A reference standard {e.g. a control), is typically needed in order to detect whether the arginase activity is increased. For example, in order to detect whether a candidate compound upregulates arginase activity, the activity of arginase in the presence of a candidate compound may be compared with the activity of arginase in the absence of a candidate compound.

The reference may have been determined before performing the method of the invention, or may be determined during {e.g. in parallel) or after the method has been performed. It may be an absolute standard derived from previous work.

Candidate compounds

Typical candidate compounds for use in all the screening methods of the invention include, but are not restricted to, peptides, peptoids, proteins, lipids, metals, small organic molecules, RNA aptamers, antibiotics and other known pharmaceuticals, polyamines, antibodies (as used herein, the term "antibody" refers to intact molecules as well as to fragments thereof, such as Fab, F(ab')2 and Fv, which are capable of binding to the antigenic determinant in question) or antibody derivatives {e.g. antigen-binding fragments, single chain antibodies including scFvs, etc.), and combinations or derivatives thereof. Small organic molecules have a molecular weight of about more than 50 and less than about 2,500 daltons, and most preferably between about 300 and about 800 daltons. Candidate compounds may be derived from large libraries of synthetic or natural compounds. For instance, synthetic compound libraries are commercially available from MayBridge Chemical Co. (Revillet, Cornwall, UK) or Aldrich (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts may be used. Additionally, candidate compounds may be synthetically produced using combinatorial chemistry either as individual compounds or as mixtures.

In some instances, it may be desirable to conduct a preliminary screening step to reduce the number of candidate compounds used in the methods of the invention.

In vivo confirmation of function of compounds identified

Once a compound has been identified, it may be desirable to perform further experiments to confirm the in vivo function of the compound.

The invention therefore provides a method of assessing the in vivo effect of a compound obtained or obtainable by any of the methods described above comprising administering the compound to a test animal and assessing the effect on pregnancy, including birth defects, changes to the gestation period and changes in birth weight. Tests in non-human animals, for example non-human mammals or non- human primates may be used.

Compounds identified by screening methods

The invention provides a compound that increases arginase activity, increases polyamine synthesis, decreases arginine biosynthesis, decreases arginine transporter activity or downregulates the expression of CD3ζ, obtained or obtainable by any of the methods described above. Preferably, the compounds of the invention are organic compounds.

Preferably a compound as identified above has an established safety profile in pregnant women.

Pharmaceutical uses of compounds identified

Once a compound has been identified using one of the methods of the invention, it may be necessary to conduct further work on its pharmaceutical properties. For example, it may be necessary to alter the compound to improve its pharmacokinetic properties or bioavailability. The invention extends to any compounds obtained or obtainable by the methods of the invention which have been altered to improve their pharmacokinetic properties.

Pharmaceutical compositions

The invention provides pharmaceutical compositions comprising any of the compounds of the invention or a combination of two or more (e.g. three or more, four or more, five or more etc.) of compounds of the invention.

The compositions should preferably comprise a therapeutically effective amount of compounds of the invention. The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targeted disease or condition, preferably spontaneous miscarriage or preeclampsia, or to exhibit a detectable therapeutic or preventative effect. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

The precise effective amount for a human subject will depend upon the severity of the risk of miscarriage and/or other pregnancy-related complications, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.

A pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent. Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.

Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable carriers is available in reference 38.

Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

The composition may be sterile.

Compositions of the invention are preferably non-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose.

Compositions of the invention are preferably gluten free. Compositions will generally have an osmolality of between 200 mθsm/kg and 400 mθsm/kg, preferably between 240-360 mθsm/kg, and will more preferably fall within the range of 290-300 mθsm/kg. Compositions may be substantially isotonic with respect to humans.

Compositions may include sodium salts (e.g. sodium chloride) to give tonicity. A concentration of 10+2mg/ml NaCl is typical.

Compositions of the invention may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. A phosphate buffer is typical. Buffers will typically be included in the 5-2OmM range.

The pH of a composition of the invention will generally be between 5.0 and 7.5, and more typically between 5.0 and 6.0 for optimum stability, or between 6.0 and 7.0.

Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.

Targeted delivery of the compositions will generally be accomplished any one of a number of methods such as hydrogels, pastes, vaginal tablets, pessaries/suppositories, particulate systems, local injection, intravaginal tampon devices, sponges and rings. Dosage treatment may be a single dose schedule or a multiple dose schedule.

If the target is involved in the synthesis or transport of arginine, one approach comprises administering to a subject an inhibitor compound (antagonist) as described above, along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.

When the target is involved in the catabolism of arginine, biosynthesis of polyamines or downstream signalling between arginine and the suppression of the immune response, one approach comprises administering to a subject a therapeutically effective amount of a compound that activates the target, i.e., an agonist as described above, to decrease the amount of available arginine or to suppress the immune system.

Where the invention includes the delivery of a protein, it may be modified to improve in vivo characteristics, such as improving its pharmacokinetic properties. For example, proteins may be modified by the addition of PEG. In particular, a PEG 5 kD conjugate of arginase has been shown to have an increased plasma half life in mice [39] and may be more effective than the native enzyme in vitro [40].

Methods of treatment

The invention provides a method of maintaining or providing increased arginase activity or reducing the level of arginine before or during pregnancy to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication. Such conditions include, but are not limited to, premature labour, intrauterine growth retardation, eclampsia, preeclampsia, or other complications of pregnancy and/or fetal development.

The method comprises the step of administering to a patient before or during pregnancy a compound and/or composition of the invention. Preferably the compound or composition is compatible with pregnancy e.g. has previously been certified by a regulatory authority, such as the FDA or EMEA, to be safe for administration to pregnant females.

The invention also provides the use of a compound and/or composition of the invention, in therapy

The invention also provides the use of a compound and/or compositions of the invention, in the manufacture of a medicament for treating or preventing spontaneous miscarriage and/or other pregancy-realted complication.

Nutritional supplements

Various nutritional supplements are known that are considered to improve chances of conception or to improve the health of pregnant women and their unborn fetuses. Pregnant women require an increased intake of iron, calcium, folate, and sometimes protein and vitamin D during pregnancy.

One example of a commercially available supplement designed for pregnant women, FertilityBlend, contains folic acid; vitamins E, B6 and B 12; iron; magnesium; zinc; L-arginine; chasteberry, a herb that is thought to optimize ovulation; and the antioxidants green tea and selenium. Similarly, Materna and S-26 Mama, manufactured by Wyeth contain folic acid, iron, B-complex vitamins, vitamin C, vitamin D, vitamin E, and zinc; and protein, docosahexaenoic acid, folic acid, iron, calcium and vitamins and minerals respectively.

The invention provides nutritional supplements suitable for use before or during pregnancy, with the aim of reducing arginine levels, comprising folate and one or more free amino acids, that is free of arginine. Preferably the nutritional supplement may further comprise one or more of iron, calcium, protein, zinc, docosahexaenoic acid, vitamin A, vitamin B, vitamin C, vitamin D and vitamin E.

Manganese (Mn²⁺) has been identified as a cofactor for arginase. Therefore the nutritional supplement preferably also comprises a source of Mn²⁺. Preferably the supplement may also comprise a source OfNi²⁺ and/or Co²⁺. The supplement may be zinc free.

General

The term "spontaneous miscarriage" means the spontaneous loss of a pregnancy before 24 weeks of gestation.

The term "recurrent miscarriage" is defined as more than one consecutive miscarriage, for example three or more consecutive miscarriages.

The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y. The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

The term "about" in relation to a numerical value x means, for example, x+10%.

Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure IA shows arginase activity in mononuclear cells. Arginase activity was measured as described in the modes for carrying out the invention. Highest arginase activity is localised in the placenta. Data are for: • PIaCs; o maternal PBMCs; α control PBMCs. Statistical differences via student's t test are: *P=0.01, ** P=0.02.

Figure IB shows arginase activity in peripheral neutrophils. Arginase activity was measured as for Figure IA. Data are for: o maternal neutrophils (from 8 individuals); G control neutrophils (from 9 individuals). Statistical difference via student's ttest is: * P=0.02.

Figure 1C shows arginase activity in sera. The activity of arginase was measured in maternal (o) and non-pregnant control (D) sera, as described for Figure IA. Similarly, Figure ID shows urea levels.

Figure IE shows a western blot of arginase in various cell lysates.

Figure 2 shows results of CD3ζ assays in arginase-expressing cells in the placenta.

Figures 3A shows local downregulation of CD3ζ chain in the placenta. The solid line shows data for PIaCs, whereas the dashed line shows data for maternal PBMCs. Figure 3B shows the MFI (mean fluorescence intensity) of CD3ζ in peripheral TCR+ cells (•) and paired placental TCR+ cells (o) from 13 separate patients, and % reduction in PIaCs compared to maternal PBMCs is shown in 3C.

Figure 4A shows CD3ζ chain expression in TCR+ cells isolated from the placenta, and Figure 4B shows the proliferation of placental T lymphocytes activated through anti-CD3/anti-CD28. The X-axes show the number of days post-stimulation. Arginine was either present (•) or absent (o).

Figures 5A and 5B show that downregulation of CD3ζ chain is restored by inhibition of arginase or by addition of L-arginine. The dotted lines show results using maternal PBMCs + PIaCs. In Figure 5A the solid line shows results when nor-NOHA was included. In Figure 5B, the solid line shows results when L-arginine was included. The addition of nor-NOHA or L-arginine shifts the line to the right in both cases. Figure 5C shows the % of dividing TCR+ cells is increased in the presence of nor-NOHA (middle bar) or exogenous L-arginine (right-hand bar) compared to controls (left). MODES FOR CARRYING OUT THE INVENTION

Assay ofarginase activity in placental cells and peripheral neutrophils

The arginase enzyme activity in mononuclear cells isolated from healthy human placenta (PIaCs) immediately after parturition was determined, and compared with maternal peripheral blood mononuclear cells (PBMCs) and PBMCs from aged-match female non-pregnant controls. The arginase enzyme activity in peripheral neutrophils isolated from the blood of pregnant individuals was determined, and compared the arginase activity in peripheral neutrophils from non-pregnant controls.

PIaCs, maternal and control PBMCs, and maternal and control neutrophils were isolated by Ficoll, the single cell suspensions were frozen and arginase activity was measured in the lysates as previously described [41]. Briefly, 25ml of the lysate was incubated with 12ml of 0.1% triton X-IOO, 12ml of 25mM Tris-HCl and 1.2ml of 2OmM MnCl₂ for 7min at 56°C. Arginase activity was conducted by adding 50ml of 0.5mM L-arginine pH9.7 and incubating for 15min at 37°C. The reaction was stopped with 400ml of H₂SO₄ (96%)/H₃PO₄ (85%)/H₂O (1/3/7, v/v/v). Urea concentrations were measured at 540nm after addition of 20ml of α-isonitrosopropiophenone (dissolved in 100% ethanol) followed by heating at 100⁰C for 30min. All the incubation were performed in a thermocycler. One unit of enzyme activity is defined as the amount of enzyme that catalyses the formation of lmmol urea/min.

In all cases, arginase activity in PIaCs was significantly higher than in paired PBMCs, or in PBMCs from non-pregnant controls (Figure IA). Although arginase activity in PBMCs from pregnant women at the time of birth was notably lower than in PIaCs, it was still significantly higher than in PBMCs from non-pregnant controls (Figure IA). Arginase activity in peripheral neutrophils isolated from pregnant women was higher than in neutrophils from non-pregnant controls (Figure IB). Similar results were obtained when the mean fluorescent activity of arginase 1 was compared in CD14- CD15+ cells isolated from pregnant and control women by flow cytometry. Determination of arginase protein by western blot confirmed that higher arginase I protein expression was found in PIaCs (Figure IE), but arginase II expression was not seen. Sera prepared from maternal blood collected at the time of birth also contained significantly higher urea (p=0.001; Figure ID) and arginase activity (p=0.03; Figure 1C) than sera from controls, indicating an increased arginine turn over in vivo.

Assay for alternatively-activated macrophages

The type of arginase-expressing cells in the placenta was determined by a combination of intracellular protein staining and cell surface labelling. The majority of arginase-expressing cells were neutrophils (CD15⁺CD14- and CD15⁺CD14^low, Figures 2A & 2B); CD14^high cells did not express arginase. Another small population of arginase-expressing cells was identified within the CD14^low population. These were alternatively activated macrophages, as characterised by the co-expression of the mannose receptor CD206 (Figure 2C). Thus two populations of cells express arginase in the placenta: neutrophils and alternatively-activated macrophages.

Figure 2D shows the percentage of alternatively-activated macrophages in three types of cell.

Assay for CD3ζ expression

The bioavailability of L-arginine can affect T cell functions and the consumption of this amino acid by alternatively activated macrophages leads to modulation of CD3ζ chain expression in T cells [42]. Downregulation of CD3ζ expression results in uncoupling of the TCR signal transduction pathways with consequent T cell hyporesponsiveness [43].

To examine the functional effect of placental arginase, PIaCs and Jurkat cells were co-cultured; the latter have been used to assay arginase-induced modulation of T cell responsiveness [7,42]. PIaCs or maternal PBMCs (2xlO⁵ cells) were incubated with IxIO⁵ Jurkat cells in a final volume of 200μl in the presence or in the absence of lOμl 5.6 μM nor-NOHA or 2μl of 10OmM L-arginine. Two days later, mean fluorescence intensity (MFI) of CD3ζ in TCR⁺ cells (i) or frequency of TCR⁺ cells incorporating BrdU (ii) were determined. Jurkat cells were identified by increased side/forward scatter. 100% response represented the value of the MFI of CD3ζ in Jurkat cells cultured alone.

Co-culture of PIaCs with Jurkat cells resulted in both downregulation of CD3ζ chain expression (Figure 2E) and decreased proliferation of Jurkat cells (Figure 2F). Importantly, CD3ζ chain expression and T cell proliferation could be restored by addition of either a competitive arginase inhibitor, N^ω-hydroxy-nor-L-arginine (nor-NOHA [44]) or by the addition of exogenous L-arginine (Figures 2E & 2F). In contrast, maternal PBMC had no effect on CD3ζ chain expression or proliferation of Jurkat cells. These results demonstrate that PIaCs can mediate CD3ζ chain downregulation and T cell hyporesponsiveness through arginase-mediated L-arginine depletion.

To determine the functional consequences of local arginase activity on T cells within the placenta, the expression of CD3ζ in placental TCR⁺ cells was examined directly ex vivo. Mean fluorescence intensity of CD3ζ chain expression was determined in maternal TCR+ PBMCs and TCR+ PIaCs. In all individuals examined, CD3ζ chain expression was significantly reduced in TCR⁺ cells derived from the placenta as compared to maternal T lymphocytes in peripheral blood (average reduction: 40.8% ± 2.8) (Figures 3A & 3C). These results demonstrate that CD3ζ chain expression is downregulated in TCR⁺ cells in the placenta, a compartment with high arginase activity.

As TCR⁺ cells are likely to circulate between placenta and periphery, it was thought that this downregulation might be reversible, to allow the maternal immune system to respond to other antigenic challenges whilst maintaining a state of non-responsiveness against the fetus. To test this possibility, the reversibility of CD3ζ downregulation of placental TCR⁺ was tested. PIaCs (4xlO⁵) were stimulated with plate-bound anti-CD3 and anti-CD28 mAb in a final volume of 200μl in complete DMEM in the absence of L-arginine, or in complete DMEM containing 0.1, 0.4, 1 or 2mM L-arginine. Cells were harvested daily after post-stimulation, and the MFI of CD3ζ in TCR⁺ cells or the frequency of TCR⁺ cells incorporating BrdU were determined. Similar results were obtained with all the concentrations of L-arginine tested. In the presence of L-arginine, TCR-mediated stimulation of PIaCs resulted in increased CD3ζ chain expression in TCR⁺ cells; in the absence of exogenous L- arginine no recovery was observed (Figure 4A). This shows that CD3ζ chain downregulation of TCR⁺ placental cells is reversible. A functional correlate of CD3ζ chain expression is the ability of cells to proliferate. Placental TCR⁺ cells stimulated in the presence of L-arginine retain this ability (Figure 4B). These experiments demonstrate that the functional phenotype of placental TCR⁺ cells is reversible and that the reduced responsiveness of placental T cells can not be due to apoptosis. This reveals a role for the metabolism of L-arginine in the regulation of T cell responses in the placenta.

Finally, to confirm the role of placental arginase and L-arginine in mediating T cell hyporesponsiveness, autologous maternal PBMCs were stimulated in the presence of PIaCs and the effects of arginase inhibition or addition of exogenous L-arginine on maternal TCR⁺ cells were assessed. PIaCs (2x10⁵) were stimulated with plate-bound anti-CD3 mAb and anti-CD28 mAb in a final volume of lOOμl of DMEM (O.lmM L-arginine). lOμl nor-NOHA (5.6μM) was added to some of the wells, 30min later, maternal PBMCs (IxIO⁵, labeled with CFSE to differentiate maternal TCR⁺ from placental TCR⁺) were added in a final volume of lOOμl. L-arginine (2μl of 10OmM) was added to some of the wells. Two days later, the cells were harvested and the expression of CD3ζ in maternal TCR⁺ cells and the frequency of maternal TCR⁺ cells undergoing division were determined.

Competitive inhibition of arginase (nor-NOHA) resulted in a clear upregulation of CD3ζ chain in maternal TCR⁺ cells (Figure 5A). Similarly, addition of exogenous L-arginine also enhanced CD3ξ expression (Figure 5B). In agreement with these data, the proliferative response of maternal TCR⁺ was also increased in the presence of nor-NOHA or exogenous L-arginine (Figure 5C). These results demonstrate that PIaCs can mediate local and reversible T cell hyporesponsiveness in the placenta, directly linking arginase and the bioavailability of L-arginine to feto-maternal tolerance.

Conclusions

The survival of the semi-allogeneic fetus is critically dependent upon the induction of unresponsiveness of the maternal immune system. According to the invention, there is a physiological mechanism whereby arginase can mediate suppression of maternal T cell responses. High arginase activity is present in cells isolated from placenta, and the sources of arginase are neutrophils and a population of alternatively activated macrophages.

The arginase levels seen in placental cells are comparable to those in the liver, the organ with the highest arginase concentration in the human body [45]. High arginase levels in the placenta imply a high rate of substrate consumption and decreased levels of extracellular L-arginine. This view is supported by downregulation of CD3ζ chain expression in placental TCR⁺ cells, a finding consistently associated with L-arginine depletion [7,42,46]. Importantly, T cell hyporesponsiveness has been shown to is reversible, and T cells in the placenta retain their capacity to respond to stimulation. In addition, placental cells co-cultured with peripheral blood lymphocytes induce arginase-dependent, L-arginine-mediated T cell hyporesponsiveness, indicating a novel pathway through which immune privilege can be mediated at the feto-maternal interface.

It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

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Claims

1. A method for reducing the risk of a pregnancy disorder in a pregnant female patient, by (i) maintaining or increasing the patient's arginase activity, (ii) decreasing the patient's arginine levels, and/or (iii) increasing the patient's arginine breakdown.

2. The method of claim 1, wherein the arginase is arginase I.

3. The method of any preceding claim, wherein the increase of arginase activity, decrease in arginine levels and/or arginine breakdown occurs in the patient's uterus, endometrium or placenta.

4. The method of claim 3, wherein the increase of arginase activity, decrease in arginine levels and/or arginine breakdown is localised to the patient's uterus, endometrium or placenta.

5. The method of claim 3 or claim 4, wherein the method involves administering a medicament to the patient's vagina or uterus.

6. The method of any preceding claim, wherein the pregnancy disorder is miscarriage, intrauterine growth retardation, preterm labour, eclampsia or preeclampsia.

7. The method of any preceding claim, wherein arginase activity is increased by administering arginase to the patient.

8. The method of any preceding claim, wherein arginase activity is increased by administering to the patient a medicament that up-regulates their arginase expression.

9. The method of any preceding claim, wherein arginase activity is increased by administering to the patient a medicament that includes a source of Mn⁴⁺, Ni⁺⁺ and/or Co⁺*.

10. The method of any preceding claim, wherein arginine levels are decreased by one or more of: (i) reducing their dietary arginine supply; (ii) reducing their dietary supply of an arginine precursor; (iii) administering to the patient a medicament that down-regulates expression of an enzyme involved in arginine biosynthesis; (iv) administering to the patient a medicament that inhibits an enzyme involved in arginine biosynthesis; (v) administering to the patient a medicament that increases a system y⁺ cationic amino acid transporter that transports arginine into a cell, such as CAT-I ; (vi) administering to the patient a medicament that up-regulates expression of a system y⁺ cationic amino acid transporter that transports arginine into a cell; and/or (vii) administering to the patient a medicament that increases their NOS activity.

11. A method for improving the likelihood of pregnancy in a non-pregnant female, by (i) maintaining or increasing the patient's arginase activity, and/or (ii) decreasing the patient's arginine levels.

12. The method of claim 1 1, comprising one or more of the features from claims 2 to 10.

13. The method of claim 11 or claim 12, wherein the female has previously had a spontaneous miscarriage.

14. A method for decreasing the likelihood of pregnancy, or of increasing the likelihood of miscarriage, by (i) decreasing the patient's arginase activity, and/or (ii) increasing the patient's arginine levels.

15. A method for assessing a patient's risk of suffering a pregnancy disorder, in which their arginase activity, and/or arginase I genotype are determined.

16. The method of claim 15, wherein the patient is a female.

17. The method of claim 16, wherein the patient is pregnant.

18. The method of claim 16 or claim 17, wherein arginase activity is measured in T cells.

19. The method of claim 17 or claim 18, wherein arginase activity is determined before and during pregnancy.

20. The method of claim 14, wherein the patient is a male.

21. The method of claim 20, wherein arginase activity is measured in spermatozoa.

22. A method for assessing a patient's risk of suffering a pregnancy disorder, in which in which their CD3ζ levels are determined.

23. A method for assessing a patient's risk of suffering a pregnancy disorder, in which activity is determined of a system y⁺ cationic amino acid transporter that transports arginine into a cell.

24. A method for assessing a patient's risk of suffering a pregnancy disorder, in which in which their polyamine levels are determined.

25. The method of any one of claims 22 to 24, wherein the patient is a female.

26. The method of claim 26, wherein the patient is pregnant.

27. The method of claim 16 or claim 17, wherein the determination is made for T cells.

28. The method of any preceding claim, wherein the patient is human.

29. A nutritional supplements for use before or during pregnancy, comprising folate and one or more free amino acids, but that is free of arginine.

30. The supplement of claim 29, including one or more of: iron, calcium, protein, zinc, docosahexaenoic acid, vitamin A, vitamin B, vitamin C, vitamin D and vitamin E.

31. The supplement of claim 29 or claim 30, including a source of Mn^+"1", Ni^+"1" and/or Co^+"1"..

32. The supplement of any one of claims 29 to 31, which is free from Zn⁺⁺.

33. An assay method for identifying if a compound that can be used for (a) maintaining or increasing arginase activity in a patient or (b) reducing arginine levels in a patient, wherein arginase activity is assessed in the presence and absence of the compound, wherein an increase in arginase activity in the presence of the compound indicates that the compound is suitable.

34. An assay method for identifying if a compound that can be used for (a) maintaining or increasing arginase activity in a patient or (b) reducing arginine levels in a patient, wherein the compound is tested for its effect the activity of polyamine biosynthetic enzymes that act downstream of L-arginine, such as L-ornithine decarboxylase.

35. An assay method for identifying if a compound that can be used to modulate the activity of an arginine transporter, such as CAT-I, comprising a step of assessing the activity of the transporter in the presence and absence of a candidate compound.

36. An assay method for identifying if a compound that can be used to modulate expression of CD3ζ expression, comprising a step of assessing expression of CD3ζ in the presence and absence of a candidate compound.

37. A method for identifying a compound for use in suppressing the immune system during pregnancy, wherein the compound interacts with mTOR to prevent the induction of the p70 S6 kinase by arginine, comprising a step of assessing if the compound binds to mTOR.

38. The assay method of claim 33 or claim 34, further including at least one of the following steps: (i) testing the compound for its ability to reduce T-cell activation in vitro; (ii) testing the compound for its ability to reduce T-cell activation in vivo, particularly T cells obtained from a placenta; (iii) testing the compound for its ability to downregulate alloantigen responses in vivo; and/or (iv) testing the compound in vivo for side effects that would be undesirable in pregnancy.

39. A method for reducing the risk of miscarriage and/or premature birth in a pregnant female patient, by (i) administering to the patient a medicament that up-regulates expression of a system y⁺ cationic amino acid transporter that transports arginine into a cell; (ii) administering to the patient a medicament that increases a system y⁺ cationic amino acid transporter that transports arginine into a cell; (iii) administering to the patient a medicament that binds to mTOR, such as rapamycin or wortmanin; (iv) administering to the patient a medicament that decreases CD3ξ expression; (v) administering to the patient a medicament that inhibits CD3ζ activity; (vi) administering to the patient a medicament that increase polyamine production expression; (vii) administering to the patient a medicament that inhibits polyamine breakdown; (vii) administering to the patient a medicament comprising a polyamine; (viii) administering to the patient a medicament that increases GCN2kinase activity;