AU2005205750B2 - Immunoregulator - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Immunoregulator The following statement is a full description of this invention, including the best method of performing it known to us: 004691676vl.doc Title: Immunoregulator.

FIELD OF THE INVENTION The invention relates to the field of immunology, more specifically to the field of immune-mediated disorders such as allergies, auto-immune disease, transplantation-related disease or inflammatory disease.

BACKGROUND OF THE INVENTION The immune system produces cytokines and other humoral factors to protect the host when threatened by inflammatory agents, microbial invasion, or injury. In most cases this complex defence network successfully restores normal homeostasis, but at other times the immunological mediators may actually prove deleterious to the host. Some examples of immune disease and immune system-mediated injury have been extensively investigated including anaphylactic shock, autoimmune disease, and immune complex disorders.

Recent advances in humoral and cellular immunology, molecular biology and pathology have influenced current thinking about auto-immunity being a component of immunemediated disease. These advances have increased our understanding of the basic aspects of antibody, B-cell, and T-cell diversity, the generation of innate (effected by monocytes, macrophages, granulocytes, natural killer cells, mast cells, y6 T cells, complement, acute phase proteins, and such) and adaptive (T and B cells and antibodies) or cellular and humoral immune responses and their interdependence, the mechanisms of (self)-tolerance induction and the means by which immunological reactivity develops against auto-antigenic constituents.

Since 1900, the central dogma of immunology has been that the immune system does not normally react to self.

However, it as recently become apparent that auto-immune responses are not as rare as once thought and that not all auto-immune responses are harmful; some responses play a distinct role in mediating the immune response in general. For example, certain forms of auto-immune response such as recognition of cell surface antigens encoded by the major histocompatibility complex (MHC) and of anti-idiotypic responses against self idiotypes are important, indeed essential, for the diversification and normal functioning of the intact immune system.

Apparently, an intricate system of checks and balances is maintained between various subsets of cells T-cells) of the immune system, thereby providing the individual with an immune system capable of coping with foreign invaders. In that sense, auto-immunity plays a regulating role in the immune system.

However, it is now also recognised that an abnormal auto-immune response is sometimes a primary cause and at other times a secondary contributor to many human and animal diseases. Types of auto-immune disease frequently overlap, and more than one auto-immune disorder tends to occur in the same individual, especially in those with auto-immune endocrinopathies. Auto-immune syndromes may be mediated with lymphoid hyperplasia, malignant lymphocytic or plasma cell proliferation and immunodeficiency disorders such as hypogammaglobulinaemie, selective Ig deficiencies and complement component deficiencies.

Auto-immune diseases, such as systemic lupus erythematosus, diabetes, rheumatoid arthritis, postpartum thyroid dysfunction, auto-immune thromocytopenia, to name a few, are characterised by auto-immune responses, for example directed against widely distributed self-antigenic determinants, or directed against organ- or tissue specific antigens. Such disease may follow abnormal immune responses against only one antigenic target, or against many self antigens. In many instances, it is not clear whether auto-immune responses are directed against unmodified self-antigens or selfantigens that have been modified (or resemble) any of numerous agents such as viruses, bacterial antigens and haptenic groups.

There is as yet no established unifying concept to explain the origin and pathogenesis of the various autoimmune disorders. Studies in experimental animals support the notion that auto-immune diseases may result from a wide spectrum of genetic and immunological abnormalities which differ from one individual to another and may express themselves early or late in life depending on the presence or absence of many superimposed exogenous (viruses, bacteria) or endogenous (hormones, cytokines, abnormal genes) accelerating factors.

It is evident that similar checks and balances that keep primary auto-immune disease at bay are also compromised in immune mediated disorders, such as allergy (asthma), acute inflammatory disease such as sepsis or septic shock, chronic inflammatory disease (i.e rheumatic disease, Sj6grens syndrome, multiple sclerosis), transplantation-related immune responses (graft-versushost-disease, post-transfusion thrombocytopenia), and many others wherein the responsible antigens (at least initially) may not be self-antigens but wherein the immune response to said antigen is in principle not wanted and detrimental to the individual. Sepsis is a syndrome in which immune mediators, induced by for example microbial invasion, injury or through other factors, induce an acute state of inflammation which leads to abnormal homeostasis, organ damage and eventually to lethal shock. Sepsis refers to a systemic response to serious infection. Patients with sepsis usually manifest fever, tachycardia, tachypnea, leukocytosis, and a localised site of infection.

Microbiologic cultures from blood or the infection site are frequently, though not invariably, positive. When this syndrome results in hypotension or multiple organ system failure (MOSF), the condition is called sepsis or septic shock. Initially, micro-organisms proliferate at a nidus of infection. The organisms may invade the bloodstream, resulting in positive blood cultures, or might grow locally and release a variety of substances into the bloodstream. Such substances, when of pathogenic nature are grouped into two basic categories: endotoxins and exotoxins. Endotoxins typically consist of structural components of the micro-organisms, such as teichoic acid antigens from staphylococci or endotoxins from gramnegative organisms tlike LPS). Exotoxins toxic shock syndrome toxin-l, or staphlococcal enterotoxin A, B or C) are synthesised and directly released by the microorganisms.

As suggested by their name, both of these types of bacterial toxins have pathogenic effects, stimulating the release of a large number of endogenous host-derived immunological mediators from plasma protein precursors or cells (monocytes/macrophages, endothelial cells, neutrophils, T cells, and others) It is in fact generally these immunological mediators which cause the tissue and organ damage associated with sepsis or septic shock. Some of these effects stem from direct mediator-induced injury to organs. However, a portion of shock-associated-organ dysfunction is probably due to mediator-induced abnormalities in vasculature, resulting in abnormalities of systemic and regional blood flow, causing refractory hypotension or MOSF (Bennett et al.).

The non-obese diabetic (NOD) mouse is a model for auto-immune disease, in this case insulin-dependent diabetes mellitus (IDDM) which main clinical feature is elevated blood glucose levels (hyperglycemia). Said elevated blood glucose level is caused by auto-immune destruction of insulin-producing g cells in the islets of Langerhans of the pancreas (Bach et al. 1991, Atkinson et al. 1994). This is accompanied by a massive cellular infiltration surrounding and penetrating the islets (insulitis) composed of a heterogeneous mixture of CD4+ and CD8+ T lymphocytes, B lymphocytes, macrophages and dendritic cells (O'Reilly et al. 1991) The NOD mouse represents a model in which autoimmunity against beta-cells is the primary event in the development of IDDM. Diabetogenesis is mediated through a multifactorial interaction between a unique MHC class II gene and multiple, unlinked, genetic loci, as in the human disease. Moreover, the NOD mouse demonstrates beautifully the critical interaction between heredity and environment, and between primary and secondary autoimmunity, its clinical manifestation is for example depending on various external conditions, most importantly of the micro-organism load of the environment in which the NOD mouse is housed.

As for auto-immunity demonstrable in NOD mice, most antigen-specific antibodies and T-cell responses are measured after these antigens were detected as selfantigens in diabetic patients. Understanding the role these auto-antigens play in NOD diabetes may further allow to distinguish between pathogenic auto-antigens and auto-immunity that is an epiphenomenon.

In general, T lymphocytes play a pivotal role in initiating the immune mediated disease process (Sempe et al. 1991, Miyazaki et al. 1985, Harada et al. 1986, Makino et al. 1986). CD4+ T-cells can be separated into at least two major subsets Thl and Th2. Activated Thl cells secrete IFN-y and TNF-a, while Th2 cells produce IL- 4, IL-5 and IL-10. Thl cells are critically involved in the generation of effective cellular immunity, whereas Th2 cells are instrumental in the generation of humoral and mucosal immunity and allergy, including the activation of eosinophils and mast cells and the production of IgE (Abbas et al. 1996). A number of studies have now correlated diabetes in mice and human with Thl phenotype development (Liblau et al. 1995, Katz et al. 1995). On the other hand, Th2 T cells are shown to be relatively innocuous. Some have even speculated that Th2 T cells in fact, may be protective. Katz et al. have shown that the ability of CD4+ T cells to transfer diabetes to naive recipients resided not with the antigen specificity recognised by the TCR per se, but with the phenotypic nature of the T cell response. Strongly polarised Thl T cells transferred disease into NOD neonatal mice, while Th2 T cells did not, despite being activated and bearing the same TCR as the diabetogenic Thl T cell population. Moreover, upon co-transfer, Th2 T cells could not ameliorate the Thl-induced diabetes, even when Th2 cells were co-transferred in 10-fold excess (Pakala et al. 1997).

The incidence of sepsis or septic shock has been increasing since the 1930's, and all recent evidence suggests that this rise will continue. The reasons for this increasing incidence are many: increased use of invasive devices such as intravascular catheters, widespread use of cytotoxic and immunosuppressive drug therapies for cancer and transplantation, increased longevity of patients with cancer and diabetes who are prone to develop sepsis, and an increase in infections due to antibiotic-resistant organisms. Sepsis or septic shock is the most common cause of death in intensive care units, and it is the thirteenth most common cause of death in the United States. The precise incidence of the disease is not known because it is not reportable; however, a reasonable annual estimate for the United States is 400,000 bouts of sepsis, 200,000 cases of septic shock, and 100,000 deaths from this disease.

Various micro-organisms, such as Gram-negative and Gram-positive bacteria, as well as fungi, can cause sepsis and septic shock. Certain viruses and rickettsiae probably can produce a similar syndrome. Compared with Gram-positive organisms, Gram-negative bacteria are somewhat more likely to produce sepsis or septic shock.

Any site of infection can result in sepsis or septic shock. Frequent causes of sepsis are pyelonephritis, pneumonia, peritonitis, cholangitis, cellulitis, or meningitis. Many of these infections are nosocomial, occurring in patients hospitalised for other medical problems. In patients with normal host defences, a site of infection is identified in most patients. However, in neutropenic patients, a clinical infection site is found in less than half of septic patients, probably because small, clinically inapparent infectious in skin or bowel can lead to bloodstream invasion in the absence of adequate circulating neutrophils. Clearly there is a need to protect against sepsis or septic shock in patients running such risks.

Recently, considerable effort has been directed toward identifying septic patients early in their clinical course, when therapies are most likely to be effective. Definitions have incorporated manifestations of the systemic response to infection (fever, tachycardia, tachypnea, and leukocytosis) along with evidence of organ system dysfunction (cardiovascular, respiratory, renal, hepatic, central nervous system, hematologic, or metabolic abnormalities). The most recent definitions use the term systemic inflammatory response syndrome (SIRS) emphasising that sepsis is one example of the body's immunologically-mediated inflammatory responses that can be triggered not only by infections but also by noninfectious disorders, such as trauma and pancreatitis (for interrelationships among systemic inflammatory response (SIRS), sepsis, and infection, see Crit. Care Med. 20:864, 1992; For a review of pathogenic sequences of the events in sepsis or septic shock see N Engl J Med 328:1471, 1993).

Toxic shock syndrome toxin (TSST-1) represents the most clinically relevant exotoxin, identified as being the causative agent in over 90% of toxic shock syndrome cases (where toxic shock is defined as sepsis or septic shock caused by super-antigenic exotoxins). Super antigens differ from "regular" antigens in that they require no cellular processing before being displayed on a MHC molecule. Instead they bind to a semi-conserved region on the exterior of the TCR and cause false "recognition" of self antigens displayed on MHC class II (Perkins et al.; Huber et al. 1993). This results in "false" activation of both the T cell and APC leading to proliferation, activation of effector functions and cytokine secretion. Due to the superantigen's polyclonal activation of T cells, a systemic wide shock results due to excessive inflammatory cytokine release.

(Huber et al. 1993, Miethke et al. 1992).

The inflammatory cytokines involved in sepsis are similar. These immunological mediators are tumor necrosis factor (TNF), interferon gamma (IFN-gamma), nitric oxide (Nox) and interleukin l(IL-1), which are massively released by monocytes, macrophages and other leukocytes in response to bacterial toxins (Bennett et al., Gutierrez-Ramos et al 1997). The release of TNF and other endogenous mediators may lead to several pathophysiological reactions in sepsis, such as fever, leukopenia, thrombocytopenia, hemodynamic changes, disseminated intravascular coagulation, as well as leukocyte infiltration and inflammation in various organs, all of which may ultimately lead to death. TNF also causes endothelial cells to express adhesion receptors (selectins) and can activate neutrophils to express ligands for these receptors which help neutrophils to adhere with endothelial cell surface for adherence, margination, and migration into tissue inflammatory foci (Bennett et Blocking the adhesion process with monoclonal antibodies prevents tissue injury and improves survival in certain animal models of sepsis or septic shock (Bennett et al.).

These findings, both with auto-immune disease, as well as with acute and chronic inflammatory disease, underwrite the postulated existence of cells regulating the balance between activated Th-sub-populations.

Possible disturbances in this balance that are induced by altered reactivity of such regulatory T cell populations can cause immune-mediated diseases, which results in absence or over-production of certain critically important cytokines (O'Garra et al. 1997). These Th-subpopulations are potential targets for pharmacological regulation of immune responses.

In general, immune mediated disorders are difficult to treat. Often, broad-acting medication is applied, such as treatment with corticosteroids or any other broad acting anti-inflammatory agent that in many aspects may be detrimental to a treated individual.

In general there is a need for better and more specific possibilities to regulate the checks and balances of the immune system and treat immune mediated disorders.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment, or any form of suggestion, that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

SUMMARY OF THE INVENTION The invention provides among others an immunoregulator (NMPF) obtainable or derivable from a urinary metabolite of hCG, in particular from nicked forms of bhCG, or (synthetic) peptide homologues or analogues thereof. These forms of b-hCG have peptide bond cleavages within the b-subunit (Birken et al, Endocrinology 133:1390-1397, 1993). Surprisingly, it has been found that a range of beta-HCG breakdown products provides a cascade of immunoregulators (NPMF) with a host of functions. Even more surprisingly, said immunoregulators are interrelated and derived from one another. The invention provides use of such an NMPF in preparing a pharmaceutical composition for treating an immunemediated disorder, a pharmaceutical composition and a method for treating an immune-mediated disorder. Immunemediated disorders as described herein include chronic inflammatory disease, such as diabetes type I or II, rheumatic disease, Sj6grens syndrome, multiple sclerosis), transplantation-related immune responses such as graft-versus-host-disease, post-transfusion thrombocytopenia, chronic transplant rejection, preeclampsia, atherosclerosis, asthma, allergy and chronic auto-immune disease, and acute inflammatory disease, such as (hyper)acute transplant rejection, septic shock and acute autoimmune disease. Autoimmune 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 autoimmune 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.

In one embodiment the invention seeks to provide a method for modulating expression of a gene in a cell comprising providing said cell with a tri- or tetrameric peptide.

In another embodiment the invention seeks to provide a method for identifying or obtaining a signalling molecule comprising a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprising providing said cell with a tri- or tetrameric peptide and determining the activity and/or nuclear translocation of a gene transcription factor.

In yet another embodiment there is a method for identifying or obtaining a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprising providing said cell with a tri- or tetrameric peptide or derivative or analogue thereof and determining relative up-regulation and/or down-regulation of at least one gene expressed in said cell. In a further embodiment there is a method for identifying or obtaining a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprising providing a tri- or tetrameric peptide or derivative or analogue thereof and determining binding of said peptide or derivative or analogue thereof to a factor related to gene control. The invention also provides tri-or tetrameric peptides useful in modulating expression of a gene in a cell and identifiable or obtainable by employing a method of the invention. Preferably, the invention provides use of a tri-or tetrameric peptide capable of modulating expression of a gene in a cell, in a pharmaceutical composition.

DETAILED DESCRIPTION OF THE INVENTION As used herein, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude other additives, components, integers or steps.

Autoimmune diseases can be classified as organ specific or non-organ specific depending on whether the response is primarily against antigens localised in particular organs, or against wide-spread antigens. The current mainstay of treatment of autoimmune 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 autoimmune disease and other inflammations. Based on the immunoregulatory properties, e.g. the capacities to regulate the Thl/Th2 ratio, to modulate dendritic cell differentiation, their low side-effect profile, and the beneficial clinical effects, etc., it shows these urinary metabolite preparations or synthetic analogues thereof to be very helpful in the treatment of patients with immune-mediated inflammation, such autoimmune disease.

A non-limiting list of an immune diseases includes: Hashimoto's thyroditis, primary mysxoedema thyrotoxicosis, pernicious anaemia, autoimmune 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, autoimmune haemolytic anaemia, idiopathic thrombocytopenic purpura, idiopathic leucopenia, primary biliary cirrhosis, active chronic hepatitis, cryptogenic cirrhosis, ulcerative colitis, Sj6gren's syndrome, rheumatoid arthritis, dermatomyositis, polymyositis, scleroderma, mixed connective tissue disease, discoid lupus erythematosus, and systemic lupus erythematosus.

In one embodiment, the invention provides an immunoregulator capable of down-regulating Thi cell levels and/or upregulating Th2 cell levels, or influencing their relative ratio in an animal, said 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 said NMPF from said source, NMPF 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, dendritic or antigen presenting cell subsetpopulations in a diseased animal human), preferably where these lymphocyte subset-populations comprise Thl or Th2, or DC1 or DC2 populations. In general, naive CD4' helper T lymphocytes (Th) develop into functionally mature effector cells upon stimulation with relevant antigenic peptides presented on the major histocompatibility complex (MHC) class II 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: Thl cells producing exclusively interleukin-2 interferon-gamma (IFN-y) and lymphotoxin, while Th2 cells produce IL-4, IL-5, IL6, IL10 and IL-13. These Thl and Th2 subsets appear to be extremes in cytokine production profiles and within these polarized subsets, individual Th cells exhibit differential rather than co-ordinated cytokine gene expression. These subsets develop from common Th precursor cells (Thp) after triggering with relevant peptides into ThO cells producing an array of cytokines, including IL-2, IL-4, IL-5 and IFN-y. These activated ThO cells subsequently polarize into the Thl 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 Thl or Th2 subset development. The Thl-TH2 subsets appear to cross-regulate each other's cytokine production profiles, mainly through IFN-y 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 IL-12 is a dominant factor promoting Thl subset polarization and dendritic cells and macrophages produce IL-12. Moreover, IL-12 induces IFN-y production by T cells and natural killer (NK) cells.

Recently, it was reported that IL-18 acts synergistically with IL-12 to induce Thl development. Polarization of Th2 cells is critically dependent on the presence of IL-4 produced by T cells or basophils and mast cells. APCderived IL-6 has also been shown to induce small amounts of IL-4 in developing Th cells. IL-10 and APC-derived prostaglandin E 2

(PGE

2 inhibit IL-12 production and Thl priming.

The Thl-Th2 paradigm has been useful in correlating the function of Thl 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 Thl response. Thl 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 neutralisation 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 Thl 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 in to Thl 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. Lymhoid precursors give rise to DC2 cells after CD40Ligand 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 while selective differentiation to Thl 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 Thl 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 NMPF as provided by the invention is capable of regulating or modulating DC activity and differentiation, thereby allowing selective differentiation and activity of Thl and/or Th2 cells.

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

In another embodiment, the invention provides an immunoregulator comprising an active component obtainable from a mammalian chorionic gonadotropin preparation said active component 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 Thl and/or Th2 cells, in case of acute inflammation, such as seen with shock or (hyper)acute transplantation rejection wherein said active component is capable of reducing ASAT or other relevant plasma enzyme levels after or during organ failure, as commonly seen with shock.

Although said immunoregulator according to the invention is easily obtained as urinary gonadotropin metabolite or break down product from urine, for example wherein said mammalian chorionic gonadotropin preparation is derived from urine, other sources, such as serum, cells or tissues comprising gonadotropin are applicable as well. Also from said sources an immunoregulator according to the invention capable of for example regulating Thl and/or Th2 cell activity, and/or capable of modulating dendritic cell differentiation, is provided. In particular, as immunoregulator a (synthetic) peptide is provided obtainable of derivable from beta- HCG, preferably from nicked beta-HCG. Of course, such a peptide, or functional equivalent thereof is obtainable or derivable from other mammalian gonadotropins, as explained herein earlier. Said peptide is for example capable of protecting against septic shock or other immune-mediated disorders. Preferably, said peptide immunoregulator is obtained from a peptide having at least 10 amino acids such as a peptide having an amino acid sequence MTRVLQGVLPALPQVVC or functional fragment a breakdown product) or functional analogue thereof. Functional fragments herein relates to the immunoregulatory effect or activity as for example can be measured in the septic shock or NOD mouse experimental model. Fragments can be somewhat 1 or 2 amino acids) smaller or larger on one or both sides, while still providing functional activity.

The invention further provides a method for selecting an immunoregulator comprising determining therapeutic effect of an immunoregulator by subjecting an animal prone to show signs of diabetes to a peptide composition or fraction thereof, and determining the development of diabetes in said animal. Similarly, a method for selecting an immunoregulator comprising determining therapeutic effect of an immunoregulator by subjecting an animal prone to show signs of septic shock to a peptide composition or fraction thereof and determining the development of septic shock in said animal is provided herewith, the septic shock model also being a fast read-out model for the determination of anti-diabetic activity. Preferably, peptide compositions tested in a method according to the invention are obtained from a peptide having at least 10 amino acids such as a peptide having an amino acid sequence MTRVLQGVLPALPQVVC or functional fragment a breakdown product) or functional analogue thereof.

Functional fragments herein relates to the immunoregulatory effect or activity as for example can be measured in the septic shock or NOD mouse diabetes experimental model. Fragments can be somewhat 1 or 2 amino acids) smaller or larger on one or both sides.

Surprisingly, it has been found in the animal test systems as provided herein that a range of beta-HCG breakdown products provides a cascade of peptide immunoregulators with a host of functions. Even more surprisingly, said immunoregulator peptides are interrelated and derived from one another and can also be produced synthetically. The invention provides use of such an immunoregulating peptide in preparing a pharmaceutical composition for treating an immunemediated disorder, a pharmaceutical composition and a method for treating an immune-mediated disorder. A useful peptide found in a method according to the invention can be further modified or improved for one or more characteristics by peptide synthesis skills known, for example by identification of functional analogues with replacement mapping techniques, by binding-site (PEPSCAN) detection technology and so on, and can comprise D- or Lamino acids or modified amino acids at one or more (or all) places in the desired sequence. Also, peptide derivatives can be made, such as by circularization (for example by providing with (terminal) cysteines, dimerisation or multimerisation, by linkage to lysine or cystein or other side-chains that allow linkage or multimerisation, repeated, brought in tandem configuration, conjugated or otherwise linked to carriers known in the art, if only by a labile link that allows dissociation. Of course, newly developed peptide compositions or derivatives can be tested according to 'a method as provided herein.

Functional analogue herein not only relates to analogues or homologues peptides from MIF or MIF-like proteins, from LH or PMSG, or gonadotropin-like proteins, be it modified by glycosylation or modification with unidentified amino acids or non-protein amino acids, but also to synthetic peptide analogues that can be made with peptide synthesis skills known, for example by identification of functional analogues with replacement mapping techniques, PEPSCAN detection technology and so on, and can comprise D- or L-amino acids or modified amino acids at one or more (or all) places in the desired sequence. Also, peptides can be circularised (for example by providing with (terminal) cysteines, dimerised or multimerised, by linkage to lysine or cystein or other side-chains that allow linkaage or multimerisation, repeated, brought in tandem configuration, conjugated or otherwise linked to carriers known in the art, if only by a labile link that allows dissociation.

Preferably, an immunoregulator as provided by the invention is obtainable or derivable from a gonadotropin 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, or pregnant mouse uterus extract (PMUE) extracted from uteri of gravid mice or human chorionic gonadotropin (hCG or HCG) found in blood or urine of pregnant women.

An NMPF as provided by the invention can be associated with or without gonadotropin as for example present in the urine of first trimester of pregnancy (NMPF) and in commercial hCG preparations (NMPF) has immune regulatory effects.

In particular, NMPF can inhibit or regulate autoimmune 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 NMPF has the ability to regulate T-cell subpopulations and inhibit TNF and IFN-gamma, NMPF 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 erythematosus, diabetes, rheumatic disease, Sj6grens syndrome, multiple sclerosis, post-partum thyroid dysfunction and thyroid dysfunction related dementia's such as Alzheimer's disease, auto-immune thromocytopenia and others, such as allergies and chronic inflammatory disease and transplantation related immune responses.

Furthermore, the invention provides detection of genetic predisposition for immune-mediated disorders, whereby individuals with particular isoforms or amino acid variations in HCG or HCG derived peptides or immunoregulators are predisposed for certain disorders.

Once known, it is provided by the invention to provide the genetically predisposed individual with the proper peptide immunoregulator via gene therapy In particular, an immunoregulator according to the invention is provided wherein said functional fragment comprises a peptide having at least 10 amino acids such as having an amino acid sequence, LQGVLPALPQVVC (945 S48), or VLPALPQVVC (i 48) or LQGVLPALPQ (S 45), or a functional analogue thereof, herein also called NMPF-K.

Said immunoregulator comprising said peptide (or mixtures of peptides) having the desired length of about at least amino acids (and especially when bound to a larger molecule such as when bound via its cysteine to another beta-HCG fragment) generally regulates Thl/Th2 balance as well as innate immunity during an immune mediated disorder. For example septic shock, LPS induced proliferation of splenocytes or diabetes is accelerated or aggravated. Similar activity is provided by a relative short-chain peptide (third immunoregulator, 3-5 amino acids long) that comprises MTRV or MTR or QVVC or VVC or CLQG or LQGV or LQG (and especially when bound to a larger molecule such as when bound via its cysteine to another beta-HCG fragment).

More in particular, a first immunoregulator is provided comprising a functional fragment comprising an amino acid sequence VLPALPQVVC or LQGVLPALPQ or functional analogue thereof which counteracts the regulatory activities of another, second immunoregulator according to the invention wherein said functional fragment comprises an amino acid sequence of from 9 to 6 amino acids (herein also called NMPF-Kb), such as VLPALPQ or GVLPALPQ or GVLPALP or VLPALP or functional analogue thereof, which for example is capable of regulating Thl/Th2 balance as well as innate immunity during an immune mediated disorder such that it is capable to reduce the clinical symptoms seen with immune-mediated disorders, such as septic shock, LPS induced proliferation of splenocytes or diabetes, instead of accelerating or aggravating these symptoms of immunemediated disease, as for example is shown in the detailed description where NMPF-Kb is capable of protecting a mouse against a lipopolysaccharide induced septic shock, or other acute or chronic immune-mediated disorder as explained herein. As there is an overlap between (45 and 348 peptide (345; LQGVLPALPQ 348: VLPALPQWC), we also tested denaturated 345+P48 (LQGVLPALPQVVC) peptide for its effect on LPS induced proliferation (in vitro) and anti-shock activity (in vivo) in BALB/c mice. Our results showed that denaturated 345+P48 peptide inhibits LPS induced proliferation and in vivo septic shock. Breakdown products are generated via proteolysis, for example by lysis with leucocyte elastate, and can undergo further notification such as by the activity of (glutathion) transferases. One of the possible breakdown product of P45+p48 peptide is LQG which resembles glutathione (tripeptide of G, C, and Q with L-glutamate having an isopeptide bond with the amino moiety of L-cysteine). We have shown that NMPF also inhibits (toxin) streptozotocin (SZ) induced diabetes in mice through destruction of beta-cells. One of the mechanisms involved in the destruction of pancreatic beta cells is the formation of reactive radicals (ROS, NO etc.) that also play an important role in the pathogenesis of many other diseases like nephropathy, obstructive nephropathy, acute and chronic renal allograft rejection, auto-immune diseases (like SLE, rheumatoid arthritis, diabetes, MS), AIDS, diseases related to angiogenesis, atherosclerosis, thrombosis and type II diabetes mellitus. So, it is likely that NMPF also acts as 'anti-oxidant'. For example breakdown products of p45+p48 such as LQG or CLQG peptides alone or in combination with certain carbohydrates or modified with unidentified amino acids or with nonprotein amino acids such as P-alanine, y-Aminobutyric acid, Ornithine, etc. posses immunomodulatory activity (NMPF).

Not wishing to be bound by theory, NMPF-K and NMPF- Kb activity can be described as maintaining a Thl/Th2 balance, whereby NMPF-K acts as if binding to an appropriate receptor but not activating it whereas NMPF- Kb is binding to said receptor and activating it to modulate the Thl/Th2 balance in a beneficial way. NMPF-K and NMPF-Kb are therein both ligands of the same or at least a conformationally similar or alike receptor molecule. Said receptor molecule is now also provided, since it and its acitivity are defined herein by said ligands.

For example, our results show that NMPF-Kb inhibits sepsis or septic shock caused by endotoxin or by exotoxin. NMPF-Kb 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 (NMPF), for example for stimulating or regulating lymphocyte action comprising an active component said active component capable of stimulating splenocytes obtained from a female non-obese diabetes (NOD) mouse, said stimulated splenocytes delaying the onset of diabetes in a NODsevere-combined-immunodeficient (NOD.scid) mouse reconstituted at 8 weeks old with said splenocytes, or comprising an active component functionally related thereto.

In one embodiment, the invention provides an pharmaceutical composition or immunoregulator wherein said active component is capable of inhibiting gammainterferon production or stimulating interleukine-4 production of splenocytes obtained from a female non-obese diabetes (NOD) mouse. Clinical grade preparations of gonadotropins such as hCG and PMSG have since long been used to help treat reproductive failure in situations where follicular growth or stimulation of ovulation is desired. Said 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 a immunoregulator comprising an active component obtainable or derivable from a mammalian CG preparation said active component capable of stimulating splenocytes obtained from a non-obese diabetes (NOD) mouse, or comprising an active component functionally related to said active compound, for example wherein said stimulated splenocytes are capable of delaying the onset of diabetes in a NODsevere-combined-immunodeficient mouse reconstituted with said splenocytes.

The invention also provides an immunoregulator wherein said active component is capable of inhibiting gamma-interferon production obtained from a non-obese diabetes (NOD) mouse. The invention also provides an immunoregulator wherein said active component is capable of stimulating interleukine-4 production of splenocytes obtained from a non-obese diabetes (NOD) mouse.

An immunoregulator as provided by the invention (NMPF) has immune regulatory effects. In particular, NMPF 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 NMPF has the ability to regulate T-cell sub-populations and inhibit TNF and IFN-gamma, NMPF 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 erythematosus, diabetes, rheumatoid arthritis, post-partum thyroid dysfunction, auto-immune thromocytopenia and others, such as allergies and chronic inflammatory disease rheumatic disease, Sj6grens syndrome, multiple sclerosis) and transplantation related immune responses. Our results for example show that NMPF- Kb inhibit sepsis or septic shock caused by endotoxin or by exotoxin. NMPF-Kb as provided by the invention inhibits or counters immune mediated auto-immune diseases, chronic inflammatory diseases as well as acute inflammatory diseases.

The invention thus provides use of an immunoregulator according to the invention for the production of a pharmaceutical composition for the treatment of an immune-mediated-disorder, for example wherein said immune-mediated disorder comprises chronic inflammation, such as diabetes, multiple sclerosis or chronic transplant rejection, wherein said immunemediated disorder comprises acute inflammation, such as septic or anaphylactic shock or acute or hyper acute transplant rejection, wherein said immune-mediated disorder comprises auto-immune disease, such as systemic lupus erythematosus or rheumatoid arthritis, wherein said immune-mediated disorder comprises allergy, such as asthma or parasitic disease, in particular wherein said immune-mediated disorder comprises an overly strong immune response directed against an infectious agent, such as a virus or bacterium or wherein said immunemediated disorder comprises pre-eclampsia or another pregnancy related immune-mediated disorder. Use of NMPF-K as contraceptive as morning-after-pill or contraceptive vaccine eliciting contraceptive or sterilising antibodies in the vaccinated female mammal) is also provided. Use of NMPF-Kb is provided for facilitating fertility, especially in case where improved implantation is required. Especially, use is provided wherein said treatment comprises regulating innate immunity and/or relative ratios and/or cytokine activity of lymphocyte, dendritic or antigen presenting cell subset-populations in a treated individual, in particular wherein said subset populations comprise Thl or Th2, or DC1 or DC2 cells. Thus the invention provides a method for treating an immune-mediated-disorder comprising subjecting an animal to treatment with at least one immunoregulator according to the invention, in particular wherein said disorder comprises diabetes or sepsis.

The invention provides also a method for diagnosing or determining the risk of non-pregnancy related immune disorders associated with Thl/Th2 misbalance as demonstrable by a misbalance between NMPF-K and NMPF-Kb, as for example produced or derived from pituitary derived gonadotropin, especially in age-related disease such as auto-immune and chronic inflammatotory disease, such as type II diabetes, rheumatic disease, thyroid dysfunction related mental disease such as dementia's like Alzheimers and others, and atherosclerosis and related disease, said method comprising determining in a sample, preferably a blood or urine sample, the relative ratio of a relative long-chain peptide versus a relative shortchain peptide, said peptides derivable from breakdown of beta-HCG, in particular comprising determining the relative ratio of a relative long-chain peptide versus a relative short-chain peptide derived from breakdown a peptide having an amino acid sequence MTRVLQGVLPALPQVVC, for example wherein said relative long-chain peptide comprises an amino acid sequence LQGVLPALPQ or GVLPALPQ or VLPALPQ or GVLPALP or VLPALP, in particular wherein said relative short-chain peptide comprises MTRV or MTR or PALP or QVVC or VVC or LQGV or LQG. Detection of said long-chain peptides and short chain peptides, be it modified by glycosylation or modification with unidentified amino acids or non-protein amino acids is preferably achieved by immunological detection methods as known in the art.

The invention provides also a method for diagnosing or determining the risk of a pregnancy related immunemediated disorder such as pre-eclampsia, or other immunemediated disorder and the outcome of pregnancy and/or pregnancy related immune disease (such as gestation diabetes mellitus (GDM)) comprising determining in a sample, preferably a urine sample, the relative ratio of a relative long-chain peptide versus a relative shortchain peptide, said peptides derivable from breakdown of beta-HCG, in particular comprising determining the relative ratio of a relative long-chain peptide versus a relative short-chain peptide derived from breakdown a peptide having an amino acid sequence MTRVLQGVLPALPQVVC, for example wherein said relative long-chain peptide comprises an amino acid sequence LQGVLPALPQ or GVLPALPQ or VLPALPQ or GVLPALP, in particular wherein said relative short-chain peptide comprises MTRV or MTR or QVVC or VVC,or LQGV or LQG.

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 prohematopoetic effect on immunodeficient patients (Lunardi-Iskandar et al., Nature 375, 64-68; Gill et al., New. Eng. J. Med. 335, 1261-1269, 1996; US patent 5677275), and a direct inhibitory antiviral effect on human and simian immunodeficiency virus (HIV and SIV) (Lunardi-Iskandar et al., Nature Med. 4, 428-434, 1998, US patent 5700781). Said 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 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 (a or P) hCG were killing but intact recombinant hCH not. It was also found that the killing effect was also seen with lymphocytes.

Therapy of KS has recently been directed at using betahCG for its anti-tumour 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-tumour 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 1-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-tumour activity through the selective induction of apoptosis, besides direct cytotoxic effects on the tumour cells.

Furthermore, they postulated that the anti-tumour activity could not be due to an immune-mediated response, since there was no infiltration of the tumour 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 immunemediated disorder obtainable from a hCG preparation or a fraction derived thereof. The effects of said 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. Said treatment can be direct, for example treatment can comprise providing said individual with a pharmaceutical composition, such as a hCG or PMSG preparation, comprising an immunoregulator as provided by the invention. It is also possible to provide said pharmaceutical composition with a fraction or fractions derived from a pregnant animal by for example sampling urine or serum or placental (be it of maternal or foetal origin) or other tissue or cells and preparing said immunoregulator comprising said active component from said urine or serum or tissue or cells by fractionation techniques known in the art (for example by gel permeation chromatograpy) and testing for its active component by stimulating a NOD mouse or its splenocytes as described. In particular, said 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 said individuals normally are more susceptible to immune diseases than male individuals, especially autoimmune diseases, during pregnancy they are more resistant to these diseases.

The invention also provides a method for in vitro stimulation of lymphocytes and transferring said stimulated lymphocytes as a pharmaceutical composition to an animal for treating said 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, said disorder comprises diabetes, yet other immune mediated disorders, such as acute and chronic inflammation, can also be treated. In yet another preferred embodiment, said disorder comprises sepsis or septic shock. The invention provides a method of treatment for an animal, preferably wherein said animal is human.

In a particular embodiment, a method provided by the invention is further comprising regulating relative ratios and/or cytokine activity or cytokine expression or marker expression of lymphocyte, dendritic or antigen presenting cell subset-populations in said animal, such as subset-populations that comprise Thl or Th2 cells, or Th3 or Th8 cells, or DC1 or DC2 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 said immunoregulator, preferably obtainable from a pregnant mammal, for the production of a pharmaceutical composition for the treatment of an immune-mediateddisorder, preferably selected from a group consisting of allergies, auto-immune disease (such as systemic lupus erythematosus or rheumatoid arthritis), transplantationrelated 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 wherein said 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 autoimmune 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.

Said overly strong immune response is kept at bay with an immunoregulator as provided by the invention. Yet other 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 NMPF according to the invention; whereby NMPF also acts as anti-oxidant directly or indirectly. For example, a determining event in the pathogenesis of diabetes I is the destruction of insulinproducing pancreatic beta cells. There is strong evidence that the progressive reduction of the beta-cell mass is the result of a chronic autoimmune 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 are potent beta-cell toxic effector molecules. The reactive radical NO mediates its deleterious effect mainly through the induction of widespread DNA strand breaks, other radicals, such as oxygen, through their effects on lymfocyte subpopulations such as Thl 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 (non-specific) 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 costimulatory 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. So these two great arms of 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, or for it has 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 autoimmunity, 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 well known that the cytokine micro-environment plays a key role in T helper cell differentiation toward the Thl or Th2 cell type during immune responses. IL-12 induces Thl differentiation, whereas IL-4 drives Th2 differentiation.

Recently it has also been shown that subsets of dendritic cells (DC1, DC2) provide different cytokine microenvironments that determine the differentiation of either Thl 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 Thl or Th2 responses. Moreover, development of Thl responses can be antagonized directly by IL-4 and indirectly by which inhibits the production of IL-12 and interferon-ginducing 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 Thl to Th2 switch may prevent the development of organ-specific autoimmune pathologies and required for the maintance of pregnancy. Recently it has become clear that distinct subsets of regulatory T cells are responsible for regulating both Thl 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 the action of TGF-beta; this would be in keeping with the ability of TGF-beta to inhibit both Thl and Th2 development while IL-10 could preferentially inhibit Thl alone.

The selective outgrowth of Thl 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 in to Thl 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 or endotoxin, and results in high production of IL-12. Lymhoid precursors give rise to DC2 cells after stimulation, and produced IL-1, IL-6 and IL- 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 Thl 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 Thl type cells, while DC2 produce IL-10 and selectively promote Th2 development in the presence of exogenous IL-4.

NMPF as provided by the invention is able to regulate the Thl/Th2 balance in vivo (BALB/c, NOD) and in vitro. In dominant Thl phenotype models like NOD, NMPF (like NMPF-P and its fractions) amongst others downregulates 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 Trl by NMPF.

These regulatory cells may play role in the therapeutic effects of NMPF in immune and inflammatory diseases and immune tolerance. 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 said for the preparation of a pharmaceutical composition for the treatment of an immune-mediated disorder.

Purified NMPF is used to produce monoclonal antibodies and/or other specific reagents thereby facilitating the design of an NMPF-specific quantitative immuno-assay. Also single chain Fv 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.

The invention further provides a method and a pharmaceutical composition for modulating cardiovascular or circulatory disorders, such as heart failure, brain infarctions, Alzheimer's disease, thrombosis, arteriosclerosis, pregnancy related cardiovascular or circulatory disorders and the like. It has been found that an immunoregulator as described supra has a very beneficial effect on animals, including humans, suffering from a cardiovascular disorder.

An immunoregulator according to the invention also widens the scope of possibilities of dotter treatments.

In cases where conventionally such a treatment could not be performed because of risks of an oxygen tension becoming too low, a dotter treatment is now feasible when combined with treatment with an immunoregulator described above. Accordingly, expensive and difficult bypass surgery may in many cases be avoided.

Gene control is generally thought to occur at four levels: 1) transcription (either initiation or termination), 2) processing of primary transcripts, 3) stabilization or destabilization of mRNAs, and 4) mRNA translation. The primary function of gene control in cells is to adjust the enzymatic machinery of the cell to its nutritional, chemical and physical environment.

It is generally thought that gene expression is regulated at both the levels of transcription and translation. Modulation or regulation of gene expression requires factors called transcriptional factors. The term "gene control or regulation" also refers to the formation and use of mRNA. Although control can be exerted at a number of different molecular steps, differential transcription probably most frequently underlies the differential rate of protein synthesis in prokaryotes as well as eukaryotes. It is generally thought that activator proteins (also called transcription factors or transcriptional activators) bind to DNA and recruit the transcriptional machinery in a cell to a promotor, thereby stimulating gene expression.

Further, differential processing of RNA transcripts in the cell nucleus, differential stabilization of mRNA in the cytoplasm, and differential translation of mRNA into protein are also important in eukaryotic gene control.

These steps define the regulatory decisions in a transcriptional circuit and misregulation at any stage can result in a variety of diseases.

Where in unicellular organisms, be it of prokaryotic or eukaryotic origin, a cell's response to its environment is influenced by many stimuli from the outside world, reflecting the often widely variable environment of the single cell, most cells in multicellular organisms experience a fairly constant environment. Perhaps for this reason, genes that are devoted to responses to environmental changes constitute a much smaller fraction of the total number of genes in multicellular organisms than in single-cell organisms.

As said above, cells react to environmental changes, which they perceive through extracellular signals. These signals can be either physical light, temperature, pressure and electricity) or chemical food, hormones and neurotransmitters). Cells can both sense and produce signals. This makes it possible for them to communicate with each other. In order to achieve this, there are complex signal-sensing and -producing mechanisms in uni- and multi-cellular organisms.

Two groups of chemical signals can be distinguished: membrane-permeable and -membrane-impermeable signals. The membrane-permeable signal molecules comprise the large family of steroid hormones, such as estrogens, progesterone and androgens. Steroids pass the plasma membrane and bind to specific receptors, which are localized in the cytoplasm or nucleus of the cell. After binding of the hormone, the receptor undergoes a conformational change. The receptor is then able to bind to DNA itself or to proteins which can in turn interact with DNA. In general, steroid hormones can directly regulate gene expression by means of this process. The membrane-impermeable signal molecules include acetylcholine, growth factors, extracellular matrix components, (peptide)-hormones, neuropeptides, thrombin, lysophosphatidic acid, the yeast mating factors and, for the social amoeba Dictyostellium discoideum, folic acid and cyclic AMP. They may be membrane-permeable in themselve but act only outside the cell, i.e. they are recognized by receptors, which are localized on the plasma membrane of the cell. The receptors are specific for one particular signal molecule or a family of closely related signal molecules. Upon binding of their ligands, these receptors transduce the signals by several mechanisms.

The most characteristic and exacting requirement of gene control on multicellular organisms is the execution of precise developmental decisions so that the right gene is activated in the right cell at the right time. These developmental decisions include not only those related to the development of an organism oer se, as for example can be seen during embryogenesis and organogenesis or in response to disease, but also relate to the differentiation or proliferation or apoptosis of those cells that merely carry out their genetic program essentially without leaving progeny behind.

Such cells, such as skin cells, precursors of red blood cells, lens cells of the eye, and antibodyproducing cells, are also often regulated by patterns of gene control that serve the need of the whole organism and not the survival of an individual cell.

It is generally reasoned that there are at least three components of gene control: molecular signals, levels and mechanisms. Firstly, it is reasoned that specific signalling molecules exist to which a specific gene can respond. Secondly, control is exerted on one or more levels the step or steps) in the chain of events leading from the transcription of DNA to the use of mRNA in protein synthesis. Thirdly, at each of those levels, specific molecular mechanisms are employed to finally exert the control over the gene to be expressed.

Many genes are regulated not by a signalling molecule that enters the cells but by molecules that bind to specific receptors on the surface of cells.

Interaction between cell-surface receptors and their ligands can be followed by a cascade of intracellular events including variations in the intracellular levels of so-called second messengers (diacylglycerol, Ca 2 cyclic nucleotides). The second messengers in turn lead to changes in protein phosphorylation through the action of cyclic AMP, cyclic GMP, calcium-activated protein kinases, or protein kinase C, which is activated by diaglycerol.

Many of the responses to binding of ligands to cellsurface receptors are cytoplasmatic and do not involve immediate gene activation in the nucleus. Some receptorligand interactions, however, are known to cause prompt nuclear transcriptional activation of a specific and limited set of genes. For example, one proto-oncogene, cfos, is known to be activated in some cell types by elevation of almost every one of the known second messengers and also by at least two growth factors, platelet-derived growth factor and epidermal growth factor. However, progress has been slow in determining exactly how such activation is achieved. In a few cases, the transcriptional proteins that respond to cellsurface signals have been characterized.

One of the clearest examples of activation of a preexisting inactive transcription factor following a cellsurface interaction is the nuclear factor (NF)-kappaB, which was originally detected because it stimulates the transcription of genes encoding immunoglobulins of the kappa class in B-lymphocytes. The binding site for NKkappaB in the kappa gene is well defined (see for example P.A. Baeuerle and D. Baltimore, 1988, Science 242:540), providing an assay for the presence of the active factor.

This factor exists in the cytoplasm of lymphocytes complexed with an inhibitor. Treatment of the isolated complex in vitro with mild denaturing conditions dissociates the complex, thus freeing NK-kappaB to bind to its DNA site. Release of active NF-kappaB in cells is now known to occur after a variety of stimuli including treating cells with bacterial lipopolysaccharide (LPS) and extracellular polypeptides as well as chemical molecules phobol esters) that stimulate intracellular phosphokinases. Thus a phosphorylation event triggered by many possible stimuli may account for NF-kappaB conversion to the active state. The active factor is then translocated to the cell nucleus to stimulate transcription only of genes with a binding site for active NF-kappaB.

The inflammatory response involves the sequential release of mediators and the recruitment of circulating leukocytes, which become activated at the inflammatory site and release further mediators (Nat. Med.

7:1294;2001). This response is self-limiting and resolves through the release of endogenous anti-inflammatory mediators and the clearance of inflammatory cells. The persistent accumulation and activation of leukocytes is a hallmark of chronic inflammation. Current approaches to the treatment of inflammation rely on the inhibition of pro-inflammatory mediator production and of mechanisms that initiate the inflammatory response. However, the mechanisms by which the inflammatory response resolves might provide new targets in the treatment of chronic inflammation. Studies in different experimental models of resolving inflammation have identified several putative mechanisms and mediators of inflammatory resolution. We have shown that cyclopentenone prostaglandins (cyPGs) may be endogenous anti-inflammatory mediators and promote the resolution of inflammation in vivo. Others have shown a temporal shift to the production of anti-inflammatory lipoxins during the resolution of inflammation. In recent years, apoptosis has been identified as an important mechanism for the resolution of inflammation in vivo. It has been postulated that defects in leukocyte apoptosis are important in the pathogenesis of inflammatory disease. In addition, the selective induction of apoptosis in leukocytes may offer a new therapeutic approach to inflammatory disease.

Considering that NF-kappaB is thought by many to be a primary effector of disease Baldwin, J. Clin.

Invest., 2001, 107:3-6), numerous efforts are underway to develop safe inhibitors of NF-kappaB to be used in treatment of both chronic and acute disease situations.

Specific inhibitors of NF-kappaB should reduce side effects associated with drugs such as NSAIDS and glucocorticoids and would offer significant potential for the treatment of a variety of human and animal diseases.

Specific diseases or syndromes where patients would benefit from NF-kappaB inhibition vary widely and range from rheumatoid arthritis, atherosclerosis, multiple sclerosis, chronic inflammatory demyelinating polyradiculoneuritis, asthma, inflammatory bowel disease, to Helicobacter pylori-associated gastritis and other inflammatory responses, and a variety of drugs that have effects on NF-kappaB activity, such as corticosteroids, sulfasalazine, 5-aminosalicylic acid, aspirin, tepoxalin, leflunomide, curcumin, antioxidants and proteasome inhibitors. These drugs are considered to be nonspecific and often only applicable in high concentrations that may end up toxic for the individual treated.

Inactive cytoplasmatic forms of transcription factors can thus be activated by removal of an inhibitor, as is the case with NF-kappaB, or, alternatively, by association of two (or more) proteins, neither of which is active by itself as in the case of interferon-alphastimulated factor Levy et al., 1989, Genes and Development 3:1362). After interferon-alpha attaches to its cell-surface receptor, one of the proteins is changed within a minute or less, and the two can combine. The active (combined) factor is then translocated to the cell nucleus to stimulate transcription only of genes with a binding site for the protein. Considering that interferon-alpha is a mediator of responses of the body directed at pathogens and self-antigens, modulating regulation of genes that are under influence of the interferon-alpha-stimulated factor would contribute to the treatment of a variety of human and animal diseases.

Other typical examples of signalling molecules that affect gene expression via cell-surface receptor interaction are polypeptide hormones such as insulin, glucagon, various growth factors such as EGF, VEGF, and so on.

The steroid hormones and their receptors represent one of the best understood cases that affect transcription. Because steroid hormones are soluble in lipid membranes, they can diffuse into cells. They affect transcription by binding to specific intracellular receptors that are site-specific DNA-binding molecules.

Other examples of signalling molecules that enter the cell and act intra-cellularly are thyroid hormone (T 3 vitamin D and retinoic acid, and other small lipidsoluble signalling molecules that enter cells and modulate gene expression. The characteristic DNA-binding sites for the receptors for these signalling molecules are also known as response elements.

Another example of a small molecule that is involved in regulation of gene expression is ethylene, a gas that for example induces the expression of genes involved in fruit ripening. Also, small plant hormones, known as auxines and cytokinins regulate plant growth and differentiation directly by regulating gene expression.

Given the critical role of regulatory factors in gene regulation, the development of artificial or synthetic counterparts that could be used in methods to rectify errors in gene expression has been a longstanding goal at the interface of chemistry and biology.

The inventors have now unearthed an insight in the biology and physiology of the nature of regulatory factors in gene regulation in cellular organisms that allows for an unexpected fast progress in the identification and development of an artificial or synthetic compound acting as a gene regulator, and its use as new chemical entity for the production of a pharmaceutical composition or its use in the treatment of disease. The insight is herein provided that many of small peptides that are derivable by proteolytic breakdown of endogenous proteins of an organism, or that are derivable by proteolytic breakdown of proteins of a pathogen, i.e. during the presence of said pathogen in a host organism, can exert an often very specific gene regulatory activity on cells of said organism. In a particular embodiment, the present invention has major value for investigators in furthering the quality and quantity of knowledge regarding the mechanisms controlling NFKB-initiated gene expression under a variety of different conditions and circumstances.

With these insights the invention provides among others a screening method for identifying or obtaining a signaling molecule comprising a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell, be it in vitro or in vivo in an experimental animal such as a monkey or a small laboratory animal such as a rat or mouse, comprising providing said cell (or animal) with at least one lead peptide or derivative or analogue thereof and determining the activity and/or nuclear translocation of a gene transcription factor, in particular wherein said lead peptide is 3 to 15 amino acids long, more preferably, wherein said lead peptide is 3 to 9 amino acids long, preferably wherein said lead peptide is 4 to 6 amino acids long, most preferred wherein said peptide is 3 or 4 amino acids long.

The invention provides a method for modulating expression of a gene in a cell comprising providing said cell with a tri- or tetrameric peptide other than QVVC, PALP, LQGV, MTRV, VVC, MTR, LQG and other than QVVC, PALP, LQGV, MTRV, VVC, MTR, LQC wherein one acid has been replaced by an alanine residue, and the invention also provides pharmaceutical composition comprising a tri- or tetrameric peptide capable of modulating expression of a gene in a cell and a pharmaceutically acceptable carrier wherein said peptide is other than QWC, PALP, LQGV, MTRV, VVC, MTR, LQG and other than QVVC, PALP, LQGV, MTRV, VVC, MTR, LQC wherein one acid has been replaced by an alanine residue.

Functional derivative or analogue herein relates to the signalling molecular effect or activity as for example can be measured by measuring nuclear translocation of a relevant transcription factor, such as NF-kappaB in an NF-kappaB assay, or AP-1 in an AP-1 assay, or by another method as provided herein. Fragments can be somewhat 1 or 2 amino acids) smaller or larger on one or both sides, while still providing functional activity.

A screening method according to the invention is also provided wherein the method further comprises determining whether said gene transcription factor regulates the transcription of a cytokine, as for example measured by detecting cytokine transcript levels or the actual presence as such in the treated call or animal, or wherein said gene transcription factor comprises a NFkappaB/Rel protein, or by determining relative upregulation and/or down-regulation of at least one gene of interest expressed in said cell or of a multitude of genes expressed in said cell, as easily can be done with gene chip technology or any of other methods herein explained.

The invention also provides a screening method for identifying or obtaining a signaling molecule comprising a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell be it in vitro or in vivo in an experimental animal such as a monkey or a small laboratory animal such as a rat or mouse, comprising providing said cell (or animal) with a lead peptide or derivative or analogue thereof and determining relative up-regulation and/or downregulation of at least one gene expressed in said cell, especially wherein said lead peptides are sufficiently small as identified herein. Such as method as provided herein for identifying or obtaining a signaling molecule comprising a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell may also comprise providing (be it in vitro or in vivo) a lead peptide or derivative or analogue thereof and determining binding of said peptide or derivative or analogue thereof to a factor related to gene control, such as a transcription factor, in particular wherein said transcription factor regulates the transcription of a cytokine, or determining the activity and/or nuclear translocation of a gene transcription factor in said cell provided with said peptide.

Advantageously, a screening method according to the invention is provided wherein said lead peptide is one of a member of a library of peptides or derivatives or analogues thereof, in particular, wherein said library is composed of peptides that are selected based on their occurrence in a naturally occurring protein. For investigations aimed at finding new chemical entities useful in human or veterinary therapy, based on using the lead peptide technology as provided herein, it is preferred that said protein is a mammalian protein, a human protein is most preferred. Protein sequences can be obtained from commonly available databases, such as for example are provided for the human genome. Other useful proteins from which libraries of lead peptides can be taken are those derived from pathogen proteins. For identifying peptides useful in crop cultivation, several plant protein data bases are available.

In a preferred embodiment, a screening method according to the invention is provided wherein lead peptides in said library are selected under guidance of proteolytic site prediction, such as peptides that are predicted or deemed to be recognized in a MHC context, by way of example such as hCG-derived peptides that are predicted to be recognized as antigenic determinants and presented in the context of HLA-molecules: Other sets or libraries of useful lead peptides can be designed by making the overlaps very stringent, e.g, that all but 1 or 2 amino acids overlap, such as is given here by way of example for LQGV, QGVL, GVLP, and so on.

The invention provides new chemical entities based on trimeric or tetrameric peptides that act as a signaling molecule useful in modulating expression of a gene in a cell and identifiable or obtainable by employing a screening method according to the invention as provided herein. Useful signaling molecules are already provided herein as modulators of NF-kappaB/Rel protein, as detailed further on. The invention also provides use of a signaling molecule as thus provided for the production of a pharmaceutical composition for the modulation of gene expression, for example by inhibiting NF-kappaB/Rel protein activation, or its use for the production of a pharmaceutical composition for the treatment of a primate or domestic animal.

That small peptides, and even breakdown products, can have biological activity, is already known.

Proteolytic breakdown products of endogenous or pathogen derived proteins are for example routinely generated by the proteasome system and presented in the context of class I or II major histocompabilility complex (MHC).

Also, it has been recognized that classically known neuropeptides (also known as peptide neurotransmitters) or small peptide hormones, such as antidiuretic hormone, oxytocin, thyrotropin-releasing hormone, gonadotropinreleasing hormone, somatostatinsgastrin, cholecystokinin, substance-P, enkephalins, neurotensin, angiotensins, and derivatives or equivalents thereof have distinct biological activity which is in general mediated by cellsurface receptor interaction. Furthermore, it is now known that certain small and arginine- or lysine- or proline-rich peptides, i.e. having more than 50% of arginine, or 50% of lysine or 50% of proline, or having more than 50% arginine and lysine, or more than arginine and proline, or more than 50% lysine and proline, or more than 50% arginine and lysine and proline residues, have distinct membrane-permeation properties that may result in biological activity.

However, the present invention relates to small peptides other than classically known neuropeptides or peptide hormones, and other than the above identified arginine- or lysine- or proline-rich peptides. It is preferred that the peptides of the invention and for use as lead peptide in a screening method as provided by the invention are small. A most preferred size is 3 or 4 amino acids.

As said, the invention provides the insight that trimeric or tetrameric peptides that are derivable or obtainable by proteolytic breakdown of endogenous proteins of an organism, or that are derivable or obtainable by proteolytic breakdown of proteins of a pathogen, i.e. during the presence of said pathogen in a host organism, can exert an often very specific gene regulatory activity on cells of said organism. This insight produces an immediate incentive for systematic approaches to practice or execute a method as provided herein to identify a signalling molecule, by obtaining information about the capacity or tendency of an small (oligo)peptide, or a modification or derivative thereof, (herein jointly called lead peptide) to regulate expression of a gene. Such a method as provided herein for example comprises the steps of contacting said peptide, or a modification or derivative thereof, with at least one cell and determining the presence of at least one gene product in or derived from said cell. Such a method is particularly useful when said lead peptide comprises an amino acid sequence corresponding to a fragment of a naturally occurring polypeptide. Exemplary of course herein is a method wherein said naturally occurring polypeptide comprises an hormone such as human chorionic gonadotropic hormone (hCG). However, other proteins, selected from classes as widely varying as immunoglobulins, heat shock proteins, Cys proteases, cytochrome p450 enzymes, (serine/threonine, or tyrosine) kinases, receptor proteins, protein phosphates, come to mind first when selecting polypeptide sequence from which lead peptides are designed. Other proteins can be taken as starting point as well. Specific mention deserve pathogen proteins as starting point. For example, it is worthwhile selecting such proteins from parasite pathogens, especially of those organisms that live for a certain time in a particular endobiontic relationship with their host, such as is the case with Taenia spp, leading to cysticercosis in man and animals, as with Schistosoma spp, as with malaria, or Trypanosoma, Giardia spp, Dictiocaulus spp, etc. Other pathogens that deserve attention are intracellular organisms such as found among (myco)bacteria and viruses.

In one embodiment is preferred to perform such testing as provided herein systematically, based for example on a combinatorial chemistry format wherein a multitude of lead peptides (in a so-called peptide library) is tested, and promising individual lead peptides, or groups of lead peptides are further tested in subsequent rounds of testing, whereby such lead peptides can be modified to ones desire, as for example as described herein by replacement or substitution of amino acids with other or non-naturally- or naturally occurring) amino acids or modifications or derivatives thereof. Especially by including such subsequent rounds of optimisation, the invention herewith provides a systemic method for identifying or obtaining a signalling molecule comprising a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprising providing said cell with a peptide or derivative or analogue thereof and determining the activity and/or nuclear translocation of a gene transcription factor and then synthesising the molecule with the desired activity.

In that way it is first possible to obtain in a systematic way information on the tendency or capacity of a leadpeptide, or a modification or derivative thereof, to regulate expression of a gene, and to improve on that capacity in subsequent rounds of lead optimalisation, until the lead compound is developed into a chemical entity useful in a pharmaceutical composition or method of treatment aimed at regulating a gene or genes under study. Provided herein is a method for obtaining information about the capacity or tendency of a lead peptide, or a modification thereof, to regulate expression of a gene comprising the steps of contacting said lead peptide, or a modification thereof, with at least one cell and determining the presence of at least one gene product derived from said cell.

As said, lead peptides can be derived from naturally occurring polypeptides such as natural protein molecules.

Lead peptides containing 3 to 4 amino acids are preferably used. They may be tested in a random fashion, being derived from the proteome of the organism under study. Lead peptides may comprise overlapping amino acid sequences as well as peptides that are the result of a predicted chemical or enzymatic cleavage digestion of a polypeptide. Lead peptides can be linear peptides as well as cyclic peptides. A naturally occurring protein can be an endogenous protein such as human chorionic gonadotropic hormone (hCG). Further, it can for example be a peptide derived from a pathogen polypeptide such as Bordetella, Yersinia, Toxoplasma gondii and African Swine Fever Virus. As said, many pathogens have evolved mechanisms to counteract or escape the host immune response by inhibiting NF-kappaB activation and suppressing the upregulation of proinflammatory cytokines. On the other hand, some viruses, including HIV-I, CMV and SV-40, take advantage of NF-kappaB as a host factor that is activated at sites of infection. A method is provided for studying host-pathogen interactions comprising determining the effect of a lead peptide derived from a polypeptide of said pathogen on the gene expression of said host.

In one embodiment, a linear scan is performed which is the systematic screening of overlapping lead peptides derived from a protein sequence with an appropriate bioassay. Such a bioassay comprises an assay for obtaining information about the capacity or tendency of a lead peptide, or a modification thereof, to regulate expression of a gene. A scan with 4-mer or a 3-mer peptides can yield valuable information on the linear stretch of amino acids that form an interaction site and allows identification of lead peptides that have the capacity or tendency to regulate gene expression. Lead peptides can be modified to modulate their capacity or tendency to regulate gene expression, which can be easily assayed in an in vitro bioassay such as a reporter assay.

For example, some amino acid at some position can be replaced with another amino acidsof similar or different properties. Alanine (Ala)-replacement scanning, involving a systematic replacement of each amino acid by an Ala residue, is a suitable approach to modify the amino acid composition of a lead peptide when in a search for a signaling molecule capable of modulating gene expression.

Of course, such replacement scanning or mapping can be undertaken with amino acids other than Ala as well, for example with D-amino acids. In one embodiment, a peptide derived from a naturally occurring polypeptide is identified as being capable of modulating gene expression of a gene in a cell. Subsequently, various synthetic Alamutants of this lead peptide are produced. These Alamutants are screened for their enhanced or improved capacity to regulate expression of a gene compared to lead polypeptide.

Furthermore, a lead peptide, or a modification or analogue thereof, can be chemically synthesised using Dand or L-stereoisomers. For example, a lead peptide that is a retro-inverso of an oligopeptide of natural origin is produced. The concept of polypeptide retroinversion (assemblage of a natural L-amino acidcontaining parent sequence in reverse order using D-amino acids) has been applied successfully to synthetic peptides. Retro-inverso modification of peptide bonds has evolved into a widely used peptidomimetic approach for the design of novel bioactive molecules which has been applied to many families of biologically active peptide.

The sequence, amino acid composition and length of a peptide will influence whether correct assembly and purification are feasible. These factors also determine the solubility of the final product. The purity of a crude peptide typically decreases as the length increases. The yield of peptide for sequences less than residues is usually satisfactory, and such peptides can typically be made without difficulty. The overall amino acid composition of a peptide is an important design variable. A peptide's solubility is strongly influenced by composition. Peptides with a high content of hydrophobic residues, such as Leu, Val, Ile, Met, Phe and Trp, will either have limited solubility in aqueous solution or be completely insoluble. Under these conditions, it can be difficult to use the peptide in experiments, and it may be difficult to purify the peptide if necessary. To achieve a good solubility, it is advisable to keep the hydrophobic amino acid content below 50% and to make sure that there is at least one charged residue for every five amino acids. At physiological pH Asp, Glu, Lys, and Arg all have charged side chains. A single conservative replacement, such as replacing Ala with Gly, or adding a set of polar residues to the N- or C-terminus, may also improve solubility.

Peptides containing multiple Cys, Met, or Trp residues can also be difficult to obtain in high purity partly because these residues are susceptible to oxidation and/or side reactions. If possible, one should choose sequences to minimize these residues. Alternatively, conservative replacements can be made for some residues.

For instance, Norleucine can be used as a replacement for Met, and Ser is sometimes used as a less reactive replacement for Cys. If a number of sequential or overlapping peptides from a protein sequence are to be made, making a change in the starting point of each peptide may create a better balance between hydrophilic and hydrophobic residues. A change in the number of Cys, Met, and Trp residues contained in individual peptides may produce a similar effect. In another embodiment of the invention, a lead peptide capable of modulating gene expression is a chemically modified peptide. A peptide modification includes phosphorylation (e.g on a Tyr, Ser or Thr residue), N-terminal acetylation, C-terminal amidation, C-terminal hydrazide, C-terminal methyl ester, fatty acid attachment, sulfonation (tyrosine), N-terminal dansylation, N-terminal succinylation, tripalmitoyl-S- Glyceryl Cysteine (PAM3 Cys-OH) as well as farnesylation of a Cys residue. Systematic chemical modification of a leadpeptide can for example be performed in the process of leadpeptide optimalization.

Synthetic peptides can be obtained using various procedures known in the art. These include solid phase peptide synthesis (SPPS) and solution phase organic synthesis (SPOS) technologies. SPPS is a quick and easy approach to synthesize peptides and small proteins. The C-terminal amino acid is typically attached to a crosslinked polystyrene resin via an acid labile bond with a linker molecule. This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products.

Generally, an amino acid consists of a central carbon atom (called the a-carbon) that is surrounded by four other groups: a hydrogen, an amino group, carboxyl group, and a side chain group. The side chain group, designated R, defines the different structures of the amino acids. Certain side chains contain functional groups that can interfere with the formation of the amide bond. Therefore, it is important to mask the functional groups of the amino acid side chain. The N-terminus can be protected with a Fmoc or Boc group, which is stable in acid, but removable by base. Any side chain functional groups are protected with base stable, acid labile groups. To begin each coupling, the masking group on the resin bound amino acid/peptide is removed with piperidine in N,N-dimethyl formamide (DMF). It is then rinsed and a protected amino acid is added which has been activated at its 'alpha' carboxyl group. The activation is achieved by creating the N-hydroxybenzotriazole (HOBt) ester in situ. The activated amino acid and the resin bound amico acid are allowed to react in the presence of base to form a new peptide bond. This process is repeated until the desired peptide is assembled at the resin. Once the peptide is complete, it is ready to be cleaved from the resin. This is accomplished using a mixture of trifluoroacetic acid (TFA) and scavangers. Scavangers serve to neutralize cations which are formed during the removal of the side chain protecting groups. The solution is at least 82% TFA, and the rest a mixture of phenol, thioanisol, water, ethanedithiol (EDT), and triisopropylsilane (TIS). The lead peptide on the resin is allowed to react with the cleavage mixture for several hours, which then affords the peptide in solution. It can then be precipitated and washed in tert-butyl methyl ether, and analyzed or purified as desired.

An important link in any polypeptide chain is the amide bond, which is formed by the condensation of an amine group of one amino acid and a carboxyl group of another. The replacement of key amide bonds in peptide fragments by isosteric groups has recently received considerable attention as a possible means of generating novel bio-active substances with improved stability. In one embodiment of the invention, an lead peptide comprises a synthetic molecule in which at least one amide bond has been replaced by an isosteric group such as a ketomethylene or a trans-alkene group. Classically, well-defined molecules were systematically modified and the product compounds analyzed for improved biological activity. Newer combinatorial chemistry methods allow the synthesis of a large population of similar compounds.

This is generally followed by the selection, or screening, of peptides for biological activity such as the capacity to regulate gene expression. In one embodiment of the invention, lead peptides are synthesized in a random fashion using a combinatorial chemistry approach. Combinatorial chemistry, combined with recent advances in robotic screening, enables the testing of a large number of compounds in a short period of time. This technique involves the preparation of a large number of structurally related compounds either as mixtures in the same reaction vessel or individually by parallel synthesis. In this manner large pools of similar compounds can be synthesized within a short period of time. Combinatorial libraries can be prepared using both solution chemistry and by solid phase synthesis; however, solid phase synthesis allows the use of excess reagents to drive the reaction to completion and easy removal of the reagents and side-products by simple filtration of the polymeric support and washing with solvent.

Therefore, solid phase synthesis offers a more attractive approach to the generation of chemical libraries for screening purposes.

Combinatorial chemistry is well suited to peptides.

Lead peptide libraries can be easily synthesized using solid-phase chemistry. Sequence degeneracy can be incorporated during the synthesis using either split synthesis or parallel synthesis. In the split synthesis approach, the solid support is divided into portions prior to each coupling step. A different molecular unit (synthon), like an amino acid, is then coupled to each portion. All portions are recombined after coupling and the synthesis cycle is completed. This "split and mix" approach has the advantage of yielding a unique sequence on each support bead and variability in synthon reactivity can be corrected by varying the coupling conditions. Peptide synthesis, where variations in reactivity between amino acids are significant, requires the "split and mix" approach. Head-to-tail cyclization of peptides on the resin provides a facile route to cyclic compounds. In addition to general advantages of solid phase synthesis, such as high efficiency and easy purification, head-to-tail cyclization of peptides on polymer supports provides minimal risk of intermolecular reactions dimerization and oligomerization), even under high concentration. This is another advantage over solution chemistry which requires high dilution conditions to avoid intermolecular side reactions of the linear peptide.

In one embodiment, a method is provided for identifying or obtaining a signaling molecule comprising a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprising providing said cell with a peptide or functional derivative or analogue thereof and determining the activity and/or nuclear translocation of a gene transcription factor.

The thus developed chemical entity can be administered and introduced in-vivo systemically, topically, or locally. The peptide, or is modification or derivative, can be administered as the entity as such or as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with an inorganic acid (such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid); or with an organic acid (such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid); or by reaction with an inorganic base (such as sodium hydroxide, ammonium hydroxide, potassium hydroxide); or with an organic base (such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines). A selected peptide and any of the derived entities may also be conjugated to sugars, lipids, other polypeptides, nucleic acids and PNA; and function in-situ as a conjugate or be released locally after reaching a targeted tissue or organ.

Going back to lead peptide detection, it is for example possible to generate such a lead peptide library at a purely random basis, the library comprising tri- or tetrameric peptide fragments as test entities (herein also called lead peptides) for each and every combination of amino acids or amino acid derivatives known, and then contacting each of said lead peptides, or a modification or derivative thereof, with at least one cell, and than determining the presence of at least one gene product in or derived from said cell.

It is however preferred to start with a more selective peptide library wherein for example lead peptides are selected on the basis of their occurence in endogenous (host or pathogen) proteins sequences from which (preferably 3-4 amino acids long) lead peptides are predicted and then synthesized on the basis of proteolytic site prediction. Several of such models exist, it is for example preferred to use a computer model allowing the prediction of a MHC-I or MHC-II specific proteolytic breakdown sequences. In yet another example of such a peptide library, lead peptides to be tested in said library are derived from a protein sequence by selecting and synthesizing peptide fragments in an overlapping fashion from the protein in question whereby the overlap can for example by 1, 2 or 3 amino acids or whereby all but 1 amino acid overlap in the consecutive sequences. Even more preferred is a method whereby the peptide library is composed of lead peptides that are derived by first selecting longer peptide sequences under guidance of proteolytic site prediction from a protein, as above, and from those longer sequences designing 3 to 4 amino acids long peptide fragments that are derived in an overlapping fashion from the predicted longer sequences. In short, the invention provides a method for modulating expression of a gene in a cell comprising providing said cell with a tri- or tetrameric peptide, in particular wherein said peptide is derived, either by direct proteolysis or based on the (predicted) amino acid sequence and then synthesised from free amino acids or by proteolysis of a recombinant protein)from a naturally occuring protein.

A further non-limiting list of proteins from which peptide secquences may be derived for further testing as lead peptide includes collagen, PSG, CEA, MAGE (malanoma associated growth antigen), Thrombospondin-l, Growth factors, MMPs, Calmodulin, Olfactory receptors, Cytochrome p450, Kinases, Von Willebrand factor (coagulation factors), Vacuolar proteins (ATP sythase), Glycoprotein hormones, DNA polymerase, Dehydrogenases, Amino peptidases, Trypsin, Viral proteins (such as envelope protein), Elastin, Hibernation associated protein, Antifreeze glycoprotein, Proteases, Circumsporozoite, Nuclear receptors, Transcription actors, Cytokines and their receptors, Bacterial antigens, Nramp, RNA polymerase, Cytoskeletal proteins, Hematopoietic (neural) membrane proteins, Immunoglobulins. HLA/MHC, G-coupled proteins and their receptors, TATA binding proteins, Transferases, Zinc finger protein, Spliceosmal proteins, HMG (high mobility group protein), ROS (reactive oxygen species), superoxidases, superoxide dismutase, Protooncogenes/tumor suppressor genes, Apolipoproteins.

A further method is provided for obtaining information about the capacity or tendency of a lead peptide, or a modification or derivative thereof, to regulate gene expression wherein information is obtained using microarray technology. Microarray technology makes use of the sequence resources created by genome sequencing projects and other sequencing efforts to answer the question, what genes are expressed in a particular cell type of an organism, at a particular time, under particular conditions. Microarrays exploit the preferential binding of complementary single-stranded nucleic acid sequences. Microarrays allow a systematic examination of the gene expression profile in cells.

There are several names for this technology DNA microarrays, DNA arrays, DNA chips, gene chips, and others. A microarray is typically a glass (or some other material) slide, onto which nucleic acid molecules, such as DNA or RNA, are attached at fixed locations (spots).

There may be tens of thousands of spots on an array, each containing a huge number of identical DNA molecules (or fragments of identical molecules), of lengths from twenty to hundreds of nucleotides. For gene expression studies, each of these molecules ideally should identify one gene or one exon in the genome. The spots are either printed on the microarrays by a robot, or synthesized by photolithography (similarly as in computer chip productions) or by ink-jet printing.

In one embodiment of the invention, microarray technology is used to determine the capacity of a peptide to control the relative upregulation and/or downregulation of at least one gene in a cell. Also, a method is provided to exploit microarray technology to determine the modulatory effect of one or more lead peptides or derivatives thereof on the up-regulation and/ or downregulation of a multitude of genes expressed in a cell. In a further embodiment, a lead peptide with the desired activity, as determined in a for example microarray, is synthesized. There are several ways how microarrays can be used to measure gene expression levels. One of the most popular microrarray applications allows comparing gene expression levels in two different samples, the same cell type in a non-treated and a treated condition. The total mRNA from the cells in two different conditions is extracted and labelled with two different dyes: for example a green dye for cells at condition 1 and a red dye for cells at condition 2 (to be more accurate, the labelling is typically done by synthesizing single stranded DNAs that are complementary to the extracted mRNA by an enzyme called reverse transcriptase). Both extracts are washed over the microarray. Labelled gene products from the extracts hybridise to their complementary sequences in the spots due to the preferential binding complementary single stranded nucleic acid sequences tend to attract to each other and the longer the complementary parts, the stronger the attraction. The dyes enable the amount of sample bound to a spot to be measured e.g. by the level of fluorescence emitted when a fluorescent dye is excited by a laser. If the RNA from the sample in condition 1 is in abundance, the spot will be green, if the RNA from the sample in condition 2 is in abundance, it will be red. If both are equal, the spot will be yellow, while if neither are present it will not fluoresce and appear black. Thus, from the fluorescence intensities and colours for each spot, the relative expression levels of the genes in both samples can be estimated.

As is exemplified in the detailed description, microarray technology is an attractive approach to monitor the capacity of a lead peptide to upregulate or downregulate gene expression in a cell. A typical experiment comprises a series of parallel samples, each sample containing at least one cell that is provided with a different lead peptide, or a modification or derivative thereof. Also included is a control sample containing at least one cell but no lead peptide. A cell can be a primary cell, such as a peripheral blood mononuclear cell (PBMC) or a cell derived from a laboratory cell line such as a Jurkat, COS-7, MCF-7, 293T cell. At a certain time period following addition of a peptide to a sample, an aliquot is taken. From each aliquot containing a cell that is incubated during a certain time period in the presence of an lead peptide, an inventory is made of (the clusters of) induced and repressed genes using microarray technology. This time period can be 3 hours, or a shorter time period such as 2 hours or 1 hour following addition.

Remarkably, even shorter time periods can be chosen to determine the modulatory effect of a peptide or derivative or analogue thereof on gene expression, like minutes or even less than 20 minutes.

The detectable effect of lead peptides according to the invention on gene control is surprisingly fast when compared to other regulators of gene transcription, which typically induce detectable changes in gene expression upon prolonged incubation times in the range of several, e.g. 6-24 hours. In a preferred embodiment, a cell is first exposed to a compound known to alter the gene expression program in a cell and subsequently provided with a peptide according to the invention. Compounds include receptor agonists, receptor antagonists and other compounds known to induce altered gene transcription.

Also included are compounds which mimic intracellular signals that occur during natural responses to receptor agonists or antagonists, for example a combination of ionomycin and PMA. In another embodiment, a cell is contacted with a microbial agent such as bacterial lipopolysaccharide (LPS) or even intact bacteria (e.g.

Escherichia coli, Bortadella pertussis, Staphylococcus aureus) to induce an immune response. A typical experiment involves a series of parallel samples comprising at least one cell, wherein each sample provided with a different lead peptide, or a modification or derivative thereof, in combination with a compound known to alter the gene expression program in said cell.

In another embodiment, such a compound and a peptide are added to a cell simultaneously. In yet another embodiment, a peptide or a functional derivative thereof is added to a cell prior to or after providing a cell with a compound known to alter the gene expression profile in a cell. Then, an inventory is made of induced and repressed genes using a microarray. From this inventory, the modulatory effect of a peptide on gene control can be readily determined.

Provided is also a method for identifying or obtaining a signalling molecule comprising a peptide or a functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprising providing said cell with a peptide or a derivative or analogue thereof and determining the activity of a gene transcription factor. In one embodiment of the invention, a signalling molecule comprising a peptide or functional derivative or analogue thereof capable of modulating gene expression is identified using a reporter gene assay.

Reporter genes are generally nucleic acid sequences encodingeasily assayed proteins. They are used to replace other coding regions whose protein products are more difficult to assay. A reporter gene is fused downstream of a promotor of interest, so that transcripts initiating at the promotor proceed through the reporter gene. Commonly used reporter genes encode enzymes such as chloramphenicol acetyltransferase (CAT), betagalactosidase, beta-glucuronidase and luciferase.

Interesting reporter genes also comprise fluorescent proteins which fluoresce on irradiation with UV. These include green fluorescent protein (GFP) and spectral variants thereof, such as yellow fluorescent protein (YFP), red fluorescent protein (RFP) and cyano fluorescent protein (CFP). Reporter genes can be attached to other sequences so that only the reporter protein is made or so that the reporter protein is fused to another protein (fusion protein). Reporter genes can "report" many different properties and events: the strength of promoters, whether native or modified for reverse genetics studies; the efficiency of gene delivery systems; and the efficiency of translation initiation signals. A reporter gene construct can be transfected into a cell, e.g. a laboratory cell line, using one of the many transfection techniques known in the art including those using DEAE-dextran, calcium phosphate precipitation, adenovirus- or retrovirus-mediated gene transduction, cationic liposome transfection systems using Lipofectin, Lipofectamine, DOTAP or Fugene reagent) and electroporation techniques. Mixing a liposomal transfection reagent with DNA results in spontaneously formed stable complexes that can directly be added to the tissue culture medium with or without serum. These complexes adhere to the cell surface, fuse with the cell membrane and release the DNA into the cytoplasm. This method of DNA transfer is very gentle, avoiding cytotoxic effects, so that cells can be transfected with high efficiency. Transfection of a cell with a reporter gene construct can be transient or stable. Provided is a method to determine the modulatory effect of an lead peptide on gene expression by exposing a transfected cell to an lead peptide according to the invention, or a mixture thereof, and assaying for reporter gene activity in said cell. The modulatory effect can be a inhibitory or a stimulatory effect. In a preferred embodiment, a reporter gene assay is used to assay for NFkappaB activity, but reporter gene assays designed to assay the activity of one or more other transcription factors may also be used. A luciferase reporter gene construct can be placed under the control of an NFkappaB-driven promotor. Transfected cells are exposed to a series of lead peptides, such as peptide fragments of different lengths derived from naturally occurring polypeptides or synthetic peptides in which amino acids are systematically replaced e.g. by Ala residues or D-amino acids. After a certain incubation time, luciferase activity is assayed to determine the effect of an lead peptide on NFkappaB activity. From this analysis, it is clear whether a peptide has any gene modulatory effect and, if so, whether this effect is inhibitory or stimulatory.

Reporter gene assays allow analysis of a large number of different samples in a relatively short time.

It can easily be performed using multiwell plates such as 96-well plates. The activity of many reporter genes can be assayed using colorimetric or fluorescense detection in for example an automated plate reader. Thus, a reporter gene assay can be used in a high -throughput format. This is especially advantageous when performing several rounds in screening for gene regulatory effects of a peptide, for example in the process of lead peptide optimalization. In these types of assays, it is preferred to focus on a small number of different promotor elements. For example, cells can be transfected with a reporter gene construct for the detection of NFkappaB activity, or with a reporter gene construct for AP-I activity or with a reporter construct designed to readily determine NFAT-I activity. Cells can be transfected in parallel with one reporter gene construct but it also possible to provide a cell with more than one reporter gene construct. Of course, to allow discrimination between activities of the different promotors, it is preferred that each promotor construct contains a different reporter gene. In one embodiment of the invention, a cell is provided with more than one reporter gene construct to determine the effect of a peptide or a derivative or analogue thereof on transcriptional activity. For example, a cell is cotransfected with two or even three different plasmids, each containing a distinct fluorescent reporter gene fused downstream of a distinct promotor of interest. From this, the effect of said peptide on each promotor is determined. In such an experimental set-up, it is obviously preferred that reporter gene products of the co-transfected reporter constructs are easily distinguished from each other. Interesting reporter genes that can be used in co-transfection reporter gene assays include GFP or EGFP (enhanced green fluorescence protein) and spectral variants thereof, such as RFP, YFP and CFP.

Following exposure of said cell to a peptide according to the invention, the activities of each fluorescent reporter gene is measured by fluorescence detection using a suitable optical filter set, like a multi-band filter set. Multi-band sets are used for multiple labelling and simultaneous viewing of multiple fluorophores. Each set of exciters, dichroics, and emitters yields isolated bands of excitation and emission energy.

To detect an effect of a lead peptide on gene expression, especially to detect an inhibitory effect, it is preferred to have a significant level of basal gene transcriptional activity in a cell. To facilitate detection of an inhibitory effect of a peptide on gene expression, a cell can be treated with a compound known to induce a profound increase in gene expression. This compound can be added before, after or at the same time at which a cell is provided with an lead peptide. For instance, a cell containing a luciferase reporter gene under the control of an NF-kappaB-driven promotor is exposed to LPS. This will induce an increase in luciferase activity compared to untreated control cells, in which there may only be a low basal level of NFkappaB-dependent transcriptional activity. Subsequently, or simultaneously, at least one peptide suspected of being capable to modulate gene expression is added. Then, the effect of said peptide on LPS-induced luciferase activity is assayed and compared to the luciferase activity in a parallel sample comprising cells which only received LPS but no peptide.

Using a method for identifying or obtaining a signalling molecule comprising a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprising providing said cell with a peptide or derivative or analogue thereof and determining the activity and/or nuclear translocation of a gene transcription factor as provided herein furthermore allows at random testing of a multiplicity of oligo- or leadpeptides, leading to automated combinatorial chemistry formats, wherein a great many of candidate signal molecules are being tested in a (if so desired at random) pattern for their reactivity with a molecular library of synthetic peptides representing potential signal molecules allowing the rapid detection of particularly relevant molecules out of tens of thousands of (combinations of) molecules tested. In a preferred embodiment, the invention provides a method wherein said lead peptides, or at least their activities, are positionally or spatially addressable, e.g. in an array fashion, if desired aided by computer directed localisation. In an array, said pluralities are for example addressable by their positions in a grid or matrix.

Also provided herein is a method for identifying or obtaining a signalling molecule comprising a peptide or a functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprising providing said cell with a lead peptide or a derivative or analogue thereof and determining the nuclear translocation of a gene transcription factor. Cytoplasm to nucleus translocation is an early measure of transcription factor activation. It is regarded to be a critical step in the coupling of extracellular stimuli to the transcriptional activation of specific target genes.

Various methods are available to a person skilled in the art to analyse the subcellular localization of a transcription factor nuclear translocation of a transcription factor. Conventional techniques to analyse the presence of a transcription factor in a nuclear fraction include EMSA (electrophoretic mobility shift assay) and immunocytochemistry. It is also possible to follow the translocation of a fluorescently-tagged transcription factor, such as GFP-NFkB, using fluorescence microscopy. However, to assay a large number of samples for the ability of an lead peptide to modulate nuclear translocation of a transcription factor, it is preferred to use a less elaborate approach. For example, commercial kits ('translocation kits') have recently become commercially available (Cellomics, Inc; www.cellomics.com) which can be used to conveniently measure the cytoplasm to nucleus translocation of a transcription factor. The assay involves detection of a transcription factor by a specific primary antibody, followed by detection of said primary antibody by a fluorochrome-conjugated secondary antibody. Translocation kits are available to measure activation of various transcription factors, including NF-kappaB, STAT and ATF- 2. Together with specialized software and instrumentation, a kit comprises a fully automated screen to identify compounds, such as peptides or derivatives thereof, that inhibit or induce transcriptional activation on a cell-by-cell basis. Assays are performed in standard, high-density microplates, where measurements of the rate and extent of transcription factor translocation are made in intact cells, providing biologically representative information. Kits are available in various sizes. A kit containing reagents for 480 assays can for instance be used in a phase of evaluating the gene modulatory activity of a relatively small amount (like in the range of 10 to 15 of peptide fragments derived from a naturally occurring polypeptide.

This can result in the identification of a few leadpeptides which can modulate the activity of a gene transcription factor. In a subsequent round of screening, such a lead peptide is used for the development of more effective derivatives or homologues. In such a process of lead peptide optimalization, a kit may be used that contains reagents for 4800 individual test samples. In a further embodiment, a kit containing reagents for 19200 individual test samples is used for the screening of a library of synthetic peptides, for example for determining the modulatory effect of thousand of random peptides generated by combinatorial chemistry. A kit combines fluorescent reagents and protocols for optimized sample preparation and assays, and requires no cell lysis, purification, or filtration steps. After fixation, the plates are stable for extended periods, ranging from one week to several months, depending on the cell type and dye, when stored light-protected at The present invention also has a variety of other different applications and uses. Of clinical and medical interest and value, the present invention provides the opportunity to selectively control NFKB-dependent gene expression in tissues and organs in a living subject, preferably in a primate, allowing upregulating essentially anti-inflammatory responses such as and downregulating essentially pro-inflammatory responses such as mediated by TNF-alpha, nitric oxide IL-Ibeta. The invention thus provides use of a NFKB regulating peptide or derivative thereof for the production of a pharmaceutical composition for the treatment of an ischhemic event in a primate, and provides a method of treatment of an ischemic event in a primate. In one such instance as provided herein, such a subject has suffered from ischemic events or has undergone anoxia or infarction. A typical clinical instance is the myocardial infarction or chronic myocardial ischemia of heart tissue in various zones or areas of a living human subject, or, likewise a cerebrovascular infarct.

In response to a variety of pathophysiological and developmental signals, the NFkB/Rel family of transcription factors are activated and form different types of hetero- and homodimers among themselves to regulate the expression of target genes containing kappaB-specific binding sites. NF-kB transcription factors are hetero- or homodimers of a family of related proteins characterized by the Rel homology domain. They form two subfamilies, those containing activation domains RELB, and c-REL) and those lacking activation domains (p50, p52). The prototypical NFkB is a heterodimer of p65 (RELA) and p50 (NF-kBl). Among the activated NFkB dimers, p50-p65 heterodimers are known to be involved in enhancing the transcription of target genes and p50-p50 homodimers in transcriptional repression. However, p65-p65 homodimers are known for both transcriptional activation and repressive activity against target genes. KappaB DNA binding sites with varied affinities to different NFB dimers have been discovered in the promoters of several eukaryotic genes and the balance between activated NFkB homo- and heterodimers ultimately determines the nature and level of gene expression within the cell. The term "NFkBregulating peptide" as used herein refers to a peptide or a modification or derivative thereof capable of modulating the activation of members of the NFkB/Rel family of transcription factors. Activation of NFkB can lead to enhanced transcription of target genes. Also, it can lead to transcriptional repression of target genes.

NFkB activation can be regulated at multiple levels. For example, the dynamic shuttling of the inactive NFkB dimers between the cytoplasm and nucleus by IkappaB proteins and its termination by phosphorylation and proteasomal degradation, direct phosphorylation, acetylation of NFkB factors, and dynamic reorganization of NFkB subunits among the activated NFkB dimers have all been identified as key regulatory steps in NFkB activation and, consequently, in NFkB-mediated transcription processes. Thus, a NFkB-regulating peptide is capable of modulating the transcription of genes that are under the control of NFkB/Rel family of transcription factors. Modulating comprises the upregulation or the downregulation of transcription.

Preferably, a lead peptide for use in generating a gene expression inventory or database according to the invention is 2, 3, 4, 5, or 6 amino acids long, and capable of modulating the expression of a gene, such as a cytokine, in a cell. In a preferred embodiment, a peptide is a signaling molecule that is capable of traversing the plasma membrane of a cell or, in other words, a peptide that is membrane-permeable. Also, a useful peptide for treating an acute or chronic inflammatory disease comprising is a peptide capable of reducing the production NO and or TNF alpha by a cell. A reduction in the production of NO and or TNF alpha in a cell can be achieved by inhibiting a transcription factor of the NF-kB family. This inhibition occurs at several different levels, including direct binding of a peptide to a transcription factor. Also, a peptide as provided herein can inhibit the nuclear translocation of a peptide, as is shown in the detailed description.

Use of a NFkB-regulating peptide is provided, or a mixture of at least two of such peptides, for the treatment of disease that affect or is affected by the NF-kB pathway. In a preferred embodiment, a peptide according to the invention is suitably used in a strategy to modulate the production of one or more cytokines in a cell. Specifically attractive is the use of a peptide for the production of a pharmaceutical composition to inhibit the production of a cytokine in a cell, for example via the suppression of cytokine expression that is under the control of a transcription factor of the NF-kB/Rel family. For example, use of a NFkB-regulating peptide for the production of a pharmaceutical composition is provided for the treatment of sepsis. Furthermore, use of a NFkB-regulating peptide for the production of a pharmaceutical composition for the treatment of anthrax, for instance via the modulation of the production of inflammatory cytokines such as interleukin or via reducing the production of NO and or TNF alpha in a cell. Seemingly unrelated disorders such as asthma, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, chronic obstructive pulmonary disease, allergic rhinitis and cardiovascular disease all have inflammatory elements. As mentioned before, NF-KB is a master regulator of a broad set of inflammatory genes, including TNF, IL-I and cell adhesion molecules, which give rise to immune-inflammatory diseases. Rheumatoid arthritis (RA) is a prototype of chronic inflammatory disease. Studies in animal models of RA demonstrated crucial involvement of NF-kB in regulation of inflammation, apoptosis, and proliferation in the arthritic synovium. Thus, NF-kB emerges as very attractive target for therapeutic intervention in RA and other chronic inflammatory conditions. A logical way to inhibit NF-kB activation is to modulate the signaling cascades which controls transcriptional activity of NFkB.

The invention provides a method for modulating expression of a gene in a cell comprising providing the cell with a signalling molecule comprising an small peptide i.e. oligopeptide or functional analogue or derivative thereof. Such a molecule is herein also called NMPF or referenced by number. Since small peptides, and functional analogues and derivatives of such relatively short amino acid sequences, are easily synthesized these days, the invention provides a method to modulate gene expression with easily obtainable synthetic compounds such as synthetic peptides or functional analogues or derivatives thereof.

A functional analogue or derivative of a peptide is defined as an amino acid sequence, or other sequence monomers, which has been altered such that the functional properties of the sequence are essentially the same in kind, not necessarily in amount. An analogue or derivative can be provided in many ways, for instance, through conservative amino acid substitution. Also peptidomimetic compounds can be designed that functionally or structurally resemble the original peptide taken as the starting point but that are for example composed of non-naturally occurring amino acids or polyamides. With conservative amino acid substitution, one amino acid residue is substituted with another residue with generally similar properties (size, hydrophobicity), such that the overall functioning is likely not to be seriously affected. However, it is often much more desirable to improve a specific function. A derivative can also be provided by systematically improving at least one desired property of an amino acid sequence. This can, for instance, be done by an Ala-scan and/or replacement net mapping method. With these methods, many different peptides are generated, based on an original amino acid sequence but each containing a substitution of at least one amino acid residue. The amino acid residue may either be replaced by alanine (Ala-scan) or by any other amino acid residue (replacement net mapping). This way, many positional variants of the original amino acid sequence are synthesized. Every positional variant is screened for a specific activity. The generated data are used to design improved peptide derivatives of a certain amino acid sequence.

A derivative or analogue can also for instance be generated by substitution of an L-amino acid residue with a D-amino acid residue. This substitution, leading to a peptide which does not naturally occur in nature, can improve a property of an amino acid sequence. It is for example useful to provide a peptide sequence of known activity of all D-amino acids in retro inversion format, thereby allowing for retained activity and increased half-life values. By generating many positional variants of an original amino acid sequence and screening for a specific activity, improved peptide derivatives comprising such D-amino acids can be designed with further improved characteristics.

A person skilled in the art is well able to generate analogous compounds of an amino acid sequence. This can for instance be done through screening of a peptide library. Such an analogue has essentially the same functional properties of the sequence in kind, not necessarily in amount. Also, peptides or analogues can be circularized, for example, by providing them with (terminal) cysteines, dimerized or multimerized, for example, by linkage to lysine or cysteine or other compounds with side-chains that allow linkage or multimerization, brought in tandem- or repeatconfiguration, conjugated or otherwise linked to carriers known in the art, if only by a labile link that allows dissociation.

The invention also provides a signalling molecule for modulating expression of a gene in a cell comprising a small peptide or functional analogue or derivative thereof. Surprisingly, the inventors found that a small, notably tri-, tetra-, penta-, or hexameric peptide acts as a signalling molecule that can modulate signal transduction pathways and gene expression. It is preferred to use a tri- or tetrameric peptide. Such a small peptide, or functional analogue or derivative of such a small peptide that acts as such a signalling molecule for modulating expression of one or more genes in a cell can be identified or obtained by at least one of various methods for finding such a signalling molecule as provided herein.

For example, one method as provided herein for identifying or obtaining a signalling molecule comprising a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprises providing the cell with a peptide or derivative or analogue thereof and determining the activity and/or nuclear translocation of one or more gene transcription factors. Such activity can be determined in various ways using means and/or methods honed to the specific transcription factor(s) under study. In the detailed description, it is provided to study NFkappaB/Rel protein translocation and/or activity, but it is, of course, also easily possible to study translocation and/or activity of any other transcription factor for which such tools are available or can be designed. One such other transcription factor is for example the interferon-alpha-stimulated factor as discussed above. Other useful transcription factors to study in this context comprise c-Jun, ATF-2, Fos, and their complexes, ELK-l, EGR-I, IRF-I, IRF-3/7, AP-I, NF- AT, C/EBPs, Spl, CREB, PPARgamma, and STAT proteins to name a few. Considering that many proteins are subject to proteolytic breakdown whereby oligopeptide fragments are generated, many already before the full protein even has exerted a function, it is hereby established that oligopeptide fragments of such proteins (of which a nonextensive list is given in the detailed description, but one can for example think of MAPKK-2 that can give rise to a peptider MLAR or VLPAL, but also of nitric oxide synthase that can give rise to peptides FPGC or PGCP, LPA, or PAVP after proper proteolysis) are involved in feedback mechanisms regulating gene expression, likely by modulating the effect of transcription factors on gene expression. In addition, oligopeptide fragments of proteins (of which a non-extensive list is given in the detailed description) can also modulate the activity of extracellular components such as factor XIII (examples of oligopeptide fragments obtained from factor XIII are LQGV, GVVP, PRG, PRGV) or activated protein C (APC), thereby eventually leading to the modulation of intracellular signal transduction pathways and gene(s) expression.

As said, the invention provides active tri- or tetrameric peptides acting as a signalling molecule. To allow for improved bio-availability of such a signalling molecule (which is useful as a pharmacon, especially when produced artificially), the invention also provides a method for determining whether a small peptide or derivative or analogue thereof can act as a functional signalling molecule according to the invention, the method further comprising determining whether the signalling molecule is membrane-permeable, and, as explained above, after passage through a plasma membrane and not via binding with a cell-surface receptor, exerts its gene-regulatory effect. Such a signalling molecule, i.e. synthetic compound being a small peptide or functional analogue or derivative thereof as provided herein thus preferably interacts not via cell-surfacereceptor mediated signalling followed by a cascade of intracellular events but has direct intracellular actitivity.

Using a method for identifying or obtaining a signalling molecule comprising a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprising providing said cell with a peptide or derivative or analogue thereof and determining the activity and/or nuclear translocation of a gene transcription factor as provided herein furthermore allows (if required at random) testing of a multiplicity of oligo- or leadpeptides, leading to automated combinatorial chemistry formats, wherein a great many of candidate signal molecules are being tested in a (if so desired at random) pattern for their reactivity with a molecular library of synthetic peptides representing potential signal molecules allowing the rapid detection of particularly relevant molecules out of tens of thousands of (combinations of) molecules tested.

In a preferred embodiment, the invention provides a method wherein said leadpeptides are positionally or spatially addressable, e.g. in an array fashion, if desired aided by computer directed localisation. In an array, said pluralities are for example addressable by their positions in a grid or matrix. It is useful that such peptide fragments or oligopeptides to be tested (herein also called leadpeptides) be 3 to 4 amino acids long.

The invention for example provides a process or method for obtaining information about the capacity or tendency of a tri- or tetrameric peptide, or a modification or derivative thereof, to regulate expression of a gene comprising the steps of: a) contacting the peptide, or a modification or derivative thereof, with at least one cell; b) determining the presence of at least one gene product in or derived from the cell. It is preferred that the peptide comprises an amino acid sequence corresponding to a fragment of a naturally occurring polypeptide, such as hCG, or MAPKK, or another kinase, be it of plant or animal cell, or of eukaryotic or prokaryotic origin, or a synthase of a regulatory protein in a cell, such as wherein the regulatory protein is a (pro-) inflammatory mediator, such as a cytokine. Several candidate proteins and peptide fragments are listed in the detailed description which are a first choice for such an analysis from the inventors' perspective, but the person skilled in the art and working in a specific field of interest in biotechnology shall immediately understand which protein to select for such analyses for his or her own purposes related to his or her field. The invention for example provides a process or method for obtaining information about the capacity or tendency of an oligopeptide, or a modification or derivative thereof, to regulate expression of a gene wherein said method allows (if required at random) testing of a multiplicity of tri- or tetrameric peptides, leading to automated combinatorial chemistry formats, wherein a great many of candidate signal molecules are being tested in a (if so desired at random) pattern for their reactivity with a molecular library of synthetic peptides representing potential signal molecules allowing the rapid detection of particularly relevant molecules out of tens of thousands of (combinations of) molecules tested. In a preferred embodiment, the invention provides a method wherein said tri- or tetrameric peptides are positionally or spatially addressable, e.g. in an array fashion, if desired aided by computer directed localisation. In an array, said pluralities are for example addressable by their positions in a grid or matrix.

In particular, it is provided to perform a process according to the invention further including a step c) comprising determining the presence of the gene product in or derived from a cell which has not been contacted with the tri- or tetrameric peptide, or a modification or derivative thereof, and determining the ratio of gene product found in step b to gene product found in step c, as can easily been done with the present-day genechip technology (see for example the detailed description herein) and related methods of expression profiling known in the art.

Another method provided herein for identifying or obtaining information on a signalling molecule (or for that matter the signalling molecule itself, considering that the next step of synthesizing the molecule, generally being a short peptide, is whole within the art) comprising a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprises providing the cell with a peptide or derivative or analogue thereof and determining relative up-regulation and/or down-regulation of at least one gene expressed in the cell. The up-regulation can classically be studied by determining via for example Northern or Western blotting or nucleic acid detection by PCR or immunological detection of proteins whether a cell or cells make more (in the case of up-regulation) or less (in the case of down-regulation) of a gene expression product such as mRNA or protein after the cell or cells have been provided with the peptide or derivative or analogue thereof. Of course, various methods of the invention can be combined to better analyze the functional analogue of the peptide or derivative or analogue under study. Furthermore, relative up-regulation and/or down-regulation of a multitude or clusters of genes expressed in the cell can be easily studied as well, using libraries of positionally or spatially addressable predetermined or known relevant nucleic acid sequences or unique fragments thereof bound to an array or brought in an array format, using for example a nucleic acid library or so-called gene chip expression analysis systems. Lysates of cells or preparations of cytoplasma and/or nuclei of cells that have been provided with the peptide or derivative or analogue under study are then contacted with the library and relative binding of for example mRNA to individual nucleic acids of the library is then determined, as further described herein in the detailed description.

A functional analogue or derivative of a tri- or tetrameric peptide that can act as a signalling molecule for modulating expression of a gene in a cell can also be identified or obtained by a method for identifying or obtaining a signalling molecule comprising an oligopeptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprising providing a peptide or derivative or analogue thereof and determining binding of the peptide or derivative or analogue thereof to a factor related to gene control. Such a factor related to gene control can be any factor related to transcription (either initiation or termination), processing of primary transcripts, stabilization or destabilization of mRNAs, and mRNA translation.

Binding of a peptide or derivative or analogue thereof to such a factor can be determined by various methods known in the art. Classically, peptides or derivatives or analogues can be (radioactively) labelled and binding to the factor can be determined by detection of a labelled peptide-factor complex, such as by electrophoresis, or other separation methods known in the art. However, for determining binding to such factors, array techniques, such as used with peptide libraries, can also be employed, comprising providing a multitude of peptides or derivatives or analogues thereof and determining binding of at least one of the peptides or derivatives or analogues thereof to a factor related to gene control.

In a preferred embodiment, the factor related to gene control comprises a transcription factor, such as an NF-kappaB-Rel protein or another transcription factor desired to be studied. When binding of a functional analogue according to the invention to such factor has been established, it is, of course, possible to further analyze the analogue by providing a cell with the peptide or derivative or analogue thereof and determining the activity and/or nuclear translocation of a gene transcription factor in the cell, and/or by providing a cell with the peptide or derivative or analogue thereof and determining relative up-regulation and/or downregulation of at least one gene expressed in the cell.

The invention thus provides a signalling molecule useful in modulating expression of a gene in a cell and/or useful for reducing NO production by a cell and identifiable or obtainable by employing a method according to the invention. Useful examples of such a signalling molecule can be selected from the group of oligopeptides LQG, AQG, LQGV, AQGV, LQGA, LAGV, LPGC, MTRV, MTR, WVVC, QVVC and functional analogues or derivatives thereof. Surprisingly, the invention provides here the insight that gene expression can be modulated or regulated by small, tri- or tetrameric peptides, which are most likely breakdown products of larger polypeptides such as chorionic gonadotrophin (CG) and growth hormones or growth factors such as fibroblast growth factor, EGF, VEGF, RNA 3' terminal phosphate cyclase and CAP18. In principle, such regulating peptide sequences can be derived from a part of any protein of polypeptide molecule produced by prokaryotic and/or eukaryotic cells, and the invention provides the insight that breakdown products of polypeptides, preferably oligopeptides at about the sizes as provided herein, are naturally involved as signalling molecules in modulation of gene expression. In particular, as signalling molecule, a (synthetic) peptide is provided obtainable or derivable from beta-human chorionic gonadotrophin (beta-hCG), preferably from nicked beta-HCG. It was thought before that breakdown products of nicked-beta hCG were involved in immuno-modulation (PCT International Patent Application W099/59671) or in the treatment of wasting syndrome (PCT International Patent Application W097/49721, US Patent 6,583,109) but a relationship with modulation of gene expression was not forwarded in these publications. Of course, such an oligopeptide, or functional equivalent or derivative thereof, is likely obtainable or derivable from other proteins that are subject to breakdown or proteolysis and that are close to a gene regulatory cascade. Preferably, the peptide signalling molecule is obtained or derived from a peptide having at least 10 amino acids, examples of which are given in the detailed description.

Not wishing to be bound by theory, it is postulated herein that an unexpected mode of gene regulation has been uncovered. Polypeptides, such as endogenous CG, EGFetc., but also polypeptides of pathogens such as viral, bacterial or protozoal polypeptides, are subject to breakdown into distinct oligopeptides, for example by intracellular proteolysis. Distinct proteolytic enzymes are widely available in the cell, for example in eukaryotes in the lysosomal or proteasomal system. Some of the resulting breakdown products are oligopeptides of 3 to 4 amino acids long that are surprisingly not without any function or effect to the cell, but as demonstrated herein may be involved, possibly via a feedback mechanism in the case of breakdown of endogenous polypeptides, as signalling molecules in the regulation of gene expression, as demonstrated herein by the regulation of the activity or translocation of a gene transcription factor such as NF-kappaB by for example peptides LQGV, LQG, VVC, MTRV, and MTR. Synthetic versions of these oligopeptides as described above, and functional analogues or derivatives of these breakdown products, are herein provided to modulate gene expression in a cell and be used in methods to rectify errors in gene expression or the treatment of disease. Oligopeptides such as LQG, AQG, LQGV, AQGV, LQGA, LAGV, LPGC, MTRV, MTR, WC, or functional tri-or tetrameric peptide analogues thereof, are particularly effective.

By using the insight as expressed herein, in a preferred embodiment, the invention provides a method for modulating expression of a gene in a cell comprising providing the cell with a tri- or tetrameric peptide or functional analogue or derivative thereof wherein the peptide is membrane-permeable in that it enters the cell.

Most small peptides as described herein already have an inherent propensity to become intracellularly involved, for that purpose, however, it is preferred to select those peptides with a molecular weight smaller than 500 dalton, preferably having a molecular structure with less than 5 H-bond donors, more preferred with less than 4 or even 3 H-bond donors (expressed as the sum of OHs and NHs) and/or less than 10 H-bond acceptors, more preferred less than 8 or even 6 H-bond acceptors (expressed as the sum of Ns and Os). It is preferred that the peptides according to the invention have a lypophilicity below Clog P 5, as expressed as a ratio of octanol solubility to aqueous solubility (Testa et al, Pharm. Res. 13, 335- 343, 1996), or alternatively, have a lypophilicity below Mlog P 4.15, as calculated according to Moriguchi et al (Chem. Pharm. Bull. 40, 127-130, 1992).

In a preferred embodiment, the invention provides a method for modulating expression of a gene in a cell comprising providing the cell with a signalling molecule comprising a small peptide (amino acid sequence) or functional analogue or derivative thereof, wherein the signalling molecule modulates NF-kappaB/Rel protein conversion or translocation. As said, NF-KB was originally identified as a gene transcription factor that bound to an enhancer element in the gene for the IgK light chain and was believed to be B cell-specific.

However, subsequent studies revealed that NF-kappaB/Rel proteins are ubiquitously expressed and play a central role as transcription factor in regulating the expression of many genes, particularly those involved in immune, inflammatory, developmental and apoptotic processes. NF- KB related gene transcription factors can be activated by different stimuli such as microbial products, proinflammatory cytokines, T- and B-cell mitogens, and physical and chemical stresses. NF-KB in turn regulates the inducible expression of many cytokines, chemokines, adhesion molecules, acute phase proteins, and antimicrobial peptides.

NF-KB represents a group of structurally related and evolutionarily conserved gene transcription factors. So far, five mammalian NF-KB proteins named Rel (c-Rel), RelA (p65), RelB, NF-kappa-Bl (p50 and its precursor p105), and NF-Kappa-B2 (p52 and it precursor p100) have been described. NF-KB proteins can exist as homo- or heterodimers, and although most NF-KB dimers are activators of transcription, the p50/p50 and p52/p52 homodimers often repress the transcription of their target genes. In Drosophila, three NF-KB homologs named Dorsal, Dif, and Relish have been identified and characterized. Structurally, all NF-KB/Rel proteins share a highly conserved NH 2 -terminal Rel homology domain (RHD) that is responsible for DNA binding, dimerization, and association with inhibitory proteins known as IKBs. In resting cells, NF-KB/Rel dimers are bound to IKBs and retained in an inactive form in the cytoplasm. Like NF- KB, IkBs are also members of a multigene family containing seven known mammalian members including IKBa, IKB, IKby, IKBS, Bcl-3, the precursor Rel-proteins, p100 and p105, and one Drosophila IKB named Cactus. The IKB family is characterized by the presence of multiple copies of ankyrin repeats, which are protein-protein interaction motifs that interact with NF-KB via the RHD.

Upon appropriate stimulation, IKB is phosphorylated by IKB kinases (IKKs), polyubiquitinated by a ubiquitin ligase complex, and degraded by the 26S proteosome.

Consequently, NF-KB is released and translocates into the nucleus to initiate gene expression.

NF-KB related transcription factors regulate the expression of a wide variety of genes that play critical roles in innate immune responses. Such NF-KB target genes include those encoding cytokines IL-1, IL-2, IL-6, IL-12, TNF-a, LTa, LT3, and GM-CSF), adhesion molecules ICAM, VCAM, endothelial leukocyte adhesion molecule [ELAM]), acute phase proteins SAA), and inducible enzymes iNOS and COX-2). In addition, it has been demonstrated recently that several evolutionary conserved antimicrobial peptides, P-defensins, are also regulated by NF-KB, a situation similar to Drosophila. Besides regulating the expression of molecules involved in innate immunity, NF-KB also plays a role in the expression of molecules important for adaptive immunity, such as MHC proteins, and the expression of critical cytokines such as IL-2, IL-12 and IFN-y. Finally NF-KB plays an important role in the overall immune response by affecting the expression of genes that are critical for regulating the apoptotic process, such as c-IAP-1 and c-IAP-2, Fas ligand, c-myc, p53, and cyclin D1.

Under normal conditions, NF-kappaB is rapidly activated upon microbial and viral invasion, and this activation usually correlates with resistance of the host to infection. However, persistent activation of NF-kappaB may lead to the production of excessive amounts of proinflammatory mediators such as IL-12 and TNF-alpha, resulting in tissue damage, as in insulin-dependent diabetes mellitus, atherosclerosis, Crohn's disease, organ failure, and even death of the host, as in bacterial infection-induced septic shock. It is interesting to note that in order to survive in the host, certain pathogens, such as Schistosoma japonica, Bordetella, Yersinia, Toxoplasma gondii and African Swine Fever Virus have evolved mechanisms to counteract or escape the host system by inhibiting NF-kappaB activation. On the other hand, some viruses, including HIV-1, CMV and SV-40, take advantage of NF-kappaB as a host factor that is activated at sites of infection.

Furthermore, the invention provides a method to explore alterations in gene expression in antigenpresenting cells such as dendritic cells in response to microbial exposure by analyzing a gene-expression profile of dendritic cells in response to microorganisms such as for example bacteria such as Escherichia coli, or other pathogenic bacteria, fungi or yeasts such as Candida albicans, viruses such as influenza virus and the effect of (simultaneous) treatment of these diseases with a signalling molecule according to the invention. For example, human monocyte-derived dendritic cells are cultured with one or more pathogens for 1-36 hours, and gene expression is analyzed using an oligonucleotide array representing a (be it large or small) set of genes.

When the pathogens regulate the expression of a core set of a distinct number of genes, these genes may be classified according to their kinetics of expression and function. Generally, within 4 hours of pathogen exposure, genes associated with pathogen recognition and phagocytosis will be down-regulated, whereas genes for antigen processing and presentation are up-regulated 8 hours post-exposure. Treatment of such dendritic cells with a signalling molecule according to the invention (be it simultaneous or before or after the treatment of the cells with the pathogen) allows studying the effect a signalling molecule according to the invention has on the effect a pathogen has on an antigen-presenting cell.

In short, the invention surprisingly provides a signalling molecule capable of modulating expression of a gene in a cell, the molecule being a tri- or tetrameric peptide, or a functional tri- or tetrameric amino acid analogue or derivative thereof. Of course, such signalling molecule can be longer, for example by extending it and/or C-terminally), with more amino acids or other side groups, which can for example be (enzymatically) cleaved off when the molecule enters the place of final destination. Such extension may even be preferable to prevent the signalling molecule from becoming active in an untimely fashion; however, the core or active fragment of the molecule comprises the aforementioned tri- or tetrameric peptide or analogue or derivative thereof. Such a peptide according to the invention exerts its biological function by regulating gene expression in an other way than a classically known membrane-impermeable signalling molecule acts, such as acetylcholine, growth factors, extracellular matrix components, (peptide)-hormones, neuropeptides, thrombin, i.e. not by cell-surface receptor mediated signalling.

In particular, the invention provides a modulator of NF-kappaB/Rel protein activation comprising a signalling molecule according to the invention. Such modulators are widely searched after these days. Furthermore, the invention provides use of a signalling molecule according to the invention for the production of a pharmaceutical composition for the modulation of gene expression.

Also, the invention provides a method for the treatment of bone disease such as osteoporosis comprising administering to a subject in need of such treatment a molecule comprising an oligopeptide peptide or functional analogue thereof, the molecule capable of modulating production of NO and/or TNF-alpha by a cell. Such a method of treatment is particularly useful in postmenopausal women that no longer experience the benefits of being provided with a natural source of several of the signalling molecules as provided herein, as physiologically derived from hCG and its breakdown products. Furthermore, the invention provides a method for the treatment of an inflammatory condition associated with TNF-alpha activity of fibroblasts, such as seen with chronic arthritis or synovitis, comprising administering to a subject in need of such treatment a molecule comprising an oligopeptide peptide or functional analogue thereof wherein the molecule is capable of modulating translocation and/or activity of a gene transcription factor present in a cell, in particular of the NF-kappaB factor. Such a treatment can be achieved by systemic administration of a signalling molecule according to the invention, but local administration in joints, bursae or tendon sheaths is provided as well. The molecule can be selected from table 6 or identified in a method according to the invention. It is preferred when the treatment comprises administering to the subject a pharmaceutical composition comprising an oligopeptide or functional analogue thereof also capable of reducing production of O NO by a cell, for example, wherein the composition Scomprises at least two oligopeptides or functional O(i1 analogues thereof, each capable of reducing production of ;NO and/or TNF-alpha by a cell, in particular wherein the at least two oligopeptides are selected from the group LQGV, AQGV and VLPALP.

Furthermore, the invention provides use of an Soligopeptide or functional analogue thereof capable of t reducing production of NO and/or TNF-alpha by a cell for (1 10 the production of a pharmaceutical composition for the treatment of an inflammatory condition or a post-meno- (I pausel condition, or a bone disease such as osteoporosis, or for the induction of weight loss. The term "pharmaceutical composition" as used herein is intended to cover both the active signalling molecule alone or a composition containing the signalling molecule together with a pharmaceutically acceptable carrier, diluent or excipient. Acceptable diluents of an oligopeptide as described herein in the detailed description are for example physiological salt solutions or phosphate buffered salt solutions. In one embodiment of the present invention, a signal molecule is administered in an effective concentration to an animal or human systemically, e.g. by intravenous, intra-muscular or intraperitoneal administration. Another way of administration comprises perfusion of organs or tissue, be it in vivo or ex vivo, with a perfusion fluid comprising a signal molecule according to the invention.

Topical administration, e.g. in ointments or sprays, may also apply, e.g. in inflammations of the skin or mucosal surfaces of for example mouth, nose and/or genitals.

Local administration can occur in joints, bursae, tendon sheaths, in or around the spinal cord at locations where nerve bundles branch off, at the location of hernias, in or around infarcted areas in brain or heart, etc. The administration may be done as a single dose, as a discontinuous sequence of various doses, or continuously for a period of time sufficient to permit substantial modulation of gene expression. In the case of a continuous administration, the duration of the administration may vary depending upon a number of factors which would readily be appreciated by those skilled in the art.

The administration dose of the active molecule may be varied over a fairly broad range. The concentrations of an active molecule which can be administered would be limited by efficacy at the lower end and the solubility of the compound at the upper end. The optimal dose or doses for a particular patient should and can be determined by the physician or medical specialist involved, taking into consideration well-known relevant factors such as the condition, weight and age of the patient, etc.

The active molecule may be administered directly in a suitable vehicle, such as e.g. phosphate-buffered saline (PBS) or solutions in alcohol or DMSO. Pursuant to preferred embodiments of the present invention, however, the active molecule is administered through a single dose delivery using a drug-delivery system, such as a sustained-release delivery system, which enables the maintenance of the required concentrations of the active molecule for a period of time sufficient for adequate modulation of gene expression. A suitable drug-delivery system would be pharmacologically inactive or at least tolerable. It should preferably not be immunogenic nor cause inflammatory reactions, and should permit release of the active molecule so as to maintain effective levels thereof over the desired time period. A large variety of alternatives are known in the art as suitable for purposes of sustained release and are contemplated as within the scope of the present invention. Suitable delivery vehicles include, but are not limited to, the following: microcapsules or microspheres; liposomes and other lipid-based release systems; viscous instillates; absorbable and/or biodegradable mechanical barriers and implants; and polymeric delivery materials, such as polyethylene oxide/polypropylene oxide block copolymers, polyesters, cross-linked polyvinylalcohols, polyanhydrides, polymethacrylate and polymethacrylamide hydrogels, anionic carbohydrate polymers, etc. Useful delivery systems are well known in the art.

A highly suitable formulation to achieve the active molecule release comprises injectable microcapsules or microspheres made from a biodegradable polymer, such as poly(dl-lactide), poly(dl-lactide-co-glycolide), polycaprolactone, polyglycolide, polylactic acid-coglycolide, poly(hydroxybutyric acid), polyesters or polyacetals. Injectable systems comprising microcapsules or microspheres having a diameter of about 50 to about 500 micrometers offer advantages over other delivery systems. For example, they generally use less active molecules and may be administered by paramedical personnel. Moreover, such systems are inherently flexible in the design of the duration and rate of separate drug release by selection of microcapsule or microsphere size, drug loading and dosage administered. Further, they can be successfully sterilized by gamma irradiation.

The design, preparation and use of microcapsules and microspheres are well within the reach of persons skilled in the art and detailed information concerning these points is available in the literature. Biodegradable polymers (such as lactide, glycolide and caprolactone polymers) may also be used in formulations other than microcapsules and microspheres; e.g. premade films and spray-on films of these polymers containing the active molecule would be suitable for use in accordance with the present invention. Fibers or filaments comprising the active molecule are also contemplated as within the scope of the present invention.

Another highly suitable formulation for a singledose delivery of the active molecule in accordance with the present invention involves liposomes. The encapsulation of an active molecule in liposomes or multilamellar vesicles is a well-known technique for targeted drug delivery and prolonged drug residence. The preparation and use of drug-loaded liposomes is well within the reach of persons skilled in the art and well documented in the literature.

Yet another suitable approach for single-dose delivery of an active molecule in accordance with the present invention involves the use of viscous installates. In this technique, high molecular weight carriers are used in admixture with the active molecule, giving rise to a structure which produces a solution with high viscosity. Suitable high molecular weight carriers include, but are not limited to, the following: dextrans and cyclodextrans; hydrogels; (cross-linked) viscous materials, including (cross-linked) viscoelastics; carboxymethylcellulose; hyaluronic acid; and chondroitin sulfate. The preparation and use of drug-loaded viscous instillates is well known to persons skilled in the art.

Pursuant to yet another approach, the active molecule may be administered in combination with absorbable mechanical barriers such as oxidized regenerated cellulose. The active molecule may be covalently or non-covalently ionically) bound to such a barrier, or it may simply be dispersed therein.

A pharmaceutical composition as provided herein is particularly useful for the modulation of gene expression by inhibiting NF-kappaB/Rel protein activation.

NF-kappaB/Rel proteins are a group of structurally related and evolutionarily conserved proteins (Rel). Well known are c-Rel, RelA (p65), RelB, NF-kappaB1 (p50 and its precursor p105), and NF-kappaB2 (p52 and its precursor p100). Most NF-kappaB dimers are activators of transcription; p50/p50 and p52/p52 homodimers repress the transcription of their target genes. All NF-kappaB/Rel proteins share a highly conserved NH2-terminal Rel homology domain (RHD). RHD is responsible for DNA binding, dimerization, and association with inhibitory proteins known as IkappaBs. In resting cells, NFkappaB/Rel dimers are bound to IkappaBs and retained in an inactive form in the cytoplasm. IkappaBs are members of a multigene family (IkappaBalpha, IkappaBbeta, IkappaBgamma, IkappaBepsilon, Bcl-3, and the precursor Rel-proteins, pl00 and p105. Presence of multiple copies of ankyrin repeats interact with NF-kappaB via the RHD (protein-protein interaction. Upon appropriate stimulation, IkappaB is phosphorylated by IkappaB Kinase (IKKs), polyubiquitinated by ubiquitin ligase complex, and degraded by the 26S proteosome. NF-kappaB is released and translocates into nucleus to initiate gene expression.

NF-kappaB regulation of gene expression includes innate immune responses: such as regulated by cytokines IL-1, IL-2, IL-6, IL-12, TNF-alpha, LT-alpha, LT-beta, GM-CSF; expression of adhesion molecules (ICAM, VCAM, endothelial leukocyte adhesion molecule [ELAM]), acute phase proteins (SAA), ilnducible enzymes (iNOS and COX-2) and antimicrobial peptides (beta-defensins). For adaptive immunity, MHC proteins IL-2, IL-12 and IFN-alpha are regulated by NF-kappaB. Regulation of overall immune response includes the regulation of genes critical for regulation of apoptosis (c-IAP-1 and c-IAP-2, Fas Ligand, c-myc, p53 and cyclin Dl.

Considering that NF-kappaB and related transcription factors are cardinal pro-inflammatory transcription factors, and considering that the invention provides a signalling molecule, such as an oligopeptide and functional analogues or derivatives thereof that are capable of inhibiting NF-kappaB and likely also other pro-inflammatory transcription factors, herein also called NF-kappaB inhibitors, the invention provides a method for modulating NF-kappaB activated gene expression, in particular for inhibiting the expression and thus inhibiting a central pro-inflammatory pathway.

The consequence of this potency to inhibit this proinflammatory pathway is wide and far-reaching. The invention for example provides a method to mitigate or treat inflammatory airway disease such as asthma.

Generally, asthma patients show persistent activation of NF-kappaB of cells lining the respiratory tract.

Providing these patients, for example, by aerosol application, with a signalling molecule according to the invention, such as LQGV or AQGV or MTRV or functional analogue or derivative thereof, will alleviate the inflammatory airway response of these individuals by inhibiting NF-kappaB activation of the cells. Such compositions can advantageously be made with signalling molecules that are taken up in liposomes.

As said, inflammation involves the sequential activation of signalling pathways leading to the production of both pro- and anti-inflammatory mediators.

Considering that much attention has focused on proinflammatory pathways that initiate inflammation, relatively little is known about the mechanisms that switch off inflammation and resolve the inflammatory response. The transcription factor NF-kB is thought to have a central role in the induction of pro-inflammatory gene expression and has attracted interest as a new target for the treatment of inflammatory disease. However NF-kB activation of leukocytes recruited during the onset of inflammation is also associated with pro-inflammatory gene expression, whereas such activation during the resolution of inflammation is associated with the expression of anti-inflammatory genes and the induction of apoptosis. Inhibition of NF-kB during the resolution of inflammation protracts the inflammatory response and prevents apoptosis. This shows that NF-kB has an antiinflammatory role in vivo involving the regulation of inflammatory resolution. The invention provides a tool to modulate the inflammation at the end phase, a signalling molecule or modulator as provided herein allows the modulation of the NF-kappaB pathway at different stages of the inflammatory response in vivo, and in a particular embodiment, the invention provides a modulator of NFkappaB for use in the resolution of inflammation, for example through the regulation of leukocyte apoptosis.

Useful oligopeptides can be found among those that accelerate shock.

The invention also provides a method to mitigate or treat neonatal lung disease, also called chronic lung disease of prematurity, a condition often seen with premature children who develop a prolonged pulmonary inflammation or bronchopulmonary dysplasia. Treating such premature children with an NF-kappaB inhibitor, such as oligopeptide LQGV, or functional analogue or derivative thereof, as provided herein allows such lung conditions to be prevented or ameliorated as well.

Recent advances in bone biology provide insight into the pathogenesis of bone diseases. The invention also provides a method of treatment of a post-menopausal condition such as osteoporosis comprising modulation and inhibition of osteoclast differentiation and inhibiting TNF-alpha induced apoptosis of osteoblasts, thereby limiting the dissolve of bone structures, otherwise so prominent in post-menopausal women that have no longer a natural source of hCG and thus lack the modulatory effect of the signal molecules that are derived of hCG as shown herein. The invention thus also provides a method of treatment of a bone disease, such as osteoporosis (which is often, but not exclusively, seen with post-menopausal women). Furthermore, NO and TNF-alpha modulators as provided herein inhibit the inflammatory response and bone loss in periodontitis. Furthermore, considering that there is a correlation between TNF-alpha activity and severity of clinical manifestations in ankylosing spondylitis, the invention provides the treatment of spondylitis by use of a signalling molecule as provided herein.

Furthermore, considering that an important pathogenic component in the development of insulindependent diabetes mellitus (type 1) comprises overactivation of the NF-kappaB pathway as seen in dendritic cells, treatment with an NF-kappaB inhibitor according to the invention will lead to reduced symptoms of diabetes, or at least to a prolonged time to onset of the disease.

Particularly effective oligopeptide signalling molecules according to the invention in this context are LQGV, MTRV, LPGC, which were shown herein to postpone onset of diabetes in an Non-obese Diabetic Mouse (NOD). Another approach to treatment of diabetes, in particular insulin -independent diabetes (type comprises inhibition of the PPARgamma cascade with an oligopeptide signalling molecule or functional analogue or derivative thereof.

Another use that is provided relates to a method for combating or treating auto-immune disease. A non-limiting list of immune diseases includes: Hashimoto's thyroiditis, primary myxoedema thyrotoxicosis, pernicious anaemia, autoimmune 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, autoimmune haemolytic 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 erythematosus, and systemic lupus erythematosus.

Another use that is provided relates to a method for combating or treating infections caused by microorganisms, in particular those infections that are caused by micro-organisms that activate the NF-kappaB pathway during infections.

Such microorganisms are manifold, including bacteria, viruses, fungi, and protozoa, but other pathogens worms) can have the same effect.

Activation of the NFkappaB pathway by a microbial infection in general occurs via activation of the Tolllike receptor pathway. The invention provides a method to modulate and in particular to inhibit parts of gene expression that are related to the inflammatory responses of an organism that are generally activated through one of the Toll-like receptor pathways.

Toll-like receptor-mediated NF-kappaB activation is central in recognition of pathogens by a host. Such recognition of pathogens generally occurs through germline-encoded molecules, the so-called pattern recognition receptors (PRRs). These PRRs recognize widespread pathogen-associated molecular patterns (PAMPs). The pattern recognition receptors are expressed as either membrane-bound or soluble proteins. They include CD14, beta2-integrins (CD11/CD18), C-type lectins, macrophage scavenger receptors, complement receptors (CR1/CD35, CR2/CD21) and Toll-like receptors (TLRs). TLRs are distinguished from other PRRs by their ability to recognize and discriminate between different classes of pathogens. TLRs represent a family of transmembrane proteins that have an extracellular domain comprising multiple copies of leucine-rich repeats (LRRs) and a cytoplasmic Toll/IL-1R (TIR) motif that has significant homology to the intracellular signalling domain of the type I IL-1 receptor (IL-1RI). Therefore, TLRs are thought to belong to the IL-1R superfamily.

Pathogen-associated molecular patterns (PAMPS) are not expressed by hosts but are components of the pathogenic micro-organism. Such PAMPS comprise bacterial cell wall components such as lipopolysaccharides (LPS), lipoproteins (BLP), peptidoglycans (PGN), lipoarabinomannan (LAM), lipoteichoic acid (LTA), DNA containing unmethylated CpG motifs, yeast and fungal cell wall mannans and beta-glucans, double-stranded RNA, several unique glycosylated proteins and lipids of protozoa, and so on.

Recognition of these PAMPS foremost provides for differential recognition of pathogens by TLRs. For example, TLR2 is generally activated in response to BLPs, PGNs of gram-positive bacteria, LAM of mycobacteria, and mannans of yeasts, whereas TLR4 is often activated by LPS of gram-negative bacteria and LTA of gram-negative bacteria; also a secreted small molecule MD-2 can account for TLR4 signalling.

Several oligopeptides capable of modulating gene expression according to the invention have earlier been tested, both ex vivo and in vivo, and in small animals, but a relationship with modulation of gene expression was not brought forward. A beneficial effect of these oligopeptides on LPS-induced sepsis in mice, namely the inhibition of the effect of the sepsis, was observed.

Immunomodulatory effects with these oligopeptides have been observed in vitro and in ex vivo such as in T-cell assays showing the inhibition of pathological Thl immune responses, suppression of inflammatory cytokines (MIF), increase in production of anti-inflammatory cytokines (IL-10, TGF-beta) and immunomodulatory effects on antigen-presenting cells (APC) like dendritic cells, monocytes and macrophages.

Now that the insight has been provided that distinct synthetic oligopeptides or functional analogues or derivatives thereof, for example those that resemble breakdown products which can be derived by proteolysis from endogenous proteins such as hCG, can be used to modulate gene expression, for example by NF-kappaB inhibition, such oligopeptides find much wider application. Release of active NF-kappaB in cells is now known to occur after a variety of stimuli including treating cells with bacterial lipopolysaccharide (LPS) and the interaction with a Toll-like receptor (see for example Guha and Mackman, Cell. Sign. 2001, 13:85-94). In particular, LPS stimulation of dendritic cells, monocytes and macrophages induces many genes that are under the influence of activation by transcription factors such as NF-kappaB, p50, EGR-1, IRF-1 and others that can be modulated by a signalling molecule according to the invention. Considering that LPS induction of EGR-1 is required for maximal induction of TNF-alpha, it is foreseen that inhibition of EGR-1 considerably reduces the effects of sepsis seen after LPS activation. Now knowing the gene modulatory effect of the signalling molecules such as peptides as provided herein allows for rational design of signal molecule mixtures that better alleviate the symptoms seen with sepsis. Administration of such a signalling molecule or mixture preferably occurs systematically, e.g. by intravenous or intraperitoneal administration. In a further embodiment, such treatment also comprises the use of for example an antibiotic, however, only when such use is not contraindicated because of the risk of generating further toxin loads because of lysis of the bacteria subject to the action of those antibiotics in an individual thus treated.

Other use that is contemplated relates to a method for combating or treating viral infections, in particular those infections that are caused by viruses that activate the NF-kappaB pathway during infections. Such virus infections are manifold; classical examples are hepatitis B virus-induced cell transformation by persistent activation of NF-kappaB. Use of a signalling molecule according to the invention is herein provided to counter or prevent this cell transformation.

Other disease where persistent NF-kappaB activation is advantageously inhibited by a signalling molecule according to the invention is a transplantation-related disease such as transplantation-related immune responses, graft-versus-host-disease, in particular with bone-marrow transplants, acute or chronic xeno-transplant rejection, and post-transfusion thrombocytopenia.

Another case where persistent NF-kappaB activation is advantageously inhibited by a signalling molecule according to the invention is found in the prevention or mitigation of ischemia-related tissue damage seen after infarcts, seen for example in vivo in brain or heart, or ex vivo in organs or tissue that is being prepared or stored in preparation of further use as a transplant.

Ischemia-related tissue damage can now be mitigated by perfusing the (pre)ischemic area with a signalling molecule according to the invention that inhibits NFkappaB activation. Examples of conditions where ischemia (also called underperfusion) plays a role include eclampsia which can be ascribed to focal cerebral ischemia resulting from vasoconstriction, consistent with the evidence of changes detected by new cerebral imaging techniques. The liver dysfunction intrinsic to the HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome could also be attributed to the effects of acute underperfusion. Other conditions of ischemia are seen after coronary occlusion, leading to irreversible myocardial damage produced by prolonged episodes of coronary artery occlusion and reperfusion in vivo, which has already been discussed in PCT/NL01/00259 as well.

Now that the insight has been provided that distinct synthetic oligopeptides, for example those that resemble breakdown products which can be derived by proteolysis from endogenous proteins such as hCG, can be used to modulate gene expression, for example by NF-kappaB inhibition, the oligopeptides find much wider application. For example, the invention provides a method for perfusing a transplant with a perfusing fluid comprising at least one signalling molecule according to the invention; ischemic or pre-implantation damage due to activation of NF-kappaB in the transplant can then be greatly diminished, allowing a wider use of the transplants.

The invention provides a signalling molecule useful in modulating expression of a gene in a cell. Several examples of the use of such a signalling molecule for the production of a pharmaceutical composition for the treatment of medical or veterinary conditions are herewith given. In one embodiment, the invention provides such use in the treatment of an immune-mediated disorder, in particular of those cases whereby a central role of NF-kappaB/Rel proteins in the immune response is found.

However as said, modulating gene expression via modulating activity of other transcription factors, such as AP-l or PPARgamma, and others is also provided, now that the gene modulating role of signalling molecules such as the oligopetides or analogues or derivatives thereof is understood. As also said, now knowing that oligopeptides, likely breakdown products, play such a central role in modulation of gene expression, the invention provides straightforward ways for identifying further gene expression modulating oligopeptides, and provides synthetic versions of these, and analogues and derivatives thereof for use in a wide variety of disorders and for use in the preparation of a wide variety of pharmaceutical compositions. Examples of such treatment and useful pharmaceutical compositions are for example found in relation to conditions wherein the immune-mediated disorder comprises chronic inflammation, such as diabetes, multiple sclerosis or acute or chronic transplant rejection, in particular in those cases whereby antigen-presenting cells (APC's or dendritic cells (DCs) are enhanced by (overactive) and persistent NF-kappaB expression or wherein the immune-mediated disorder comprises acute inflammation, such as septic or anaphylactic shock or acute transplant rejection. Other immune-mediated disorders that can be treated with a pharmaceutical composition comprising a signalling molecule according to the invention comprise auto-immune disease, such as systemic lupus erythematosus or rheumatoid arthritis (in particular by inhibiting IL-8 and/or IL-15 production by inhibiting NF-kappaB activity on the expression of these genes), allergy, such as asthma or parasitic disease, overly strong immune responses directed against an infectious agent, such as a virus or bacterium (in particular responses that include rapid hemorrhagic disease caused by infection with organisms such as Yersinia pestis, Ebola-virus, Staphylococcus aureus in cases of tampon-disease), bacterial (such as meningococcal) or viral meningitis and/or encephalitis, and other life-threatening conditions). Such overly strong responses are seen with for example pre-eclampsia, recurrent spontaneous abortions (RSA) or preterm parturition or other pregnancy-related disorders. Especially with forms of eclampsia/pre-eclampsia that are associated with genetically programmed increased production of tumourgrowth factor beta-l, treatment according to the invention is recommended. Also, in situations where RSA is likely attributable to increased IL-10 levels during pregnancy, or to increased TNF-alpha activity, for example due to the presence of an unfavourable allele, in particular of a G to A polymorphism in the promotor of the gene encoding TNF-alpha, treatment with a pharmaceutical composition as provided herein is recommended. Treatment directed at such pregnancy-related immune disorders is herein also provided by inhibiting NF-kappaB activity directed at activating natural killer (NK) cell activity. Also, LPS-induced reduced fertility, or abortions, seen in pregnant sows can be reduced by applying a signalling molecule or method as provided herein.

Such use in treatment of an immune-mediated disorder preferably comprises regulating relative ratios and/or cytokine activity of lymphocyte, dendritic or antigenpresenting cell subset-populations in a treated individual, in particular wherein the subset populations comprise Thl or Th2, or DC1 or DC2 cells. Other embodiments of the invention comprise use of a signalling molecule according to the invention for the manufacture of a medicament for modulating a cardiovascular or circulatory disorder, such as coronary arterial occlusion and also in a pregnancy related cardiovascular or circulatory disorder.

Furthermore, the invention provides a pharmaceutical composition for modulating a cardiovascular or circulatory disorder, in particular a pregnancy related cardiovascular or circulatory disorder, comprising a signalling molecule according to the invention or mixtures thereof. Such a composition finds its use in a method for modulating a cardiovascular or circulatory disorder, in particular a pregnancy related cardiovascular or circulatory disorder, comprising subjecting an animal (in particular a mammal) to treatment with at least one signalling molecule according to the invention. Non-pregnancy related disorders that are for example related to hypercholesterolemia are susceptible to treatment with a signalling molecule according to the invention as well. For example, apolipoprotein E (apo E) deficiency is associated with a series of pathological conditions including dyslipidemia, atherosclerosis, Alzheimer's disease, increased body weight and shorter life span. Inheritance of different alleles of the POLYMORPHIC apoE gene is responsible for 10% of the variation in plasma cholesterol in most populations. Individuals HOMOZYGOUS for one variant, apoE2, can develop type III dysbetalipoproteinaemia if an additional genetic or environmental factor is present.

Some much rarer alleles of apoE produce dominant expression of this disorder in heterozygous individuals.

ApoE is a ligand for the LDL receptor and its effects on plasma cholesterol are mediated by differences in the affinity of the LDL receptor for lipoproteins carrying variant apoE proteins. The factors that regulate apoE gene transcription have been investigated extensively by the expression of gene constructs in transgenic mice and involve complex interactions between factors that bind elements in the 5' promoter region, in the first intron and in 3' regions many kilobases distant from the structural gene. Deletion of the apoE gene is associated with changes in lipoprotein metabolism (plasma total cholesterol), HDL cholesterol, HDL/TC, and HDL/LDL ratios, esterification rate in apo B-depleted plasma, plasma triglyceride, hepatic HMG-CoA reductase activity, hepatic cholesterol content, decreased plasma homocyst(e)ine and glucose levels, and severe atherosclerosis and cutaneous xanthomatosis. The invention provides a method and a signalling molecule for the treatment of conditions that are associated with dysfunctional LDL receptors such as ApoE and other members of the apolipoprotein family. In particular, use of a signalling molecule comprising GVLPALPQ and/or VLPALP or a functional analogue or derivative thereof is preferred.

The invention also provides use of a signalling molecule for the preparation of a pharmaceutical composition or medicament and methods of treatment for various medical conditions that are other than use in the preparation of a pharmaceutical composition for the treatment of an immune-mediated disorder or a method of treatment of an immune-mediated disorder. For example, the invention provides topical application, for example in an ointment or spray comprising a signal molecule according to the invention, for the prevention or mitigation of skin afflictions, such as eczemas, psoriasis, but also of skin damage related to overexposure to UV-light.

Also, use is contemplated in palliative control, whereby a gene related to prostaglandin synthesis is modulated such that COX2 pathways are effected.

Furthermore, the invention also provides use of a signalling molecule for the preparation of a pharmaceutical composition or medicament and methods of treatment for various medical conditions that are other than use in the preparation of a pharmaceutical composition for the treatment of wasting syndrome, such as the treatment of particular individuals that are suffering from infection with HIV or a method of treatment of wasting syndrome of such individuals.

In one embodiment, the invention provides the use of a signalling molecule according to the invention for the preparation of a pharmaceutical composition or medicament for modulating angiogenesis or vascularization, in particular during embryonal development, or after transplantation to stimulate vascularization into the transplanted organ or inhibit it in a later phase.

Signalling molecules that effect angiogenesis are disclosed herein in the detailed description.

Use as provided herein also comprises regulating TNF-alpha receptor CD27) expression on cells, thereby modulating the relative ratios and/or cytokine activity of lymphocyte, dendritic or antigen presenting cell subset-populations in a treated individual. As for example described in the detailed description, the particular oligopeptide according to the invention is capable of down-regulating CD27 expression on cells of the T-cell lineage.

Down-regulating TNF-alpha itself is also particularly useful in septic-shock-like conditions that not only display increased TNF-alpha activity but display further release of other inflammatory compounds, such as NO. NO production is a central mediator of the vascular and inflammatory response. Our results show that inflammatory cells like macrophages stimulated with an inflammatory active compound such as LPS produce large amounts of NO. However, these cells co-stimulated with most of the NMPF peptides (NMPF peptide 1 to 14, 43 to 66 and 69), even in a very low dose (1 pg/ml), inhibited production of NO. Typical septic-shock-like conditions that can preferably be treated by down-regulating TNFalpha and NO production comprise disease conditions such as those caused by Bacillus anthracis (anthrax) and Yersinia pestis toxins or infections with these microorganisms likely involved in bio-terrorism. Anthrax toxin is produced by Bacillus anthracis, the causative agent of anthrax, and is responsible for the major symptoms of the disease. Clinical anthrax is rare, but there is growing concern over the potential use of B. anthracis in biological warfare and terrorism. Although a vaccine against anthrax exists, various factors make mass vaccination impractical. The bacteria can be eradicated from the host by treatment with antibiotics, but because of the continuing action of the toxin, such therapy is of little value once symptoms have become evident. Thus, a specific inhibitor of the toxin's action will prove a valuable adjunct to antibiotic therapy. The toxin consists of a single receptor-binding moiety, termed "protective antigen" and two enzymatic moieties, termed "edema factor" (EF) and "lethal factor" (LF).

After release from the bacteria as nontoxic monomers, these three proteins diffuse to the surface of mammalian cells and assemble into toxic, cell-bound complexes.

Cleavage of PA into two fragments by a cell-surface protease enables the fragment that remains bound to the cell, PA63, to heptamerize and bind EF and LF with high affinity. After internalization by receptor-mediated endocytosis, the complexes are trafficked to the endosome. There, at low pH, the PA moiety inserts into the membrane and mediates translocation of EF and LF to the cytosol. EF is an adenylate cyclase that has an inhibitory effect on professional phagocytes, and LF is a protease that acts specifically on macrophages, causing their death and the death of the host.

Furthermore, the invention provides use of a signalling molecule according to the invention for the production of a pharmaceutical composition for the modulation of gene expression and use in a method of treatment by modulating gene expression. Particular genes that may be modulated by particular peptides are provided herein.

Down-regulating TNF-alpha itself, and/or a receptor for TNF-alpha, as is herein also provided, is also beneficial in individuals with Chagas cardiomyopathy.

Also, use of a signalling molecule according to the invention for the preparation of a pharmaceutical composition for modulation of vascularization or angiogenesis in wound repair, in particular of burns, is herein provided. Also, use of a pharmaceutical composition as provided herein is provided in cases of post-operative physiological stress, whereby not only vascularization will benefit from treatment, but the general well-being of the patient is improved as well.

Another use of a signalling molecule according to the invention comprises its use for the preparation of another pharmaceutical composition for the treatment of cancer. Such a pharmaceutical composition preferably acts via modulating and up-regulating apoptotic responses that are classically down-regulated by NF-kappaB activity.

Inhibiting the activity with a signalling molecule according to the invention allows for increased cell death of tumorous cells. Another anti-cancerous activity of a signalling molecule as provided herein comprises down-regulation of c-myb, in particular in the case of hematopoietic tumors in humans. In this context, downregulation of 14.3.3 protein is also provided.

A further use of a signalling molecule according to the invention comprises its use for the preparation of a further pharmaceutical composition for the treatment of cancer. Such a pharmaceutical composition preferably acts via modulating and down-regulating transferrine receptor availability, in particular on tumorous cells.

Transferrine receptors are classically up-regulated by NF-kappaB activity. Inhibiting the activity with a signalling molecule according to the invention allows for reduced iron up-take and increased cell death of tumorous cells. In particular, erythroid and thromboid cells are susceptible to the treatment.

Yet a further use of a signalling molecule according to the invention comprises its use for the preparation of yet another pharmaceutical composition for the treatment of cancer, in particular of cancers that are caused by viruses, such as is the case with retroviral-induced malignancies and other viral-induced malignancies. Such a pharmaceutical composition preferably acts via modulating and down-regulating cell-proliferative responses that are classically up-regulated by virus-induced transcriptional or NF-kappaB activity. Inhibiting the activity with a signalling molecule according to the invention allows for decreased proliferation and increased cell death of tumorous cells. Such a pharmaceutial composition may also act via modulating angiogenic responses induced by IL-8, whereby for example inhibition of IL-8 expression via inhibition of transcription factor AP-I or NF-kappaB expression results in the inhibition of vascularization-dependent tumor growth.

Furthermore, the invention provides the use of a signalling molecule for the preparation of a pharmaceutical composition for optimizing human or animal fertility and embryo survival, and a method for optimizing fertility and embryo survival. In particular, the invention provided for a method and composition allowing the down-regulation of TNF-alpha in the fertilized individual, optimally in combination with a composition and method for up-regulating IL-10 in the individual. Such a composition and method find immediate use in both human and veterinary medicine.

Also, the invention provides the use of a signalling molecule for the preparation of a pharmaceutical composition for modulating the body weight of an individual, in particular by modulating gene expression of a gene under influence of peroxisome proliferatoractivated receptor gamma (PPARgamma) activation and lipid metabolism by applying a signalling molecule according to the invention, and a method for modulating body weight comprising providing an individual with a signalling molecule according to the invention.

A further use of a signalling molecule as provided herein lies in the modulation of expression of a gene in a cultured cell. Such a method as provided herein comprises subjecting a signalling molecule according to the invention to the cultured cell. Proliferation and/or differentiation of cultured cells (cells having been or being under conditions of in vitro cell culture known in the art) can be modulated by subjecting the cultured cell to a signalling molecule according to the invention. It is contemplated that for example research into proliferation or differentiation of cells, such as stemcell research, will benefit greatly from understanding that a third major way of effecting gene modulation exists and considering the ease of application of synthetic peptides, and analogues or derivatives thereof.

Furthermore, it is contemplated that a signalling molecule as provided herein finds an advantageous use as a co-stimulatory substance in a vaccine, accompanying modern day adjuvants or replacing the classically used mycobacterial adjuvants, especially considering that certain mycobacteria express hCG-like proteins, of which it is now postulated that these bacteria have already made use of this third pathway found in gene modulation as provided herein by providing the host with breakdown products mimicking the signalling molecules identified herein. Treatment and use of the compositions as provided herein is not restricted to animals only, plants and other organisms are also subject to this third pathway as provided herein. Furthermore, now that the existence of such a pathway has been demonstrated, it is herein provided to make it a subject of diagnosis as well, for example to determine the gene modulatory state of a cell in a method comprising determining the presence or absence of a signalling molecule as provided herein or determining the presence or absence of a protease capable of generating such a signalling molecule from a (preferable endogenous) protein.

Cells react to environmental and intrinsic changes, which they perceive through extracellular and inter- as well as intracellular signals. The nature of these signals can be either for example physical or chemical.

Moreover, different classes of molecules present in blood react to each other and induce a cascade of reactions that have direct effects on other molecules and/or eventually lead to cellular responses, for example complement system and blood coagulation proteins.

Many genes are regulated not by a signalling molecule that enters the cells but by molecules that bind to specific receptors on the surface of cells for example receptors with enzymatic activity (receptor tyrosine kinases, receptor-like protein tyrosine phosphatases, receptor serine/threonine kinases, histidine kinases, guanylyl cyclases) and receptors without enzymatic activity (cytokine receptors, integrins, G-proteincoupled receptors). Interaction between cell-surface receptors and their ligands can be followed by a cascade of intracellular events that modulate one or more intracellular-transducing proteins, including variations in the intracellular levels of so-called second messengers (diacylglycerol, Ca 2 cyclic nucleotides, inositol(1,4,5) trisphosphate, phosphatidylinositol(3,4,5) trisphosphate, phosphatidylinositol transfer protein (PITP)). This leads to the activation or inhibition of a so-called "effector protein". The second messengers in turn lead to changes in protein for example protein phosphorylation through the action of cyclic AMP, cyclic GMP, calcium-activated protein kinases, or protein kinases (for example AGC group serine/threonine protein kinases, CAMK group serine/threonine protein kinases, CMGC group serine/threonine kinases, protein tyrosine kinase group, or others like MEK/Ste7p). Phosphorylation by protein kinases is one of the regulatory mechanisms in signal transmission that modulate different cellular pathways such as Ras/MAPK pathway, MAP kinase pathway, JAK-STAT pathway, wnt-pathway. In many instances, this all results in altered gene expression (for example genes for the regulation of other genes, cell survival, growth, differentiation, maturation, functional activity).

Many of the responses to binding of ligands to cellsurface receptors are cytoplasmatic and do not involve immediate gene activation in the nucleus. In some instances, a pre-existing inactive transcription factor following a cell-surface interaction is activated that leads to immediate gene activation. For example, the protein NF-kappaB, which can be activated within minutes by a variety of stimuli, including membrane receptors (for example pattern recognition receptors like Toll-like receptor binding to pathogen-associated molecular patterns), inflammatory cytokines such as TNF-a, IL-1, Tcell activation signals, growth factors and stress inducers.

Our genomic experiment with NMPF peptide LQGV showed very immediate effects on signal transduction and gene regulation since the cells were treated with the peptide for only four hours. In this short period of time LQGV down-regulated at least 120 genes and up-regulated at least 6 genes in the presence of a strong stimulator (PHA/IL-2 stimulated T-cell line demonstrating the profound effect on signalling molecules according to the invention and modulatory effect on gene expression.

The genes affected by LQGV include oncogenes, genes for transcription factors, intracellular enzymes, membrane receptors, intracellular receptors, signal transducing proteins (for example kinases) and some genes for unkown molecules. This shows that LQGV as an example of the synthetic signalling molecule (oligopeptide or functional analogue or derivative thereof) as described here, has a broad spectrum of effects at different extracellular and intracellular levels. In addition, our HPLC/MS data have shown the presence of LQGV in the nucleus of a macrophage cell line (RAW267.4) within a half hour and also indicates the direct effects on DNA level as well as at an intracellular level, which is further supported by NFkappaB experiments. The ultimate modulatory effect of LQGV is dependent on, for example, type of the cell, differentiation and maturation status of the cell, the functional status and the presence of other regulatory molecules. This was evident by a shock experiment in which different peptides had similar or different effects on the disease. The same results were obtained with DC, fertilized chicken egg experiments, and CAO experiments; effects were dependent on type of co-stimulation (GM-CSF alone or in combination with LPS, or VEGF) and time of the treatment. Due to this, tri- or tetrameric peptides according to the invention have the ability to modulate cellular responses at different levels.

The invention is further explained in the following examples and accompanying discussion without limiting the invention thereto. It is to be noted that these examples discuss implications of the invention of which it will be clear to the skilled person that they provide a general teaching applicable over a broad scope.

EXAMPLE I Introduction The immune system has two arms: the non-specific (innate) and specific (adaptive) immune defense, both of which have cellular and humoral components. T and B cells account for the antigen-specific cellular and humoral (antibodies) immune defense. On the other hand, monocytes/macrophages, granulocytes, NK cells, mast cells and likely also gd T cells are the cellular components of the innate immune system, while complement, acute phase proteins, lysozyme and mannose-binding lectin (MBL) are major humoral components of the innate immune system. The adaptive system has been studied most because of its specificity and lasting effectiveness in eliminating infections. The innate system is thought to play a critical role in the most fundamental immune challenge in mammals: 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, the so called signal 1. Full responses often require adjuvants (such as endotoxin), which, through interaction with the innate immune system, produce signal 2, in the form of costimulatory surface molecules or cytokines. Signal 2 appears to determine the biological significance of antigens and communicates this information to the adaptive system. In fact, it is believed that this signal 2 instructs the adaptive system to either respond or not (Immunology Today 20, 114-118).

So, the innate system is an integral part of the specific immune defense.

During pregnancy there are increased numbers of monocytes and granulocytes from the first trimester onwards. It has been found that, in normal pregnancy, circulating monocytes and granulocytes have activated phenotypes, in some ways comparable with changes observed in systemic sepsis (Am. J. Obstet. Gynecol. 179, 80-86).

Others have shown increased monocyte phagocytosis and respiratory burst activity. Monocyte surface expression of the endotoxin receptor CD14 is increased, and in response to endotoxin monocytes from normal pregnant women produce more of the proinflammatory type I cytokine IL-12 (Immunology Today 20, 114-118). Other studies have similarly found granulocyte activation in pregnancy as well as changes in plasma levels of soluble innate factors typical of an acute phase response (Am. J.

Reprod. Immunol. Microbiol. 15, 19-23).

During pregnancy the maternal immune system is modulated, resulting in suppression of maternal immune responses against the fetus, while maintaining the mother's resistance to infection. We have shown the presence of immunoregulator (IR, W99-59617) which we named in this document NMPF (Natural immuno-Modulatory Pregnancy-Factor(s)) that regulate both innate and adaptive immune systems in a stimulatory and antagonistic way (W099-59617). These factors include, but are not limited to, commercial hCG preparations derived from human pregnancy urine, b-hCG preparations, certain peptides of b-hCG, certain combinations of b-hCG peptides and certain gel filtration chromatography fractions of commercial hCG preparations and human pregnancy urine.

Balance in these factors is crucial for proper regulation of the maternal immune system. For example, the overactivation of the innate system can cause problems in the progression of the pregnancy itself. Pre-eclampsia is one of such condition characterized by hyperactivation of the innate immune system. Recently it has been also suggested that the chronic misbalance between the two immune systems could be the basis of type II diabetes (noninsulin dependent diabetes mellitus) and other diseases as well (W99-59617) Several cytokines have been proposed to play an important role in balancing the immune system. One such cytokine which plays an important role in the innate immune defense and in the regulation of inflammatory responses is macrophage migration inhibitory factor

(MIF)

MIF was originally identified by its ability to prevent the migration of macrophages out of capillary tubes. Since then, the expression of MIF activity has been found at a variety of inflammatory loci, suggesting its role in regulating the function of macrophages in host defense (Science 153, 80-82; J. Exp. Med. 137, 275- 288). First described as a T-cell cytokine, recently, MIF is identified to be a peptide also released by pituitary cells in response to infection and stress (Nature 365, 756-759; Nature 377, 68-71). Originally considered to be the target of MIF action, monocytes and macrophages have been found to be a main source of MIF that is released after exposure to bacterial endo- and exotoxins and to cytokines. Once released, MIF induces the expression of proinflammatory mediators by macrophages and activated T cells, thereby strongly promoting inflammatory and immune responses (Nature Medicine 6,164-170). The critical regulatory role of MIF within the immune system is further underscored by the finding that MIF is induced by glucocorticoids and has the unique ability to override the anti-inflammatory and immunosuppressive effects of glucocorticoids on macrophages and T cells. Thus, MIF and glucocorticoids function as a physiological counterregulatory dyad that controls host inflammatory and immune responses (Proc. Natl. Acad. Sci. USA 93, 7849- 7854). Anti-MIF antibodies reduce the inflammation in experimental models of glomerulonephritis, arthritis, and allograft rejection, confirming the role of MIF in the regulation of inflammatory responses. Elevated concentrations of MIF have also been detected in alveolar air spaces of patients with the adult respiratory distress syndrome (ARDS). Recent studies have also shown that MIF is an important mediator of lethal endotoxemia and staphylococcal toxic shock, playing a critical role in the pathogenesis of septic shock. Besides the functions in the immune system, MIF has also other activities. For instance, MIF mRNA and protein are expressed in brain, embryonic eye lens and differentiating epidermal cells, suggesting its pivotal role in the regulation of the neuroendocrine system, cell growth and differentiation. A number of reports showed the presence of MIF in various organs and tissues: dermal vessels constitutively express MIF and can be strongly activated to express MIF in acute/chronic inflammations such as eczema and psoriasis. MIF expression on endothelium may provide an important differentiogenic signal for mononuclear phagocytes on their way to the tissue site.

One of the mechanisms of immune regulation that we detect during pregnancy is through modulation of the innate and adaptive immune defenses by NMPF. By way of example, but not limited to, acting directly or indirectly on regulatory cells of the APC compartment (such as DC1, DC2) or on lymphocytes (regulatory T cells), NMPF biases activated T lymphocytes towards Th2 immune response. The suppression of Thl immune responses may be compensated by the stimulation of the innate immune defense by NMPF which could explain the maintenance of maternal resistance to infection.

Recently, it has been shown that in some instances such compensatory mechanism (stimulation of innate immunity) could be more dominant and may account for abnormal pregnancy: pre-eclampsia.

Pre-eclampsia is a common, pregnancy-specific syndrome defined by clinical findings of elevated blood pressure combined with proteinuria and edema. The incidence has been reported to be between two and seven per cent of all pregnancies. The clinical findings become manifested mostly late in pregnancy. The disease can progress rapidly, at times without warning, to a lifethreatening disease. Expedient delivery initiates the resolution of pre-eclampsia but is a major cause of fetal and maternal morbidity and mortality.

Roberts et al in their classic article gathered the evidence to invoke activation of maternal endothelium as an underlying process. Generalized maternal endothelial cell dysfunction allowed most, if not all, clinical aspects to be potentially explained by a single, unifying process: hypertension through disturbed endothelial control of vascular tone, fluid retention by increased endothelial permeability, and clotting dysfunction resulting from abnormal endothelial expression of procoagulant. Eclampsia can be ascribed to focal cerebral ischemia resulting from vasoconstriction, consistent with the evidence of changes detected by new cerebral imaging techniques. The liver dysfunction intrinsic to the HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome could also be attributed to the effects of acute underperfusion.

Endothelial cells can be activated in several different ways that are potentially relevant to the origins of pre-eclampsia, and several candidate factors have emerged, including free fatty acids, lipoproteins, oxidized lipoproteins or lipid peroxides, tumor necrosis factor alpha (TNF-a), fibronectin degradation products, and deported syncytiotrophoblastic microvillous fragments. The source of the factors that lead to endothelial cell dysfunction has not been determined with certainty, but the evidence points to the placenta.

In addition to endothelial dysfunction there is substantial published evidence that there is systemic activation of the maternal inflammatory cell responses in pre-eclampsia. Both granulocytes and monocytes are activated. There is increased release of the proinflammatory cytokines TNF-a and its 2 soluble receptors, interleukin 6 (IL-6) and soluble phospholipase A2 (an important mediator of inflammatory reactions) into the circulation. It is well known that the clotting system is abnormally activated, and complement systems are similarly affected. Postmortem observations indicate that in some circumstances the lethal pathologic condition resembles that of the Shwartzmann reaction, a particular form of inflammatory response to endotoxin that has been characterized in experimental animals.

Since the above mentioned characteristics of preeclampsia resemble that of septic shock, we identified that also NMPF (IR) factor(s) are involved in preeclampsia that can worsen septic shock or sepsis. We addressed this by using a high dose LPS animal model for septic shock. Since in the urine of pre-eclamptic patients high levels of nicked hCG b-subunits are present, we also tested these nicked subunits to find out whether they worsen septic shock and so behave like MIF, which is an important mediator of lethal endotoxemia and staphylococcal toxic shock.

Material and Methods NMPF purification: To analyse the NMPF from commercial hCG preparations, we used a Shimadzu HPLC system equipped with Alltech macrosphere size exclusion (GPC) column of 60A, 100A or 300A (250 x 4.6 mm and 300 x 7.5 mm). The separation ranges of the columns were 28,000 250, 2500 350,00 and 1,200,000 7,500 Dalton, respectively. External molecular weight standards were employed to calibrate the column elution positions. The markers used were: aprotinin (6,500 Da), cytochrome C (12,400), carbonic anhydrase (29,000), albumin (66,000) and blue dextran (2,000,000).

To analyze NMPF, three different hCG preparations were used: NMPF-PG (Pregnyl; Organon; OSS, The Netherlands), NMPF-A (APL; Weyth Ayerst; Philadelphia, USA) and NMPF-PR (Profasi; Serono, Rome, Italy). As running buffer 50mM ammonium bicarbonate buffer containing ethanol vol/vol) was used. Sample load volume was 10-50 ml for the 250 x 4.6 mm column and 200 ml for the 300 x 7.5 mm column. The flow rate for the 250 x 4.6 mm and 300 x 7.5 mm columns were 0.5 ml/min for min. and 1-2 ml/min for 45 min, respectively.

First trimester pregnancy urine (2 litres) was collected in a bottle from a healthy volunteer and was refrigerated until delivered at the laboratory within 2 days. Upon delivery, 1 gram per litre of sodium azide was added and the pH was adjusted to 7.2-7.4 with sodium hydroxide and allowed to sediment for 1 hour at room temperature Approximately, 75% of the supernatant was decanted and the remainder close to the precipitate was centrifuged (10 min at 25,000 rpm at 40 0 C) to remove sediment and added to the rest of the supernatants. The supernatants were filtered through 0.45 mm in a Minitan (Millipore) transversal filtration set-up. Subsequently, the filtrate (2 litre) was concentrated in an Amicon ultrafiltration set-up equipped with an YM Diopore membrane with a 10 kDa cut-off. The final volume (250 ml) was dialysed against 2 changes of 10 litres of Milli Q water. Next the sample was further concentrated by 10 kDa cut-off in an Amicon ultrafiltration system to a final volume of 3 ml.

Mice used in sepsis or septic shock experiments: Female BALB/c mice of 8-12 weeks of age were used for all experiments. The animals were bred in our facility under specific pathogen-free conditions according to the protocols described in the Report of European Laboratory Animal Science Associations (FELASA) Working group on Animal Health (Laboratory Animals 28: 1-24, 1994).

Injection protocols: For the endotoxin model, BALB/c mice were injected i.p. with 150-300 pg LPS coli 026:B6; Difco Lab., Detroit, MI, USA). Control groups were treated with PBS i.p. only. To test the effect of NMPF, we treated BALB/c with an optimized dose of 700 IU of different hCG preparations, thereof derived fractions (10-50 mg) or from first trimester pregnancy urine (NMPF- U) for 3 days and then injected with LPS i.p..

In order to determine whether NMPF inhibited shock even after the shock induction, we also treated BALB/c mice with NMPF i.p. after 3, 12, 24 and 36 h of injection with LPS. At different time points semi-quantitative sickness scores and survival rates were noted.

Semi-quantitative sickness measurements: Mice were scored for sickness severity using the following measurement scheme: 1 Percolated fur, but no detectable behaviour differences compared to normal mice.

2 Percolated fur, huddle reflex, responds to stimuli (such as tap on cage), just as active during handling as healthy mouse.

3 Slower response to tap on cage, passive or docile when handled, but still curious when alone in a new setting.

4 Lack of curiosity, little or no response to stimuli, quite immobile.

Laboured breathing, inability or slow to self-right after being rolled onto back (moribund, sacrificed) b-hCG peptide and anti-MIF treatment: Most urinary metabolites of hCG are a nicked form of b-hCG. These forms of b-hCG have peptide bond cleavages within the bsubunit. b48 (VLPALPQVVC) is one such peptide which has been shown to be associated with a natural urinary metabolite of hCG. To test the effect of this peptide on septic shock, we injected BALB/c mice with LPS and treated them 2 h later i.p. with b48-peptide (100 mg). In order to see whether possible breakdown products also have effect on septic shock, we incubated b48-peptide at 37 0 C for three h before testing the peptide in the septic shock model in BALB/c mice.

Previously (WO 99-59617), we have shown that NMPF (IR) has also anti-diabetic effect. So in order to test whether b48 peptide has anti-diabetic effect, we performed transfer experiments. Total spleen cells were recovered from diabetic NOD mice and stimulated in vitro in RPMI+ supplemented with 10% FBS with coated anti-CD3 (145-2c11; 25 mg/ml) and IL-2 (50 U/ml) along with 300 IU/ml NMPF (Pregnyl) or b48 peptide (20 mg/ml). Culture flasks were then incubated at 370C in 5% of CO 2 in air for 48 h. After 48 h cells were twice washed with PBS and x 106 cells were i.p. transferred into an 8-wk-old NOD.scid mouse In vitro/ ex vivo LPS stimulated proliferation of splenocytes: After 48 h of septic shock induction in BALB/c mice by high dose LPS injection, spleen cells (1 x 106 cells/ml) were recovered and restimulated in vitro with LPS (10 U/ml) in 96-well plates (round bottom).

After 24 hours of culture, the LPS stimulated proliferation of splenocytes was measured via 3 H]TdR incorporation during the last 16 hours in culture. In other experiments splenocytes from non-treated BALB/c mice were isolated and in vitro stimulated (1 x 106 cells/ml) with LPS in the presence or absence of different sources of NMPF (37.5-600 IU/ml)(Pregnyl, Organon; APL, Wyeth Ayerst; Profasi, Serono), NMPF fractions (10-20 mg/ml), b-48 peptide or its breakdown products, anti-MIF or combinations of these products each at 10 mg/ml. After 24 hours of culture, the LPS stimulated proliferation of splenocytes was measured.

Results NMPF purification: Samples of NMPF from different sources (Pregnyl, APL, Profasi, Pregnancy urine) were applied on the Macroshere GPC 300 A column and eluted with ammonium bicarbonate. Three selected areas were fractionated, NMPF-1 which elutes apparently with molecular weight of >25 kDa, NMPF-2 which elutes apparently with molecular weight between the 25kDa-6kDa, and NMPF-3 which elutes apparently with molecular weight <6kDa (figure All these fractions were lyophilized and were tested for anti-shock activity (shown elsewhere 122 in this document). The lower molecular weight fraction (NMPF-3) which elutes after the column volume was further fractionated on the Macrosphere GPC 60 A column (figure All fractions were lyophilized and were also tested for anti-shock activity.

NMPF treatment in LPS-induced septic shock: To determine the effect of high-dose LPS treatment in NMPF treated mice, BALB/c mice were injected intraperitoneally with LPS (150 mg/kg) and survival was assessed daily for 5 days. PBS-treated BALB/c mice succumbed to shock from day 1 after high-dose LPS injection, with lower than 10% of mice alive on day (figure In contrast, 100% of the mice treated with NMPF from source Pregnyl, or its fractions NMPF-1 or NMPF-3 obtained from GPC 300 A column, were alive on day (P<0.001) (figure while groups of mice treated with NMPF-2 from source Pregnyl or Dexamethasone (data not shown) demonstrated around 25% of survivors (figure Not all commercial hCG preparations showed NMPF activity; for example NMPF from source Profasi showed only partial anti-shock activity (around 40% survival).In addition, variability in NMPF activity between different batches of the same source as well as variability of activity of same batch in time was observed. Treatment of BALB/c mice with APL before or after the shock induction, showed in a number of experiments acceleration of shock and early death.

In order to determine whether there are factor(s) present in hCG preparation that also accelerate shock and inhibit or counteract NMPF activity, we further fractionated NMPF-3 from a pretested active batch (containing anti-shock activity) and a non-active batch from source Pregnyl on GPC 60 A column. Three selected areas were fractionated, NMPF-3.1 which elutes apparently with molecular weight of >2000 Da, NMPF-3.2 which elutes apparently with molecular weight between 2000-300 Da and NMPF-3.3 elutes apparently with molecular weight lower then 300 Da (figure All fractions were tested for anti-shock activity.

Results from these experiments revealed that antishock activity in a pretested active batch resided in a fraction NMPF-3.2, while NMPF-3.3 fraction from both (active and non-active) batches accelerated shock (figure In order to determine whether NMPF-3.3 inhibits the anti-shock activity of NMPF-3.2, we added NMPF-3.3 into NMPF-3.2 in 10:1 ratio (100:10 mg) and injected the mixture i.p. in mice two hours after LPS injection Data from these experiments showed that in all mice treated with NMPF-3.2 fraction alone, septic shock was inhibited and they had sickness scores lower than 2 (figure while this anti-shock activity of NMPF-3.2 fraction was inhibited with NMPF-3.3. NMPF-3.3 treatment alone accelerated shock and the treated mice died even earlier than PBS treated mice (figure Same trend of results were obtained in experiments, in which active and non-active batches from Pregnyl were mixed and injected in BALB/c mice after septic shock induction (data not shown).

Ratio between NMPF-3.2 and NMPF-3.3: Next, we further purified NMPF-3.2 and NMPF-3.3 on GPC 60 A column from active and non-active Pregnyl batches, and from first trimester pregnancy urine and determined the ratio.

We found that first trimester pregnancy urine having anti-shock activity had around 1:2.2 ratio (NMPF-3.2 NMPF-3.3) (figure and non-active batch of Pregnyl had 1:3.4 ratio (figure while the active batch of Pregnyl had around 1:1 ratio (Figure Ex vivo LPS stimulated splenocytes proliferation: After 48 hours of LPS shock induction, splenocytes from PBS treated and NMPF treated mice(from mice treated with either active Pregnyl, thereof derived NMPF-3.2 or NMPF- 3.3 fractions, or APL preparation) were isolated and restimulated with LPS. After 24 hours of culture, LPS stimulated proliferation of splenocytes was measured.

Reduction in LPS induced proliferation was observed after culture of splenocytes from NMPF (active batch of Pregnyl) and thereof derived NMPF-3.2 (1600 vs 1350 cpm) fraction treated BALB/c mice as compared to PBS treated mice (3500 cpm), while treatment by NMPF(APL) or NMPF-3.3 increased the LPS stimulated proliferation (6000 vs 7200 cpm). Comparable results were obtained when splenocytes from untreated BALB/c mice were in vitro stimulated with LPS in the presence of above mentioned additions(data not shown).

In vitro treatment with NMPF from different sources, b-48 peptide, denaturated b-48 peptide and anti-MIF: The major characteristics of pre-eclampsia resemble that of septic shock. Therefore we hypothesized that there might be also NMPF (IR) factor(s)that are involved in preeclampsia and also worsen septic shock or sepsis. Above we have shown that NMPF-3.3 is one such fraction which accelerates septic shock and increases in vitro/ex vivo LPS induced splenocytes proliferation, which is correlated with increase in the disease severity. In the urine of pre-eclamptic patients high levels of nicked hCG b-subunits are present. Therefore we also tested whether these nicked subunits worse septic shock and so resemble NMPF-3.3 fraction. Furthermore, MIF is an important mediator of lethal endotoxemia and staphylococcal toxic shock, so we also compared the effects of b-48 peptide and NMPF on proliferation with anti-MIF and MIF.

These experiments revealed that anti-MIF has a trend to decrease LPS induced proliferation, similar as a pretested Pregnyl batch that shows anti-shock activity (NMPF-PG') (figure Moreover, anti-MIF and NMPF-PG together work synergistically and decrease proliferation (figure NMPF from APL (NMPF-A), non-active Pregnyl batch (NMPF-PG-; without anti-shock activity) and b-48 peptide (NMPF-K) increased the LPS induced proliferation as compared to LPS only (figure 8-12).On the other hand, NMPF-PG+ or denaturated b-48 peptide (NMPF-Kb) inhibited and decreased the LPS induced proliferation at least till the level of anti-MIF treatment alone (figure In vivo treatment of BALB/c mice with NMPF-PG-, NMPF-K or NMPF-A after septic shock induction accelerated the disease severity (at t=48 hrs 0-25% survival rate) as compared to PBS treated mice (at t=72 hrs 15% survival rate), while septic shock in BALB/c mice was completely inhibited by NMPF-PG or NMPF-Kb.

In addition, our NOD spleen cells transfer experiments revealed that 22 days after transferring, NOD.scid mice receiving b48-peptide and PBS treated spleen cells were positive for diabetes and within a week they reached a blood glucose level above 30 mmol/l, while NOD.scid mice receiving NMPF (pregnyl) treated spleen cells remained normal (blood glucose <8 mmol/l). 6 weeks after transferring, the PBS and b48 reconstituted NOD.scid mice looked very uncomfortable, while NMPF mice group remained healthy. Mice from all groups were killed at this time.

There are many physiological conditions and immune pathologies where adaptive and innate immune systems are involved separately or in combination. For example, it has been shown that in pregnancy the maternal innate immune system is more stimulated, and it has been proposed that type II diabetes mellitus is due to chronic hyperactivation of the 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 organspecific autoimmunity, allergy, asthma etc, and the adaptive immune system can also play a role in the maintenance of pregnancy and in the prevention of "allograft" rejection and chronic inflammation, presumably including atherosclerosis and related diseases.

As shown in our previous (Immunoregulator; W099- 59617) NMPF (IR) is able to regulate the Thl/Th2 balance in vivo (BALB/c, NOD) and in vitro. In dominant Thl phenotype models like NOD, NMPF (like NMPF-PG and its fractions) amongst others promote the IL-10 and TGF-beta production, which indicates the induction of regulatory cells like Th3 and Trl by NMPF. These regulatory cells may play role in the beneficial effects of NMPF in immune and inflammatory diseases and immune tolerance. While NMPF and several of its fractions are able to inhibit the production of IFN-gamma in vitro and in vivo, this was not observed for NMPF-3 (IR-P3) and recombinant hCG (rhCG). NMPF-3 (IR-P3) and rhCG separately show no to moderate inhibition of the IFN-gamma production, but the combination of NMPF-3 and rhCG gives a strong inhibition of the IFN-gamma production. This implies the need of NMPF-3 for rhCG for at least its IFN-gamma inhibition capacity in these models, while NPMPF-1 and NMPF-2 alone are capable to inhibit IFN-gamma production. This holds also for the anti-CD3 stimulated spleen cells obtained from in vivo treated NOD mice and for the polarization of T-helper cells to the Th2 phenotype. In our previous work we have also shown that NMPF (IR) has the potential to inhibit acute inflammatory responses, like in sepsis or septic shock. So, chronic as well as acute immune responses are modulated by NMPF.

By way of example and not wishing to bound to theory, in pregnancy a fetus has to survive potential maternal immune rejection, which is in part achieved through deviation of the maternal immune system towards Th2-type immune responses. But in this way maternal immune suppression carries the attendant risk of infection, as is observed in transplant patients receiving corticosteroids or other immunosuppressive therapy. NMPF (IR) factor(s) obtainable at least from pregnancy urine and thereof derived hCG preparations have the potential to modulate immune responses in such a way that the maternal rejection of the fetus is suppressed and that the mother maintains or even increases her resistance to infection. These and related factors are also responsible for the inhibition of immune diseases, particularly Thl-mediated immune diseases, during pregnancy.

By way of example and not wishing to bound to theory, pregnancy apparently demands incompatible immune adjustments. On the one hand, adaptive immune responses during pregnancy are modulated at different cellular levels towards immune tolerance state (such as Th2-type) and on the other hand the maternal innate immune system is modulated for resistance to infection. The evidence is that components of the maternal innate immune system are systemically activated. There are increased numbers of monocytes and granulocytes from the first trimester onwards. It has also been found that in normal pregnancy circulating monocytes and granulocytes in the maternal blood have an activated phenotype, in some ways comparable with changes observed in systemic sepsis.

Others have shown increased monocyte phagocytosis and respiratory burst activity, and an increased expression of endotoxin receptor CD14 on monocytes as well as an increased response to endotoxin: monocytes from normal women produce more of the proinflammatory cytokines like in septic shock. Many studies have similarly found granulocyte activation in pregnancy as well as changes in.

plasma levels of soluble innate factors typical of an acute phase response. Not all components of the innate system are activated in the maternal circulation. Most notably, cytotoxic activity and IFN-gamma production by NK cells are suppressed.

By way of example and not wishing to bound to theory, we propose that one of the mechanisms of NMPF to modulate the immune response during pregnancy is the following: some NMPF factors during pregnancy can ensure that if T cells are activated, there is a bias to a Th2 response. This could be achieved by effecting different cell populations like macrophages, DC, T cells and their regulatory subsets. Other or similar NMPF factors could activate monocytes and hence other innate cells. So, the balance between different NMPF factors is crucial for a balanced regulation of different immune responses. We propose that in pre-eclampsia there is a misbalance between different NMPF factors. Over-activation of innate cells by NMPF factor(s) and/or a decrease in adaptive immune response (particularly Thl-type) inhibiting NMPF factor(s) could cause Thl/Th2 misbalance towards the Thl phenotype, in some ways comparable with changes observed in systemic sepsis. Our results showed that there are also NMPF factor(s) (NMPF-3.3) that can stimulate innate immunity and accelerate septic shock, while other NMPF factor(s) like NMPF-3.2 inhibit septic shock and the activity of NMPF-3.3. NMPF-3.2 factor(s) present in NMPF- 3 fraction in combination with for example hCG modulate the adaptive immune response towards Th2-type (W099- 59617; inhibition of IFN-gamma by NMPF-3 (IR-P3) in combination with hCG) and is essential for normal pregnancy and inhibition of Thl autoimmune diseases, induction of tolerance etc.

Analysis of hCG preparation (Pregnyl) and pregnancy urine have shown that hCG preparation and pregnancy urine having anti-shock activity contain NMPF-3.2 and NMPF-3.3 fractions in about an 1:2 ratio or higher, while hCG preparations without anti-shock activity or that worse septic shock have an NMPF-3.2 and NMPF-3.3 ratio of 1:3 or lower. This also explains why not all commercial hCG preparations have anti-shock activity. Moreover, we showed that hCG preparation possessing a high ratio of NMPF-3.3:NMPF-3.2 and so having no anti-shock activity, mixed with an active hCG preparation could gain antishock activity. So, the ratio between different NMPF factors or fractions like NMPF-3.2 and NMPF-3.3 can be used as a diagnostic marker not only for the prediction of successful pregnancy, but also for different immunopathology such as pre-eclampsia, sepsis or septic shock etc. In addition, in abnormal pregnancy like preeclampsia, one can also use NMPF factor(s) or NMPFfraction(s) NMPF-3.2) as a treatment. Our experiments also showed that NMPF (NMPF-3.2) inhibited septic shock even 30 h after shock induction, this shows that NMPF not only inhibits early mediators of endotoxin lethality like TNF-alpha, IL-lb, MIF, but also inhibits late mediators such as recently characterized high mobility group-i (HMG-1) protein (Science 285, 248-251).

hCG is a member of the structural superfamily of cysteine knot growth factors like NGF, PDGF-B and TGFbeta and a members of the glycoprotein hormone family which also includes LH, FSH and TSH. They each consist of two noncovalently associated protein subunits, a common kD alpha chain and a hormone specific 23 kD beta chain (Annu. Rev. Biochem. 50, 465-495). hCG is produced by placental trophoblasts of normal pregnancy, and in gestational trophoblastic disease. It is also produced in much smaller quantities by the pituitary (Endocrinology 137, 1402-1411) in both pre- and postmenopausal women and in men (Trends in Endocrinology and Metabolism 1, 418- 421), in many non-gestational malignant tumors and other tissues. hCG possesses a complex structure as a family of isoforms with structural, immunological and biological differences. The chemical basis for this heterogeneity is not known with certainty but differences in the amino acid composition, carbohydrate residues or both have been proposed. More recently it was also shown that oxidation of specific methionine residues may also be responsible.

Different forms of hCG, alpha and beta-subunits, their nicked fragments, beta-core fragment and multiple isoforms of hCG have been reported in different tissues and body fluids (Journal of Endocrinology 161, 99-106; Endocrinology 129, 1541-1550; Obstet. Gynecol. 77, 53-59; Journal of Biochemistry 107, 858-862; Obstet. Gynecol.

223-228; Endocrinology 133, 985-989; 129, 1551-1558; 130, 2052-2058; Journal of Endocrinology 135, 175-188; 139, 519-532; Molecular and Cellular Endocrinology 125, 93-131).

Since all commercial hCG preparations are derived from pregnancy urine and contain different breakdown products of hCG, we speculated whether these products have NMPF activity. The most known breakdown products of hCG are beta-core hCG, a peptide bond nick in the betasubunit between residues 44-45, 46-47 and 47-48. b48 (NMPF-K) is found in approximately 10-20% of the molecules in pregnancy urine and is associated with a natural urinary metabolite of hCG. Our experiments showed that NMPF-K accelerates septic shock (like MIF) and LPS induced proliferation of splenocytes alone or in combination with a non-active hCG preparation. This effect is inhabitable with anti-MIF, active hCG preparation, NMPF-3.2 and denaturated b48 (NMPF-Kb) peptide. This shows that NMPF-K activity resembles with NMPF-3.3 and the NMPF-Kb activity resembles to NMPF-3.2.

In addition, there are also other peptide bond cleavages in hCG and its subunits as well as heterogeneity of the beta-core fragment. For example b45 bond cleavage, mainly found in hCG preparation and in urine, possibly derive from the action of bacterial proteases. In addition, Medeiros et. al. showed that HPLC separation of beta-core in its reduced and S-carboxymethylated forms showed three peptides, but only two of them could be sequenced and was demonstrated to be the previously reported b6-40 and 92 peptides of bhCG, while the third peak did not give any clear sequence because of the low signal due to several unidentified amino acids. We showed that breakdown products of NMPF-K share activity with NMPF- 3.2. This NMPF-K peptide lies between two beta-core fragments (b6-40 and b55-92) and partially derived from beta-core b55-92 fragment. It is possible that there are also other single and/or double cleavage products of beta-core fragments or of not yet identified beta-core peptides (like Medeiros et. al. showed beta-core faction with a unidentified amino acids) responsible for NMPF activity in hCG preparations and pregnancy urine.

Breakdown products of b48-peptide with additional unidentified amino acids from beta-core and/or with additional glycosylation possess among other antidiabetic and anti-chronic inflammatory activity.

In short, the invention provides among others an immunoregulator (immunoregulating peptide) obtainable or derivable from a urinary metabolite of hCG, in particular from (nicked) forms of beta-hCG, or (synthetic) peptide homologues or analogues thereof. These forms of beta-hCG have peptide bond cleavages within the beta-subunit (Birken et al, Endocrinology 133:1390-1397, 1993), and herein it is provided that the breakdown products, especially those from the beta-44 to beta-49 regions provide significant immunoregulatory effects by using the animal model test systems as provided.

It was found for example herein in animal experiments as described below that peptides obtainable from hCG react in a septic shock model with strong immunoregulatory effects.

EXAMPLE II Materials and Methods 132 Gel permeation: We fractionated commercial hCG preparation (c-hCG, Pregnyl, Organon, Oss, The Netherlands) as follows: we used Shimadzu HPLC system equipped with Alltech macrosphere size exclusion (GPC) 60A column (4.6 mm ID x 250 mm L) in 50mM ammonium bicarbonate buffer. The separation range for this column was 28,000 250 Dalton. Sample (20,000 IU hCG/ml) load volume was 10-50 ml. The flow rate was 0.3 ml/min for minutes. External molecular weight standards were also employed to calibrate the column elution positions. The markers used were: aprotinin (6,500 Da), cytochrome C (12,400) and carbonic anhydrase (29,000). In addition, the concentrated urine (see urine purification) obtained from Pelicon system was filtered through 0.45 mm filter and 40,000 IU c-hCG (Pregnyl) dissolved in 50 mM ammonium bicarbonate were analysed on Shimadzu HPLC system equipped with Superdex G25 (30 mm ID x 990 mm L) desalted column in 50mM ammonium bicarbonate buffer supplemented with 5% methanol. The separation range for column were 5000 1000 Dalton. Sample load volume was 7-10 ml. The flow rate was 3 ml/min for 250 minutes. External molecular weight standards were also employed to calibrate the column elution positions. 100 ml fractions were collected, lyophilised and were further tested for anti-shock activity.

Urine purification: First trimester pregnancy urine (2 litres) was collected in a bottle from a healthy volunteer and was refrigerated until delivered at the laboratory within 2 days. Upon delivery, 1 gram per litre of sodium azide was added and the pH was adjusted to 7.2- 7.4 with sodium hydroxide and allowed to sediment for 1hour at room temperature Approximately, 75% of the supernatant was decanted and the remainder close to the precipitate was centrifuged (10 min at 25000 rpm at 4 0 C) to remove sediment and added to the rest of the supernatants. The supernatant (about 2 litre) was concentrated in a Pellicon ultrafiltration set-up equipped with a Pellicon XL filter (Millipore, cat. No.

PXC010C50) with a 5 kDa cut-off. The final volume was 150 ml. Urine from healthy non-pregnant women, and from women in their first trimester pregnancy with autoimmune disease (SLE, Sjogren) were treated with same method as mentioned above.

Endotoxin shock model: For the endotoxin model, BALB/c mice were injected i.p. with 8-9 mg/kg LPS (E.

coli 026:B6; Difco Lab., Detroit, MI, USA). Control groups (PBS) were treated with PBS i.p. only. To test the effect of NMPF, we treated BALB/c with a dose of 300-700 IU of different hCG preparations (PG23;Pregnyl batch no.

235863, PG25; Pregnyl batch no. 255957), with peptides mg/kg) or with different fractions (0.5-1 mg/kg) after two hours of LPS injection.

LPS induced proliferation: In order to determine whether treatment of LPS injected BALB/c mice with different fractions, peptides or commercial hCG (c-hCG) alter the proliferative response of spleen cells, we also isolated splenocytes from above mentioned LPS shock experiments. Total spleen cells (1 x 106 cells/ml) from treated BALB/c mice were restimulated in RPMI' supplemented with 10% FBS with different concentrations of LPS 10, 20 mcg/ml) in round bottom 96-well plates.

Plates were incubated at 37 0 C in 5% C02 in air for 24hrs.

The proliferation was measured via 3 H]TdR incorporation by adding 0.5 mCi/well during the last 12 hrs in culture.

Flow cytometry: In some experiments, after 48 hours of septic shock induction spleen cells were isolated for flow cytometry analysis. The cell surface markers analysed in these experiments were CD19, CD80, B220, CD4, F4/80, NK1.1, DX-5 and CD25. For analysis of these marker FITC or PE conjugated mABs were purchased from BD PharMingen. Shortly, spleen cells (2x10 5 were washed twice with FACS buffer and incubated with mABs according to the manufacturer's instructions. Hereafter, cells were washed and analysed on FACSort flow cytometer (Becton Dickinson). Based on their forward and side scatter characteristics, live cells were gated and analyzed.

Coronary Artery Occlusion (CAO) experiments: NMPF have immunoregulatory effects in chronic inflammatory as well as acute inflammatory mice models. Since certain cytokines like TGF-betal, TNF-alpha, IL-1 and ROS (reactive oxygen species) have been implicated in irreversible myocardial damage produced by prolonged episodes of coronary artery occlusion and reperfusion in vivo that leads to ischaemia and myocardial infarct, we tested the cardio-protective properties of peptides in ad libitum fed male Wistar rats (300 The experiments were performed in accordance with the Guiding principles in the Care and Use of Animals as approved by the Council of the Amcerican Physiological Society and under the regulations of the Animal Care Committee of the Erasmus University Rotterdam. Shortly, rats were stabilized for 30 minutes followed by i.v. 1 ml of peptide treatment (0.5 mg/ml) in 10 minutes. Five minutes after completion of treatment, rats were subjected to a coronary artery occlusion (CAO). In the last minutes of CAO, rats were again treated over 10 minutes i.v. with 1 ml of peptide (0.5 mg/ml) followed by 120 minutes of reperfusion Experimental and surgical procedures are described indetail in Cardiovascular Research 37(1998) 76-81. At the end of each experiment, the coronary artery was re-occluded and was perfused with ml Trypan Blue Sigma Chemical Co.) to stain the normally perfused myocardium dark blue and delineate the nonstained area at risk The heart was then quickly excised and cut into slices of 1 mm from apex to base.

From each slice, the right ventricle was removed and the left ventricle was divided into the AR and the remaining 135 left ventricle, using micro-surgical scissors. The AR was then incubated for 10 min in 370C Nitro-Blue-Tetrazolium (Sigma Chemical Co.; 1 mg per 1 ml Sorensen buffer, pH which stains vital tissue purple but leaves infarcted tissue unstained. After the infarcted area (IA) was isolated from the noninfarcted area, the different areas of the LV were dried and weighed separately.

Infarct size was expressed as percentage of the AR.

Control rats were treated with PBS.

NOD experiments: We treated NOD mice at the age of 8-10 weeks with PBS or peptide 1 (VLPALPQVVC), or recombinant hCG (rhCG, Sigma) and rhCG in combination with peptide.1 each with 10-40 mcg i.p. for three days.

In order to determine the effect of the treatment on Thl polarisation, we isolated CD4+ cells and performed Thl polarisation assays as follows: Purified CD4+ T cells from the spleen were obtained by negative selection due to complement depletion with antibodies specific for B cells, NK cells, monocytes/macrophages and granulocytes.

Cells were further purified using magnetic activated cell sorting with a cocktail of biotinylated mAbs against CDllb, B220, CD8 and CD40, followed by incubation with streptavidin-conjugated microbeads (Milteny Biotech, Bergisch Gladbach, Germany). CD4+ T-cells used for experiments were always 90-95% purified as determined by flow cytometry. For primary stimulation, purified CD4+ T cells were cultured at 1 x 105 cells/well in flat bottom 96-well plates (Nalge Nunc Int., Naperville, IL, USA), and stimulated with plate-bound anti-CD3 mAb (145-2C11, 25 mg/ml), anti-CD28, and IL-2 (50 U/ml). For differentiation of Thl cells, anti-IL-4 mAb (11B11; mg/ml) and IL-12 (10 ng/ml) were added to the cultures.

Unprimed cultures contained only anti-CD3, anti-CD28 and IL-2. All doses were optimised in preliminary experiments. After 4 days of culture, the cells were washed 3 times and transferred to new anti-CD3-coated 96well plates and restimulated in the presence of IL-2 U/ml) and anti-CD28 (10 mcg/ml). Forty-eight hours later, supernatants were collected and assayed for IFN-gamma production by ELISA as readout for Thl polarization.

Cytokine ELISA: IFN-gamma was detected using monoclonal anti-IFN-gamma antibody (XMG1.2) as the capture antibody and revealed with biotinylatedconjugated rat anti-mouse IFN-gamma monoclonal antibody (R46A2). ABTS substrate was used for detection.

Flat bottom microplates (96-wells, Falcon 3912, Microtest II Flexible Assay Plate, Becton Dickinson, Oxnard, USA) were coated with IFN-gamma specific capture antibodies (XMG1.2, 5 mg/ml) diluted in PBS and stored at 4 0 C for 18 hrs. After coating, plates were washed (PBS, 0.1% BSA, 0.05% Tween-20) and blocked with PBS supplemented with 1% BSA at room temperature for 1 hr.

After washing, samples and standards were added and incubation was continued for at least 4 hrs at room temperature. Thereafter, plates were washed and biotinylated detection antibodies were added (R46A2, 1 mcg/ml) and incubated overnight at 4 0 C. After washing, streptavidin-peroxidase (1/1500 diluted, Jackson Immunoresearch, West Grove, PA, USA) was added. After 1 hr, plates were washed and the reaction was visualized using 2,2'-azino-bis-3-ethylbenz-thiazoline-6-sulfonic acid (ABTS, 1 mg/ml, Sigma, St. Louis, MO, USA). Optical density was measured at 414 nm, using a Titertek Multiscan (Flow Labs, Redwood City, USA).

Peptide synthesis: The peptides were prepared by solid-phase synthesis (Merrifield, 1963) using the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl-based methodology (Atherton, 1985) with 2-chlorotrityl chloride resin (Barlos, 1991) as the solid support. The side-chain of glutamine was protected with a trityl function. The peptides were synthesized manually. Each coupling consisted of the following steps: removal of the alpha-amino Fmoc-protection by piperidine in dimethylformamide (DMF) (ii) coupling of the Fmoc amino acid (3 eq) with diisopropylcarbodiimide (DIC)/1hydroxybenzotriazole (HOBt) in DMF/N-methylformamide (NMP).(iii) capping of the remaining amino functions with acetic anhydride/diisopropylethylamine (DIEA) in DMF/NMP.

Upon completion of the synthesis, the peptide resin was treated with a mixture of trifluoroacetic acid (TIS) 95:2.5:2.5. After minutes TIS was added until decouloration. The solution was evaporated in vacuo and the peptide precipitated with diethylether. The crude peptides were dissolved in water (50-100 mg/ml) and purified by reverse-phase highperformance liquid chromatography (RP-HPLC). HPLC conditions were: column: Vydac TP21810C18 (10 x 250 mm); elution system: gradient system of 0.1% TFA in water v/v and 0.1% TFA in acetonitrile (ACN) v/v flow rate 6 ml/min; absorbance was detected from 190-370 nm. There were different gradient systems used. For peptides LQG and LQGV: 10 minutes 100% A followed by linear gradient 0-10% B in 50 minutes. For peptides VLPALP and VLPALPQ: minutes 5% B followed by linear gradient 1% B/minute. The collected fractions were concentrated to about 5 ml by rotation film evaporation under reduced pressure at 40 0

C.

The remaining TFA was exchanged against acetate by eluation two times over a column with anion exchange resin (Merck II) in acetate form. The eluate was concentrated and lyophilized in 28 hours.

EAE models: Female SJL/J mice 8-12 weeks of age; obtained from Harlan, Zeist, The Netherlands, or bred at the Erasmus University Rotterdam) were immunized with 100 mcg of proteolipid protein peptide 139-151 (PLP 139-151; H-His-Ser-Leu-Gly-Lys-Trp-Leu-Gly-His-Pro-Asp- Lys-Phe-OH; obtained from either Peptides International, Louisville, KY, or TNO Prevention and Health, Leiden, The Netherlands), emulsified in CFA containing 4 mg/ml of Mycobacterium tuberculosis H37 Ra (Difco, St. Louis, MO).

distinct model of EAE were induced by injection of either 200 mg pertussis toxin (Sigma) in 50 mcl PBS i.v. on day 0 and 2 post immunization, or 1010 Bordetella pertussis bacteria (RIVM, Bilthoven, The Netherlands) in 200 mcl PBS i.v. on day 1 and 3 after immunization. Mice were examined for clinical signs of EAE and weighed daily.

clinical symptoms of EAE were scored on scale of 0 to with graduations of 0.5 for intermediate scores: no clinical signs; flaccid tail; mild paraparesis; dual hind limb paralysis; moribund; or death due to EAE. Starting from day 7 post immunization, 60-F2 or 60-F3 fractions (20 mcg) from c-hCG (10,000 IU) in a total volume of 200 mcl PBS was injected i.p. for two weeks at alternate day. Control mice were treated with PBS only.

Results Gel permeation of urine and commercial hCG preparation: c-hCG (figure 16) and first trimester pregnancy urine were fractionated on HPLC system equipped with GPC 60A column. Three selected areas were fractionated, 60A-fraction 1 (60A-F1) which elutes apparently with molecular weight of >10 kDa, 60A-F2 which elutes apparently with molecular weight between the and 60A-F3 which elutes apparently with molecular weight <1kDa. These fractions were tested further for anti-shock activity.

In addition, urine from healthy pregnant women in their first trimester pregnancy, urine from women in their first trimester pregnancy with autoimmune disease and urine from healthy non-pregnant women were analysed on superdex G25 column. All 100 ml fractions were also tested for anti-shock activity. Figure 17 and 18 show the chromatograms of c-hCG and urine from healthy pregnant women in their first trimester of pregnancy.

Survival curve: To determine the effect of highdose LPS treatment in c-hCG and urine fractions treated mice, BALB/c mice were injected intraperitoneally with LPS (8-9 mg/kg) and survival was assessed daily for 5 days. In our previous patent (W09959617) and in this patent application we have shown that three selected areas were fractionated on GPC 60A column: 60A-F1 which elutes apparently with molecular weight of >10 kDa, F2 which elutes apparently with molecular weight between the 10kDa-lkDa, and 60A-F3 which elutes apparently with molecular weight <1kDa. All these activities were tested for anti-shock activity and they all had anti-shock activity (presumably the lower molecular weight activity also elutes along with the high molecular weight fractions). PBS-treated BALB/c mice succumbed to shock between days 1 and 2 after the high dose LPS injection, with less than 10% of the mice alive on day 5. In contrast 100% of 60A-F1 and 60A-F3 treated mice were alive on day 5 (p<0.001), while 60A-F2 treated mice demonstrated only around 70% of survival. Since the lower molecular weight fraction had also anti-shock activity, we fractionated c-hCG, urine of first trimester pregnancy, urine from women with autoimmune diseases in their first trimester of pregnancy and urine from healthy non-pregnant women on G25 superdex column. All 100 ml fractions were tested for anti-shock activity.

PBS-treated BALB/c mice succumbed to shock between days 1 and 2 after the high-dose LPS injection, with less than 10% of mice alive on day 5. In contrast, 100% of the mice treated with c-hCG, first trimester pregnancy urine fraction V from healthy individuals or urine from individuals with autoimmune disease in their first trimester of pregnancy were alive on day 5 (P<0.001).

However, some fractions which were eluted before (fraction II and IV) and after (the anti-shock) fraction V fraction VI) had accelerated shock and all treated mice died even much earlier (within 24 hours after septic shock induction) than PBS treated mice. In addition, the anti-shock activity of fraction III and V was inhibited by the addition of fraction II, IV or VI in at least ratio of 1:6. This applied also for fractions obtained from commercial hCG preparations and pregnancy urine from healthy individuals. Moreover, we have noticed that very less amount of anti-shock fractions from the urine of pregnant individuals with autoimmune disease were need to inhibit septic shock in BALB/c mice. These women also showed clinical improvement in the autoimmune disease during pregnancy.

Illness kinetics: Visible signs of sickness were apparent in all of the experimental animals, but the kinetics and obviously the severity of this sickness were significantly different. After treatment of BALB/c mice with LPS (endotoxin) and either first trimester pregnancy urine fraction V from healthy individuals, first trimester pregnancy urine from individuals with autoimmune disease or commercial hCG preparation the clinical symptoms of the LPS treated BALB/c mice did not exceed the sickness level 2. In addition, these fractions even inhibited the symptoms of shock and mortality when administered 32 hours after LPS injection.

Peptides data (NMPF): The table below shows survival percentages of mice over the time period of 72 hours. For the LPS (endotoxin) model, BALB/c mice were injected i.p.

with 8-9 mg/kg LPS coli 026:B6; Difco Lab., Detroit, MI, USA). Control groups (PBS) were treated with PBS i.p.

only. We treated BALB/c mice with a dose of 300-700 IU of different hCG preparations (PG23;Pregnyl batch no.

235863, PG25; Pregnyl batch no. 255957) or with peptides mg/kg) after two hours of LPS injection.

These experiments showed (table that peptides 4 and 6 inhibited shock completely (all mice had in first 24 hours sickness scores not higher than 2; shortly thereafter they recovered completely and had sickness scores of while peptides 2, 3 and 7 accelerated shock (all mice had in first 24 hours sickness scores of 5 and most of them died, while the controle mice treated with LPS+PBS had sickness scores of 3-4 in first 24 hours and they died after 48 hours with sickness scores of In addition, peptides 1, 5, 8, 9, 11, 12, 13 and 14 showed in number of different experiments variability in effectiveness as well as in the kind (inhibitory vs accelerating) of activity. This variability is likely attributable to the rate of breakdown of the various peptides, and the different effects the various peptides and their breakdown products have in vivo. Similar to the above mentioned shock experiments with fractions, the shock inhibiting activity was inhibitable by the addition of shock accelerating activity and visa versa.

These data are representative of at least separate experiments.

Table 1.

Test substance

(HRS)

SURVIVAL IN TIME 0 16 40 72 100 100 67 17 100 100 100 100 100 83 83 83

PBS

PG2 3 PG2 5

PEPTIDE

NO.

1 2 3 4 6 7 8 9 11 12 13 14

SEQUENCE

VLPALPQVVC

LQGVLPALPQ

LQG

LQGV

GVLPALPQ

VLPALP

VLPALPQ

GVLPALP

XTVC

MTRV

MTR

LQGVLPALPQVVC

(CYCLIC) LQGVLPALPQVVC 100 100 100 100 100 100 100 100 100 100 100 100 100 100 67 83 100 100 100 83 100 100 100 100 100 83 50 0 20 100 80 100 0 83 50 67 67 100 83 17 0 17 100 17 100 0 67 100 83 Table 2.

SEQUENCE ID:

LQGV

AQGV

LQGA

VL PAL P AL PAL P

VAPALP

AL PAL PQ

VLPAAPQ

VLPAIIAQ

anti-shock effect shock accelerating effect

LAGV

LQAV

VLAALP

VLPAAP

VLPALA

VLPALPQ

VLAALPQ

VLPALPA

In table 2 we see the effect of ALA-replacement (PEPSCAN) in peptide LQGV, VLPALP, VLPALPQ in septic shock experiments. We conclude, that the change in even one amino acid by a neutral amino acid can lead to different activity. So, genomic differences as well as polymorphism in these peptides can regulate the immune response very precise. Derivates of these peptides, for example (but not limited to) by addition of non-classical amino acids or derivatives that are differentially modified during or after synthesis, for example benzylation, amidation, glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand etc. could also lead to a better effectiveness of the activity.

Since MIF is one of the major inducers of sepsis, we also restimulated spleen cells from Peptide 1 group mice with LPS in vitro and then measured the MIF production.

Figure 19 shows that in vivo treatment with LPS increased MIF production as compared to PBS treated mice, while Peptide 1 treatment after the shock induction inhibited MIF production (Figure 19). No effect on MIF production was found in mice treated with Peptide 1 alone; this shows the specificity of the peptide 1. In addition, LPS restimulated proliferation was also studied in splenocytes from peptide 1 and c-hCG-V (fraction V from c-hCG) treated mice. These data showed that after restimulation with LPS in vitro, splenocytes from LPS treated mice have a greater capacity to proliferate in vitro as compared to PBS treated mice (figure 20). On the other hand, splenocytes from LPS+peptide 1 and LPS+c-hCG- V treated mice showed a much higher capacity to proliferate as compared to the LPS treated control mice (Figure 20). No differences in LPS induced proliferation was observed in mice treated with PBS, peptide 1 or chCG-V alone.

Figure 21 shows the effect of restimulation of splenocytes from in vivo treated mice with different doses of LPS in vitro. These data are also consistent with the above mentioned proliferation data. In these experiments restimulation of splenocytes from mice treated with peptide 1(anti-shock activity), c-hCG (containing anti-shock activity) and c-hCG-V (anti-shock fraction from c-hCG) after the septic shock induction showed higher capacity to proliferate as compared to LPS+PBS treated mice. On the other hand, splenocytes from mice treated with peptide 2 (shock accelerating peptide) showed the same capacity to proliferate as compared to LPS+PBS treated mice (figure 21). In this figure it is important to notice that the kinetics of the proliferation of spleen cells from peptide 1 and c-hCG-V fraction treated mice were the same.

All together, in vitro stimulation of splenocytes from BALB/c mice treated with LPS and peptide or fraction with anti-septic shock activity decreased proliferation which is associated with inhibition of septic shock in vivo with these peptides or fractions. On the other hand, in vitro restimulation with LPS of splenocytes from in vivo LPS+anti-septic shock activity treated BALB/c mice increased proliferation which is associated with the inhibition of septic shock.

Flow cytometry: flow cytometry analysis of splenocytes from treated BALB/c mice revealed that the septic shock inhibitory and septic shock accelerating effects correlated with a characteristic pattern of surface makers of the spleen cells. Figure 22 shows that the shock inhibitory activities (c-hCG, peptide 1, peptide 4 and peptide 6) increased the expression of molecule on CD19 cells as compared to PBS+LPS control group, whereas minor effect was observed with peptide 7 which accelerates shock. Figure 23 shows decreased number of CD19/CD40 cells in the spleens of shock inhibitory activities as compared to PBS+LPS group, while no effect was observed with peptide 7 in shock experiments. Figure 24 and 25 shows septic shock inhibitory activity deactivates B220 positive and F4/80 positive cells as compared to the PBS+LPS treated groups. While the number of activated CD4+ T cells (figure 26) were increased with septic shock inhibitory activities. No differences were observed in the activation of B220-, F4/80- and CD4 positive cells with shock accelerating activity (peptide 7) (figures 24-26). In addition, decrease in Nkl.1 cell membrane marker experssion was observed after treatment with LPS and peptide with septic shock inhibitory activities as compared to PBS+LPS group, while no effect was found after treatment with shock accelerating activity (figure 27) An increased number of Dx-5 (pan-NK cells marker) was observed with septic shock inhibitory as well as shock accelerating activity (figure 28). These results suggest that the septic shock inhibitory activity might be correlated with the deactivation of macrophages and B-cells, increased number activated CD4+ T cells and NK cells, while the septic shock accelerating activity correlates with increased number of activated NK cells (activation and number of macrophages, B and T cells compare to LPS+PBS).

hCG bioactivity: hCG binds to a LH receptor and induce signalling through cAMP. We determined whether peptides 1, 2, 4, 6 and low molecular weight anti-shock fraction c-hCG-V could bind to LH receptor and posses hCG bioactivity. Figure 29 shows that hCG and c-hCG bind to 293-hLHRwt/CREluc cells and induce dose-dependent luciferase activity, while no effect was observed in luciferase activity with peptide 1, 2, 4, 6 and low molecular weight fraction c-hCG-V (figure 30). Moreover, addition of peptide 1, 2, 4, 6 and fraction c-hCG-V in the presence of hCG also did not show effect on luciferase activity induce by hCG itself (figure 31).

These data show that these peptides and fraction chCG-V do not have hCG bioactivity and they do not bind to LH receptor, nor that they disturb the binding of hCG to the LH receptor.

NOD experiments: In our previous patent (WO9959617) we have shown that high levels of IFN-gamma and dominant Thl cells are associated with autoimmune diseases, we tested whether lower molecular weight fractions from a column (60A-F3) of c-hCG and first trimester pregnancy urine can suppress dominant Thl activity. To determine whether 60A-F3 needed an additional factor, such as hCG, to exert its full activity, we also treated NOD mice with 60A-F3, rhCG, and 60A-F3 in combination with rhCG and then evaluated the Thl polarisation. Figure 32 shows that there was moderate inhibition of IFN-gamma production found under Thi polarisation conditions with 60A-F3 (c-hCG) and rhCG alone, while the outgrowth of Thl cells was completely blocked with the combination of rhCG and 60A-F3(c-hCG) (figure 32). Similar results were found with the lower molecular weight fraction 60A-F3 of first trimester pregnancy urine (data not shown).

We also stimulated spleen cells from these treated mice with anti-CD3 in vitro and then measured the IFNgamma production at different time points. Figure 33 shows that in vivo treatment with c-hCG and its fractions 60A-F1 (IR-P1) and 60A-F2 (IR-P2) inhibited the anti-CD3 stimulated IFN-gamma production, while a moderate increase in IFN-gamma production was found with rhCG and 60A-F3. In addition, fraction 60A-F3 (IR-P3) in combination with rhCG was able to inhibit the production of IFN-gamma (figure 33).

Anti-CD3 stimulated proliferation studies showed that anti-CD3 stimulated splenocytes from NOD mice treated with c-hCG, and 60A-F1 have a smaller capacity to proliferate in vitro (figure 34). Furthermore, splenocytes from 60A-F3 (IR-P3) and rhCG treated mice showed a higher capacity to proliferate as compared to the PBS treated control mice (CTL), while 60A-F3(IR-P3) in combination with rhCG caused the same decrease in proliferation as c-hCG and 60A-F1 (IR-P1) (figure 34).

Moderate effect was found in the anti-CD3 stimulated proliferation of splenocytes from 60A-F2 treated NOD mice. Similarly, we also tested the effect of peptide 1 in combination with rhCG on Thl differention and IFNgamma production and proliferation. We observed that there was an increase of IFN-gamma production found under Thl polarisation conditions with peptide 1 (27 ng/ml) and r-hCG alone (25 ng/ml) as compared to PBS (20 ng/ml), while the outgrowth of Thl cells was completely blocked with the combination of rhCG and peptide 1 (7 ng/ml).

Furthermore, splenocytes from peptide 1 and rhCG treated mice showed a higher capacity to proliferate as compared to the PBS treated control mice (CTL), while peptide 1 in combination with rhCG caused the same decrease in proliferation as c-hCG and 60A-F1 and 60A-F2.

EAE model: Figure 35 shows the effect of 60A-F1 (IR-P1), 60A-F2 (IR-P2) and 60A-F3 (IR-P3) in EAE model induced by PLP+PTX. Here, we see that the treatment of mice with 60A-F1 and 60A-F2 reduced the disease severity as well as delayed the induction of EAE. While 60A-F3 treatment only delayed the onset of the disease, suggesting that it needs an additional factor(s) from 60A-F2. These result are also consistant with weight results shown in figure 35. In this figure, mice treated with active NMPF fractions after EAE induction, lost less weight than PBS treated mice. In addition, treatment of mice with c-hCG and 60A-F3 fraction from c-hCG showed less disease severity in EAE mouse model induced by PLP/B. pertussis which is a chronic disease model for EAE (MS) (figure 36).

r CAO model: Our CAO data showed that 15 rats in controle group treated with only PBS had infarcted area of 70+2% (avearge±standard error) after 60-minutes of CAO followed by 2 hours of reperfusion. While rats treated with peptide VLPALP, LQGV, VLPALPQVVC, LQGVLPALPQ ,LAGV, LQAV and MTRV showed infarcted area of 62+6%, 55±6%, 55±5%, 67±2%, 51±4%, 62+6% and 68+2%, respectively. Here, we see that certain peptides (such as VLPALP, LQGV, VLPALPQVVC, LAGV) have protective on the area at risk for infarction. In addition, peptide LQAV showed smaller infarcted area but in some instances the area was haemorhagic infarcted. These are the same peptides that have anti-septic shock activity in vivo. It is important to note that mice treated with certain above mentioned peptides showed less viscousity of blood. Apart from immunological effect, there is a possibility that these peptides have also effect on blood coagulation system directly or indirectly and there a certain homology between CAO and sepsis models. So, in both model the circulatory system plays an important role in the pathogensis of the disease.

Discussion Human chronic gonadotropin (hCG) is the glycoprotein hormone known as the hormone of pregnancy since its detection forms the basis of all pregnancy tests. Its is produced very early in pregnancy by the developing trophoblast tissue which becomes the placenta. The hormone serves to maintain the steroid secretions of the corpus luteum (derived from the ovarian follicle after ovulation). The resultant steroids maintain the lining of the uterus in a state suitable for development of the embryo after its implantation.

HCG is a member of the glycoprotein hormone family, which also includes human luteinizing hormone human follicle-stimulating hormone (FSH), and human thyroidstimulating hormone (TSH). These hormones are heterodimeric, sharing a common alpha subunit which in humans is encoded by a single gene and each having a unique beta subunit structure that confers hormone specificity. Among the four related hormones, only hCG and its close structural homolog, LH, bind to the same receptor present within the ovary in females and the testis in males. Human LH stimulates sex steroid production in both male and female and thus, the development of sex-specific characteristics. Human FSH binds to the different receptors in ovary and testis and serves to stimulate development of ova in the female and sperm in the male. The fourth homrone, human TSH, is not directly related to reproductive function but is responsible for stimulation of the production of thyroxine which controls the rate of bodily metabolism.

Recently, after solving the three-dimensional (3D) structure of hCG, it has been shown that hCG is a member of the structural superfamily of cystine knot growth factors like NGF, PDGF-B and TGF-beta.

HCG exhibits a variety of forms, especially in urine (Birken, 1996; O'Connor, 1994; Alfthan, 1996; Cole, 1996; Wide, 1994; Birken, 1993; Cole, 1993). It appears in abundance in the urine of women during the first trimester of pregnancy. It exhibits charge heterogeneity due to variability in its sialic acid content. These forms include heterodimeric hCG with intact polypeptide backbone (hCG), heterodimeric hCG with peptide bond cleavages it its beta-loop 2 (residues 44-49) (nicked hCG), hCG beta-core fragment which is derived from hCG beta-subunit and is composed of residues 6-40 disulfide bridged to residues 55-92 and containing trimmed carbohydrate groups with no sialic acid, hCG beta-subunit derived from dissociation of hCG (beta-hCG), hCG alphasubunit derived from dissociation of hCG (alpha-hCG). In addition, there is a pituitary form of hCG and pituitary form of an hLH beta-core fragment.

The beta-subunit, like the alpha-subunit of hCG, is composed of three loops; roughtly loop l(residues 9-40), loop 2 (residues 41-54) and loop 3 (residues 55-92). The beta-subunit also has the region termed "seatbelt", which wraps the alpha-subunit. The beta-subunit contains six disulfide bridges which hold the molecule together when peptide bond cleavages take place in loop 2 resulting in nicked hCG. The beta-core fragment is missing most of loop 2 and the seatbelt region. Hence, beta-core fragment is missing the entire seatbelt region, most of loop 2, and part of the amino terminus of the beta-subunit.

Cleavages in the beta-loop 2 region (since is known to be exposed to solvent and is easily cleaved by proteases) result in biologically inactive hCG and many immunoassays fail to measure nicked hCG accurately due to diminished immunopotency after cleavages in beta-loop 2.

Here we have shown that number of selected breakdown products from loop 2 have immune regulatory effects. In our experiments peptides 4 (LQGV) and 6 (VLPALP) inhibited shock completely, while peptide 2 (LQGVLPALPQ), 3 (LQG) and 7 (VLPALPQ) accelerated shock. In addition, 1 (VLPALPQVVC), 5 (GVLPALPQ), 8 (GVLPALP), 9 (VVC), 11 (MTRV), 12 (MTR), 13 (LQGVLPALPQWC) and 14 (cyclic, CLQGVPALPQVVC) showed in number of different experiments variability in effectiveness as well as in the kind (inhibitory vs accelerating) of activity. This variability is likely attributable to the rate of breakdown of the various peptides, and the different effects the various peptides have. Nicking in amino acid residues of beta-hCG beginning at residues 44, 45, 48 and 49 by human leukocyte elastase are observed and the existence of nicked hCG (and also analogous nicking residues) had been established by several investigator (Kardana, 1991; Birken, 1991). Moreover, one or two nicked beta site are also commonly seen in a single hCG preparation as well as in LH preparations (Ward, 1986; Hartree, 1985; Sakakibara, 1990; Birken, 2000; Cole, 1991; Birken, 1993). Existence of these peptides or their homologous during pregnancy explains the immunoregulatory state of the pregnancy. We have also shown that combination of peptide 1 with recombinant hCG is able to inhibit the development of dominant Thl CD4+ T cells while alone peptide 1 as well as recombinant hCG are not.

This strongly suggests the need of additional factor from hCG in order achieved this effect. These other factors could be derived from different parts hCG or their homologous that are known to exist as fragments such as residues beta-CG6-40, beta-CG41-55, beta-CG55-92 and beta-CG90-110. In addition, anti-septic shock peptides as well as shock accelerating peptides have some homology with certain regions of the above mentioned fragments. Previously, we have shown that 60A-F2 (also known as IR-P2 with anti-diabetic activity) does not posses anti-shock effects and even in some case it accelerates septic shock. So, it is likely that indeed the peptides (table used in the septic shock model in combination with above mentioned fragments from beta-hCG have anti-chronic inflammatory (anti-diabetic) effects.

Same is true for anti-EAE activity. These various peptides act inconsert to maintain hemostasis in the immune system and to prevent and control disbalances of the immune system like in infections, sepsis, autoimmune disease and in immune-mediated diseases.

We have shown in our previous patent (W09959617) and this patent application that these NMPF activities have regulatory effects on innate and adoptive immune responses and are present in variable ratio's which explains the heterogeneity in results with commercial hCG preparations derived from pregnancy urine even from the single commercial manufacturer.

I

Number of studies have shown that hCG is not only produce by placenta, but for example pituitary as well as PBMC from non-pregnant individuals and male individuals are also able to produce hCG. Apart from the possibility that NMPF could be produce by placenta and by maternal cells, it is possible that NMPF could also be derived from hCG like molecules in the presence of various proteases and its regulation might be also tightly controlled by these proteases. Many proteases are shown to be exist in human plancenta, serum and in other parts of the body and one of their physiological roles could be the production of such immunomodulating peptides. Since we have also observed in our experiments the upregulation of pan-Nk cells (upregulation of Dx-5 marker), it is reasonable to think that that these type of cells do not have only natural killer cell activity, but are also involved in the regulation of adoptive as well as innate immune responses (Saito, 2000). For instance, Nk cells have the ability to modulate cellular response of antigen presenting cells (APC) like mcrophages, DC, and other lymphocytes population. In addition the strong cytotoxic activity of NK cells especially pan-NK cells contribute to the inhibition of tumor and tumor metastasis without inducing significant toxicity (Arai, 2000). Such immunomodulatory effect is also observed by neutrophils which are involved in different pathological insults such as sepsis. Not only are these cells crucial cell population which bridge innate resistance and acquired cell-mediated immune responses but they also play a role in driving T-cell host responses directly or indirectly through APC against the intracellular organism (Tateda, 2001). So, during pregnancy the cross talk mediated by NMPF and hCG in the maternal endocrine system, decidual immune system and trophoblast function modulate the relationship between maternal and fetal immunity and endocrine systems. On one side due to NMPF and hCG mamalian fetus perceived, as a successful allograft and successful parasite and on the other the maternal and the fetus are also optimally immuno-compitent for protection against threats like infections.

In addition, in this patent implication, we have shown that certain peptides (such as VLPALP, LQGV, VLPALPQVVC, LAGV) have protective on the area at risk for infarction. In addition, peptide LQAV showed smaller infarcted area but in some instances the area was haemorhagic infarcted. These are the same peptides that have anti-septic shock activity in vivo. It is important to note that mice treated with certain above mentioned peptides showed less viscousity of blood. Apart from immunological effect, there is a possibility that these peptides have also effect on blood coagulation system directly or indirectly and there a certain homology between CAO and sepsis models. So, in both model the circulatory system plays an important role in the pathogensis of the disease. In this CAO model we see a protective role of NMPF on myocardial infarction this also suggest the same protective effect of NMPF in other organs as well in circulatory related disease. Examples of such diseases are (but not limited to) cerebral vascular incident (CVI), circulatory diseases of the brain, retinopathies (such as associated with vascular diseases like diabetes), circulatory diseases of pregnancy, thrombosis, atherosclerosis.

Since NMPF has a immunomodulatory effect and protective effect on CAO induced infarct, it can also be used in instances where there is risk of low blood circulation leading to decrease in oxygen supply (hypoxia). Examples are (but not limited to) angiography procedures, PTCA, (coronary) bypass procedures and stenosis.

EXAMPLE III MATERIAL AND METHODS Peptide synthesis The peptides as mentioned in this document were prepared by solid-phase synthesis (Merrifield, 1963) using the fluorenylmethoxycarbonyl (Fmoc)/tert-butylbased methodology (Atherton, 1985) with 2-chlorotrityl chloride resin (Barlos, 1991) as the solid support. The side-chain of glutamine was protected with a trityl function. The peptides were synthesized manually. Each coupling consisted of the following steps: removal of the alpha-amino Fmoc-protection by piperidine in dimethylformamide (DMF), (ii) coupling of the Fmoc amino acid (3 eq) with diisopropylcarbodiimide (DIC)/1hydroxybenzotriazole (HOBt) in DMF/N-methylformamide (NMP) and (iii) capping of the remaining amino functions with acetic anhydride/diisopropylethylamine (DIEA) in DMF/NMP. Upon completion of the synthesis, the peptide resin was treated with a mixture of trifluoroacetic acid

(TFA)/H

2 0/triisopropylsilane (TIS) 95:2.5:2.5. After minutes TIS was added until decolorization. The solution was evaporated in vacuo and the peptide precipitated with diethylether. The crude peptides were dissolved in water (50-100 mg/ml) and purified by reverse-phase highperformance liquid chromatography (RP-HPLC). HPLC conditions were: column: Vydac TP21810C18 (10 x 250 mm); elution system: gradient system of 0.1% TFA in water v/v and 0.1% TFA in acetonitrile (ACN) v/v flow rate 6 ml/min; absorbance was detected from 190-370 nm. There were different gradient systems used. For example for peptides LQG and LQGV: 10 minutes 100% A followed by linear gradient 0-10% B in 50 minutes. For example for peptides VLPALP and VLPALPQ: 5 minutes 5% B followed by linear gradient 1% B/minute. The collected fractions were concentrated to about 5 ml by rotation film evaporation under reduced pressure at 40 0 C. The remaining TFA was exchanged against acetate by eluting two times over a column with anion exchange resin (Merck II) in acetate form. The elute was concentrated and lyophilised in 28 hours. Peptides later were prepared for use by dissolving them in PBS.

Transcription factor experiment Macrophage cell line. The RAW 264.7 macrophages, obtained from American Type Culture Collection (Manassas, VA), were cultured at 37 0 C in 5% C02 using DMEM containing 10% FBS and antibiotics (100 U/ml of penicillin, and 100 gg/ml streptomycin). Cells (1 x10 6 /ml) were incubated with peptide (10 [ig/ml) in a volume of 2 ml. After 8 h of cultures cells were washed and prepared for nuclear extracts.

Nuclear extracts. Nuclear extracts and EMSA were prepared according to Schreiber et al. Methods (Schriber et al. 1989, Nucleic Acids Research 17). Briefly, nuclear extracts from peptide stimulated or nonstimulated macrophages were prepared by cell lysis followed by nuclear lysis. Cells were then suspended in 400 gl of buffer (10 mM HEPES (pH 10 mM KC1, 0.1 mM KCL, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitors), vigorously vortexed for 15 s, left standing at 4 0 C for 15 min, and centrifuged at 15,000 rpm for 2 min. The pelleted nuclei were resuspended in buffer mM HEPES (pH 10% glycerol, 400 mM NaC1, 1 mM EDTA, ImM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitors) for 30 min on ice, then the lysates were centrifuged at 15,000 rpm for 2 min. The supernatants containing the solubilized nuclear proteins were stored at -70 0 C until used for the Electrophoretic Mobility Shift Assays

(EMSA).

EMSA. Electrophoretic mobility shift assays were performed by incubating nuclear extracts prepared from control (RAW 264.7) and peptide treated RAW 264.7 cells with a 32P-labeled double-stranded probe AGCTCAGAGGGGGACTTTCCGAGAG synthesized to represent the NF-kappaB binding sequence. Shortly, the probe was end-labeled with T4 polynucleotide kinase according to manufacturer's instructions (Promega, Madison, WI). The annealed probe was incubated with nuclear extract as follows: in EMSA, binding reaction mixtures (20 gl) contained 0.25 Ag of poly(dI-dC) (Amersham Pharmacia Biotech) and 20,000 rpm of 32P-labeled DNA probe in binding buffer consisting of 5 mM EDTA, 20% Ficoll, 5 mM DTT, 300 mM KC1 and 50 mM HEPES. The binding reaction was started by the addition of cell extracts (10 Ag) and was continued for 30 min at room temperature. The DNA-protein complex was resolved from free oligonucleotide by electrophoresis in a 6% polyacrylamide gel. The gels were dried and exposed to x-ray films.

RESULTS

NF-kB experiments The transcription factor NF-kB participates in the transcriptional regulation of a variety of genes. Nuclear protein extracts were prepared from LPS and peptide treated RAW264.7 cells or from LPS treated RAW264.7 cells. In order to determine whether the peptide modulates the translocation of NF-kB into the nucleus, on these extracts EMSA was performed. In this experiment tri- or tetrameric peptides that show the modulation of translocation of NF-kB are: LQG, LQGV, VVC, MTRV, MTR.

NFkB analysis in macrophages Mouse macrophage cell line: RAW 264.7 mouse macrophages were cultured in DMEM, containing 10% or 2% FBS, penicillin, streptomycin and glutamine, at 37 oC,

CO

2 Cells were seeded in a 12-wells plate (3x10 6 cells/ml) in a total volume of 1 ml for 2hours and then stimulated with LPS coli 026:B6; Difco Laboratories, Detroit, MI, USA) and/or NMPF (1 pg/ml). After minutes of incubation plates were centrifuged and cells were collected for nuclear extracts.

Nuclear Extracts: Nuclear extracts and EMSA were prepared according to Schreiber et al. Method (Schriber et al. 1989, Nucleic Acids Research 17). Cells were collected in a tube and centrifuged for 5 minutes at 2000 rpm (rounds per minute) at 4 0 C (Universal 30 RF, Hettich Zentrifuges). The pellet was washed with ice-cold Tris buffered saline (TBS pH 7.4) and resuspended in 400 il of a hypotonic buffer A (10 mM HEPES pH 7.9, 10 mM KC1, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitor cocktail (Complete T Mini, Roche) and left on ice for 15 minutes. Twenty five micro litre 10% NP-40 was added and the sample was centrifuged (2 minutes, 4000 rpm, 4 0 The supernatant (cytoplasmic fraction) was collected and stored at -70°C. The pellet, which contains the nuclei, was washed with 50 pl buffer A and resuspended in 50 il buffer C (20 mM HEPES pH 7.9, 400 mM NaC1, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitor cocktail and 10% glycerol). The samples were left to shake at 4 0 C for at least 60 minutes.

Finally the samples were centrifuged and the supernatant (nucleic fraction) was stored at -70 0

C.

Bradford reagent (Sigma) was used to determine the final protein concentration in the extracts.

EMSA: For Electrophoretic mobility shift assays an oligonucleotide representing NF-KB binding sequence AGC TCA GAG GGG GAC TTT CCG AGA was synthesized.

Hundred pico mol sense and antisense oligo were annealed and labelled with y- 32 P-dATP using T4 polynucleotide kinase according to manufacture's instructions (Promega, Madison, WI). Nuclear extract (5-7.5 tg) was incubated for 30 minutes with 75000 cpm probe in binding reaction mixture (20 microliter) containing 0.5 pg poly dI-dC (Amersham Pharmacia Biotech) and binding buffer BSB mM MgC12, 5 mM CaC1 2 5mM DTT and 20% Ficoll) at room temperature. The DNA-protein complex was resolved from free oligonucleotide by electrophoresis in a 4-6% polyacrylamide gel (150 V, 2-4 hours). The gel was then dried and exposed to x-ray film.

Results The transcription factor NF-kB participates in the transcriptional regulation of a variety of genes. Nuclear protein extracts were prepared from either LPS (1 mg/ml), NMPF (1 mg/ml) or LPS in combination with NMPF treated RAW264.7 cells. In order to determine whether the NMPF peptides modulate the translocation of NF-kB into the nucleus, on these extracts EMSA was performed. In this experiment tri- or tetrameric peptides that show the inhibition of LPS induced translocation of NF-kB are: LQG, LQGV, WC, MTRP. The peptide that in this experiment promoted LPS induced translocation of NF-kB is: MTRV.

Basal levels of NF-kB in the nucleus was decreased LQG) and LQGV while basal levels of NF-kB in the nucleus was increased by VVC, MTRV and MTR. In other experiments, QVVC also showed the modulation of translocation of NF-kB into nucleus (data not shown).

Nuclear location of peptide experiment Reverse-phase high-performance liquid chromatography (RP-HPLC) method was used to prove the presence of synthetic tetrameric-peptide in the nuclear extracts. We used a Shimadzu HPLC system equipped with Vydac monomeric C18 column (column218MS54, LC/MS C18 reversed phase, 300A, 5 microm, 4.6mm ID x 250mm elution system: gradient system of 0.01% TFA and 5% acetonitrile (CAN) in water v/v and 0.01% TFA in 80% acetonitrile (ACN) v/v flow rate 0.5 ml/min; absorbance was detected from 190-370 nm. The gradient time programme was as follows: Time (min) Buffer B concentration 0.01 0 5.0 0 30.0 40.0 100 60.0 100 65.0 0 70.0 0 The elution time of peptide LQGV was determined by injecting 2 microgram of the peptide in a separate run.

Mass spectrometry (MS) analysis of fraction which contained possible LQGV (elution time was determined by injecting the peptide in the same or separate run) was performed on LCQ Deca XP (Thermo Finnigan).

RESULTS

Nuclear location of peptide experiment The nuclear protein extracts used in EMSA experiments were also checked for the presence of LQGV by means of HPLC and MS. Since HPLC profile of LQGV showed that the peptide elutes at around 12 minutes (data not shown), fraction corresponding to region 10-15 minutes was collected and analysed for the presence of this peptide in MS. The MSn data and MS-sequence confirm the presence of LQGV peptide in the nuclear protein extract obtained from LQGV+LPS stimulated RAW264.7 cells.

Endotoxin shock model (Sepsis) Sepsis. For the endotoxin model, BALB/c mice were injected i.p. with 8-9 mg/kg LPS coli 026:B6; Difco Lab., Detroit, MI, USA). Control groups (PBS) were treated with PBS i.p. only. To test the effect of NMPF from different sources (synthetic, commercial hCG preparation we treated BALB/c with a dose of 300-700 IU of different hCG preparations (PG23;Pregnyl batch no. 235863, PG25; Pregnyl batch no. 255957) and with synthetic peptides (5 mg/kg) after two hours of LPS injection. In other experiments BALB/c mice were injected i.p. either with 10 mg/kg or with 11 mg/kg LPS coli 026:B6; Difco Lab., Detroit, MI, USA). Subsequently mice were treated after 2 hours and 24 hours of LPS treatment with NMPF peptides.

Semi-quantitative sickness measurements. Mice were scored for sickness severity using the following measurement scheme: 1 Percolated fur, but no detectable behaviour differences compared to normal mice.

2 Percolated fur, huddle reflex, responds to stimuli (such as tap on cage), just as active during handling as healthy mouse.

3 Slower response to tap on cage, passive or docile when handled, but still curious when alone in a new setting.

4 Lack of curiosity, little or no response to stimuli, quite immobile.

Laboured breathing, inability or slow to selfright after being rolled onto back (moribund) 6 Sacrificed

RESULTS

Endotoxin shock model (Sepsis) Sepsis experiments. To determine the effect of synthetic peptides (NMPF) in high-dose LPS shock model, BALB/c mice were injected intraperitoneally with different doses of LPS and survival was assessed daily for 5 days. In this experiment (for the LPS endotoxin model) BALB/c mice were injected i.p. with 8-9 mg/kg LPS coli 026:B6; Difco Lab., Detroit, MI, USA). Control groups (PBS) were treated with PBS i.p. only. We treated BALB/c mice with a dose of 300-700 IU of different hCG preparations (PG23;Pregnyl batch no. 235863, Pregnyl batch no. 255957) or with peptides (5 mg/kg) after two hours of LPS injection.

These experiments showed (table that NMPF peptides 4, 6, 66 and PG23 inhibited shock completely (all mice had in first 24 hours sickness scores not higher than 2; shortly thereafter they recovered completely and had sickness scores of while peptides 2, 3 and 7 accelerated shock (all mice had in first 24 hours sickness scores of 5 and most of them died, while the control mice treated with LPS+PBS had sickness scores of 3-4 in first 24 hours and they most of them died after 48 hours with sickness scores of 5 (17% survival rate at 72 hours). In addition, peptides 1, 5, 8, 9, 11, 12, 13, 14 and 64 showed in number of different experiments variability in effectiveness as well as in the kind (inhibitory vs accelerating) of activity. This variability is likely attributable to the rate of breakdown of the various peptides, and the different effects the various peptides and their breakdown products have in vivo. In addition, these experiments also showed the variability in anti-shock activity in c-hCG preparations that is likely attributable to the variation in the presence of anti-shock and shock accelerating NMPF. Visible signs of sickness were apparent in all of the experimental animals, but the kinetics and obviously the severity of this sickness were significantly different. These data are representative of at least separate experiments.

In Table 2 we see the effect of ALA-replacement (PEPSCAN) in peptide LQG, LQGV, VLPALP, VLPALPQ in septic shock experiments. We conclude that the change in even one amino acid by a neutral amino acid can lead to different activity. So, genomic differences as well as polymorphism in these peptides can regulate the immune response very precise. Derivatives of these peptides, for example (but not limited to) by addition of classical and non-classical amino acids or derivatives that are differentially modified during or after synthesis, for example benzylation, amidation, glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand etc. could also lead to a better effectiveness of the activity.

To determine whether treatment of BALB/c mice with NMPF inhibit septic shock at different stages of disease, synthetic peptides (NMPF) were injected i.p. at 2 and 24 hours after the induction of septic shock with high dose LPS (10 mg/kg).

As shown in Tables 3 and 4, control mice treated PBS after the shock induction, reached a sickness score of at 14 and 24 hours, and remained so after the second injection with PBS. The survival rate in control group mice was 0% at 48 hours. In contrast to control mice, mice treated with NMPF 9, 11, 12, 43, 46, 50 and reached a maximum sickness score of 2-3 at 24 hours after the induction of septic shock and further reached a maximum sickness score of 1-2 at 48 hours after the second injection of NMPF. In addition, mice treated with NMPF 5, 7, 8, 45, 53 and 58 reached a sickness score of and after the second injection with NMPF all mice returned to a sickness score of 1-2 and survival rates in NMPF groups were 100%. Mice treated with NMPF 3 reached sickness scores of 3-4 and the second NMPF injection did save these mice. These experiments show that NMPF peptides have anti-shock activity at different stages of the disease and NMPF have anti-shock activity even at disease stage when otherwise irreversible damage had been done. This indicates that NMPF have effects on different cellular levels and also have repairing and regenerating capacity.

Dendritic cells experiments Mice. The mouse strain used in this study was BALB/c (Harlan, Bicester, Oxon, GB). All mice used in experiments were females between 8 and 12 weeks of age.

Mice were housed in a specific-pathogen-free facility. The Animal Use Committee at the Erasmus University Rotterdam, The Netherlands approved all studies.

In vivo treatment. At least six mice per group were injected intraperitonally with LPS (10 mg/kg; Sigma). After 2 and 24 hrs of LPS induction, mice were injected i.p. with either NMPF (5 mg/kg) or Phosphate Buffered Saline (PBS), in a volume of 100 pl. LPS induced shock in this model had more than 90% mortality at 48 hrs.

Bone marrow cell culture. From treated mice, bonemarrow cells were isolated and cultured as follows.

BALB/c mice were killed by suffocation with CO 2 The femurs and tibiae were removed and freed of muscles and tendons under aseptic conditions. The bones were placed in R10 medium (RPMI 1640, supplemented with 50 U/ml penicillin, 50 pg/ml streptomycin, 0.2 M Na-pyruvate, 2 mM glutamine, 50 pM 2-mercaptoethanol and 10% fetal calf serum (Bio Whittaker, Europe)).

The bones were then cleaned more thoroughly by using an aseptic tissue and were transferred to an ice cold mortier with 2 ml of R10 medium. The bones were crushed with a mortel to get the cells out of the bones. Cells were filtered through a sterile 100 pM filter (Beckton Dickinson Labware) and collected in a 50 ml tube (FALCON). This procedure was repeated until bone parts appeared translucent.

The isolated cells were resuspended in 10 ml of and 30 ml of Geys medium was added. The cell suspension was kept on ice for 30 minutes, to lyse the red blood cells. Thereafter, the cells were washed twice in medium. Upon initiation of the culture, the cell concentration was adjusted to 2 x 10 5 cells per ml in medium supplemented with 20 ng/ml recombinant mouse Granulocyte Monocyte-Colony Stimulating Factor (rmGM-CSF; BioSource International, Inc., USA) and seeded in 100 mm non-adherent bacteriological Petri dishes (Falcon). For each condition six Petri dishes were used and for further analysis, cells were pooled and analysed as described ahead. The cultures were placed in a 5% C0 2 -incubator at 37 0 C. Every three days after culture initiation, 10 ml fresh R10 medium supplemented with rmGM-CSF at 20 ng/ml was added to each dish.

Nine days after culture initiation, non-adherent cells were collected and counted with a Coulter Counter (Coulter).

Alternatively, BM cells from untreated mice were isolated and cultured as described above and were in vitro treated with the following conditions: NMPF 4, NMPF 46, NMPF 7, NMPF 60 (20 ig/ml) were added to the culture either at day 0 or day 6 after culture initiation. Or LPS (1 pg/ml) was added to the culture at day 6 with or without the NMPF.

Immunofluorescence staining. Cells (2 x 105) were washed with FACS-buffer (PBS with 1% BSA and 0.02% sodium azide), and transferred to a round-bottomed 96-well plate (NUNC). The antibodies used for staining were against MHC-II PE and CDllc/CD18 FITC (PharMingen/Becton Dickinson, Franklin Lakes, NJ, US).

Cells were resuspended in 200 [l FACS-buffer containing both of the antibodies at a concentration of ng/pl per antibody. Cells were then incubated for min at 4 0 C. Thereafter, cells were washed 3 times and finally resuspended in 200 (1 FACS-buffer for flowcytometric analysis in a FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany). All FACS-data were analyzed with CellQuest software (Becton Dickinson, Heidelberg, Germany).

Statistical analysis All differences greater than 20% are considered to be significant.

RESULTS

Dendritic cells experiments Cell yield of ex vivo bone-marrow cell cultures. To determine the in vivo effect of LPS and NMPF treatment on the cell yield obtained from a nine-day culture of bonemarrow with rmGM-CSF, cells were isolated from the BM of treated mice and cultured, harvested and counted as described. The cell yield of the bone-marrow cultures of LPS (10 mg/kg) treated mice is significantly decreased compared to PBS treated mice. Mice treated with NMPF 4, NMPF 7, NMPF 46 and NMPF 60 after LPS shock induction, had a significantly increased cell yield compared to LPS in the presence of rmGM-CSF. In addition, BM cultures from NMPF 46 treated mice gave a significantly increased cell yield even compared to the PBS group.

Immunofluorescence staining of in vivo treated bonemarrow derived DC. Culture of BM cells in the presence of rmGM-CSF gave rise to an increased population of cells that are positive for CDllc and MHC-II. Cells positive for these cell membrane markers are bone-marrow derived dendritic cells DC are potent antigen presenting cells (APC) and modulate immune responses. In order to determine the maturation state of myeloid derived DC, cells were stained with CDllc and MHC-II.

The expression of the MHC-II molecule was significantly decreased on CDllc-positive cells from LPS treated mice as compared to the PBS group. This decrease in MHC-II expression was further potentiated by the in vivo treatment with NMPF 4 and NMPF 46. However, treatment of mice with NMPF 7 and NMPF 60 significantly increased the expression of the MHC-II molecule even as compared to the PBS group.

Cell yields of in vitro bone-marrow cell cultures.

To determine the effect of LPS and NMPF in vitro on the cell yield of a nine-day culture of bone-marrow cells, we isolated the BM cells from untreated BALB/c mice and cultured in the presence of rmGM-CSF. In addition to rmGM-CSF, cultures were supplemented with NMPF at either day 0 or day 6 with or without the addition of LPS at day 6.

There is a significant decrease in cell yield in LPS treated BM cells as compared to PBS. BM cells treated with NMPF 4, 7, 46 or 60 at time point t=0 or t=6 without LPS, showed a significant increase in cell yield as compared to the PBS group. However, BM cell cultures treated with NMPF 4 at time point t=6 showed significant decrease in cell yield as compared to the PBS group and this effect is comparable with the effect of LPS. In addition, BM cells treated with NMPF 4, 7, 46 or 60 at time point t=6 in combination with LPS showed a significant increase in cell yield as compared to the LPS group and even in the group of NMPF 7 the cell yield was significantly increased as compared to the PBS group.

Immunofluorescence staining of in vitro treated bone-marrow derived DC. To determine the maturation state of DC, CDllc positive cells were stained for MHC-II antibody. There is an opposite effect of LPS on MHC-II expression as compared to in vivo experiments, namely, MHC-II expression is significantly increased with LPS treatment in vitro as compared to PBS. NMPF 4 with LPS further potentiated the effect of LPS, while NMPF 7 with or without LPS significantly inhibited the expression of MHC-II as compared to LPS and PBS, respectively. However, cells treated with NMPF 46 without LPS showed significantly increased expression of MHC-II on CDllc positive cells. Furthermore, no significant differences were found in the group NMPF with or without LPS on MHC-II expression as compared to LPS and PBS treated cells.

To determine the in vivo effect of LPS and NMPF treatment on the cell yield obtained from a nine-day culture of bone-marrow with rmGM-CSF, cells were isolated from the BM of treated mice and cultured, harvested and counted as described. The cell yield of 'attached' cells was significant increased with NMPF 4, 7, 9, 11, 43, 46, 47, 50, 53, 58 60 and even in the group of NMPF 7, 46 and the cell yield was significant increased as compared to the PBS group. In addition, cell yield of 'unattached' cells was significant increased with NMPF 4, 7, 9, 11, 46, 50, 53, 58 60 and agin in the group of NMPF 46 the cell yield was significant increased as compared to the PBS group.

To determine the effect of LPS and NMPF in vitro on the cell yield of a nine-day culture of bone-marrow cells of female NOD mice, we isolated the BM cells from untreated NOD mice and cultured in the presence of rmGM- CSF. In addition to rmGM-CSF, cultures were supplemented with NMPF. In these experiments the bone-marrow cell yield of 'un-attached' cells was significant increased with NMPF 1,2,3,4,5,6,7,8,9, 12 and 13 as compared to PBS group and no effect was observed with NMPF 11. The 'attached' bone-marrow cells of these experiments showed different yield than the 'un-attached' cells, namely there was a significant increased in cell yield in cultures treated with NMPF 3 and 13, while cultures treated with NMPF 2 and 6 showed significant decrease in the cell yield as compared to PBS (more additional results are summarised in table Coronary Artery Occlusion (CAO) experiments CAO induction and treatment. NMPF have immunoregulatory effects in chronic inflammatory as well as acute inflammatory mice models. Since certain Scytokines like TGF-betal, TNF-alpha, IL-1 and ROS Ji (reactive oxygen species) have been implicated in Sirreversible myocardial damage produced by prolonged episodes of coronary artery occlusion and reperfusion in vivo that leads to ischaemia and myocardial infarct, we tested the cardio-protective properties of peptides in ad Slibitum fed male Wistar rats (300 The experiments V were performed in accordance with the Guiding principles C( 10 in the Care and Use of Animals as approved by the Council Sof the American Physiological Society and under the C( regulations of the Animal Care Committee of the Erasmus University Rotterdam. Shortly, rats were stabilised for 30 minutes followed by i.v. 1 ml of peptide treatment (0.5 mg/ml) in 10 minutes. Five minutes after completion of treatment, rats were subjected to a 60-min coronary artery occlusion (CAO). In the last 5 minutes of CAO, rats were again treated over 10 minutes i.v. with 1 ml of peptide (0.5 mg/ml) followed by 120 minutes of reperfusion Experimental and surgical procedures are described in detail in Cardiovascular Research 37(1998) 76-81. At the end of each experiment, the coronary artery was re-occluded and was perfused with ml Trypan Blue Sigma Chemical Co.) to stain the normally perfused myocardium dark blue and delineate the nonstained area at risk The heart was then quickly excised and cut into slices of 1 mm from apex to base.

From each slice, the right ventricle was removed and the left ventricle was divided into the AR and the remaining left ventricle, using micro-surgical scissors. The AR was then incubated for 10 min in 370C Nitro-Blue-Tetrazolium (Sigma Chemical Co.; 1 mg per 1 ml Sorensen buffer, pH which stains vital tissue purple but leaves infarcted tissue unstained. After the infarcted area (IA) was isolated from the noninfarcted area, the different areas of the LV were dried and weighed separately.

Infarct size was expressed as percentage of the AR.

Control rats were treated with PBS.

RESULTS

Coronary Artery Occlusion (CAO) experiments Our CAO data showed that 15 rats in control group treated with only PBS had infarcted area of 70+2% (average+standard error) after 60-minutes of CAO followed by 2 hours of reperfusion. While rats treated with peptide VLPALP, LQGV, VLPALPQVVC, LQGVLPALPQ, LAGV, LQAV and MTRV showed infarcted area of 62+6%, 55±6%, 55+5%, 67±2%, 51±4%, 62+6% and 68+2%, respectively. Here, we see that certain peptides (such as VLPALP, LQGV, VLPALPQVVC, LAGV) have a protective effect on the area at risk for infarction. In addition, peptide LQAV showed smaller infarcted area but in some instances the area was haemorhagic infarcted. In addition NMPF-64 (LPGCPRGVNPWS) had also protective effect in CAO experiment. It is important to note that mice treated with certain above mentioned peptides showed less viscousity of blood. Apart from immunological effect, these peptides may have also effect on blood coagulation system directly or indirectly since there is certain homology with blood coagulation factors for additional results of NMPF peptides see table So, in both models the circulatory system plays an important role in the pathogenesis of the disease.

Chicken eggs experiments In vivo treatment of fertilised chicken eggs with NMPF. Fertile chicken eggs (Drost Loosdrecht BV, the Netherlands) were incubated in a diagonal position in an incubator (Pas Reform BV, the Netherlands) at 37 OC and 32% relative humidity.

Solutions of NMPF peptides (Img/ml) and VEGF were made in PBS. At least ten eggs were injected for every condition. The treatment was performed as follows: on day 0 of incubation, a hole was drilled into the eggshell to open the air cell. A second hole was drilled 10 mm lower and right from the first hole for injection. The holes in the eggshell were disinfected with jodium. The NMPF peptides (100 ug/egg) and/or VEGF (100 ng/ml) were injected in volume of 100 l. The holes in the eggshell weresealed with tape (Scotch Magic T M Tape, 3M) and the eggs were placed into the incubator.

Quantification of angiogenesis. On day 7 of incubation, the eggs were viewed under an UV lamp to check if the embryos were developing in a normal way and the dead embryos were counted. On day 8 of incubation, the embryos were removed from the eggs by opening the shell at the bottom of the eggs. The shell membrane was carefully dissected and removed. The embryos were placed in a 100-mm Petri dish. The embryo and the blood vessels were photographed (Nikon E990, Japan) in vivo with the use of a microscope (Zeiss Stemi SV6, Germany). One overview picture was taken and 4 detail pictures of the blood vessels were taken. Only eggs with vital embryos were evaluated.

Data analysis. Quantification of angiogenesis was accomplished by counting the number of blood vessels branches. Quantification of vasculogenesis was accomplished by measuring the blood vessel thickness. The number of blood vessel branches and the blood vessel thickness were counted in the pictures (4 pictures/egg) using Corel Draw 7. Thereafter, the number of blood vessel branches and the thickness of the blood vessels were correlated to a raster of microscope (10 mm 2 for comparison. The mean number of branches and the mean blood vessel thickness of each condition (n=10) were calculated and compared to the PBS control eggs using a Student's T-test.

i

RESULTS

Chicken eggs experiments In order to determine the effect of NMPF on angiogenesis and vasculogenesis we treated fertilized chicken eggs with NMPF or NMPF in combination with VEGF as described in materials and methods section. NMPF 3, 4, 9 and 11 promoted angiogenesis while NMPF VEGF, 7, 43, 44, 45, 46, 51 and 56 inhibited angiogenesis NMPF 6, 7, 12, 45, 46 and 66 were able to inhibit angiogenesis induced by VEGF. Moreover, NMPF 6 itself did not show any effect on angiogensis, but it inhibited (p<0.05) NMPF 3 induced angiogenesis. NMPF 1, 2,3, 4, 6, 7, 8, 12, 50, 51, and 52 had vasculogenesis inhibiting (p<0.05) effect, while only NMPF 44 promoted (p<0.05) vasculogenesis.

NO experiment Cell culture. The RAW 264.7 murine macrophage cell line, obtained from American Type Culture Collection (Manassas, VA, USA), were cultured at 37 0 C in 5% C02 using DMEM containing 10% fetal calf serum (FCS), 50 U/ml penicillin, 50 pg/ml streptomycin, 0.2 M Na-pyruvate, 2 mM glutamine and 50 iM 2-mercaptoethanol (Bio Whittaker, Europe). The medium was changed every 2 days.

Nitrite measurements. Nitrite production was measured in the RAW 264.7 macrophage supernatants. The cells (7.5 x10 5 /ml) were cultured in 48-well plates in 500 microliter of culture medium. The cells were stimulated with LPS (10 microg/ml) and/or NMPF (1 pg/ml, 1 ng/ml, 1 microg/ml) for 24 hours, then the culture media were collected. Nitrite was measured by adding 100 microl of Griess reagent (Sigma) to 100 microl samples of culture medium. The OD 540 was measured using a microplate reader, and the nitrite concentration was calculated by comparison with the ODs 40 produced using standard solutions of sodium nitrite in the culture medium.

RESULTS

NO experiment NO production is a central mediator of the vascular and inflammatory response. Our results show that macrophages (RAW 264.7) stimulated with LPS produce large amount of NO. However, these cells co-stimulated with most of the NMPF peptides (NMPF peptide 1 to 14, 43 to 66 and 69) even in a very low dose (1 pg/ml) inhibited the production of NO.

Results Analysis of different peptides in data bases Examples of different data bases in which peptides were analysed are: Proteomics tools: Similarity searches BLAST data base (ExPasy, NCBI) SMART (EMBL) PATTINPROT (PBIL) Post-translational modification prediction SignalP (CBS) Primary structure analysis HLA Peptide Binding Predictions (BIMAS) Prediction of MHC type I and II peptide binding

(SYFPEITHI)

Amino acid scale representation (Hydrophobicity, other conformational parameters, etc.) (PROTSCALE) Representations of a protein fragment as a helical wheel(HelixWheel HelixDraw)

RESULTS

A non-extensive list of relevant tri- or tetrameric peptides with predicted gene-regulatory activity.

pdbilDE711DE7-A INTERACTION OF FACTOR XIII ACTIVATION PEPTIDE WITH ALPHA- THROMBIN

LQGV

pdbI1QMHI1QMH-A CRYSTAL STRUCTURE OF RNA 3'-TERMINAL PHOSPHATE CYCLASE, AN UBIQUITOUS ENZYME

LQTV

pdb iLYPjiLYP CAP18 (RESIDUES 106 137) IQG, IQGL, LPKL, pdbllB90llB90-A HUMAN ALPHA-LACTALBUNIN

LPEL

pdb I GLU1I1GLU-A GLUCOCORTICOID RECEPTOR (DNA-BINDING

DOMAIN)

PARP

pdbI2KINI2KTN-B KINESIN (MONOMERIC) FROM RATTUS

NORVEGICUS

MTRI

pdbi1SMPI1SMP-I MOL_-ID: 1; MOLECULE: SERRATIA METALLO PROTEINASE; CHAIN: A LQKL, PEAP pdbI1ES7IlES7-B COMPLEX BETWEEN BMP-2 AND TWO BMP RECEPTOR IA ECTODOMAINS LPQ, PTLP pdbI1BHXI1BHX-F X-RAY STRUCTURE OF THE COMPLEX OF HUMAN ALPHA THROMBIN WITH THE INHIBITOR SDZ 229-357 LQV, LQVV pdbll VCB I1VCB-A THE VHL-ELONGINC-ELONGINB STRUCTURE

PELP

pdbI1CQKI1CQK-A CRYSTAL STRUCTURE OF THE CH3 DOMAIN FROM THE MAK33 ANTIBODY

PAAP

pdb I FCB1I1FCB-A FLAVOCYTOCHROME

LQG,

pdb I LDC1I1LDC-A L-LACTATE DEHYDROGENASE: CYTOCHROME C OXIDOREDUCTASE (FLAVOCYTOCHROME 1.1.2.3) MUTANT WITH TYR 143 REPLACED BY PHE (Y143F) COMPLEXED WITH PYRUVATE

LQG

pdbllBFB 1BFB BASIC FIBROBLAST GROWTH FACTOR COMPLEXED WITH HEPARIN TETRAMER FRAGMENT LPAL, PALP pdb|1MBFI1MBF MOUSE C-MYB DNA-BINDING DOMAIN REPEAT 1

LPN

pdblIR2AI1R2A-A THE MOLECULAR BASIS FOR PROTEIN KINASE A

LQG,

pdb|lCKAI1CKA-B C-CRK (N-TERMINAL SH3 DOMAIN) (C-CRKSH3- N) COMPLEXED WITH C3G PEPTIDE (PRO-PRO-PRO-ALA-LEU-PRO-

PRO-LYS-LYS-ARG)

PALP

pdbl RLQI1RLQ-R C-SRC (SH3 DOMAIN) COMPLEXED WITH THE PROLINE-RICH LIGAND RLP2 (RALPPLPRY) (NMR, MINIMIZED AVERAGE STRUCTURE) LPPL, PPLP pdb|1TNTI1TNT MU TRANSPOSASE (DNA-BINDING DOMAIN) (NMR, 33 STRUCTURES) LPG, LPGL, LPK pdbllGJSI1GJS-A SOLUTION STRUCTURE OF THE ALBUMIN BINDING DOMAIN OF STREPTOCOCCAL PROTEIN G

LAAL

pdb lGBRI1GBR-B GROWTH FACTOR RECEPTOR-BOUND PROTEIN 2 (GRB2, N-TERMINAL SH3 DOMAIN) COMPLEXED WITH SOS-A PEPTIDE (NMR, 29 STRUCTURES) LPKL, PKLP pdbllISA1 ISA-A IRON(II) SUPEROXIDE DISMUTASE (E.C.1.15.1.1) LPAL, PALP pdbllFZV 1FZV-A THE CRYSTAL STRUCTURE OF HUMAN PLACENTA GROWTH FACTOR-1 (PLGF-1), AN ANGIOGENIC PROTEIN AT 2.OA

RESOLUTION

PAVP

pdb 1JLI 1JLI HUMAN INTERLEUKIN 3 (IL-3) MUTANT WITH TRUNCATION AT BOTH N- AND C-TERMINI AND 14 RESIDUE CHANGES, NMR, MINIMIZED AVERAGE LPC, LPCL, PCLP pdblI3CRX1I3CRX-A CRE RECOMBINASE/DNA COMPLEX INTERMEDIATE

I

LPA, LPAL, PALP pdbIlPRXI1PRX-A HORF6 A NOVEL HUMAN PEROXIDASE ENZYME

PTIP

pdbl1A3Z 1A3Z REDUCED RUSTICYANIN AT 1.9 ANGSTROMS

PGFP

pdbl GERI 1GER-A GLUTATHIONE REDUCTASE 6.4.2) COMPLEXED WITH FAD

PALP

pdbI1PBWI1PBW-A STRUCTURE OF BCR-HOMOLOGY (BH) DOMAIN

PALP

AI188872 11.3 366 327 18 382 [Homo sapienslqd27cOl.xl Soaresyplacenta_8to9weeks_2NbHP8Lo9W Mm.42246.3 Mm.42246 101.3 837 304 28 768 GENE=Pck1 PROTSIM=pir: T24168 phosphoenolpyruvate carboxykinase 1, cytosolic; translated LDSL, LPQ Mm.22430.1 Mm.22430 209.4 1275 157 75 1535 GENE=Askpending PROTSIM=pir:T02633 activator of S phase kinase; translated LQA, PSAP, LPS Hs.63758.4 Hs.63758 93.8 3092 1210 51 2719 GENE=TFR2 PROTSIM=pir:T30154 transferrin receptor 2; translated KVLQGRLPAVAQAV, LQG, LPA, LPAV Mm.129320.2 Mm.129320 173.0 3220 571 55 2769 GENE= PROTSIM=pir:T16409 Sequence 8 from Patent W09950284; translated LVQ, LPRL, PMLP Mm.22430.1 Mm.22430 209.4 1275 157 75 1535 GENE=Askpending PROTSIM=pir:T02633 activator of S phase kinase; translated LQA, PSAP P20155 IAC2_HUMAN Acrosin-trypsin inhibitor II precursor (HUSI-TI) [SPINK2] [Homo sapiens] LPG, LPGC Rn.2337.1 Rn.2337 113.0 322 104 1 327 GENE= PROTSIM=PRF:1402234A Rat pancreatic secretory trypsin inhibitor type II (PSTI-II) mRNA, complete cds; minus strand; translated LVG, LVGC Hs.297775.l Hs.297775 43.8 1167 753 31 1291 GENE= PROTSIM=sp:O00268 ESTs, Weakly similar to T2D3_HUMAN TRANSCRIPTION INITIATION FACTOR TFIID 135 KDA SUBUNIT [H.sapiens]; minus strand; translated

PGCP

Mm.1359.1 Mm.1359 PROTSTM=pir.A39743 urokinase plasmiogen activator receptor PGCP, LPG, LPGC sptrembl 10561771056177 ENVELOPE GLYCOPROTEIN

PAAP

sptrembl Q9W234 IQ9W234 CG13509 PROTEIN.//:tremblIAE003458lAE003458-7 gene: "CG13509"; Drosophila melanogaster genomic scaffold LAG, PATP swissIP81272INS2B HUMAN NITRIC-OXIDE SYNTHASE TIB (EC 1.14.13.39) (NOS, TYPE II B) (NOSIIB) (FRAGMENTS) LPA, PAVP sptrembl IQ91YZ3lIQ9IYZ3 DNA POLYMERASE LPC, LPCL, PCLP sptrembl IQ9PVW5lIQ9PVW5 NUCLEAR PROTEIN NP220 PGAP, LPQ, PRGP, PNP Hs.303116.2 PROTSIM=pir;T33097 stromal cell-derived factor 2-likel; translated

GCPR

pdb IBIO i1BIO HUMAN COMPLEMENT FACTOR D IN COMPLEX WITH ISATOIC ANHYDRIDE INHIBITOR

LQHV

pdb|4NOS|4NOS-A HUMAN INDUCIBLE NITRIC OXIDE SYNTHASE WITH INHIBITOR FPGC, PGCP pdbIlFL711FL7-B HUMAN FOLLICLE STIMULATING HORMONE PARP, VPGC pdbllHR6I1HR6-A YEAST MITOCHONDRIAL PROCESSING PEPTIDASE CPRG, LKGC pdbllBFAI1BFA RECOMBINANT BIFUNCTIONAL HAGEMAN FACTOR/AMYLASE INHIBITOR FROM PPGP, LPGC, PGCP, CPRE swissnewIP01229ILSHB HUMAN Lutropin beta chain precursor MMR, MMRV, LQA, LQAV, PPLP, QVVC, WC swissnewIP07434|CGHB PAPAN Choriogonadotropin beta chain precursor MMR, MMRV, LQA, LQAG, QVVC, VVC, swissnewIQ28376ITSHB HORSE Thyrotropin beta chain precursor MTRD, LPK, QDVC, DVC, IPGC, PGCP swissnewIP95180INUOB MYCTU NADH dehydrogenase I chain B LPGC, PGCP sptrembl1Q9Z284 Q9Z284 NEUTROPHIL ELASTASE

PALP,

sptremblIQ9UCG8|Q9UCG8 URINARY GONADOTROPHIN PEPTIDE (FRAGMENT). LPG, LPGG, GGPR XP 028754 growth hormone releasing hormone [Homo sapiens] LQRG, LGQL A further non-limiting list includes collagen, PSG, CEA, MAGE (malanoma associated growth antigen), Thrombospondin-l, Growth factors, MMPs, Calmodulin, Olfactory receptors, Cytochrome p450, Kinases, Von Willebrand factor (coagulation factors), Vacuolar proteins (ATP sythase), Glycoprotein hormones, DNA polymerase, Dehydrogenases, Amino peptidases, Trypsin, Viral proteins (such as envelope protein), Elastin, Hibernation associated protein, Antifreeze glycoprotein, 179 Proteases, Circumsporozoite, Nuclear receptors, Transcription actors, Cytokines and their receptors, Bacterial antigens, Nramp, RNA polymerase, Cytoskeletal proteins, Hematopoietic (neural) membrane proteins, Immunoglobulins. HLA/MHC, G-coupled proteins and their receptors, TATA binding proteins, Transferases, Zinc finger protein, Spliceosmal proteins, HMG (high mobility group protein), ROS (reactive oxygen species), superoxidases, superoxide dismutase, Protooncogenes/tumor suppressor genes, Apolipoproteins SignalP (CBS)_ SignaiP predictions: (for example)

MTRVLQGVLPALP

QXTVC

HLA Peptide Binding Predictions (BIMAS) (For example) Half time of dissociation HLA molecule type I (A_0201): GVLPALPQV (51) VLPALPQVV (48) RLPGCPRGV (14) TMTRVLQGV (115) scores MHC II (H2-Ak 15 mers) VLQGVLPAL (84) C PT MT R VL QGV LP A PG C PR G VN PVV S HLA-DRBl*0l01 15 mers 29 T R VL QGV LP A LP L QG VL P AL PQV V HLA-DRB1*0301 (DRl7) 15 mers 26 M TRV L QGV L PA L S IRL P GC PR G VN Y AV P RG QV V C NY 14 V NPV V SY AV A LS 28 22 C PTM TR VL Q GVL PA L P QV P vv TABLE 3. Further additional results of shock experiments NNPF PEPTIDES %SURVIVAL IN TIME (HRS) Tx Tx 0 14 24 48 PBS 100 100 100 0 NMPF-3 100 100 100 0 NMPF-5 100 100 100 100 NMPF-7 100 100 100 67 NMPF-8 100 100 100 100 NMPF-9 100 100 100 100 NMPF-11 100 100 100 100 NMPF-12 100 100 100 100 NMPF-43 100 100 100 100 100 100 100 100 NMPF-46 100 100 100 100 100 100 100 100 NMPF-53 100 100 100 100 NMPF-58 100 100 100 100 100 100 100 100 TABLE 4. Further additional results NMPF PEPTIDES SICKNESS SCORES Tx Tx 0 14 24 48 PBS 0,0,0,0,0,0 5,5,5,5,4,4 5,5,5,5,5,5 tttttt NMPF-3 0,0,0,0,0,0 3,3,3,3,3,4 4,4,4,4,4,4 tttttt 0,0,0,0,0,0 5,5,5,5,5,5 5,5,5,5,5,5 2,2,2,2,2,2 NMPF-7 0,0,0,0,0,0 1,1,4,4,4,4 5,5,5,5,5,5 2,2,2,2,tt NMPF-8 0,0,0,0,0,0 3,3,5,5,5,5 5,5,5,5,5,5 2,2,4,4,4,5 NMPF-9 0,0,0,0,0,0 3,3,4,4,5,5 2,2,2,2,2,2 1,1,2,2,2,2 NMPF-1 1 0,0,0,0,0,0 1,1,3,3,4,4, 2,2,2,2,4,41,1,11 NMvPF-12 0,0,0,0,0,0 1,1,1,1,3,31,1111111111 NMPF-43 0,0,0,0,0,0 1,1,4,4,4,4 1,1,1,1,3,3 2,2,2,2,2,2 NMPF-45 0,0,0,0,0,0 5,5,5,5,4,4 3,3,4,4,5,5 2,2,4,4,5,5 NUPF-46 0,0,0,0,0,0 1,1,2,2,3,3 1,1,2,2,2,2111111 0,0,0,0,0,0 1,1,1,1,3,3 2,2,2,2,3,31,1111 NMPF-53 0,0,0,0,0,0 5,5,5,5,5,5 5,5,5,5,5,5 1,1,2,2,2,2 NMPF-58 0,0,0,0,0,0 5,5,5,5,3,3 5,5,5,5,3,31,1111 NMPF-60 0,0,0,0,0,0 1,1,4,4,2,2 2,2,2,2,4,4 1,1,1,1,1, ID SEQUENCE SEPSIS ANGIOGENSIS CAO DC NOD NMPF-i VLPALPWC-+ NMPF-2 LQGVLPALPQ NMPF-3 LQG-+ NMPF-4 LQGV GVLPALPQ NMPF-6 VLPALP NMPF-7 VLPALPQ NMPF-8 GVLPALP NMPF-9 WC

QWC

NMPF-11 MTRV NMPF-12 MTR NMPF-13 LQGVLPALPQWC NMPF-14 cydic- LQGVLPALPQWC NMPF-43 AQG NMPF-44 LAG LQA NMPF-46 AQGV NMPF-47 LAGV NMPF-48 LQAV NMPF-49 LOGA ALPALP NMPF-51 VAPALP NMPF-52 VLAALP NMPF-53 VLPMP NMPF-54 VLPALA ALPALPQ NMPF-56 VAPALPO NMPF-57 VLAALPO NMPF-58 VLPAAPQ NMPF-59 VLPALAQ NMPF-6O VLPALPA NMPF-61 WCNRDVRFESIRLPGCPRGVNPWSYAVALSCQCAL NMPF-62 WCNYRDVRFESIRLPGCPRGVNPWSYAVALSCQ NMPF-63 SIRLPGCPRGVNPWS NMPF-64 LPGCPRGVNPWS

CPRGVNPWS

NMPF-66 LPGC NMPF-67 CPRGVNP NMPF-68 PGCP NMPF-69 RPRCRPINATLAVEKEGCPVCITVNTICAGYCPT MTRVLQGVLPALPQ NMPF-71 MTRVLPGVLPALPQWC NMPF-74 CALCRRSTDCGGPKDHPLTC

SKAPPPSLPSPSRLPGPC

NMPF-76 rCDDPRQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ Table 5 Summary of results of the various peptides in the various experiments.

effects; variable effect; no entry is no effect or not yet tested when table was assembled 184 Table 6 MODULATION OF NO AND/OR TNF-A ID SEQUENCE TNF-A 0 TNF-A and NO NPF-2. VPALPQVVC NPF-2 LQGVLPALPQ I NPF-3 QG NPF-4 LQGV GVLPALPQ NMF-6 VPATJP NMF-7 VPALPQ NMF-B GVLPALP NPF-9 NC QVVC NMF-il MTRV NMF-i2 MTR NMF-13 LQGVLPALPQVVC NPF-14 cyclic- LQGVLPALPQVVC NMF-43 AQG NMF-44 LG LQA NMF-46 kQGV NMF-47 LGV NMF-48 LQAV NPF-49 LQGA PALP VAPALP NPF-52 VLAP NMF-53 VPAAP NPF-54 VPALA I APALPQ NMF-56 VAPALPQ NPF-57 VLAPQ NPF-58 VPAAPQ NMF-59 VPALAQ VPALPA VCNYRDVRFES IRLPGCPRGVNPVVSYAV NMF-61 ASCQCAL VCNYRDVRFES IRLPGCPRGVNPVVSYAV NPF-62 ASCQ NPF-63 SIRLPGCPRGVNPVVS NMF-64 PGCPRGVNPVVS 9MPF-65 CPRGVNPVVS NPF-66 LPGC NMF-67 CPRGVNP NPF-68 PGCP

RPRCRPINATLAVEK

NMF-69 GCPVCITVNTTICAGYCPT TRVLQGVLPALPQ NMF-71 MTRVLPGVLPALPQVVC NMP-74 CALCRRSTTDCGGPKDHPLTC SKAPPPSLPSPSRLPGPS

TCDDPRFQDSSSSKAPPPSLPSPSRLPGPS

NMP-76 DTPILPQ P F- 78 CRRSTTDCGGPKDHPLTC from to indicates from barely active to very active in modulating Genomic experiment PM1 T-cell line was obtained from American Type Culture Collection (Manassas, VA) and was cultured at 37 0 C in 5% C02. These cells were maintained and cultured in RPMI 1640, 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics penicillin and streptomycin. For genomic experiments cells (2 x10 6 /ml) were incubated with phytohemagglutinin (PHA, 100g/ml) and IL-2 (200 IU/ml) or PHA, IL-2 and peptide LQGV (10mg/ml) in a volume of 2 ml in 6-wells plates. After 4 h of cultures 10 x10 6 cells were washed and prepared for genechip probe arrays experiment. The genechip expression analysis was performed according to the manufacturer's instructions (Expression Analysis, Technical Manual, Affymetrix Genechip). The following major steps outline Genechip Expression Analysis: 1) Target preparation 2) Target hybridization 3) Experiment and fluidics station setup 4) Probe Array washing and staining 5) Probe array scan and 6) Data analysis.

RESULTS

Genomic experiment The gene chip expression analysis revealed that due to LQGV treatment of PM1 (T-cell line) cells for 4 hours in the presence of PHA/IL-2 down-regulated at least 120 genes more than 2 fold as compared to control PM1 cells (stimulated with PHA/IL-2) only. Moreover, at least 6 genes were up-regulated more than 2 fold in peptide treated cells as compared to control cells.

Identification of down-regulated genes due to treatment with LQGV in genomics experiment. Given are the -Fold Change and Accession number(s) or description of the gene(s).

21.2 .1 9.7 9.3 8.7 6.7 6.1 5.7 5.6 5.3 4.8 4.7 4.6 4.4 M11507 U22376 X68836 M97935 D30037 U28964 U10564; L23959 W29030 U0 8997 M9 7935 Y00638 Ras-Like Protein Tc2l; X83492 AJ 002428 Ras-Related Protein Rapib AL 080119 AF047448; D14710; X59618; D28364;;AA477898 L19161; U48736; L43821;Ras-Like Protein Tc2l; U22376 4.3 4.2 4.1 4 3.8 3.6 3.5 3.4 3.3 3.2 3.1 3 AAG75900; 2 A174 0522; 2.1 Ul18271 Fk506-Binding Protein, Alt. Splice 2 ;J05614 U08316; W28732;Y00638 AF000545 U08 997 X03484; M32886; M28209 L34075; J04088 L19161;D28423;AA442560; X98248 AB020670; W28869; Z12830;AL021546 U78082; X74262 M64174; A1862521; W27517 D13988; AL080119; M33336; L75847; M21154; M97936 U16720; M33336; U50079; U16720; X87212; M21154; X00737 AF034956; Ras Inhibitor Irif; M27749; Ras-Like Protein Tc4; X92106; D88674; H15872; L07541; V01512; L23959; Stimulatory Gdp/Gtp Exchange 2.2 W28907 2.3 Oncogene 2.4 M86667 2.6 D88357 2.7 Protein For C-Ki-Ras P21 And Sing P21; L13943; X78925; U78733 L07540; AF040958; D00596; A1659108; AF042083; AF073362; J04423; D59253; M21154; Proto- C-Myc; W26787 L12002; M55536; S75881; S75881; AFOS0llO; U17743; U90549; U31382; S81916; M64595; Serine Hydroxymethyltransferase; U88629; U72518; L14595; AB014584; A1924594; U68111; A1924594; AL009179; AF091077; M28211; Z85986; A3019435; U39318; X78711; Y09443; Z82200 X69549 Zinc Finger Protein, Kruppel-Like; D10656; M28211 2.8 2.9 W27594; X05360; V00568; L24804 L05S624 Identification of Up regulated genes due to LIQGV treatment. Depicted are the -Fold Change and the Accession number or description of the gene(s).

4.9 3.3 2.1 2.2 2.6 2.8 AF043324 L0 8096 AL031681; X87838 AW024285; D38524; L38935 Ll2 711 AF 026029 X7 0683 Further examples of use Examples of different receptor-intracellular signalling pathways involved in different disease pathogenesis where signalling molecules according to the invention find their use are: LPS stimulation of antigen presenting cells (like DC, macrophages, monocytes) through different Toll-like receptors activates different signalling pathways including, MAPK pathways, ERK, JNK and p38 pathways.

These pathways directly or indirectly phosphorylate and activate various transcription factors, including Elk-l, c-Jun, c-Fos, ATF-1, ATF-2, SRF, and CREB. In addition, LPS activates the IKK pathway of MyD88, IRAK, and TRAF6.

TAK1-TAB2 and MEKK1-ECSIT complexes phosphorylate IKKb, which in turn phosphorylates IkBs. Subsequent degradation of IkBs permits nuclear translocation of NFkB/Rel complexes, such as p50/p65. Moreover, the P13K-Akt pathway phosphorylates and activates p65 via an unknown kinase. Some of these pathways could also be regulated by other receptor signalling molecules such as hormones/growth factor receptor tyrosine kinases (PKC/Ras/IRS pathway) and cytokine receptors (JAK/STAT pathway). In the genomic experiment with the T-cell line several of these genes appeared to be downregulated or upregulated by the peptide used (LQGV). It is now clear that other peptides in T cells and the same and other peptides in other cell types similarly down-regulate or up-regulate several of these transcription factors and signalling molecules. In DC and fertilized eggs experiments NMPF had the ability to modulate growth factor (GM-CSF, VEGF) and LPS signalling. Some diseases associated with dysregulation of NF-kB and related transcription factors are: Atherosclerosis, asthma, arthritis, anthrax, cachexia, cancer, diabetes, euthyroid sick syndrome, AIDS, inflammatory bowel disease, stroke, (sepsis) septic shock, inflammation, neuropathological diseases, autoimmunity, thrombosis, cardiovascular disease, psychological disease, post surgical depression, wound healing, burn-wounds healing and neurodegenerative disorders.

PKC plays an essential role in T cell activation via stimulation of for example AP-1 and NF-kB that selectively translocate to the T cell synapse via Vav/Rac pathway. PKC is involved in a variety of immunological and non-immunological diseases as is clear from standard text books of internal medicine (examples are metabolic diseases, cancer, angiogenesis, immune mediated disorders, diabetes etc.) LPS and ceramide induce differential multimeric receptor complexes, including CD14, CDllb, Fc-gRIII, CD36, TAPA, DAF and TLR4. This signal transduction pathway explains the altered function of monocytes in hypercholesterolemia and lipid disorders.

Oxidized low-density lipoproteins contribute to stages of the atherogenic process and certain concentrations of oxidized low-density lipoproteins induce apoptosis in macrophages through signal transduction pathways. These pathways are involved in various vascular diseases such as atherosclerosis, thrombosis etc.

Bacterial DNA is recognized by cells of the innate immune system. This recognition requires endosomal maturation and leads to activation of NF-kB and the MAPK pathway. Recently it has been shown that signaling requires the Toll like receptor 9 (TLR9) and the signalling adaptor protein MyD88. Recognition of dsRNA during viral infection seems to be dependent on intracellular recognition by the dsRNA dependent protein kinase PKR. TLRs play an essential role in the immune system and they are important in bridging and balancing innate immunity and adaptive immunity. Modulation of these receptors or their down-stream signalling pathways are important for the treatment of various immunological conditions such as infections, cancer, immune-mediated diseases, autoimmunity, certain metabolic diseases with immunological component, vascular diseases, inflammatory diseases etc.

Effect of growth factor PDGF-AA on NF-kB and proinflammatory cytokine expression in rheumatoid synoviocytes; -PDGF-AA augmented NF-kB activity and mRNA expression of IL-lb, IL-8 and MIP-la. Therefore, PDGF-AA may play an important role in progression of inflammation as.well as proliferation of synoviocytes in RA.

Dendritic cell (DC) activation is a critical event for the induction of immune responses. DC activation induced by LPS can be separated into two distinct processes: first, maturation, leading to upregulation of MHC and costimulatory molecules, and second, rescue from immediate apoptosis after withdrawal of growth factors (survival). LPS induces NF-kB transcription factor.

Inhibition of NF-kB activation blocked maturation of DCs in terms of upregulation of MHC and costimulatory molecules. In addition, LPS activates the extracellular signal-regulated kinases (ERK), and specific inhibition of MEK1, the kinase which activates ERK, abrogate the ability of LPS to prevent apoptosis but do not inhibit DC maturation or NF-kB nuclear translocation. This shows that ERK and NF-kB regulate different aspects of LPS induced DC activation. Our DC data and NF-kB data also show the various effects of NMPF peptide on DC maturation and proliferation in the presence or absence of LPS. NMPF peptides modulate these pathways and are novel tools for the regulation of DC function and immunoregulation. This opens new ways for the treatment of immune diseases, particularly those in which the immune system is in disbalance (DC1-DC2, Thl-Th2, regulatory cell etc.) DC mediate NK cell activation which can result in tumour growth inhibition. DC cells and other antigen presenting cells (like macrophages, B-cells) play an essential role in the immune system and they are also important in bridging and balancing innate immunity and adaptive immunity. Modulation of these cells or their down-stream signalling pathways are important for the treatment of various immunological conditions such as infections, cancer, immune-mediated diseases, autoimmunity, certain metabolic diseases with immunological component, vascular diseases, inflammatory diseases etc. There is also evidence in the literature that mast cells play important roles in exerting the innate immunity by releasing inflammatory cytokines and recruitment of neutrophils after recognition of infectious agents through TLRs on mast cells.

In murine macrophages infected with Mycobacterium tuberculosis through JAK pathway activate STAT1 and activation of STAT1 may be the main transcription factor involved in IFN-g-induced MHC class II inhibition.

Recognition of mannose-binding lectin (MBL) through TLRs influences multiple immune mechanisms in response to infection and involved in innate immunity. Balance between innate and adoptive immunity is crucial for balanced immune system and dysregulation in immune system lead to different spectrum of diseases such as, inflammatory diseases, autoimmunity, infectious diseases, pregnancy associated diseases (like miscarriage and preeclampsia), diabetes, atherosclerosis and other metabolic diseases.

Nuclear factor-kappaB (NFkappaB) is critical for the transcription of multiple genes involved in myocardial ischemia-reperfusion injury. Clinical and experimental studies have shown that myocardial ischemia-reperfusion injury results in activation of the TLRs and the complement system through both the classical and the alternative pathway in myocardial infarction, atherosclerosis, intestinal ischaemia, hemorrhagic shock pulmonary injury, and cerebral infarction etc.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors which function as regulators of lipid and lipoprotein metabolism, glucose homeostasis, influence cellular proliferation, differentiation and apoptosis and modulation of inflammatory responses. PPAR alpha is highly expressed in liver, muscle, kidney and heart, where it stimulates the beta-oxidative degradation of fatty acids. PPAR gamma is predominantly expressed in intestine and adipose tissue, where it triggers adipocyte differentiation and promotes lipid storage. Recently, the expression of PPAR alpha and PPAR gamma was also reported in cells of the vascular wall, such as monocyte/macrophages, endothelial and smooth muscle cells. The hypolipidemic fibrates and the antidiabetic glitazones are synthetic ligands for PPAR alpha and PPAR gamma, respectively. Furthermore, fatty acid-derivatives and eicosanoids are natural PPAR ligands: PPAR alpha is activated by leukotriene B4, whereas prostaglandin J2 is a PPAR gamma ligand, as well as of some components of oxidized LDL, such as 9- and 13- HODE. These observations suggested a potential role for PPARs not only in metabolic but also in inflammation control and, by consequence, in related diseases such as atherosclerosis. More recently, PPAR activators were shown to inhibit the activation of inflammatory response genes (such as IL-2, IL-6, IL-8, TNF alpha and metalloproteases) by negatively interfering with the NFkappa B, STAT and AP-1 signalling pathways in cells of the vascular wall. Furthermore, PPARs may also control lipid metabolism in the cells of the atherosclerotic plaque. PPARs are also involved in a variety of immunological and non-immunological diseases as is clear from standard text books of internal medicine (examples are metabolic diseases, cancer, angiogenesis, immune mediated disorders, diabetes etc.) As mentioned above the nuclear receptor PPARg is important in adipogenesis, lipid storage and involved in atherosclerosis. While expressed in adipose tissue this receptor is also expressed in macrophages and in the colon. In addition, PPARg is implicated in a number of processes such as cancer and inflammation. Moreover, microbes, via its cognate receptors, typified by the TLRs, possess the capacity to regulate PPARg dependent metabolic functions and as such illustrates the intricate interplay between the microbial flora and metabolic control in the alimentary tract.

Cyclo-oxygenase 2 (COX2), an inducible isoform of prostaglandin H synthase, which mediates prostaglandin synthesis during inflammation, and which is selectively overexpressed in colon tumours, is thought to play an important role in colon carcinogenesis. Induction of COX2 by inflammatory cytokines or hypoxia-induced oxidative stress can be mediated by nuclear factor kappa B (NFkappaB). So, inhibition of NF-kB modulate COX pathway and this inhibition of NF-kB can be therapeutically useful in diseases in which COXs are involved, such as inflammation, pain, cancer (especially colorectal cancer), inflammatory bowel disease and others.

Neuronal subsets in normal brains constitutively express functionally competent C5a receptors. The functional role of C5a receptors revealed that triggered rapid activation of protein kinase C and activation and nuclear translocation of the NF-kappa B transcription factor. In addition, C5a was found to be mitogenic for undifferentiated human neuroblastoma cells, a novel action for the C5aR. In contrast, C5a protects terminally differentiated human neuroblastoma cells from toxicity mediated by the amyloid A beta peptide. This shows that normal hippocampal neurons as well as undifferentiated and differentiated human neuroblastoma cells express functional C5a receptors. These results show the role of neuronal C5aR receptors in normal neuronal development, neuronal homeostasis, and neuroinflammatory conditions such as Alzheimer's disease.

Activation of the complement system plays also an important role in the pathogenesis of atherosclerosis.

The proinflammatory cytokine interleukin (IL)-6 is potentially involved in the progression of the disease.

Here the complement system induces IL-6 release from human vascular smooth-muscle cells (VSMC) by a Gidependent pathway involving the generation of oxidative stress and the activation of the redox sensitive transcription factors NF-kB and AP-1. Modulation of complement system is important for broad ranges of disorders such as blood disorders, infections, some metabolic diseases (diabetes), vascular diseases, transplant rejection and related disorders, autoimmune diseases, and other immunological diseases.

Different transcription factors like NF-kB and intracellular signaling molecules such as different kinases are also involved in multiple drug resistance.

So, it is reasonable to believe that NMPF peptides will be effective against multiple drug resistance. Moreover, our genomic data shows that a number of genes and signalling molecules involved in tumorogenesis and metastasis are modulated. In addition since oligopeptides have also effect on angiogenesis, thus these peptides will also be used for the treatment of cancer and related diseases whereby angiogenesis requires modulation.

Proliferative diabetic retinopathy (PDR) is one of the major causes of acquired blindness. The hallmark of PDR is neovascularisation abnormal angiogenesis that may ultimately cause severe vitreous cavity bleeding and/or retinal detachment. Since NMPF peptides have angiogenesis stimulatory as well as inhibitory effects and have the ability to modulate intracellular signaling involved in growth factors (like insulin), pharmacologic therapy with certain NMPF peptides can improve metabolic control (like glucose) or blunt the biochemical consequences of hyperglycaemia (through mechanisms such as in which aldose reductase, protein kinase C (PKC), PPARs are involved). For this metabolic control or diabetes (type 2) NMPF (LQGV, VLPALP, VLPALPQ, GVLPALPQ, AQG, LAG, LQA, AQGV, VAPALP, VAPALPQ, VLPALPA, LPGC, MTR, MTRV, LQG, CRGVNPVVS are recommended. The angiogenesis in PDR could be also treated with above mentioned oligopeptides.

Osteoclastogenesis Assay A delicate balance between bone resorption and bone formation is critical for the maintenance of bone strength and integrity, wherein bone-resorbing osteoclasts and bone-forming osteoblasts play central roles. In fact, this physiologic process, termed bone remodeling, must be regulated strictly, and tipping the balance in favor of osteoclasts causes bone destruction observed in pathological conditions such as autoimmune arthritis, periodontitis, (postmenopausal) osteoporosis, Paget's disease and bone tumors.

Regulation of osteoclast differentiation is an aspect central to the understanding of the pathogenesis and the treatment of bone diseases such as autoimmune arthritis and osteoporosis. Excessive signaling by RANKL (receptor activator of NF-kappaB ligand), a member of the tumor necrosis factor (TNF) family essential for osteoclastogenesis, is thought to contribute to such pathological conditions. Joint destruction because of matrix degradation and excessive bone loss characterizes inflammatory bone diseases such as osteolysis, osteoarthritis, and rheumatoid arthritis. Accumulation of inflammatory cells and their secreted products at the inflammation site attracts osteoclasts and their precursor cells, leading to further deterioration of the bone component. Tumor necrosis factor-alpha, interleukin-1 (ILand RANKL (also known as OPGL and ODF), are abundant in sites of inflammation and are known to promote osteoclast recruitment, differentiation, and activation.

Osteoclast differentiation per se requires activation of the RANK/RANKL pathway.

The role of RANKL and TNF-alpha in osteoclast (OC) formation has been established clearly. To determine the effect of NMPF osteoclast formation, we used bone marrow (BM) cells and RAW 264.7 mouse monocytes as a model system for the differentiation of multinucleated osteoclasts.

OC Generation and Characterization: OC were generated by culturing BM cells with recombinant soluble RANKL (20 ng/ml) and M-CSF (10 ng/ml) for 7 days with or without NMPF (10 microgram/mL). OC were also generated by culturing RAW 264.7 cells with RANKL (20 ng/ml) without M- CSF or with TNF-alpha and treated with NMPF. Both culture systems generate large numbers of TRAP multinucleated cells, which express typical OC markers. Osteoclast formation was measured by quantitating the presence of multinucleated TRAP positive cells (more than three nuclei) using cytochemical staining.

Results: As anticipated, we observed a marked inhibition of ligand (RANKL; MCS-F; TNF-alpha) induced osteclastogenesis, when cells were co-incubated with those tri- or tetrameric peptides capable of inhibiting NF-kappaB activity. The ability of this set of peptides to inhibit osteoclast formation was observed in the BM as well as in the RAW 264.7 model systems, as evidenced by a reduced number of multinucleated, TRAP positive cells compared to control cells which had only received ligand.

Figure legends Figure 1: This figure shows macrosphere GPC 300 A chromatogram of NMPF (Pregnyl) sample. Three selected areas were fractionated, NMPF-1 which elutes apparently with molecular weight of >25 kDa, NMPF-2 which elutes apparently with molecular weight between the 25kDa-6kDa, and NMPF-3 which elutes apparently with molecular weight 6kDa.

Figure 2: This figure shows macrosphere GPC chromatogram of NMP-3 fraction obtained from macrosphere GPC 300 A column. Three selected areas were fractioned, NMPF-3.1 which elutes apparently with molecular weight between 2000-300 Da and NMPF-3.8 elutes apparently with molecular weight lower than 300 Da (figure All fractions were tested for anti-shock activity.

Figure 3: This figure shows that PBS-treated BALB/c mice succumbed to shock from day 1 after high-dose LPS injection, with lower than 10% of mice alive on day 5. In contrast, 100% of the mice treated with NMPF from source Pregnyl, or its fractionsNMPF-1 or NMPF-3 obtained from GPC 300 A column, were alive on day 5 (P<0.001), while groups of mice treated with NMPF-2 from source Pregnyl or Dexamethalsone (data not shown) demonstrated around of survivors. Not all commercial hCG preparations showed NMPF activity; for example NMPF from source Profasi showed only partial anti-shock activity (around survival).

Figure 4: This figure shows anti-shock activity in a pretested active batch resided in a fraction NMPF-3 and thereof derived NMPF-3.2 fraction which inhibit shock even after 24 hrs. and 36 hrs. of shock induction. In addition in all mice treated with NMPF-3.2 fraction alone, septic shock was inhibited and they had sickness scores lower than 2, while this anti-shock activity of NMPF-3.2 fraction was inhibited with NMPF-3.3. NMPF-3.3 treatment alone accelerated shock and the treated mice died even earlier than PBS treated mice.

Figure 5: This figure shows macrosphere GPC chromatogram of pooled NMPF-3.2 and NMPF-3.3 fractions from first trimester pregnancy urine (containing antishock activity). This figure shows that the ratio between fraction NMPF-3.2 and NMPF-3.3 is around 1:2.2 (see text).

Figure 6: This figure shows macrosphere GPC chromatogram of pooled NMPF-3.2 and NMPF-3.3 fractions from non-active Pergnyl batch (containing no anti-shock activity). This figure shows that the ratio between fraction NMPF-3.2 and NMPF-3.3 is around 1:3.4 (see text).

Figure 7: This figure shows macrosphere GPC chromatogram of pooled NMPF-3.2 and NMPF-3.3 fractions from active Pergnyl batch (containing anti-shock activity). This figure shows that the ratio between fraction NMPF-3.2 and NMPF 3.3 is around 1:1 (see text).

Figure 8: This figure shows LPS induced proliferation of splenocytes. Anti-MIF and NMPF (from active Pregnyl batch, NMPF-PG*) are both able to decrease LPS stimulated proliferation as compare to LPS alone, and together they show synergistically inhibitory effect on LPS stimulated proliferation.

Figure 9: This figure shows NMPF-A (APL) accelerate LPS induced proliferation, while this proliferation is inhibited by anti-MIFand NMPF-G*.

Figure 10: This figure shows that low molecular weight fraction(NMPF-PG3) from active Pregnyl batch (NMPF-PG*) as well as complete NMPF-PG+ are able to inhibit NMPF-A accelerated LPS induced proliferation.

Figure 11: This figure shows that NMPF-PG (non-active Pregnyl batch) and NMPF-A or in combination (synergistically) increase LPS induced proliferation, while NMPF-PG* inhibits this proliferation same as anti- MIF (see figure 8-9).

Figure 12: This figure shows that NMPF-K accelerates LPS induced proliferation same as NMPF-PG while in combination they synergistically increase proliferation and this increase in proliferation is inhibited with NMPF-Kb or NMPF-PG*. In addition, NMPF-Kb and NMPF-PG* synergistically decrease LPS induced proliferation.

Figures 13-15: These figures show dose dependent (300 and 600 IU/ml) inhibitory effect of NMPF-PG+ on LPS and PHA/IL-2 induced proliferation of PBMC isolated from septic shock patient. Same effect was observed in medium conditions alone.

Figure 16: This figure shows macrosphere GPC chromatogram of c-hCG. Three selected areas were fractionated, 60A-F1 which elutes apparently with molecular weight of >10 kDa, 60A-F2 which elutes apparently with molecular weight between 10 kDa-l kDa and 60A-F3 elutes apparently with molecular weight lower then 1 kDa. All fractions were tested for anti-shock activity.

Figure 17: This figure shows G25 Superdex chromatogram of c-hCG. 100 mL fractions were collected (fraction I-VII) and all fractions were tested for anti-shock activity.

Figure 18: This figure shows G25 Superdex chromatogram of first trimester pregnancy urine from healthy individuals.

100 mL fractions were collected (fraction I-VII) and all fractions were tested for anti-shock activity.

Figure 19: This figure shows that in vivo treatment with LPS increased MIF production as compared to PBS treated mice, while Peptide 1 treatment after the shock induction inhibited MIF production. No effect on MIF production was found in mice treated with Peptide 1 alone.

Figure 20: This figure shows that after restimulation with LPS in vitro, splenocytes from LPS treated mice have a greater capacity to proliferate in vitro as compared to PBS treated mice. On the other hand, splenocytes from LPS+peptide 1 and LPS+c-hCG-V treated mice showed a much higher capacity to proliferate as compared to the LPS treated control mice. No differences in LPS induced proliferation was observed in mice treated with PBS, peptide 1 or c-hCG-V alone.

Figure 21: This figure shows the effect of restimulation of splenocytes from in vivo treated mice with different doses of LPS in vitro.

Figure 22: This figure shows that the shock inhibitory activities (c-hCG, peptide 1, peptide 4 and peptide 6) increased the expression of CD80 molecule on CD19 cells as compared to PBS+LPS control group, whereas minor effect was observed with peptide 7 which accelerates shock.

Figure 23-28: These figure show flow cytometry analysis on splenocytes from treated BALB/c mice.

Figure 29-31: These figure show that hCG and c-hCG bind to 293-hLHRwt/CREluc cells and induce dose-dependent luciferase activity (fig. 29), while no effect was observed in luciferase activity with peptide 1 (VLPALPQVVC), 2 (LQGVLPALPQ), 4 (LQGV), 6 (VLPALP) and low molecular weight fraction c-hCG-V (figure Moreover, addition of peptide 1, 2, 4, 6 and fraction chCG-V in the presence of hCG also did not show effect on luciferase activity induce by hCG itself (figure 31).

Figure 32: This figure shows that there was moderate inhibition of IFN-gamma production found under Thi polarisation conditions with 60A-F3 (c-hCG) and rhCG alone, while the outgrowth of Thl cells was completely blocked with the combination of rhCG and 60A-F3(c-hCG).

Figure 33-34: These figure show that anti-CD3 stimulated splenocytes from NOD mice treated with c-hCG, and 60A-F1 have a smaller capacity to proliferate in vitro.

Furthermore, splenocytes from 60A-F3 (IR-P3) and rhCG treated mice showed a higher capacity to proliferate as compared to the PBS treated control mice (CTL), while 60A-F3(IR-P3) in combination with rhCG caused the same decrease in proliferation as c-hCG and 60A-F1 (IR-P1) (figure 34). Moderate effect was found in the anti-CD3 stimulated proliferation of splenocytes from 60A-F2 treated NOD mice.

Figure 35-36: These figure show the effect of 60A-F1 (IR- Pl), 60A-F2 (IR-P2) and 60A-F3 (IR-P3) in EAE model induced by PLP+PTX. In addition figure 36 shows the effect of treatment of mice with c-hCG and 60A-F3 fraction from c-hCG in EAE mouse model induced by PLP/B.

pertussis which is a chronic disease model for EAE (MS).

Claims (23)

1. A method for modulating expression of a gene in a cell comprising providing said cell with a tri- or tetrameric peptide.

2. A method according to claim 1 wherein said peptide is derived from a naturally occuring protein.

3. A method according to claim 1 or 2 wherein said peptide modulates translocation and/or activity of a gene transcription factor.

4. A method according to claim 3 wherein said gene transcription factor comprises a NF-kappaB/Rel protein.

A method for identifying or obtaining a signalling molecule comprising a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprising providing said cell with a tri- or tetrameric peptide and determining the activity and/or nuclear translocation of a gene transcription factor.

6. A method according to claim 5 further comprising determining whether said peptide is membrane-permeable.

7. A method according to claim 5 or 6 wherein said gene transcription factor comprises a NF-kappaB/Rel protein.

8. A method for identifying or obtaining a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprising providing said cell with a tri- or tetrameric peptide or derivative or analogue thereof and determining relative up-regulation and/or down-regulation of at least one gene expressed in said cell.

9. A method according to claim 5, 6 or 7 further comprising determining relative up-regulation and/or down-regulation of at least one gene expressed in said cell.

A method according to claim 8 or 9 further comprising determining relative up-regulation and/or down-regulation of a multitude of genes expressed in said cell.

11. A method for identifying or obtaining a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprising providing a tri- or tetrameric peptide or derivative or analogue thereof and determining binding of said peptide or derivative or analogue thereof to a factor related to gene control.

12 A method according to claim 11 further comprising providing a multitude of peptides or derivatives or analogues thereof and determining binding of at least one of said peptides or derivatives or analogues thereof to a factor related to gene control.

13. A method according to claim 11 or 12 wherein said factor related to gene control comprises a transcription factor.

14. A method according to claim 13 wherein said transcription factor comprises a NF-kappaB-Rel protein.

A method according any one of claims 11 to 14 further comprising providing a cell with said peptide or derivative or analogue thereof and determining the activity and/or nuclear translocation of a gene transcription factor in said cell.

16. A method according to any one of claims 11 to further comprising providing a cell with said peptide or derivative or analogue thereof and determining relative up-regulation and/or down-regulation of at least one gene expressed in said cell.

17. A tri- or tetrameric peptide useful in modulating expression of a gene in a cell and identifiable or obtainable by employing a method according to any one of claims 5 to 16.

18. A peptide according to claim 17 that is derived from a naturally occuring protein.

19. A pharmaceutical composition comprising a tri- or tetrameric peptide capable of modulating expression of a gene in a cell and a pharmaceutically acceptable carrier.

An inhibitor of NF-kappaB/Rel protein activation comprising a signalling molecule according to claim 17 or 18.

21. Use of a peptide according to claim 17 or 18 for the production of a pharmaceutical composition for the modulation of gene expression.

22. Use according to claim 21 for the modulation of gene expression by inhibiting NF-kappaB/Rel protein activation.

23. A method according to any one of claims 1, 5, 8 or 11 substantially as hereinbefore described with reference to any one of the examples. Erasmus Universiteit Rotterdam By its Registered Patent Attorneys Freehills Patent Trade Mark Attorneys 31 August 2005