The invention relates to the field of immunology. Specifically, the invention relates to the field of immune-mediated disorders such as allergies, auto-immune disease, transplantation-related disease or inflammatory disease. The invention provides for an immunoregulator (IR), use of an IR in preparing a pharmaceutical composition for treating an immune-mediated disorder and a method for treating an immune-mediated disorder.

Description of the Invention

The invention provides, among other things, an immunoregulator (IR), use of an IR in preparing a pharmaceutical composition for treating an immune-mediated disorder, a pharmaceutical composition and a method for treating an immune-mediated disorder. Immune mediated disorders as described herein include chronic inflammatory disease, such as diabetes type I or II, rheumatic disease, Sjogrens syndrome, multiple sclerosis, transplantation-related immune responses such as graft-versus-host-disease, post-transfusion thromboytopenia, chronic transplant rejection, pre-eclampsia, atherosclerosis, asthma, allergy and chronic auto-immune disease, and acute inflammatory disease, such as (hyper)acute transplant rejection, septic shock and acute auto-immune disease. Auto-immune diseases are a group of disorders of, in general, unknown etiology. In most of these diseases, production of autoreactive antibodies and/or autoreactive T lymphocytes can be found. An auto-immune response may also occur as manifestation of viral or bacterial infection and may result in severe tissue damage, for example destructive hepatitis because of Hepatitis B virus infection.

Auto-immune diseases can be classified as organ specific or non-organ specific depending on whether the response is primarily against antigens localized in particular organs or against wide-spread antigens. The current mainstay of treatment of auto-immune diseases is immune suppression and/or, because of tissue impairment, substitution of vital components like hormone substitution. However, immunosuppressive agents, such as steroids or cytostatic drugs, have significant side effects which limits their application. Now, the use of more specific immunoregulatory drugs is provided by the invention in the treatment of auto-immune disease and other inflammations. Based on the immunoregulatory properties as described below, e.g., by regulating the Th1/Th2 ratio, modulating dendritic cell differentiation, the low side-effect profile, the initial clinical observations, etc., it shows these preparations to be very helpful in the treatment of patients with immune-mediated inflammation, such auto-immune disease.

A non-limiting list of auto-immune diseases includes: Hashimoto's thyroiditis, primary myxedema thyrotoxicosis, pernicious anaemia, auto-immune atrophic gastritis, Addison's disease, premature menopause, insulin-dependent diabetes mellitus, stiff-man syndrome, Goodpasture's syndrome, myasthenia gravis, male infertility, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, multiple sclerosis, auto-immune hemolytic anaemia, idiopathic thrombocytopenic purpura, idiopathic leucopenia, primary biliary cirrhosis, active chronic hepatitis, cryptogenic cirrhosis, ulcerative colitis, Sjogren's syndrome, rheumatoid arthritis, dermatomyositis, polymyositis, scleroderma, mixed connective tissue disease, discoid lupus erythematosis, and systemic lupus erythematosis.

In one embodiment, the invention provides an immunoregulator capable of down-regulating Th1 cell levels and/or up regulating Th2 cell levels, or influencing their relative ratio in an animal, the immunoregulator obtainable from urine or other sources of bodily products, such as serum, whey, placental extracts, cells or tissues. "Obtainable" herein refers to directly or indirectly obtaining the IR from the source. IR is, for example, obtained via chemical synthesis or from animal or plant sources in nature.

In a preferred embodiment, the invention allows regulating relative ratios and/or cytokine activity of lymphocyte subset-populations in a diseased animal (e.g., human), preferably where these lymphocyte subset-populations comprise Th1 or Th2 populations. In general, naive CD4.sup.+helper T-lymphocytes (Th) develop into functionally mature effector cells upon stimulation with relevant antigenic peptides presented on the major histocompatibility complex (MHC) class IT molecules by antigen-presenting cells (APC). Based on the characteristic set of cytokines produced, Th cells are commonly segregated into at least two different subpopulations: Th1 cells producing exclusively interleukin-2 (IL-2), interferon-gamma (IFN-.gamma.) and lymphotoxin, while Th2 cells produce IL-4, IL-5, IL6, IL10 and IL-13. These Th1 and Th2 subsets appear to be extremes in cytokine production profiles and within these polarized subsets, individual Th cells exhibit differential rather than coordinated cytokine gene expression. These subsets develop from common Th precursor cells (Thp) after triggering with relevant peptides into Th0 cells producing an array of cytokines, including IL-2, IL-4, IL-5 and IFN-.gamma.. These activated Th0 cells subsequently polarize into the Th1 or Th2 direction based on the cellular and cytokine composition of their microenvironment. Antigen-presenting cells like the various subsets of dendritic cells besides subsets of macrophages largely determine this polarization into TH1 or Th2 subset development. The Th1-Th2 subsets appear to cross-regulate each other's cytokine production profiles, mainly through IFN-.gamma. and IL-10, and from this concept it was rationalized that disturbances in the balance between these two subsets may result in different clinical manifestations [5]. IL-12 is a dominant factor promoting Th1 subset polarization and dendritic cells and macrophages production of IL-12. Moreover, IL-12 induces IFN-.gamma. production by T-cells and natural killer (NK) cells. Recently, it was reported that IL-18 acts synergistically with IL-12 to induce Th1 development. Polarization of Th2 cells is critically dependent on the presence of IL-4 produced by T-cells or basophils and mast-cells. APC-derived IL-6 has also been shown to induce small amounts of IL-4 in developing Th cells. IL-10 and APC-derived prostaglandin E.sub.2 (PGE.sub.2) inhibit IL-12 production and Th1 priming.

The Th1-Th2 paradigm has been useful in correlating the function of Th1 cells with cell-mediated immunity (inflammatory responses, delayed type hypersensitivity, and cytotoxicity) and Th2 cells with humoral immunity. In general, among infectious diseases, resistance to intracellular bacteria, fungi, and protozoa is linked to mounting a successful Th1 response. Th1 responses can also be linked to pathology, like arthritis, colitis and other inflammatory states. Effective protection against extracellular pathogens, such as helminths, mostly requires a Th2 response, and enhanced humoral immunity may result in successful neutralization of pathogens by the production of specific antibodies.

In yet another preferred embodiment, the invention provides an immunoregulator capable of modulating dendritic cell differentiation. The selective outgrowth of Th1 vs. Th2 type cells is dependent on the interaction of precursor Th cells with antigen-presenting cells (APC) carrying the relevant peptide in conjunction with their MHC class II molecules. Cytokines released by the APC and present during the initial interaction between dendritic cells and the pertinent T-cell receptor carrying T-cells drive the differentiation into Th1 vs. Th2 subsets. Recently, two different precursors for DC (myeloid vs. lymphoid) have been described in man. Selective development of DC1 from myeloid precursors occurs after stimulation with CD40 Ligand or endotoxin and results in high production of IL-12. Lymphoid precursors give rise to DC2 cells after CD40 Ligand stimulation and produced IL-1, IL-6 and IL-10. These cytokines are of prime importance in driving the development of the activated Th cell: IL-4 is required for the outgrowth of Th2 type cells which can be greatly enhanced by the presence of IL-10, while selective differentiation to Th1 type cells is exclusively dependent on the presence of IL-12. Since DC1 are characterized by the production of IL-12, they will primarily induce outgrowth of Th1 type cells, while DC2 produce IL-10 and selectively promote Th2 development in the presence of exogenous IL-4. It is shown herein that an IR as provided by the invention is capable of regulating or modulating DC activity and differentiation, thereby allowing selective differentiation and activity of Th1 and/or Th2 cells.

In one embodiment, the invention provides an immunoregulator comprising an active component obtainable from a mammalian chorionic gonadotropin preparation. The active component is capable of stimulating splenocytes obtained from a non-obese diabetic (NOD) mouse, or comprises an active component functionally related to the active compound. For example, allowing, regulating, or modulating DC activity and differentiation, or allowing selective differentiation and activity of Th1 and/or Th2 cells in the case of chronic inflammation, such as diabetes or chronic transplant rejection as shown in the detailed description herein, the stimulated splenocytes are capable of delaying the onset of diabetes in an NOD-severe-combined-immunodeficient mouse reconstituted with the splenocytes. The active component is capable of inhibiting gamma-interferon production of splenocytes obtained from a non-obese diabetic (NOD) mouse or capable of stimulating interleukin-4 production of splenocytes obtained from a non-obese diabetic (NOD) mouse.

In another embodiment, the invention provides an immunoregulator comprising an active component obtainable from a mammalian chorionic gonadotropin preparation capable of protecting a mouse against a lipopolysaccharide-induced septic shock. For example, allowing, regulating, or modulating DC activity and differentiation or allowing selective differentiation and activity of Th1 and/or Th2 cells, in the case of acute inflammation such as seen with shock or (hyper) acute transplantation rejection, is shown in the detailed description herein. The active component is capable of reducing ASAT or other relevant plasma enzyme levels after or during organ failure as commonly seen with shock.

In one embodiment, the immunoregulator according to the invention comprises, as further detailed in the detailed description, an active component residing in a fraction which elutes with an apparent molecular weight of 58 to 15 kilodalton as determined in gel-permeation chromatography, where associating, inhibiting or synergistic components are found as well. In another embodiment, the invention provides an immunoregulator, as further detailed in the detailed description, where the active component is present in a fraction which elutes with an apparent molecular weight of smaller than 15 kilodalton as determined in gel-permeation chromatography, for example, where the active component is present in a fraction which elutes with an apparent molecular weight of <1 kilodalton as determined in gel-permeation chromatography. Although the immunoregulator according to the invention is easily obtained from urine, for example, where the mammalian chorionic gonadotropin preparation is derived from urine, other sources, such as serum, cells or tissues comprising gonadotropin are applicable as well. From the sources of an immunoregulator according to the invention capable of, for example, regulating Th1 and/or Th2 cell activity, and/or capable of modulating dendritic cell differentiation, is also provided.

Preferably, an immunoregulator as provided by the invention is obtainable from a pregnant mammal, preferably a human, for example, obtainable from a pharmacological preparation prepared to contain placental gonadotropins such as pregnant mare serum gonadotropin (PMSG) found in serum of pregnant mares (IR-S), or pregnant mouse uterus extract (PMUE) extracted from uteri (IR-UE) of gravid mice, or human chorionic gonadotropin (hCG or HCG) found in blood or urine of pregnant women. An IR as provided by the invention can be associated with or without gonadotropin as, for example, is present in the urine during the first trimester of pregnancy (IR-U), and in commercial hCG preparations (IR-P) has immune regulatory effects. In particular, IR can inhibit or regulate auto-immune and acute- and chronic-inflammatory diseases. TNF and IFN-gamma are pathologically involved in acute inflammatory disease such as sepsis or septic shock and also in auto-immune and chronic inflammatory diseases. Since IR has the ability to regulate T-cell sub-populations and inhibit TNF and IFN-gamma, IR can be used to treat, suppress or prevent immune mediator disorders such as sepsis or septic shock (acute inflammatory disease) as well as auto-immune disease or chronic inflammatory diseases such as systemic lupus erythematosis, diabetes, rheumatoid arthritis, post-partum thyroid dysfunction, auto-immune thrombcytopenia and others, such as allergies and chronic inflammatory disease (i.e., rheumatic disease, Sjogrens syndrome, multiple sclerosis) and transplantation-related immune responses. Our results, for example, show that IR inhibits sepsis or septic shock caused by endotoxin or by exotoxin. IR, as provided by the invention, inhibits or counters immune-mediated auto-immune diseases, chronic inflammatory diseases, as well as acute inflammatory diseases.

The invention provides a pharmaceutical composition for treating an immune-mediated disorder such as an allergy, auto-immune disease, transplantation-related disease or acute or chronic inflammatory disease and/or provides an immunoregulator (IR), for example, for stimulating or regulating lymphocyte action comprising an active component. The active component is capable of stimulating splenocytes obtained from a 20-week-old female non-obese diabetic (NOD) mouse, the stimulated splenocytes delaying the onset of diabetes in an NOD-severe-combined-immunodeficient (NOD. scid) mouse reconstituted at 8 weeks old with the splenocytes or comprising an active component functionally related thereto.

In one embodiment, the invention provides a pharmaceutical composition or immunoregulator where the active component is capable of inhibiting gamma-interferon production or stimulating interleukin-4 production of splenocytes obtained from a 20-week-old female non-obese diabetic (NOD) mouse. Clinical grade preparations of gonadotropins, such as hCG and PMSG, have long since been used to help treat reproductive failure in situations where follicular growth or stimulation of ovulation is desired. The preparations are generally obtained from serum or urine, and often vary in degree of purification and relative activity, depending on initial concentration in serum or urine and depending on the various methods of preparation used.

In a particular embodiment, the invention provides an immunoregulator comprising an active component obtainable from a mammalian CG preparation. The active component is capable of stimulating splenocytes obtained from a non-obese diabetic (NOD) mouse, or comprising an active component functionally related to the active compound. For example the stimulated splenocytes are capable of delaying the onset of diabetes in an NOD-severe-combined-immunodeficient mouse reconstituted with the splenocytes.

The invention also provides an immunoregulator where the active component is capable of inhibiting gamma-interferon production obtained from a non-obese diabetic (NOD) mouse. The invention also provides an immunoregulator where the active component is capable of stimulating interleukin-4 production of splenocytes obtained from a non-obese diabetic (NOD) mouse.

An immunoregulator (IR) as provided by the invention with or without hCG as, for example, present in the urine during the first trimester of pregnancy (IR-U) and in commercial hCG preparations (IR-P), has immune regulatory effects. In particular, IR can inhibit or regulate auto-immune and acute- and chronic-inflammatory diseases. TNF and IFN-gamma are pathologically involved in acute inflammatory disease such as sepsis or septic shock and also in auto-immune and chronic inflammatory diseases. Since IR has the ability to regulate T-cell sub-populations and inhibit TNF and IFN-gamma, IR can be used to treat, suppress or prevent immune mediator disorders such as sepsis or septic shock (acute inflammatory disease) as well as auto-immune disease or chronic inflammatory diseases such as systemic lupus erythematosis, diabetes, rheumatoid arthritis, post-partum thyroid dysfunction, auto-immune thrombcytopenia and others, such as allergies and chronic inflammatory disease (i.e., rheumatic disease, Sjogrens syndrome, multiple sclerosis) and transplantation-related immune responses. Our results for example show that IR inhibits sepsis or septic shock caused by endotoxin or by exotoxin. IR, as provided by the invention, inhibits or counters immune-mediated auto-immune diseases, chronic inflammatory diseases as well as acute inflammatory diseases.

Anecdotal observations and laboratory studies indicated previously that hCG might have an anti-Kaposi's sarcoma and anti-human-immunodeficiency-virus effect (Treatment Issues, July/August 1995, page 15). It has been observed that hCG preparations have a direct apoptotic (cytotoxic) effect on Kaposi's sarcoma (KS) in vitro and in immunodeficient patients and mice and a prohematopoietic effect on immunodeficient patients (Lunardi-Iskandar et al., Nature 375, 64-68; Gill et al., New. Eng. J. Med. 335, 1261-1269, 1996; U.S. Pat. No. 5,677,275), and a direct inhibitory anti-viral effect on human and simian immunodeficiency virus (HIV and SIV) (Lunardi-Iskandar et al., Nature Med. 4, 428-434, 1998, U.S. Pat. No. 5,700,781). The cytotoxic and anti-viral effects have also been attributed to an unknown hCG mediated factor (HAF) present in clinical grade preparations of hCG. However, commercial hCG preparations (such as CG-10, Steris Profasi, PREGNYL, Choragon, Serono Profasi, APL), have various effects. Analysis of several of these (AIDS, 11: 1333-1340, 1997), for example, shows that only some (such as CG-10, Steris Profasi) are KS-killing whereas others (PREGNYL, Choragon, Serono Profasi) were not. Secondly, recombinant subunits of .alpha. or .beta. hCG were killing but intact recombinant hCH were not. It was also found that the killing effect was also seen with lymphocytes. Therapy of KS has recently been directed at using beta-hCG for its anti-tumor effect (Eur. J. Med Res.21:155-158,1997), and it was reported that the beta-core fragment isolated from urine had the highest apoptotic activity on KS cells (AIDS, 11:,713-721, 1997).

Recently, Gallo et. al. reported anti-Kaposi's Sarcoma, anti-HIV, anti-SIV and distinct hematopoietic effects of clinical grade crude preparations of human chorionic gonadotropin (hCG) (Lunardi-Iskandar et al. 1995, Gill et al. 1996, Lunardi-Iskandar et al. 1998). In contrast to their previous studies, it is also claimed that the anti-tumor and anti-viral activity of hCG preparation is not due to the native hCG heterodimer, including its purified subunits or its major degradation product, the .beta.-core; instead the active moiety resides in an as yet unidentified hCG mediated factor (HAF). Whatever the true factor may be, these unidentified factors in several hCG preparations have anti-tumor activity through the selective induction of apoptosis, besides direct cytotoxic effects on the tumor cells. Furthermore, they postulated that the anti-tumor activity could not be due to an immune-mediated response, since there was no infiltration of the tumor with mononuclear cells.

Moreover, the reported pro-hematopoietic effect of clinical grade hCG was noted in clinical studies in humans infected with HIV (Lunardi-Iskandar et al. 1998), indicating that the hematopoietic effect is indirect and caused by rescuing CD4+ cells otherwise killed by HIV through the anti-HIV activity of hCG.

The invention provides an immunoregulator or a pharmaceutical composition for treating an immune-mediated disorder obtainable from an hCG preparation or a fraction derived thereof. The effects of the immunoregulator include a stimulating effect on lymphocyte populations (such as found in peripheral lymphocytes, thymocytes or splenocytes), instead of cytotoxic or anti-viral effects. The invention provides a method for treating an immune-mediated-disorder comprising subjecting an animal to treatment with at least one immunoregulator obtainable from a pregnant mammal. The treatment can be direct, for example treatment can comprise providing the individual with a pharmaceutical composition, such as an hCG or PMSG preparation, comprising an immunoregulator as provided by the invention. It is also possible to provide the pharmaceutical composition with a fraction or fractions derived from a pregnant animal by, for example, sampling urine, serum, placental (be it of maternal or foetal origin) or other tissue or cells and preparing the immunoregulator comprising the active component from the urine, serum, tissue, or cells by fractionation techniques known in the art (for example, by gel permeation chromatography) and testing for its active component by stimulating an NOD mouse or its splenocytes as described. In particular, the preparation or component is preferably derived from a pregnant animal since an embryo has to survive a potentially fatal immunological conflict with its mother: developing as an essentially foreign tissue within the womb without triggering a hostile immune attack. So, to prevent this rejection "allograft" the immunological interaction between mother and fetus has to be suppressed, either, for instance, through lack of fetal-antigen presentation to maternal lymphocytes, or through functional "suppression" of the maternal lymphocytes. If fetal antigens are presented, maternal immune responses would be biased to the less damaging, antibody-mediated T helper 2 (Th2)-type. This would suggest that pregnant women are susceptible to overwhelming infection, which is not the case. Female individuals during pregnancy maintain or even increase their resistance to infection. Moreover, while the individuals normally are more susceptible to immune diseases than male individuals, especially auto-immune diseases, during pregnancy they are more resistant to these diseases.

The invention also provides a method for in vitro stimulation of lymphocytes and transferring the stimulated lymphocytes as a pharmaceutical composition to an animal for treating the animal for an immune-mediated disorder. In a particular embodiment of the invention, a pharmaceutical composition is provided comprising lymphocytes stimulated in vitro with an immunoregulator provided by the invention.

In a preferred embodiment of the invention, the disorder comprises diabetes, yet other immune mediated disorders, such as acute and chronic inflammation, can also be treated. In yet another preferred embodiment, the disorder comprises sepsis or septic shock. The invention provides a method of treatment for an animal, preferably where the animal is human.

In a particular embodiment, a method provided by the invention further comprises regulating relative ratios and/or cytokine activity or cytokine expression or marker expression of lymphocyte subset-populations in the animal, such as subset-populations that comprise Th1 or Th2 cells, or Th3 or Th8 cells, or other effector or regulatory T-cell populations.

The invention also provides an immunoregulator for use in a method according to the invention, and use of the immunoregulator, preferably obtainable from a pregnant mammal, for the production of a pharmaceutical composition for the treatment of an immune-mediated-disorder, preferably selected from a group consisting of allergies, auto-immune disease (such as systemic lupus erythematosis or rheumatoid arthritis), transplantation-related disease and acute (such as septic or anaphylactic shock or acute or hyper-acute transplant rejection) and chronic inflammatory disease (such as atherosclerose, diabetes, multiple sclerosis or chronic transplant rejection). Furthermore, the invention provides a use according to the invention where the immune-mediated disorder comprises allergy, such as asthma or parasitic disease, or use according to the invention wherein said immune-mediated disorder comprises an overly strong immune response directed against an infectious agent, such as a virus or bacterium. Often, in most of these diseases, production of autoreactive antibodies and/or autoreactive T-lymphocytes can be found mounting or being part of a too strong immune response. This is, for example, seen with parasitic disease, where IgE production is overly strong or which disease is Th2 dependent, and detrimental for the organism, but also with myco bacterial infections such as TBC or leprosy. An auto-immune response may also occur as manifestation of viral or bacterial infection and may result in severe tissue damage, for example, destructive hepatitis because of Hepatitis B virus infection, or as seen with lymphocytic choriomeningitis virus (LCMV) infections. The overly strong immune response is kept at bay with an immunoregulator as provided by the invention. Yet another use as provided by the invention relates to treatment of vascular disease, whereby radical damage (damage caused by radicals) to cells and tissue is prevented or repaired by treatment with IR according to the invention and whereby IR also acts as an anti-oxidant directly or indirectly. For example, a determining event in the pathogenesis of diabetes I is the destruction of insulin-producing pancreatic beta cells. There is strong evidence that the progressive reduction of the beta-cell mass is the result of a chronic auto-immune reaction. During this process, islet-infiltrating immune cells, islet capillary endothelial cells and the beta cell itself are able to release cytotoxic mediators. Cytokines and, in particular, nitric oxide (NO), are potent beta-cell toxic effector molecules. The reactive radical NO mediates its deleterious effect mainly through the induction of widespread DNA strand breaks and other radicals, such as oxygen, through their effects on lymphocyte sub-populations such as Th1 and Th2 cells. This initial damage triggers a chain of events terminating in the death of the beta cell and disarray of the immune response.

Furthermore, an immunoregulator according to the invention is capable of regulating radical induced or directed cell-cell interactions or cell responses, specifically those interactions or responses of an immunological nature, e.g., related to regulating interactions of the innate or adaptive immune system. Not wishing to be bound by theory, there are two arms of the immune system: the innate (nonspecific) and adaptive (specific) systems, both of which have cellular and humoral components. Examples of cellular components of the innate immune system are monocytes, macrophages, granulocytes, NK cells, mast-cells, gd T-cell etc., while, examples of humoral components are lysozyme, complement, acute phase proteins and mannose-binding lectin (MBL). The major cellular components of the adaptive immune system are T- and B-cells, while examples of humoral components are antibodies. The adaptive system has been studied most because of its specificity, effectiveness at eliminating infection and exclusive presence in higher multicellular organisms. The innate system is often considered primitive and thought to be "unsophisticated." However, the innate system not only persists but could also play a critical role in one of the most fundamental immune challenges--viviparity. The innate system instigates an immune response by processing and presenting antigen in association with major histocompatibility complex (MHC) class I and II molecules to lymphocytes. Full response often requires adjuvant (such as endotoxin) which, through interaction with the innate immune system, produce co-stimulatory surface molecules or cytokines. This determines the biological significance of antigens and communicates this information to the adaptive system, so it instructs the adaptive system to either respond or not. These two great arms of the immune system not only influence each other but also regulate each other, at least at the cellular level, through, for example, cytokines and co-stimulatory molecules, etc.

There are many physiological conditions and immune pathologies where these two systems are involved separately or in combination. For example, it has been shown that in pregnancy the maternal innate immune system is more stimulated. It has also been proposed that type II diabetes mellitus is a disease of a chronic hyperactive innate immune system. Another example is the involvement of the innate immune system in listeriosis. Dysregulation in the adaptive immune system may also lead to immune diseases like systemic or organ-specific auto-immunity, allergy, asthma, etc., but it can also play a role in the maintenance of pregnancy and in the prevention of "allograft" rejection.

As mentioned above, the adaptive system has been studied most because of its specificity, effectiveness at eliminating infection, and exclusive presence in higher multicellular organisms. Its regulation has also been studied most. For example, it is well known that the cytokine micro-environment plays a key role in T helper cell differentiation toward the Th1 or Th2 cell type during immune responses. IL-12 induces Th1 differentiation, whereas IL-4 drives Th2 differentiation. Recently, it has also been shown that subsets of dendritic cells (DC1, DC2) provide different cytokine micro-environments that determine the differentiation of either Th1 or Th2 cells. In addition, negative feedback loops from mature T helper cell responses also regulate the survival of the appropriate dendritic cell subset and thereby selectively inhibit prolonged Th1 or Th2 responses. Moreover, development of Th1 responses can be antagonized directly by IL-4 and indirectly by IL-10, which inhibits the production of IL-12 and interferon-g-inducing factor (IGIF) by macrophages stimulated by the innate immune response. Th2 cells dependent on IL-4 to proliferate and differentiate have been implicated in allergic and atopic manifestations, and in addition through their production of IL-4 and IL-10, have been suggested to play a role in tolerance. Specifically, it has been suggested that Th1 to Th2 switch may prevent the development of organ-specific auto-immune pathologies and required for the maintenance of pregnancy. Recently, it has become clear that distinct subsets of regulatory T-cells are responsible for regulating both Th1 and Th2 responses and prevent the development of immune pathologies. One of the common features of many of these regulatory T-cells is that their function is, at least in part, due to the action of TGF-beta; this would be in keeping with the ability of TGF-beta to inhibit both Th1 and Th2 development while IL-10 could preferentially inhibit Th1 alone.

The selective outgrowth of Th1 vs. Th2 type cells is dependent on the interaction of precursor Th cells with antigen-presenting cells (APC) carrying the relevant peptide in conjunction with their MHC class II molecules. Cytokines released by the APC and present during the initial interaction between dendritic cells and the pertinent T-cell receptor carrying T-cells drive the differentiation into TH1 vs. Th2 subsets. Recently, two different precursors for DC (myeloid vs. lymphoid) have been described in man. Selective development of DC1 from myeloid precursors occurs after stimulation with CD40 Ligand or endotoxin, and results in high production of IL-12. Lymphoid precursors give rise to DC2 cells after CD40 Ligand stimulation and produce IL-1, TL-6 and IL-10. These cytokines are of prime importance in driving the development of the activated Th cell: IL-4 is required for the outgrowth of Th2 type cells which can be greatly enhanced by the presence of IL-10, while selective differentiation to Th1 type cells is exclusively dependent on the presence of IL-12. Since DC1 are characterized by the production of IL-12, they will primarily induce outgrowth of Th1 type cells, while DC2 produce IL-10 and selectively promote Th2 development in the presence of exogenous IL-4.

In a particular embodiment, the immunoregulator comprises a clinical grade hCG or PMSG preparation or a fraction derived thereof. For example, the invention provides use of an hCG preparation, or a preparation functionally equivalent thereto, for the preparation of a pharmaceutical composition for the treatment of diabetes. In yet another example, the invention provides use of an hCG preparation, or a preparation functionally equivalent thereto, for the preparation of a pharmaceutical composition for the treatment or prevention of sepsis or septic shock. For example, the invention provides a use according to the invention where the treatment comprises regulating relative ratios and/or cytokine activity of lymphocyte subset-populations, for example Th1 and/or Th2 cells in a treated individual.

The invention furthermore provides a method for selecting an immunoregulator comprising determining therapeutic effect of a candidate immunoregulator fraction. By way of example, such a method is given wherein by subjecting an animal prone to show signs of diabetes, such as an NOD mouse useful as an experimental model, to a urine fraction or fraction derived thereof, and subsequently determining the development of diabetes in the animal, one such an immunoregulator fraction or active component therein is selected or identified. In yet another embodiment, the invention provides a method for selecting an immunoregulator comprising determining therapeutic effect of an immunoregulator by subjecting an animal prone to show signs of septic shock, such as a mouse experiencing an effect of LPS or other toxin, to a urine fraction or fraction derived thereof determining the development of septic shock in the animal. A method according to the invention is preferred wherein the therapeutic effect is further measured by determining relative ratios and/or cytokine activity of lymphocyte subset-populations in the animal, or wherein the therapeutic effect is further measured by determining enzyme levels in the animal, or by measuring other clinical parameters known in the art, as shown in the detailed description herein.

Not wishing to be bound by theory, our results show that IR as provided by the invention is able to regulate the Th1/Th2 balance in vivo (BALB/c, NOD) and in vitro. In dominant Th1 phenotype models like NOD, IR (like IR-P and its fractions, amongst others, down-regulates the IFN-gamma production (in vivo/in vitro) and promote the IL-10 and TGF-beta production, in contrast to IL-4 production, which indicates the induction of regulatory cells like Th3 and Tr1 by IR. These regulatory cells may play a role in the therapeutic effects of IR in immune and inflammatory diseases and immune tolerance. We have also shown that IR and its fractions are able to inhibit the production of IFN-gamma in vitro and in vivo except for the fraction IR-P3 and rhCG that separately show no to moderate inhibition of the IFN-gamma production. The combination of IR-P3 and rhCG gives a stronger inhibition of the IFN-gamma. This implies the need of IR-P3 for rhCG for its at least its IFN-gamma inhibition in these models. This implies also to the anti-CD3 stimulated spleen cells obtained from in vivo treated NOD mice and also to polarization of T-helper cells to Th2 phenotype.

Moreover, IR-P, its fractions (IR-P1, IR-P2, IR-P3) and IR-P3 in combination with rhCG are all able to inhibit the class switch of B-cells to IgG2a, while IR-P2 and rhCG give nothing to moderate inhibition. Our results on IFN-gamma production and proliferation showed that IR-P3 alone +did not have the maximum effect as compared to IR-P, whereas, for IgG2a inhibition, we see that IR-P3 does not need rhCG to give the maximum results. However, the increase in production of IL-10 under the influence of IR-P3 is less than for IR-P1. This suggests that for maximum production of IL-10, hCG, a breakdown product thereof, or a yet unknown sub-fraction of IR-P1 in combination with IR-P3, is needed. Because IR-P3 alone is already able to promote IL-10 production, it does not need any other fraction or component to inhibit the production of IgG2a.

We have also shown that IR as provided by the invention is able to inhibit the IFN-gamma production and the promotion of IL-10, TGF-beta, IL-4 and IL-6 in the BALB/c animal model (in vitro as well as ex vivo). So it is clear that at least these cytokines are involved in the regulation of immune responses by IR and in the induction of regulatory cells. Remarkably, IR promotes the proliferation of anti-CD3 stimulated spleen cells (ex vivo) in BALB/c mice in contrast to NOD. This might reflect the difference in NOD which is an auto-immune disease model and BALB/c which is an animal model without distinct immunopathology. In both animal models (NOD/BALB/c) IR promotes LPS stimulated proliferation of spleens (in vitro and ex vivo).

Our DC experiments with NOD and BALB/c mice show that IR does not just regulate T-cell responses, but can also regulate DC maturation and function. DC that functions as professional antigen processing cells (APC) can play an important role in immune tolerance. Treatment of C57B/6 DC with IR in allo-MLR is able to down-regulate T-cell proliferation. This shows that IR can also facilitate the induction of a state of tolerance. On the basis of these data we performed MHC and non-MHC incompatible skin (C57BL/6) transplantation to recipients (BALB/c) treated with IR. Our data showed that in the control group the allograft (skin) was completely rejected within 15 days, while the skin graft of recipient mice treated with IR three times was rejected after 21 days. Accordingly, IR is able to delay graft rejection. IR, as provided by the invention, is able to inhibit the immunopathology in numerous animal models for immune diseases. IR inhibits the immunopathology and clinical symptoms in the NOD model (for diabetes), and the EAE model (for MS), inhibits allograft rejection, and delays SZT-induced diabetes. Our data also shows that IR has effects on different cell populations. IR affects T-cells and thereby regulates Th1/Th2 balance and induce regulatory cells that in turn not only just regulate T-cells but also have effects on the APC compartment. In addition, IR can regulate the APC compartment directly and can influence the innate and adaptive immune responses. By doing so, IR not only can influence diseases caused by disbalance of the adaptive immune system, but can also influence the diseases due ti the disbalance of the innate immune system or of both systems. For example, the role of cytokines and the innate immune system in the etiology of Type II diabetes is likely important. Recently it is has been suggested that unknown factors, like age and overnutrition, in genetically or otherwise predisposed subjects, cause increased secretion of cytokines from cells such as macrophages and further cytokine secretion from atherosclerotic plaques. The acute-phase response induced by cytokines includes a characteristic dyslipdemia (raised VLDL triglyceride and lowered HDL cholesterol) and other risk factors for atherosclerosis, such as fibrinogen. Cytokines also act on the pancreatic beta cell (contributing to impaired insulin secretion), on adipose tissue (stimulating leptin release) and on the brain, stimulating corticotropin-releasing hormone, ACTH and thus cortisol secretion. The latter may contribute to central obesity, hypertension and insulin resistance. A further cause of insulin resistance is the cytokine TNF-alpha, which inhibits the tyrosine kinase activity of the insulin receptor. Type II diabetic patients without microvascular or macrovascular complications have a high acute-phase response but tissue complications further increase stress reactants in Type II diabetes. In non-diabetic subjects with atherosclerosis, a "hematological stress syndrome" has been recognized for many years, consisting of high acute-phase reactants such as fibrinogen, increased blood viscosity and increased platelet number and activity. Cytokines produced by endothelium, smooth muscle cells and macrophages of the atherosclerotic plaque could contribute to this acute-phase response seen in atherosclerosis. Apart from the acute-phase proteins which are established or putative risk factors for cardiovascular disease such as fibrinogen, serum amyloid A, PAI-1, Lp(a) lipoprotein and VLDL triglyceride, pro-inflammatory cytokines produced at the sites of diabetic complications or by the diabetic process itself may also exacerbate atherosclerosis by acting on the endothelium, smooth muscle cells and macrophages. Thus, there is likely positive feedback involving cytokines and atherosclerosis, perhaps accounting for the acceleration of arterial disease in diabetes. The plaque produces cytokines, which further exacerbate the process of atherosclerosis locally but also cause an increase in circulating acute-phase proteins, many of which are themselves risk factors for atherosclerosis.

In summary, cytokines and the innate immune system play a central role in the pathophysiology of Type II diabetes and atherosclerosis. Since IR has the ability to regulate such a response, it is also beneficial to type II diabetes and atherosclerosis and its complications. In addition, IR can delay the induction of disease such as diabetes in the HD-STZ model where reactive oxygen species (ROS) play an important role, so IR can also act as an anti-oxidant directly or indirectly. Also for that reason, it is beneficial in the treatment and prevention of diabetes and related diseases. Furthermore, the invention provides an immunoregulator selected by a method according to the invention, a pharmaceutical composition comprising such a selected immunoregulator, and the use of it for the preparation of a pharmaceutical composition for the treatment of an immune-mediated disorder.

Fractions containing bioactive IR are purified to homogeneity by liquid chromatography. The direct analysis by mass spectrometry combined with database screening, using MALDI-TOF (matrix assisted laser mass desorption/ionization-time of flight), permits the characterization of IR or fractions thereof in multimolecular complexes. Nuclear magnetic resonance spectroscopy provides information on the types of bonding to the hydrogen atoms in the IR and the molecular structure of the IR. Infrared and near-ultraviolet spectroscopy aids in structural determination of the IR. MALDI-TOF and NMR analysis complements separation, if needed, and subsequent sequencing and synthesis of the bioactive IR. Chemical mutagenesis is employed to mutate the chemical composition of IR, permitting fine mapping of the interaction site with the receptor/acceptor by performing qualitative and quantitative binding analysis in appropriate detection systems like a biosensor system.

Derivatives of IR by chemical and genetic modification are again tested for bioactivity in the above methods or assays demonstrating activity of IR or IR containing mixtures. Furthermore, the present invention provides verification of the presence of a receptor of IR. Various fractions of pregnancy urine, commercial hCG preparations or fragments thereof, and recombinant hCG or fragments thereof are spiked with known amounts of IR. The mixtures are analyzed by gel permeation chromatography and compared to the mentioned samples without spiked IR and free IR. Shifts in IR peak(s) to higher molecular weight fractions indicates the presence of a receptor/acceptor. Analyzing the fractions for IR activity (after IR has been displaced from the receptor/acceptor) validates this elution profile containing the shifted IR peaks. From the fraction containing the shifted IR activity, the receptor/acceptor is purified by liquid chromatography and validated for IR function by displacement. The IR is, in addition, iodinated and spiked to fractions of first trimester pregnancy urine, commercial hCG preparations or fragments thereof, and recombinant hCG or fragments thereof and the mixtures are evaluated in appropriate detection systems like SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) under reducing and nonreducing conditions. Blots of such gels are analyzed by systems like quantitative phosphorimaging analysis using STORM technology. IR is immobilized, to e.g., AFFIGEL by the use of a chemical linker or carrier protein permitting the isolation of binding moieties by means of affinity chromatography. Subsequent elution provides purified receptor/acceptor molecules. The receptor/acceptor isolated from extracellular and intracellular sources in soluble or in membrane-bound form are immobilized to an activated biosensor surface. The IR in various concentrations will then probe this sensor surface and from the resulting binding profiles, the association rate and dissociation rate constants are determined and the affinity constant is calculated. By probing with different mixtures of IR and receptors/acceptors, epitope mapping is evaluated to obtain information on the nature of binding epitope. IR is labeled (e.g., fluorescent and radioactively) to permit detection of IR receptors in membrane-bound form to assess cellular expression and tissue distribution under non-diseased states and during the various immune and related disorders pertinent to the activity of IR. Using labeled IR and having available purified receptor, monoclonal antibodies and other specific reagents are generated allowing the design of a quantitative immuno-assay for the measurement of soluble IR receptors. Recombinant DNA technology is used to generate IR producing prokaryotic and eukaryotic expression systems. Site-directed mutagenesis is used to produce IR variants with altered binding profiles permitting the fine identification of the interaction site with the receptor/acceptor. Upon the cloning of the gene, transgenic mice with constitutive and inducible expression of the IR, as well as IR gene-deficient mice, are generated permitting the entry into the field of biotechnology and gene therapy.

Purified IR is used to produce monoclonal antibodies and/or other specific reagents, thereby facilitating the design of an IR-specific quantitative immuno-assay. Also, single chain F.sub.v fragments are isolated by using the phage display technology with the use of a phage library containing a repertoire comprising a vast number of different specificities.
 

Claim 1 of 3 Claims

1. A method for selecting an agent with a therapeutic effect against septic shock, said method comprising: providing at least a first and a second test animals of the same species prone to show signs of septic shock; administering to the first test animal a urine fraction obtained from a pregnant female mammal of the same or different species that elutes with an apparent molecular weight of 1 to 15 kilodaltons as determined by gel-permeation chromatography and administering to the second animal a control; inducing septic shock in the animals; and determining the development of septic shock in the animals, selecting a fraction as an agent with a therapeutic effect against septic shock if the first test animal shows a reduction of the development of septic shock in the first animal as compared to the second animal.
 

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