US20030165458A1

US20030165458A1 - Diagnosis, prevention, amelioration and/or treatment of disturbed immune function induced by disturbed lipid metabolism

Info

Publication number: US20030165458A1
Application number: US10/327,631
Authority: US
Inventors: Manuel Cabezas; Hans Dijk
Original assignee: Individual
Current assignee: Crossbeta Biosciences BV
Priority date: 2000-09-12
Filing date: 2002-12-20
Publication date: 2003-09-04
Also published as: WO2002022161A3; CA2421994A1; WO2002022160A3; JP2004508414A; US20030143223A1; JP2004508413A; US7498147B2; WO2002022161A2; WO2002022160A2; AU2001294402A1; CA2422032A1; AU2001294401A1; EP1318831A2; EP1186299A1; US20090162357A1; EP1318832A2

Abstract

Complement is recognized as an important, humoral defense system involved in the innate (nonspecific) recognition and elimination of microbial invaders, other foreign particles or molecules, and antigen-antibody complexes from the body. The present invention makes use of the surprising notion that the handling of lipids by the body, rather than its antimicrobial activity, is the primary and most ancient function of the complement system. Consequently, atherosclerosis as observed in disorders associated with disturbed lipid metabolism must be ascribed to either genetic or acquired defects in ancient (activatory and/or regulatory) complement components. The surprising notion is of considerable consequence to the treatment of diseases of the immune system and/or an infectious, autoimmune, neoplastic and/or hematological disease related to complement-mediated lipid metabolism and/or an underlying and/or related disease since lipids and immune complexes share the same transport pathway in the human body. Other implications of the same invention, based on the notion that lipoproteins and lymphocytes share the lymph pathway to arrive in the blood circulation, are that the lipid metabolizing system may be employed to effectively manipulate the immune system. Based on this aspect of the invention, novel oral vaccination and oral immunomodulation strategies are introduced as well.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of International Application Number PCT/NL01/00672, filed Sep. 12, 2001, designating the United States of America, International Publication No. WO 02/22160, published Mar. 21, 2002, in English.[0001]

FIELD OF THE INVENTION

The invention relates to the diagnosis, prevention, and/or treatment of immunological diseases that are induced by disturbances in lipid metabolism.

BACKGROUND

According to the classical view, immunological diseases and impaired lipid metabolism are two quite discrete disorders with distinct causalities. Auto-immune disease is an example of a disease of which the ethiology is difficult to assess. Both metabolic and inflammatory processes play a role and a relationship exists with microbiological factors.

In discussing a possible relationship between infections with pathogenic micro-organisms, MBL (mannose-binding lectin) (an innate immune-defense plasma protein) deficiency and atherosclerosis, Madsen et al. [12] suggested that unexpected non-infective mechanisms are equally likely to play a role in the development of atherosclerosis as are the possibilities of a relationship with infectious microorganisms. The role of MBL in the immune system and the use of recombinant MBL in treating deficiencies in the immune system is known in the art (e.g., WO 0070043). The present invention teaches how lipid metabolism, complement activation, atherogenic processes and immune responses are physiologically related.

SUMMARY OF THE INVENTION

The present inventors have elucidated a mechanism based on the relationship between lipid metabolism, immune adherence and immunological disorders. As a result of this new insight, a novel approach for the diagnosis, prevention, amelioration and/or therapy of immunological disorders that are induced by lipid metabolism and underlying and/or related diseases is presented, which comprises a method for the treatment and/or prophylaxis of immune diseases involving complement activation elements associated with disturbances in the complement/lipid pathway by modulating the activity of one or more elements in said pathway. By this method, a disease of the immune system and/or an infectious, autoimmune, neoplastic and/or hematological disease interconnected with complement-mediated lipid metabolism and/or an underlying and/or related disease may effectively be treated and/or its severity may be reduced. This new method may be implemented in a large range of conditions and in combination with current strategies to lower the occurrence and/or severity of immunological disorders, such as autoimmune disease and neoplastic disease.

The present invention makes use of the surprising notion that the primary and most ancient function of the complement system is the transport and targeting of lipoproteins (i.e., chylomicrons, VLDL's, LDL's, and their remnants) to the liver, rather than its antimicrobial activity. Based on this new insight, novel preventive measures and treatment modulators of immune diseases involving complement activation pathway elements are introduced.

In accordance with the invention, it has surprisingly been found that clearance of chylomicron remnants and in general clearance of all triglyceride-rich particles (chylomicrons, VLDL, IDL and their remnants) and LDL particles is positively regulated by the complement system; that is to say by the most ancient complement activation pathways, the ‘lectin’ and ‘alternative’ pathways. Delayed clearance of triglyceride-rich particles, in particular those containing apolipoprotein B as a structural protein is related to deficiencies in the ancient complement activation pathways. Moreover, in one embodiment the invention predicts that low serum levels of the intercellular matrix proteins vitronectin and/or clusterin, which function as regulators of the ‘terminal’ or ‘lytic’ pathways of complement, leads to decreased intravascular integrity of chylomicron remnants. Such a decreased integrity is typically atherogenic and can result in concomitant disease of a more immunological nature.

Accordingly, the invention relates to the use of purified or enriched physiologic complement components, physiologic complement regulators and/or extrinsic complement modulators of natural (e.g. plant-derived), synthetic, or semi-synthetic origin in the prevention and/or treatment of atherosclerosis and underlying and/or related diseases by substituting for and/or at least diminishing deficiencies in the complement activation pathways.

Because a thorough and mechanistic insight has now been achieved, the invention provides novel diagnostic tools and formulations of specific and highly effective primary and secondary prevention strategies for disturbances of immune functions influenced by the complement/lipid pathway. Dependent on what the relevant element, of an individual patient, in the specific pathways of the complement system is, a physician, based on the considerations of the invention, can modulate the activity of the complement system of the patient in order to prevent and/or treat manifestations of disease.

The present invention provides new and improved methods of prevention, amelioration and/or treatment of disturbed immune functions which are interconnected with lipid metabolism and/or underlying or related diseases. The invention further provides new and improved methods of determining the occurrence (diagnosis) of disturbed immune functions interconnected with lipid metabolism and related diseases and teaches how to classify these diseases accordingly.

The present invention provides for the coordinated design and discovery of new drugs, useful for the treatment of disturbed immune functions interconnected with lipid metabolism and related diseases. In addition, the present invention provides compositions comprising modulators of the complement activation pathways which can serve as a basis, or an ingredient of a pharmaceutical composition or a food product. Therefore, the present invention also relates to pharmaceutical products or food products that comprise such modulating compositions. The present invention provides the use of at least one complement factor or modulator for the manufacture of a medicament for the treatment, amelioration and/or prevention of disturbed immune functions interconnected with lipid metabolism or an underlying and/or related disease. Typically, such treatments would be combined with existing treatments of immune diseases. The invention may either inhibit the complement activation pathway or enhance it, depending on the disease to be treated, by modulating the lipid component of the pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the complement system in schematic representation. [0012]
FIG. 2 shows the two most ancient pathways of the complement system in a schematic representation. [0013]
FIG. 3 shows the relationship between triglyceride rich particles (TRP), their remnants (TRP-R), the position of lipoprotein lipase (LPL), free fatty-acids (FFA), acylation-stimulating protein (ASP), several complement components (C3, factor B and factor D) in relation to triglyceride (TG) uptake by adipocytes and liver-derived very low density lipoproteins (VLDL). [0014]
FIGS. 4A through E show the binding to chylomicrons of C3, MBL, clusterin and vitronectin as described in example 1. [0015]
FIGS. 5A and B show the effect of immune adherence of triglyceride-rich particles to erythrocytes in blood after staining with Sudan Black as described in example 2. [0016]
FIG. 6 shows the flow cytogram obtained after staining apo B on human erythrocytes as described in example 3. [0017]
FIG. 7 shows the internalization of triglyceride-rich particles in a blood leukocyte as described in example 4. [0018]
FIG. 8 shows the complement/lipid pathway in schematic representation. [0019]
FIG. 9 shows the potency of different substances in the activation of complement in vitro. [0020]
FIG. 10 shows the effects of glycosylated plantstanols on fasting triglycerides, plasma apoB and plasma cholesterol levels over a four month period in a patient with heterozygous Familial Hypercholesterolemia. [0021]
FIG. 11A shows the effect of vitamin A on postprandial C3 plasma concentration after 2 h in healthy lean volunteers. [0022]
FIG. 11B shows the effect of vitamin A on postprandial plasma triglycerid concentration after 2 h in healthy lean volunteers.[0023]

DETAILED DESCRIPTION OF THE INVENTION

In order to appreciate the importance of the invention, the inventors deem it necessary to explain the newly developed concept in much more detail. The surprisingly intricate relationship between the complement system and the clearance of chylomicron remnants unraveled by the inventors signifies a pathway not hitherto known. This unexpected finding gives rise to measures for treatment and prophylaxis of atherosclerosis that are themselves surprising, and that lead to the identification of additional risk factors and the development of novel therapeutic interventions which results in a significant reduction of total mortality due to CHD. [0024]
I. The Complement System: General Description [0025]
The complement system [9] is a complex signaling system comprising enzymes present in the blood. The complement system is involved in the early recognition and clearance from the circulation and tissues of foreign bodies and antigen-antibody complexes (also called immune complexes). Complement is recognized as an important, humoral defense system involved in the innate (nonspecific) recognition and elimination from the body of microbial invaders, other foreign particles or molecules, and antigen-antibody complexes. [0026]
Upon the recognition of foreign material in the tissue or blood, the most crucial and abundant complement component, C3, is activated by C3 convertases. This activation triggers a cascade of events that ultimately leads to the clearance of the foreign material. C3, consisting of an α and β chain, is activated through a split-conversion into C3b and C3a (FIG. 1). C3a represents the N-terminus (77 amino acids) of the α chain and C3b represents the C-termini of the α and β chains. C3 convertases, of which various forms exist, can be generated through three different complement activation pathways (FIG. 1) and its synthesis is well regulated. The C3-convertase-generating pathways include, in order of descending evolutionary age, the so-called ‘lectin’ pathway (LP), the ‘alternative’ pathway (AP) which is also known as the ‘amplification loop’, and the relatively young ‘classical’ pathway (CP). During evolution, an additional system known as the ‘terminal’ or ‘lytic’ pathway has developed on top of the complement activation system, which can destabilize membranes of e.g. Gram-negative bacteria, viruses, virus-infected host cells, or even tumor cells by pore formation. This pore formation kills the bacteria, virus-infected host cell (and immature virus) or tumor cell. Phylogenetic studies have pointed out that the ‘lectin’ and ‘alternative’ pathways are by far the most ancient complement activation pathways (about 700 million years; FIG. 2), whereas the ‘classical’ and ‘lytic’ pathways are relatively young (400-350 million years). [0027]
The complex nature of the complement system can be appreciated when following the fate of the split-conversion products of C3. One of the split products, C3a, is a plasminogen and anaphylatoxin, which induces the release of histamine from basophilic cells, including tissue mast cells and basophilic granulocytes. Histamine, in turn, helps phagocytes to leave the blood vessels in order to arrive at the site of complement activation, i.e., the accumulation site of foreign material or immune complex. In blood, C3a is rapidly (in about 15 min.) inactivated by serum carboxypeptidases. The most prominent serum carboxypeptidase (sCP) in blood is the constitutively expressed sCP-N. All other sCP types are inducible and are less abundant than the N type. Upon the inactivation of C3a by carboxypeptidases, the C-terminal arginine is removed, resulting in the generation of C3adesArg. This compound is (probably identical to) an acylation-stimulating protein (ASP), a hormone that can stimulate fat accumulation in the body. [0028]
C3b and its inactivated form C3bi are opsonins, which means that they can bind covalently to sugar OH groups (via ester bonds) or protein NH[0029] ₂groups (via amide bonds) on material identified as ‘foreign’. In the case of such binding events, foreign material is also termed ‘substrate’. Other complement components can also function as opsonins, among these are other C3-derivatives and the complement component C4 (see below) and derivatives thereof. Opsonins promote the clearance of foreign material by the blood-based monocytes and tissue-based macrophages, both known as mononuclear phagocytes. The mononuclear phagocytic system is present in the liver, spleen, lymph nodes, or the affected tissue itself. These specialized cells carry specific complement receptors on their surface that can bind the opsonins. Known complement receptors on phagocytes are CR1, CR3, and possibly also CR4. CR1 is an exclusive receptor for C3b whereas CR3 and CR4 are also able to bind C3bi. In contrast to mononuclear phagocytes, polymorphonuclear phagocytes (PMNs) are relatively inefficient in eliminating foreign material, at least in the absence of antibodies.
In primates, immune complexes are eliminated by the mononuclear phagocytic system in the liver, spleen and bone, after erythrocyte-mediated transport via the blood stream. The erythrocytes carry a restricted number of CR1 molecules on their surface to which C3bi-coated immune complexes can adhere. This phenomenon is called ‘immune adherence’. Erythrocytes of non-primate species are CR1 negative and consequently do not mediate the transport of immune complexes to the liver, spleen and bone. In primates suffering from systemic autoimmune diseases and neoplastic diseases (cancer), the clearance of immune complexes involves antibody-mediated activation of the complement system. [0030]
Microbial pathogens in the circulation are also cleared by the mononuclear phagocytic system, but only after MBL-mediated (‘lectin’ pathway) or antibody/C1-mediated (‘classical’ pathway) activation of complement components C4, C2, and C3. This process is known to involve erythrocyte-mediated clearance as well. [0031]
The phenomenon of ‘immune adherence’ is one of importance to the present invention, as the present inventors have found that these CR1 complement receptors not only bind immune complexes or microbial pathogens, but also chylomicrons and other triglyceride-rich particles and their remnants. Based on this finding, new methods for the treatment, amelioration and/or prophylaxis of immune diseases are presented, which are based on intervention within particular lipid elements or modulation of the complement pathways involved. [0032]
As mentioned, the complement system comprises several pathways each with a multitude of protein compounds, signaling molecules, receptors, regulators and activators. To appreciate the scope of the present invention, the various pathways of complement activation will be described in some more detail. [0033]
II. The Complement System: The ‘Lectin’ Pathway [0034]
Activation of the ‘lectin’ pathway (LP) starts with the recognition and binding of foreign bodies by a serum lectin, called mannose-binding lectin (MBL). MBL is a high-molecular-weight, sugar-binding protein, present in minute amounts (about 2 μg per ml) in blood plasma. MBL and the lung surfactant proteins A (LspA) and D (LspD), belongs to the family of the collagenous lectins (collectins). C1, the first component in the ‘classical’ pathway is a collectin-like activator of C4 and C2. Upon binding of MBL to foreign bodies, a number of MBL-associated proteins (MASPs—which are themselves esterases) become coordinately activated, ultimately leading to the generation of the active forms of the associated proteins, the LP-dependent C4, C2 and/or C3 convertases. These convertases, which have C3, C4 and C2 as their natural substrates, generate essentially five products: C3b and C3a, C4bC2a and split products C4a and C2b. Like C3b, the C4b portion of C4bC2a binds covalently to its substrate (e.g. polysaccharides or (glyco) proteins on bacteria) via ester or amide bonds, and is therefore known as an opsonin. The two split products, C4a and C2b, are released in the fluid phase. Substrate-bound C4bC2a is the LP-dependent C3 convertase, causing the conversion of C3 into C3b and C3a. Like C3a, C4a is a plasminogen and anaphylatoxin (histamine liberator), whereas C2b has kinin-like activity. Furthermore, one of the MBL-associated proteins is capable of direct activation of C3. [0035]
MBL recognizes foreign bodies by its 6 identical sugar-binding moieties with specificity for mannose, N-acetyl-glucosamine, and fucose. This makes sense, because microbial pathogens like, e.g., fungi, yeasts, and Mycobacteria, carry relatively high amounts of mannose, while peptidoglycans of Gram-positive bacteria contain N-acetyl-glucosamine as one of the major building blocks. [0036]
III. The Complement System: The ‘Alternative’ Pathway [0037]
Until the discovery of the ‘lectin’ pathway in 1989, the ‘alternative’ pathway (AP, also known as alternate pathway or alternative complement pathway), first described in 1956, was considered the most ancient complement activation route. The main function of this ‘alternative’ pathway is to increase (amplify) the number of C3-converting sites on the substrate of complement activation: the foreign body or the immune complex. This means that, once ‘non-self’ material has been identified by MBL and activation of the ‘lectin’ pathway has consequently taken place, the LP-dependent C3 convertase—C4bC2a present on the substrate—will be amplified by AP-dependent C3 convertases in the following manner (FIG. 1): Substrate-bound C3b, generated by the LP-dependent C3 convertase C4bC2a, will bind AP component factor B which, in turn, will be activated to Bb by AP component factor D (also known as adipsin) to form the AP-dependent C3 convertase (C3bBb). Along with the formation of this new C3 convertase, the factor-B part loses a split product called Ba. The enzymic function of the AP-dependent C3 convertase is stabilized upon the binding of AP component ‘properdin’ (factor P), resulting in the AP-dependent C3 convertase complex C3bBbP. Split product Ba is a leukotaxin, which helps to direct the movement of phagocytes to the site of complement activation (primary inflammation site). [0038]
The net result of AP activation is an increase in the number of C3b and inactivated C3b (C3bi) moieties on the substrate, which promotes the recognition and clearance of foreign bodies and immune complexes by, predominantly, mononuclear phagocytes (monocytes/macrophages). [0039]
IV. The Complement System: The ‘Classical’ Pathway [0040]
The ‘classical’ pathway (CP) is generally considered the youngest complement activation route, since it is dependent on antibodies (IgM and IgG), which appeared relatively late in the phylogeny (from about 350 million years ago). The CP is very similar to, and therefore probably derived from the ancient ‘lectin’ pathway, since the first CP component (C1; consisting of a complex of the collectin-like C1 q and two MASP-like proteins called C1r and C1s) is both phenotypically and functionally very much related to the MBL/MASPs complex. In addition, the ‘classical’ pathway involves ‘lectin’ pathway complement components C4 and C2. Like the sugar-bound MBL/MASPs complex, C1(composed of C1q, C1r, and C1s) bound to IgM- or IgG-type immune complexes becomes coordinately activated to form a C1-esterase which has C4 and C2 as its natural substrates and which gives rise to the generation of CP-dependent C3 convertases, which are identical to LP-dependent C3 convertases (substrate-bound C4bC2a complexes). [0041]
C4 exists in two isoforms known as C4A and C4B. C4A is involved in the clearance phenomenon, whereas C4B is mainly involved in the killing of bacteria and cell destruction (e.g. hemolysis). In the present description, C4 is understood to relate to the C4A isoform unless otherwise stated. [0042]
V. The Complement System: The Terminal or ‘Lytic’ Pathway [0043]
When a newly formed C3b molecule does not bind to the substrate directly, but to another substrate-bound C3 convertase (C4bC2a or C3bBbP), triple or quadruple complexes consisting of C4bC2aC3b or C3bBbC3bP are formed. These complexes have C5-converting activity indicating that they are able to split complement component C5 into C5b and C5a. This is the starting point of the so-called ‘terminal’or ‘lytic’ complement pathway. Like Ba, C5a is a leukotaxin, but more potent than Ba. C5b forms a complex with C6 and C7, the resultant of which is a soluble C5b-7 complex, which has affinity for membranous bilayers. Upon insertion into a membrane, e.g., a Gram-negative bacterium, complement component C8 will bind to the complex, which results in a new enzyme, the membrane-bound C9 polymerase (C5b-8). Under the influence of a C5b-8 complex, 13 C9 molecules are polymerized, resulting in a cylindrical pore in the membrane that is under attack. Depending on the total number of membrane-bound poly-C9 pores, and on whether the bacterium is encapsulated, for example, by an outer lipoprotein membrane, or not, the Gram-negative bacterium will either be killed or may survive the membrane attack. [0044]
V. The Complement System: Complement Regulation and Complement Regulators [0045]
In order to prevent unwanted activation of the complement cascade, e.g., by cells of the body itself (homologous cells, in contrast to foreign or heterologous cells), complement activation by homologous cells is heavily regulated by both cell-bound complement inhibitors and regulators in the fluid phase (e.g., serum or plasma). [0046]
V.1. The Most Important Soluble Regulators Are [0047]
For the ‘lectin’pathway, the most important soluble regulators are α[0048] ₂-Macroglobulin (α2M), serpines and C4-binding protein (C4BP), which interfere with the formation of the LP-dependent C4/C2-convertase (activated MBL/MASPs complex) and the subsequent activation of C4 and C2.
For the ‘alternative’ pathway, the most important soluble regulators are Factor H (also known as β1H) and factor H-like molecules, which act at the level of factor B binding to target-bound C3b (preventing the formation of AP-dependent C3 convertases), and C3b inactivator (factor I), which acts in conjunction with factor H, to convert C3b into its enzymatically inactive, but still active as an opsonin, C3bi form. [0049]
For the ‘classical’ pathway, the most important soluble regulators are C1INH, an inhibitor of complement component C1, which acts at the level of activated C1, and the C1-esterase (C1IN is also an inhibitor of other serine esterases such as kallikrein, the clotting factors XIa and XIIa, and the fibrinolysis product plasmin). [0050]
For the ‘lytic’ pathway, the most important soluble regulators are Vitronectin (S protein) and clusterin (also known as apolipoprotein J or apo J). These proteins act at the level of C5b-7 complexes, preventing their insertion into bilayer membranes and inhibiting C9 polymerization and consequently, the lysis of bacteria, viruses and host cells. [0051]
V.2. Cell-Bound Complement Regulators Include: [0052]
Complement receptor 1 (CR1) which has factor-H-like co-enzyme function, as apposed to factor I. CR1 is present on phagocytes, platelets, and as a carrier protein on erythrocytes. [0053]
Decay-accelerating factor (DAF, which is also known as a cluster of differentiation protein CD55) and membrane cofactor protein (MCP=CD46), both of which act at the level of AP activation. [0054]
The Homologous restriction factor with a 20-k molecular mass (HRF20=CD59) and HRF60, which both inhibit at the level of C9 polymerase (C5b-8) formation. [0055]
Sialic acid, which acts at the level of the AP-dependent C3-convertase formation (similar to CD55 and CD46) and also on C9 polymerization. [0056]
VI. The Complement System: Complement Activation and the Innate and Specific Immune System [0057]
Apart from the physiological activating and regulatory complement components mentioned above, different substances of bacterial, plant, animal, or (semi)synthetic origin are known to either activate or inhibit the complement cascade(s). These components include, e.g., bacterial lipopolysaccharides, β-glycyrrhetinic acid, phytosterols, bovine conglutinin, and polymeric substances like dextran sulphate and glucans. [0058]
Bacterial lipopolysaccharides have recently been recognized as potent activators of the ‘lectin’pathway. Likewise, β-glycyrrhetinic acid, as a possible activator of C4, was suggested to be able to activate the ‘lectin’ pathway. The phytosterols, including, for example, β-sitosterol, stigmasterol, and campesterol, have been shown to activate the ‘alternative’ pathway. Dextran sulphate functions as an acceptor site for the ‘alternative’ pathway regulatory protein factor H, and thus facilitates the ‘alternative’ pathway-mediated activation of C3 and subsequent deposition of C3b on a substrate. [0059]
Based on their complement-activating capacity, a number of these substances, including bacterial lipopolysaccharides, dextran sulphates, and glucans, as well as lipidated muramyl-dipeptides and lipophilic quaternary ammonium compounds, like dimethyldioctadecyl ammonium bromide, show potent immunological adjuvant activity, which means that they are able to stimulate antigen-specific T- and B-cell responses. [0060]
VII. Lipid Metabolism: The Physiology of Lipid Metabolism [0061]
Under physiologic conditions, about 90% of the ingested fat (triglycerides) is taken up by the epithelial cells of the small intestine, resulting in the generation of intestinally-derived triglyceride-rich lipoproteins, called chylomicrons. These chylomicrons are translocated through the epithelial cells and delivered at the basolateral side to the sub-epithelial interstitium. The structure of chylomicrons is stabilized by a large, highly glycosylated protein, called apolipoprotein B48 (apo B48), of which the most dominant sugar residues are: mannose (17.8%), N-acetyl-glucosamine (16.8%), galactose (13.4%), and fucose (3.4%), which, in fact, fully matches with the binding specificities of MBL. Apo B48 is the 5′ splice product of a larger apob gene which—in human intestinal epithelial cells—is posttranscriptionally modified by a unique editing enzyme. This modification, or editing, results in the insertion of a premature stop codon, which results in the translation of only 48% of the apob mRNA. Since the human liver lacks the unique editing enzyme, apob transcription in the liver results in the synthesis of full-length apo B100. This protein is the structural protein of the liver-derived triglyceride-rich particles known as VLDL (very low density lipoproteins) and their remnants (IDL's and LDL's). [0062]
From the sub-epithelial interstitium, chylomicrons are collected in tissue fluid (lymph). Via lymph vessels, they are transported to subsequent draining lymph nodes and, through the thoracic duct and the left subclavian vein, they finally arrive in the blood stream. Once in the circulation, chylomicrons are rapidly converted into chylomicron remnants by the action of vascular-endothelium-associated lipoprotein lipase (LPL). The resulting chylomicron remnants are present in blood in different sizes. [0063]
Chylomicrons and chylomicron remnants are efficiently cleared by the liver, where they can undergo bile-mediated excretion via the stool. However, the efficiency of the process of chylomicron and chylomicron-remnant targeting to the liver is not completely understood. In addition, the subsequent hepatic clearance of these triglyceride-rich particles has not been completely elucidated either. In the liver, it involves at least the activity of the hepatic triglyceride lipase (HTGL), interaction with specific apo E receptors, and non-receptor binding to the cellular surface in the hepatic space of Disse. Several local receptors may be involved, including low-density-lipoprotein receptor-related protein, α[0064] ₂-Macroglobulin receptor (LRP-α₂M), a parenchymal liver cell ‘chylomicron remnant receptor’, the asialoglycoprotein receptor, the lipolysis-stimulated receptor, and the LDL (low density lipoprotein) receptor. Recently, the VLDL receptor, a new member of the LDL receptor supergene family, which is not present in the liver, has been recognized as a physiological receptor for chylomicron remnants.
Cholesterol, delivered to the liver by chylomicrons and chylomicron remnants, is largely re-secreted into the circulation after incorporation into very-low density lipoproteins (VLDL). This cholesterol is further employed by the adrenals and genitals as a skeleton for their steroid-hormone synthesis. [0065]
Free fatty acids (FFA) arising from the breakdown of chylomicrons by the endothelial LPL are transported over the mucosa towards sub-endothelial fat cells (adipocytes) in which they become re-esterified into intracellular triglycerides (FIG. 3). The uptake and incorporation of FFA into adipocytes is under the positive control of a hormone called acylation-stimulating protein (ASP). [0066]
Similarly to the hydrolysis of triglycerides in chylomicrons, VLDL may become VLDL remnants, also called IDL (intermediate-density lipoproteins), by the lipolytic action of LPL, in this case, under the positive and negative control of two other apolipoproteins, apo CII and apo CIII [11], respectively. IDLs are rich in apo E which functions as the ligand for the hepatic LDL receptor and ‘remnant-receptor’(=LRP, LDL-receptor-related protein which is a member of the LDL-receptor family comprising complement repeats and is possibly older than the LDL-receptor itself). Apo E (formerly “Arginine Rich Apoprotein”) is one of the protein constituents of triglyceride-rich lipoproteins. Chylomicron remnants depend on apo E for their binding to the receptors, since the apo B48 structural protein does not contain the (carboxy-terminal) binding site for the LDL-receptor and ‘remnant receptor’. Apo E is synthesized by almost all tissues, but not by the epithelium of the intestine. The major organ responsible for apo E synthesis is the liver. As a result, chylomicrons receive apo E from HDL in the circulation, and therefore apo E is an exchangeable apoprotein. In the liver-sinusoids, hepatocytes secrete apo E resulting in an enrichment of remnant particles, thereby facilitating their removal from the circulation. There are 3 major apo E isoforms which are genetically determined: Apo E3 (the most common), apo E2 (which results in a minority of the cases in dysbetalipoproteinemia in homozygotes), and apo E4. The latter has the highest affinity for binding to the receptors, while apo E2 exhibits the lowest affinity. Apo E4-individuals are highly responsive to dietary changes and cholesterol and fat enriched diets lead to higher plasma cholesterol concentrations in these individuals, due to down-regulation of LDL-receptors. [0067]
Under physiological conditions, IDLs are taken up by LDL-receptors in the liver and the lipoproteins are degraded and cholesterol is removed from the body by excretion into the bile. [0068]
Although much is known, the metabolic pathways of the intestinally- and liver-derived triglyceride-rich particles in blood, chylomicrons and VLDL, respectively, and their remnants, have hereto only been partially identified. It has been shown that these pathways comprise common elements and show a certain overlap. However, until the present invention, the very efficient targeting to the liver of chylomicrons and chylomicron remnants under physiological conditions and their clearance, was far from understood. [0069]
VIII. Lipid Metabolism: Aberrant Lipid and/or Free-Fatty-Acid Metabolism [0070]
Chylomicron remnants are potentially atherogenic (atherosclerosis generating) particles due to their ability to directly induce foam-cell formation, without any modification. Low-density lipoprotein particles (LDL), in contrast, must be oxidized before they induce transformation of mononuclear phagocytes into foam cells. Mononuclear phagocytes have an LDL receptor by which they are able to bind, take up, internalize and subsequently degrade native LDL. As soon as the intracellular free cholesterol levels reach a threshold value the LDL receptors are down-regulated and the internalization process is stopped. Oxidized LDL particles, on the other hand, are taken up by ‘scavenger’ receptors, which are not down-regulated by cholesterol. [0071]
Since chylomicrons, VLDL, and their remnants, compete for the same metabolic pathways, patients with delayed or impaired remnant clearance may experience a temporary accumulation of chylomicrons and chylomicron remnants in the circulation, which obviously contributes to the process of atherogenesis, but also has consequences for the immunological component of the complement activation pathways. Such situations are likely to occur in patients with familial combined hyperlipidemia (FCHL), type-2 diabetes mellitus, insulin resistance, and obesity. Enhanced plasma VLDL levels in these situations are associated with delayed clearance of chylomicron remnants. [0072]
Similar mechanisms are involved in conditions in which the clearance of remnant particles is impaired due to mutations in the apo E ligand gene (type III hyperlipidemia=familial dysbetalipoproteinemia), the LDL receptor (familial hypercholesterolemia; FH), familial defective apo B100 (FDB) and after menopause. In these conditions, which are all associated with the development of (premature) atherosclerosis, but would also have an influence on immune diseases, a delayed clearance of chylomicron remnants has been established due to an impaired binding to receptors in the liver. Other disorders associated with impaired remnant clearance are apo CII deficiency, (partial) lipoprotein lipase (LPL) deficiency, and hepatic triglyceride lipase (HTGL) deficiency. In these disorders, the conversion of triglyceride-rich particles into their remnants is delayed, leading to an accumulation in the circulation of triglyceride-rich particles of different sizes and triglyceride content. [0073]
In many endocrinological disorders like hypothyroidism, growth hormone deficiency, hypercortisolism by endogenous or exogenous corticosteroids, and the postmenopausal state, a decreased clearance of chylomicron remnants has been established when compared with the control situation. Finally, in patients with premature atherosclerosis and normal fasting plasma lipids (40% of all patients with myocardial infarction below 60 years of age in males and beyond 65 years of age in females), chylomicron-remnant clearance is decreased. It has been hypothesized and it is widely accepted that this may be one of the important mechanisms underlying atherosclerosis in these groups of patients. For the first time it has now been established that it will also have an influence on the immunological part of the complement activation pathway. [0074]
Free fatty acids (FFA) arising from the breakdown of chylomicrons by the endothelial lipoprotein lipase (LPL), and their uptake by the adipocytes, stimulates these adipocytes to synthesize complement component C3 and ‘alternative’ pathway components, factors B and D (note that in healthy individuals, there is a linear relationship between total body fat and C3 levels), and according to the invention complement activation occurs. [0075]
Several important pathways involved in immune defense and lipid metabolism have been disclosed. However, a causal relationship between immune defense and lipid metabolism has not been established. Designing optimal treatment, ameliorating and/or prophylactic measures for disturbed immune functions of highly diverse nature and underlying and/or related diseases have thus far been impossible to achieve. It is now found by the present inventors that the existence of a pathway, that was hereto unknown, allows for the first time the development of such measures based on a more complete and physiological and immunological understanding of these diseases. The surprisingly intricate relationship between the complement system and the clearance of chylomicron remnants unraveled by the inventors demonstrates the presence of such a pathway, which is termed the lipid eliminating complement activation pathway or complement/lipid pathway. [0076]
Due to this new finding the identification of additional risk factors, novel therapeutic interventions and pharmaceuticals and the treatment, amelioration and prophylaxis of disturbed immune functions, which are interconnected with impaired lipid metabolism, have now become available. This invention will result in a significant reduction in the severity of such immune diseases. The novel pathway was revealed, inter alia, by three independent findings. The first finding is that not only foreign bodies, but also chylomicrons can induce complement activation. The second finding is that chylomicrons bind to erythrocytes and that this binding utilized complement factors and as a result of which, lipid transport through the blood is complement and erythrocyte mediated. This led the inventors to the finding that immune complexes and complement-activated chylomicrons compete for binding sites on the erythrocyte. The third finding relates to the glycosylation of apolipoprotein B, its kinship to MBL binding specificity and the insight that the complement-mediated lipid transport and transport of immune complexes may, thus, be modulated through interventions in the complement/lipid pathway and its individual elements or components. Such elements or components are understood to comprise all molecules, complexes and substances that play a role in the complement/lipid pathway. [0077]
It has now surprisingly been found that chylomicrons, isolated from healthy individuals after an oral fat load, carry complement components C3 (i.e., the opsonins C3b and/or C3bi) (FIG. 4A). Thus, these chylomicrons initiate complement activation. In addition, it was also surprisingly found that chylomicrons, isolated from healthy individuals after an oral fat load, also carry the ‘lectin’ pathway complement component mannose-binding lectin (MBL), and the terminal-complement-pathway inhibitors clusterin and vitronectin (FIGS. [0078] 4B-E). Thus, chylomicrons activate the ‘lectin’ pathway (MBL-binding) which may ultimately lead to opsonization with C3b(i) [see ‘lectin’ pathway]) and to binding to the CR1 receptor of phagocytes and erythrocytes [see general description of complement system]. Furthermore, the presence of clusterin and vitronectin indicates a capacity to inhibit the ‘terminal’ pathway of the human complement system.
It was found that virtually all erythrocytes of healthy volunteers carry chylomicrons and chylomicron remnants (FIG. 5A), whereas erythrocytes in the ‘fasting’ state carry considerably less chylomicrons and chylomicron remnants (FIG. 5B). This finding is in accordance with the new concept of an erythrocyte-mediated elimination of triglyceride-rich particles (and possibly also LDL particles) and complement-mediated lipid transport and can be interpreted in terms of immune adherence of remnant particles and targeting of lipids to the liver and spleen. [0079]
The pathway revealed by the present inventors provides an explanation for the observed complement activation and for a more complete physiological and immunological understanding of immunological diseases that are interconnected with lipid metabolism and/or underlying and/or related disease. The present inventors disclose that the prominent glycosylation sites of apolipoproteins B48 and B100, that are present as structural proteins on plasma chylomicrons and VLDL, respectively, match fully with the mannose, N-acetylglucosamine, and/or fucose binding specificity of MBL. This means that triglyceride-rich particles (LDL, chylomicrons, VLDL, etc.) in the blood directly activate the complement system's ‘lectin’ pathway through binding of apolipoprotein B to MBL, thereby competing with immune complexes for binding on CR1 binding sites on the erythrocytes. [0080]
As an intrinsic complement activator (of MBL), apo B is potentially very harmful (note the existence of autoantibodies against the C3 convertases F-42 and C3 nephritic factor in patients with collagen diseases). In particular, the intrinsic complement activation nature of the structural apolipoprotein B molecules of triglyceride-rich particles is now predicted to be harmful for individuals with decreased serum levels of ‘terminal’pathway inhibitors vitronectin and/or clusterin, since such a situation will, subsequent to ‘lectin’ pathway activation, allow ‘terminal’ pathway activation to occur. ‘Terminal’ pathway activation on triglyceride-rich particles may result in the release of atherogenic lipid material, particularly in patients with a genetic or acquired deficiency in the ‘terminal’ pathway regulators vitronectin or clusterin. The binding of the ‘terminal’ pathway inhibitors vitronectin and clusterin to chylomicrons can teleologically be explained in terms of protection from atherosclerosis. [0081]
Combination of chylomicron (remnant)-induced complement activation of the ‘lectin’ pathway, the matching of glycosylation sites of apolipoproteins B48 and B100, the erythrocyte-mediated elimination of triglyceride-rich particles and competition between such particles and immune complexes for CR1-mediated binding on erythrocytes predicts that increased levels of triglyceride-rich particles in blood, as occurring in FCHL and other disorders associated with atherogenic disturbances of lipid metabolism, is due to sub optimal erythrocyte-dependent clearance of chylomicrons and/or VLDL. [0082]
Also, disturbances in chylomicron- and/or VLDL- and/or chylomicron-remnant- and/or VLDL-remnant-mediated complement activation will lead to impaired lipid metabolism. Likewise, disturbances in the complement cascade, albeit subtle and e.g. acquired, may also lead to impaired lipid metabolism and, thereby to negative effects on the immune system. [0083]
This bears considerable consequences for the treatment, amelioration and prophylaxis of all diseases related to the complement/lipid pathway, specifically those immune diseases interconnected with lipid metabolism. [0084]
Diseases related to the disturbances in the complement/lipid pathway are immunological diseases that are interconnected with lipid metabolism and share an element of a common pathway. The similarity in their elimination pathways predicts that triglyceride-rich particles have to compete with soluble immune complexes and/or microbes for elimination sites on erythrocytes and in the liver and spleen, which would explain the disturbed lipid metabolism in e.g., septic shock. This bears considerable consequences for the treatment and prophylaxis of diseases such as, but not limited to, the auto-immune disorders, for example, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and paroxysmal nocturnal hemoglobinuria (PNH), virtually all infectious diseases and related disorders such as AIDS-related (secondary) lipodystrophy, septic shock, and multiple organ failure, inflammatory diseases such as Crohn's disease, inflammatory bowel syndrome (IBS), thermal injury including burns and frostbite, uveitis, psoriasis, asthma and neoplastic diseases such as cancer. This immunological aspect of the present invention holds consequences for improving the effectiveness of vaccination programs. [0085]
Disorders directly related to the complement/lipid pathway comprise: [0086]
disturbances in chylomicron-, chylomicron-remnant, VLDL- and/or VLDL-remnant-mediated complement activation; [0087]
disturbances in the complement cascade itself; [0088]
disturbances in erythrocyte-dependent chylomicron remnant and/or VLDL-remnant clearance; [0089]
disturbances in the complement-mediated lipid metabolism; and [0090]
disturbances in the regulation of lipid metabolism [0091]
Such disorders may lead to a disease which may seem to be more related to an immunological disorder or malfunction such as auto-immune diseases, infectious diseases, neoplastic diseases and/or inflammatory diseases. [0092]
Treatment, amelioration and/or prophylaxis as a benefit of the present invention occurs through modulation of the common pathway, e.g., by correction of disturbed complement function in the case of impaired complement-mediated lipid metabolism and will lead to an amelioration of lipid metabolism. By correcting the disturbed complement function, in the case of impaired complement-mediated lipid metabolism, an amelioration of disorders associated with impaired or disturbed chylomicron remnant clearance is achieved. [0093]
Further, correction of disturbed complement function, in case of impaired complement-mediated lipid metabolism, will result in an amelioration of diseases of the immune system, such as infectious, autoimmune, neoplastic or hematological diseases iterconnected with complement-dependent lipid metabolism. [0094]
Disturbances of lipid metabolism and thus of the related immune response due to delayed or disturbed erythrocyte-dependent clearance of chylomicrons and/or VLDL may have a number of possible causes, which will determine the nature of the corrective measure. There may be: [0095]
congenital defects in glycosylation of apo B48 and/or apo B100; [0096]
absolute (homozygous), or relative or acquired deficiencies of individual complement components of the ‘lectin’ and ‘alternative’ pathways (such deficiencies are known to occur for MBL [9% of the population], C4A [the defective gene frequency is 10-13% of the population], C4B [the defective gene frequency is 7-18% of the population], C2 [rare], C3 [rare], factor B [rare] and factor D [rare]); [0097]
deficiencies of serum carboxypeptidases (sCP) which exclude the conversion of C3a into C3adesArg [incidence unknown]; [0098]
absolute [rare] or relative [quite common] deficiencies of complement receptor 1 (CR1) on erythrocytes as occurring in some patients with systemic lupus erythematosus (SLE); [0099]
deficiencies of terminal-pathway regulator vitronectin [4% of the population], which may lead to the lysis of triglyceride-rich particles resulting in unwanted deposition of lipids; or [0100]
decreased serum levels of clusterin in association with exacerbations of SLE or with circulating immune complexes accompanying neoplastic diseases [deficiencies in clusterin are rare; <<1% of the population]. [0101]
The incidence of serious cardiovascular disease [37% in 1997] in the Netherlands, expressed as percentage of total numbers of fatal cases per year, matches well with the combined figures for MBL, C4A, C4B, vitronectin, and clusterin deficiencies, corrected for the incidence of double and triple deficiencies. [0102]
It is one embodiment of the present invention to provide a method for the treatment, amelioration and/or prophylaxis of immune diseases associated with the complement/lipid pathway by modulating the activity of one or more elements in said pathway. [0103]
In another embodiment according to the invention the activity of one or more elements of the lectin pathway and/or the alternative pathway of complement activation are modulated. [0104]
Modulating according to the present invention should be understood as regulating, controlling, blocking, inhibiting, stimulating, activating, mimicking, bypassing, correcting, removing, washing, administering, adding, and/or substituting one or more elements in the pathway or, in more general terms, intervening in the pathway. [0105]
It is an aspect of the invention that the elements in the pathway comprise triglyceride-rich particles and/or their remnants and their constitutive proteins, complement proteins, complement activators, complement inhibitors, complement regulators and/or complement receptors. [0106]
It is one embodiment of the present invention that the modulating the activity of one or more elements is achieved through administration of a modulator. [0107]
Modulators according to the invention are substances that can bring about a modulation in the complement/lipid pathway or the complement system and may comprise triglyceride-rich particles and/or remnants thereof and/or constitutive proteins thereof, complement proteins, complement activators, complement inhibitors such as serpines, factor H, factor I and/or C1INH, complement regulators such as α2M, their metabolic precursors, the genes encoding the proteins and/or fragments thereof and they may be of physiologic (human or primate-derived), natural (e.g., plant-derived), recombinant, synthetic and/or semi-synthetic origin and in enriched, purified and/or chemically modified, complete and/or partial form, as metabolic precursor, as biochemically functional analogue or as functional equivalent of a (physiologic) modulator and/or derivatives thereof used alone or in combination. [0108]
Functional equivalents in the context of the present invention are understood to comprise molecules having at least one function of the original compound, preferably all functions of the original compound (although not necessarily to the same extent), more preferably chemically similar compounds, most preferably compounds differing by at most three groups which are not necessary for the relevant activity and/or function of the original compound. In the context of the present invention, functional equivalents of complement factors are understood to comprise the split products of the factors. [0109]
In a preferred embodiment according to the invention, modulators may be MBL-replacement factors, which exhibit one or more functions of the mannose binding lectin such as binding to C3b or a mimetic thereof, and/or binding to the prominent apo B glycosylation sites or mimetics thereof Such an MBL-replacement factor may comprise lectins derived from plants such as e.g., concanavalin A, peanut lectin, phytohemagglutinin or wheat-germ agglutinin, and may also comprise purified or enriched physiologic MBL or synthetic, or semi-synthetic mimetics of MBL and/or functional equivalents of MBL and may be used in an aspect of the invention relating to substituting for MBL deficiencies in the complement/lipid pathway. MBL replacement compounds also comprise lipid-C3 conjugates. [0110]
In another preferred embodiment according to the invention, modulators may comprise apo B-replacement factors, which may be functional equivalents of apoB that e.g., exhibit one or more functions of apolipoprotein B48 or [0111] B 100 such as binding to MBL or mimetics thereof and an ability to form a constituent of a lipoprotein or a mimetic thereof. Such an apo B-replacement factor may be chosen, for example, from physiologic apo B 48 or B100, natural lipo-oligosaccharides, lipopolysaccharides, lipidated oligosaccharides or polysaccharides, glycoproteins, β-glycyrrhetinic acid, chylomicron-bound sialic acid, phytosterols (β-sitosterol, campesterol, and/or stigmasterol) and other amphiphilic (partially hydrophobic and partially hydrophilic) complement activator(s) (e.g., mannosylated, N-acetylglucosaminylated, and/or fucosylated phytosterols, or mannosylated, N-acetylglucosaminylated, and/or ficosylated membrane lipids, such as phosphoglycerides, glycolipids such as cerebroside or ganglioside, or sphingomyelin, phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl inositol, diphosphatidyl glycerol or sphingosine), stanols (glycosylated and non-glycosylated), lipidated dextran sulphate(s), (lipo)glucan(s), lipidated tertiary or quaternary ammonium compounds, sialylated glycolipids, combinations thereof and single and/or combined related substances. In general, suitable apo B-replacement factors comprise amphiphilic compounds or derivatives thereof wherein the hydrophilic part comprises one or more cationic, anionic and/or polar groups and wherein the hydrophobic part comprises one or more fatty-acid ester moieties. The fatty-acid ester moieties may comprise carbon chain lengths from 1 to 50 carbon atoms, they may be straight and/or branched and they may comprise saturated and/or unsaturated fatty acids.
Preferred amphiphilic modulators additionally comprise one or more sugar moieties, such as N-acetylgalactosamine, galactose and/or sialic acid, which allow interaction with a lectin binding site. In another embodiment according to the invention, the one or more sugar moieties are mannose, N-acetylglucosamine, and/or fucose moieties that allow interaction with the lectin binding site of MBL. Other suitable apo B replacement factors may comprise an IgA or IgD antibody, which is heavily mannosylated, N-acetylglucosaminylated, and/or fucosylated of either polyclonal or humanized monoclonal or combinatorial origin, directed towards one of the apolipoproteins of chylomicrons or very low-density lipoproteins (VLDL). Such antibodies may also be bi-specific antibodies reactive towards both apoB and CR1, thereby being able to, e.g., create bonds between its two antigens. [0112]
In another embodiment according to the invention, modulators may be selected from the group comprising MBL and MBL-replacement factors, C4A and functional equivalents thereof, C4B and functional equivalents thereof, C2 and functional equivalents thereof, C3 and functional equivalents thereof, IgG- and IgM-antibodies raised against triglyceride-rich particles and LDL or parts thereof, C3adesArg, factor B and functional equivalents thereof, factor D and functional equivalents thereof, factor P and functional equivalents thereof, serum carboxypeptidases such as sCP-N and functional equivalents thereof, erythrocyte-bound CR1 and functional equivalents thereof, free CR1 and functional equivalents thereof, CR1 mimetics such as C3b antibodies, vitronectin and functional equivalents thereof, clusterin and functional equivalents thereof and apo B (48 and 100) and apo B replacement factors and esterases such as one of the MASP-proteins and functional equivalents thereof [0113]
In another preferred embodiment according to the invention, modulators may comprise antibodies. In a more preferred embodiment, the modulator for the classical pathway is selected from the group of IgG and/or IgM antibodies. [0114]
In another embodiment, the group comprising apo B replacement factors also comprises an IgA or IgD antibody directed against an apo B lipoprotein, which antibody is heavily mannosylated, and/or heavily N-acetylglucosaminylated and/or heavily fucosylated. In another embodiment, the modulator for the classical pathway is selected from the group of antibodies wherein the antibody comprises a polyclonal and/or humanized monoclonal and/or combinatorial antibody and/or bi-specific antibodies reactive towards both an apo B and CR1. [0115]
Administration of a modulator may comprise oral administration, nasal administration, pulmonary administration, inhalation, anal and/or rectal administration, intravenous injection, intramuscular injection, intradermal injection, subcutaneous injection, mucous membrane diffusion, skin absorption, topical application, extracorporeal circulation-mediated administration and/or any other suitable administration route, single or in combination. [0116]
Modulators according to the invention may be administered in pure form and/or diluted form, they may be in solid, semi-solid, crystalline and/or fluidic form, dissolved and/or dispersed alone or as a constituent of a fluid, a spray, a gel, an ointment, a tablet, a suppository, a capsule (synthetic, natural or viral), a powder, an (clinical) intralipid, a (clinical) food product, a (clinical) food additive, a lipidated vaccine for oral application, slow-release and/or direct release carrier that contains the modulator and/or any other suitable formulation for administration. Furthermore, modulators may be unlabeled or labeled with signal molecules or groups such as e.g., dyes, fluorochromes, radioactive atoms or groups, enzymes or luminescent molecules or groups. [0117]
Apo B replacement factors according to the invention may be administered alone or in combination with other modulators in a natural, artificial or synthetic lipid carrier compound comprising lipoproteins, lipid micelles, lipid vesicles, artificial lipid bilayer membranes, chylomicrons, liposomes and/or other suitable and/or pharmaceutically accepted lipid substance. Clinical intralipids (fat emulsions) used in relation to the invention as parenteral feeding may comprise such a lipid carrier compound in combination with one or more modulators. In a preferred embodiment of such a parenteral feeding, the lipid carrier is selected from the group comprising mineral oil and natural oils, such as soy oil, sunflower oil, peanut oil, olive oil, palm oil and sesame oil and processed (purified and/or modified) versions thereof. In another embodiment of such a parenteral feeding, the lipid carrier is (purified) olive oil. [0118]
It is another embodiment of the present invention to administer modulators in such a manner that the modulator is generated in-vivo, e.g., by gene therapy and/or by local administration of enzymes (e.g., apo B glycosylation enzymes) their encoding gene(s) and/or gene fragments. [0119]
It is a further embodiment of the present invention to use a method for modulating the activity of one or more elements in the complement/lipid pathway for the treatment and/or prophylaxis of immune diseases associated with complement-mediated lipid metabolism. [0120]
It is a further embodiment of the present invention to use a method for modulating the activity of one or more elements in the complement/lipid pathway to prevent atherogenic processes of immune diseases that (partially) share the lipid eliminating complement activation pathway. [0121]
It is a further embodiment of the present invention to use a method for modulating the activity of one or more elements in the complement/lipid pathway to efficiently manipulate the immune system. [0122]
It is a further embodiment of the present invention to use a method for modulating the activity of one or more elements in the complement/lipid pathway to achieve optimum systemic immunosuppression by lipophilic immunosuppressants. [0123]
It is a further embodiment of the present invention to use a method for modulating the activity of one or more elements in the complement/lipid pathway to achieve optimum oral immunization. In another embodiment, a method for modulating the activity of one or more elements in the complement/lipid pathway is used as a lymph-targeting, oro-mucosal adjuvant to induce enhanced mucosal antibody (IgA) responses, T-cell reactivity, and/or systemic T-cell and/or B-cell (IgM and/or IgG) antibody responses. [0124]
It is an embodiment of the present invention to provide prophylactic measures for immune diseases associated with the complement/lipid pathway by providing improved methods for diagnosing such diseases. [0125]
It is one embodiment of the present invention to estimate the potential of plant-derived, synthetic, or semi synthetic substances for use in the treatment of immune diseases by determining their complement activation and/or consumption activity. Complement consumption should be understood as complement entering the complement cascade thereby disappearing as free component. [0126]
It is another embodiment of the present invention to estimate one or more of the complement components selected from the group comprising MBL, C4A, C4B, C2, factor B, C3adesArg, serum carboxypeptidase N, vitronectin, clusterin, and erythrocyte-bound complement receptor 1 (CR1), in blood, blood serum and/or blood plasma of a patient in order to establish the underlying or related defect of his/her disease. [0127]
It is a further embodiment of the present invention that immune diseases (infectious, autoimmune, or neoplastic) that may (partially) occupy the lipid eliminating complement activation pathway can be diagnosed more adequately so that i.a., concomitant atherogenic processes are prevented. [0128]
It is a further embodiment of the present invention to provide compositions for the treatment and/or prophylaxis of immune diseases associated with the complement/lipid pathway. Compositions according to such an embodiment of the present invention may be pharmaceutical compositions, in particular given together or in one composition comprising existing drugs for the treatment of immune diseases such as Tumor Necrosis Factor (TNF) and blockers thereof such as Humicade (CDP 571) or suppressors thereof such as thalomide (Thalidomide), interleukins such as IL-1, IL-12 and IL-15 and blockers or suppressors thereof, interferons such as IFNg and blockers or suppressors thereof, integrin and blockers or suppressors thereof, Intercellular Adhesion Molecules such as ICAM-1 and blockers or suppressors thereof and the like, probiotic strains of bacteria, additives for pharmaceutical compositions, active substances for pharmaceutical compositions, additives for clinical nutrition and/or regular food additives and that comprise modulators according to the invention, metabolic precursors of such modulators, biochemically functional analogues, functional equivalents and/or derivatives of such modulators with or without expedients such as fillers, binders, other complement activators such as vitamin A, thickening agents, preservatives, lubricants, emulsifiers, emulgators, and/or stabilizers. [0129]
It is an embodiment of the present invention that such compositions are used to modulate the activity of one or more elements of the complement/lipid pathway according to a method of the invention. [0130]
Also, the inventions provides for the use of a modulator according to the invention for the manufacture of a medicament, preferably for the treatment, amelioration and/or prophylaxis of a diseases of the immune system and/or an infectious, autoimmune, neoplastic or hematological disease interconnected with complement-mediated lipid metabolism and/or an underlying and/or related disease. [0131]

EXAMPLES

Example 1

Complement Components Associated with Chylomicrons. [0132]
Experimental procedure: Chylomicrons were isolated from plasma by ultra centrifugation and purified by column chromatography, in the following manner: For separation of lipoproteins, plasma samples were subjected to a single ultra-centrifugation step as described in Madsen et al. (12). Chylomicron (Sf>1000) and non-chylomicron (Sf<1000) fractions were separated by flotation. The chylomicron fraction contained chylomicrons and large VLDL. The non-chylomicron fraction contained chylomicron remnants, small VLDL and its remnants, LDL, HDL and the remainder of the plasma proteins. Aliquots were stored at −20° C. until use. In the fractions containing large chylomicrons (large triglyceride-rich particles) complement components C3, MBL, clusterin (exp. 1), clusterin (exp. 2) and vitronectin were measured by competitive ELISA using the purified proteins and MBL- and C3-specific polyclonal and clusterin-specific monoclonal-antibodies G7* and EB-8* and vitronectin-specific monoclonal antibody MO-24* as reagents. The presence of the complement factors could consistently be demonstrated in [0133] fractions 13 through 20 (see FIGS. 4A-E). In addition, C3 and MBL were also found in other lipoproteins isolated by one-step density gradient ultra centrifugation (Redgrave gradient) (IDL, LDL, HDL) in subjects fasting and postprandial after a fat challenge.

Example 2

Adherence of Triglyceride-Rich Particles to Erythrocytes in Whole Blood. [0134]
Experimental procedure: To observe adherence of triglyceride-rich particles to erythrocytes in whole blood, Sudan Black staining of erythrocytes in whole blood was performed. In this procedure, blood smears were prepared and Sudan Black staining was performed with a filtered and saturated solution of Sudan Black in 80% ethanol (4 grams of Sudan Black B, Electran in 200 ml of 80% ethanol) by the following procedure. The blood film on the glass slide was fixed by heat fixation (3× through a flame). The slide was soaked in Sudan Black solution for 3 min. after which the slide was rinsed with 80% ethanol. The preparation was re-hydrated by a graded ethanol series (1 min. 40% ethanol, 1 min. 20% ethanol, 1 min. demineralized water). Excess water was shaken off and the slides were dried to air. Microscopic examination of the slides revealed that virtually all erythrocytes of healthy volunteers carried chylomicrons and [0135] chylomicron remnants 4 hours after an oral fat intake (FIG. 5A), whereas erythrocytes in the ‘fasting’ state carried considerably less such particles (FIG. 5B).

Example 3

Measurement of Erythrocyte-Bound-apo B Containing Lipoproteins by Flow Cytometry. [0136]
Experimental procedure: Full capillary blood was drawn from non-fasting healthy subjects by capillary punction. The blood was washed 3 times in 10 ml of VSB buffer (Veronal Saline Buffer) by centrifugation (3,000 rpm, 10 min., 20° C.) and the cell count was adjusted to 1.5×10[0137] ⁸/ml with VSB buffer. A volume of 50 μl of the sample was pelleted and the pellet was re-suspended in 50 μl of a goat raised anti-human apo B polyclonal antibody solution (Chemicon 1:25 diluted in VSB buffer). After a 30 min. incubation of the sample at room temperature (RT) the cells were washed twice in 1 ml. of VSB buffer. The cells were pelleted and re-suspended in 50 μl of a FITC-labeled anti-goat antibody solution (Rabbit anti-goat Ig FITC, DAKO 1:10 diluted in VSB buffer). After a 30 min. incubation of the sample at RT the cells were washed twice in 1 ml. of VSB buffer, pelleted, resuspended in 0.5 ml. of VSB buffer and analyzed by flow cytometry (10,000 cells were counted). Erythrocytes were gated on forward and side scatter. It could be demonstrated that the FITC-label was associated with the side-scattering particles (erythrocytes) only in the presence of the anti-apo B antibodies (FIG. 6, bottom panel), whereas no erythrocytes-associated FITC fluorescence could be detected in the case that incubation with anti-apo B antibodies was omitted from the analysis (negative control sample, FIG. 6, top panel). It was therefore concluded that apo B was associated with the erythrocytes in whole blood.

Example 4

Binding and Internalization of Chylomicron Remnants by Leukocytes in the Blood (In Vivo). [0138]
Experimental procedure: Fasting venous blood was drawn and Sudan Black staining as described in example 2 was carried out (left panel of FIG. 7). In the right panel of FIG. 7, venous blood of the same healthy volunteer was drawn 4 hours after administration of a standardized oral fat load. In the oral RP-fat loading test, cream is used as fat source; this is a 40% (w/v) fat emulsion with a P/S ratio of 0.06, which contains 0.001% (w/v) cholesterol and 2.8% (w/v) carbohydrates. After an overnight fast of 12 h, the subjects ingest the fresh cream, to which 120.000 U of aqueous RP (Retinyl palmitate =vitamin A) had been added 18 h before the test, in a dose of 50 g per m[0139] ²body surface. After the ingestion of the fat load, subjects were only allowed to drink water or tea during the following 24 h. Peripheral blood samples were obtained in sodium EDTA (2 mg/ml) before (T=0), at hourly intervals up to 10 h and at 12 and 24 h after the meal. Tubes were protected against light by aluminum foil and centrifuged immediately for 15 min at 800×g at 4° C. Blood samples for FFA measurement were chilled and a lipase inhibitor (Orlistat) was added in order to block in-vitro lipolysis.
Increased leukocyte concentrations in the postprandial situation are involved in the process of atherosclerosis (novel finding). After having taken up surface fragments from triglyceride-rich particles or whole remnant particles (see FIG. 1), neutrophilic granulocytes become activated and induce a pro-inflammatory response which is the first step in the generation of atherosclerosis and endothelial damage. [0140]

Example 5

Magnitude and Time-Dependency of Increase of Complement Component 3 (C3) in the Postprandial Period. [0141]
Experimental procedure: Standardized oral fat loading tests (oral RP fat loading test) were performed in volunteers and patients and plasma C3 levels were determined nephelometrically at regular intervals. [0142] Complement component 3 was measured by nephelometry (Dade Behring Nephelometry type II). Maximal postprandial C3 concentrations were in most cases found after 2 hrs. (data not shown). This is consistent with the concept of chylomicron-driven complement activation (MBL mediated) followed by a compensatory C3 synthesis in-vivo. We therefore conclude that complement activation occurs in vivo during postprandial lipemia (high blood lipid concentrations).

Example 6

Binding and Internalization of Chylomicron Remnants by Leukocytes in the Blood (In Vitro). [0143]
Experimental procedure: In vitro incubations of chylomicron remnants with isolated human leukocytes were performed by methods described in example 3. Internalization of remnants in leukocytes was observed (data not shown). [0144]

Example 7

Assay for Complement-Activation cq. Complement-Consumption by Drugs/Food Components Intended for Application in Atherosclerosis or Clinical Nutrition. [0145]
Experimental procedure: In microtiter plates, one ‘classical’ pathway unit of serum or one ‘alternative’ pathway unit of serum was incubated for 0.5 hrs. with a dilution series of the substance to be investigated. In order to do so, the substance of interest is suspended in micellar form. After incubation, residual classical and alternative complement activities are estimated by conventional techniques (Klerx J. P. A. M., C. J. Beukelman, H. Van Dijk and J. M. N. Willers (1983) J. Immunol. Lett. 63:215-220; Van Dijk H, P. M. Rademaker and J. M. N. Willers (1985) J. Immunol. Meth. 85: 233-244). The degree of complement consumption is a measure of complement activation by the components. A large number of compounds were identified by this in-vitro assay for application in atherosclerosis or clinical nutrition. [0146]

Example 8

MBL-Dependent Complement Activation by Chylomicrons in Human Serum. [0147]
Experimental procedure: Chylomicrons were isolated from human serum by ultra centrifugation and purified by column chromatography. The purified chylomicron fractions were added to MBL-positive serum (from healthy human subjects) and MBL-negative serum (from MBL-deficient human subjects) and purified heterologous chicken erythrocytes were added. Complement activation was allowed to occur at 37° C. for 45 min. after which, the extend of hemolysis was evaluated by spectrophotometrical determination of hemoglobin levels in serum supernatants. It was found that hemolysis of heterologous erythrocytes was extensive in the case that an MBL-positive serum was used, whereas hemolysis was virtually absent in the case of an MBL-negative serum (data not shown). This demonstrated that chylomicrons can bring about complement activation in human serum in an MBL-dependent manner. [0148]
Using the MBL in-vitro assay, we identified, as an example, the components from olive oil and soy oil inducing Complement Lipid Pathway (CliP)-activation. [0149]
The results are summarized in FIG. 9. [0150]
From these results, two compounds were selected which show strong CLiP-induction and which are known to be safe to be administered to human (thanks to other clinical use), specifically (i) glycosylated plant sterols and (ii) vitamin A. [0151]

Example 9

Postprandial C3 Buildup. [0152]
Experimental procedure: Full capillary blood was draw from healthy subjects and the C3 levels were determined together with the leukocyte count. Postprandial (situation in blood after a meal) leukocyte increase and activation was associated with postprandial complement C3 increase. In the early postprandial phase (<4 hr) predominantly neutrophilic granulocytes were observed, whereas between 4 and 10 hrs into the postprandial period, an increase of lymphocytes was observed. These findings were consistent with the notion that leukocytes play a role in atherosclerosis by the formation of foam cells. [0153]

Example 10

Effect of Glycosylated Plant Sterols on Fasting Plasma Triglycerides and Clolesterol Levels in two MBL-Deficient Patients and in one MBL-Normal Patient with Heterozygous Familial Hypercholesterolemia. [0154]
Proof of the principle has been reached in 2 MBL-deficient patients. [0155]
These subjects were treated with a diet enriched in glycosylated plant sterols during 3 weeks. The glycosylated plant sterols were selected in the MBL in vitro test as described in example 8. This intervention resulted in a decrease of fasting plasma triglycerides and cholesterol (Table 1) [0156]

TABLE 1

effect of glycosylated plant sterols on blood

parameters of MBL-deficient patients.

Plasma TG mM Cholesterol (mM) apoB (g/L)

Before After Before After Before After

MBL def1 3.66 2.27 4.8 4.2 0.85 0.89

MBL def2 0.88 0.79 4.8 3.6 0.62 0.56
Using a different intervention with glycosylated plant stanols in a patient with heterozygous Familial Hypercholesterolemia (with relatively normal MBL activity in plasma), refractory to therapy with expanded doses of statins, in combination with a lipid lowering diet and resins, significant reductions of plasma cholesterol (from 10 to 7.8 mmol/L), fasting plasma triglycerides (from 2.3 to 1.08 mmol/L) and plasma apoB (from 1.90 to 1.62 g/L) were achieved reaching the lowest concentration ever experienced by this patient (FIG. 10). This example provides in vivo support for the Complement lipid pathway (CliP) concept developed by C-Tres, using a sub-optimal compounds. [0157]

Example 11

Effect of Vitamin A on Post Prandial CliP Stimulation. [0158]
Another series of compounds, namely vitamin A-analoguesm, were tested in 20 healthy volunteers in order to determine the CLiP stimulating potency of the compounds that had shown CLiP stimulation in vitro (Example 8). Twenty healthy volunteers were tested on 2 different occasions. Blood was drawn before and after ingestion of a standardized oral fat load with and without vitamin A (as a representative for this series of compounds) given to the participants in random order. Addition of vitamin A to the oral fat load resulted in a significantly higher postprandial plasma C3 increase, whereas the same amount of fat was ingested in both situations (FIG. 11A). [0159]
The levels of plasma trygliceride increase 2 h after acute oral fat load also showed a reduction in the volunteers, if the fat load was given with vitamin A (FIG. 11B) [0160]
This is in line with the CLiP concept, by activating the Complement system, plasma triglycerides will be reduced even in healthy normolipidemic subjects. Note: It should be stressed that the C3 increase in this group of young, healthy, lean subjects was expected to be lower due to the characteristics of the subjects. In older, insulin-insensitive subjects the postprandial C3 response is much higher. [0161]
These experiments in human gave the expected results upon administration: increase of C3-titers and decrease of triglycerides. Therefore, it is reasonable to assume that other compounds, active in the in vitro assay, will show CLiP-activities in human. [0162]
Other Analytical Methods: [0163]
Triglyceride-rich particles in plasma, chylomicrons and non-chylomicrons were determined by HPLC as described (14). TG and cholesterol were measured in duplicate by commercial calorimetric assay (GPO-PAP, and Monotest Cholesterol kit, Boehringer Mannheim) as described (14,23). Plasma apo B and apo AI were determined by immunoturbidimetry (23). Apo E genotype was determined as described (47-49). HDL2 and HDL3 cholesterol concentrations were determined by precipitation procedures as described (50). Complement [0164] factor 3 was measured immunoturbidimetry or nephelometrically. Acylation stimulating protein was determined by ELISA, as were factor B and D. Ketone bodies were measured by HPLC.
References: [0165]
All publications, patents, patent applications and other references cited herein are hereby incorporated by reference in their entirety for all purposes. [0166]
1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990's. Nature 1993; 362: 801-809. [0167]
2. Willeit J, Kiechl S, Oberhollenzer F, Rungger G, Egger G, Bonora E, Mitterer M, Muggeo M. Distinct risk profiles of early and advanced atheroclerosis. Prospective results from the Brunneck Study. Arterioscl Thromb Vasc Biol 2000; 20: 529-537. [0168]
3. Bucher H C, Griffith L E, Guyatt G H. Systematic review on the risk and benefit of different cholesterol-lowering interventions. Arterioscl Thromb Vasc Biol 1999; 19: 187-195. [0169]
4. The Heart Outcmes prevention Evaluation Study Investigators. Effects of an angiotensin-converting-enzyme inhibitor, ramipril, on cardiovascular events in high-risk patients. N Engl J Med 2000; 342:145-53. [0170]
5. The Diabetes Control and Complication Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993; 329: 977-86. [0171]
6. UK Prospective Diabetes Study (UKPDS) Group. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with [0172] type 2 diabetes (UKPDS 33). Lancet 1998; 352:837-53.
7. Haffner S M, Lehto S, Ronnemaa T, Pyorala K, Laakso M. Mortality from coronary heart disease in subjects with [0173] type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. N Engl J Med 1998; 339: 229-34.
8. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999; 340: 115-126. [0174]
9. Law S K A & K B M Reid: Complement in focus, 2nd edition, 1995. Oxford University Press, Oxford, G.B. [0175]
10. Rosenberg M E & J Silkensen: Clusterin: Physiologic and pathophysiologic considerations. Int J Biochem Cell Biol 1995; 27(7): 633-645. [0176]
11. Ishikawa Y, Akasaka Y, Ishii T, Komiyama K, Masuda S, Asuwa N, Choi-Miura N-H, Tomita M: Distribution and synthesis of apolipoprotein J in the atherosclerotic aorta. Artherioscler Thromb Vasc Biol 1998; 18:665-672. [0177]
12. Madsen H O, Videm V, Svejgaard A, Svennevig J L, Garred P: Association of mannose-binding-lectin deficiency with severe atherosclerosis. The Lancet 1998; 352:959-960. [0178]

Claims

We claim:

1. A method for the treatment or prophylaxis of immune diseases associated with the complement/lipid pathway comprising modulating the activity of one or more elements in said compliment/lipid pathway.

2. The method according to claim 1, wherein the activity of one or more elements of a pathway selected from the group consisting of the lectin, the classical and the alternative pathway for complement activation are modulated.

3. The method according to claim 1 or 2, wherein said disease is a disease selected from the group consisting of the immune system, an infectious disease, an autoimmune disease, a neoplastic disease, a hematological disease related to complement-mediated lipid metabolism, and an underlying disease or related disease.

4. The method according to any of the preceding claims, wherein said modulating the activity of one or more elements comprises administering one or more modulators.

5. The method according to any of claims 1-4, further comprising administering a second drug for the treatment of said immune disease.

6. The method according to claim 5, wherein said second drug is selected from the group consisting of Tumor Necrosis Factor (TNF) and blockers thereof, such as Humicade (CDP 571), suppressors of said immune disease, such as thalomide (Thalidomide), interleukins, such as IL-1, IL-12 and IL-15, blockers or suppressors of said interleukins, interferons, such as IfFNg, blockers or suppressors of said interferons, integrin, blockers or suppressors of said integrin, Intercellular Adhesion Molecules, such as ICAM-1, and blockers or suppressors thereof.

7. The method according to any of the preceding claims, wherein the modulator is selected from the group consisting of MBL, MBL-replacement factors, C4A and functional equivalents thereof, C4B and functional equivalents thereof, C2 and functional equivalents thereof, C3 and functional equivalents thereof, IgG- and IgM-antibodies raised against triglyceride-rich particles, LDL and parts thereof, C3adesArg, factor B and functional equivalents thereof, factor D and functional equivalents thereof, factor P and functional equivalents thereof, serum carboxypeptidases, such as sCP-N, and functional equivalents thereof, erythrocyte- bound CR1 and functional equivalents thereof, free CR1 and functional equivalents thereof, CR1 mimetics such as C3b antibodies, vitronectin and functional equivalents thereof, clusterin and functional equivalents thereof, apo B (48 and 100), apo B replacement factors and esterases, such as one of the MASP-proteins, and functional equivalents thereof.

8. The method according to any of the preceding claims, wherein said modulator is selected from the group consisting of MBL-replacement factors and apo B replacement factors.

9. The method according to claim 7, wherein said modulator is selected from the group consisting of antibodies to the classical pathway.

10. The method according to claim 9, wherein said modulator is selected from the group consisting of IgG and IgM antibodies.

11. The method according to claim 7 or 8 or 9, wherein the group comprising apo B replacement factors further comprises an IgA or IgD antibody directed against an apo B lipoprotein, wherein said antibody is heavily mannosylated.

12. The method according to claim 7 or 8 or 9, wherein the group comprising apo B replacement factors further comprises an IgA or IgD antibody directed against an apo B lipoprotein, wherein said antibody is heavily N-acetylglucosaminylated.

13. The method according to claim 7 or 8 or 9, wherein the group comprising apo B replacement factors further comprises an IgA or IgD antibody directed against an apo B lipoprotein, wherein said antibody is heavily fucosylated.

14. The method according to any of claims 9-13, wherein said antibody is selected from the group consisting of a polyclonal, humanized monoclonal, and combinatorial antibody.

15. The method according to any of claims 9-14, wherein said antibodies comprise bi-specific antibodies reactive towards both an apo B and CR1.

16. The method according to any of the preceding claims, wherein said modulator is administered by using a lipid carrier chosen from the group consisting of natural lipids, artificial lipids, synthetic lipids, mineral oil, natural oil, and processed mineral oil and natural oil, wherein said processed mineral oil and said natural oil is purified or modified.

17. The method according to claim 16, wherein said lipid carrier is olive oil.

18. The method according to any of the claims 1 to 9, wherein the modulator is generated in-vivo.

19. The method according to any of the preceding claims, wherein optimum oral immunization is achieved.

20. The method according to claim 19, wherein said optimum oral immunization comprises a response selected from the group consisting of an enhanced mucosal antibody (IgA) response, T-cell reactivity, systemic T-cell, IgM, IgG and B-cell antibody responses.

21. The method according to any of the claims 1 to 18, wherein said modulating the activity of one or more elements in said compliment/lipid pathway affects a disease associated with complement-mediated lipid metabolism.

22. The method according to any of the claims 1 to 18, wherein said modulating the activity of one or more elements in said compliment/lipid pathway affects a disease selected from the group consisting of a disease of the immune system, an infectious disease, an autoimmune disease, a neoplastic disease, a hematological disease related to complement-mediated lipid metabolism, and an underlying or related disease.

23. The method according to any of the claims 1 to 18, wherein said modulating the activity of one or more elements in said compliment/lipid pathway prevents atherogenic processes associated with infectious, autoimmune, or neoplastic diseases that at least partially occupy the lipid eliminating complement activation pathway.

24. A method for diagnosing the underlying or related defect of a disease of the immune system, an infectious disease, an autoimmune disease, a neoplastic diseas, a hematological disease related to complement-mediated lipid metabolism, and an underlying or related disease comprising carrying out an assay for at least one element of the complement/lipid pathway.

25. The method according to claim 24, wherein said assay for at least one element of the complement/lipid pathway comprises an assay for a disease selected from the group consisting of an infectious, autoimmune and a neoplastic disease, wherein said disease at least partially occupies the lipid eliminating complement activation pathway.

26. Method according to claim 24 or 25, further comprising using flow cytometry.

27. A method for discovering pharmaceutical or nutritional formulations for the treatment, amelioration or prophylaxis of a disease of the immune system, an infectious disease, an autoimmune disease, a neoplastic disease, a hematological disease related to complement-mediated lipid metabolism, and an underlying or related disease comprising testing a disease for changes in a compliment/lipid pathway.

28. The method according to any of the preceding claims, wherein modulators of the complement/lipid pathway are tested in said testing a disease for changes in a compliment/lipid pathway.

29. A composition for the treatment or prophylaxis of diseases associated with the complement/lipid pathway comprising at least one modulator capable of modulating the activity of one or more elements in said pathway.

30. The composition according to claim 29, further comprising a second drug for the treatment of an immune disease.

31. The composition according to claim 30, wherein said second drug is selected from the group consisting of Tumor Necrosis Factor (TNF), blockers of said TNF, such as Humicade (CDP 571), suppressors of said TNF, such as thalomide (Thalidomide), interleukins, such as IL-1, IL-12 and IL-15, blockers of said interleukins, suppressors of said interleukins, interferons, such as IFNg, blockers of said interferons, suppressors of said interferons, integrin, blockers of said integrin, suppressors of said integrin, Intercellular Adhesion Molecules, such as ICAM-1, blockers of said Intercellular Adhesion Molecules, and suppressors Intercellular Adhesion Molecules.

32. The composition of claim 29, wherein said at least one modulator capable of modulating the activity of one or more elements in said pathway treats, ameliorates or is prophylaxis of a disease selected from the group consisting of the immune system, an infectious disease, an autoimmune disease, a neoplastic disease, a hematological disease related to complement-mediated lipid metabolism, and an underlying or related disease.

33. The composition according to claim 32, further comprising a second drug for the treatment of an immune disease.

34. The composition according to claim 33, wherein said second drug is selected from the group consisting of Tumor Necrosis Factor (TNF), blockers of said TNF, such as Humicade (CDP 571), suppressors of said TNF, such as thalomide (Thalidomide), interleukins, such as IL-1, IL-12 and IL-15, blockers of said interleukins, suppressors of said interleukins, interferons, such as IFNg, blockers of said interferons, suppressors of said interferons, integrin, blockers of said integrin, suppressors of said integrin, Intercellular Adhesion Molecules, such as ICAM-1, blockers of said Intercellular Adhesion Molecules, and suppressors of said Intercellular Adhesion Molecules.

35. The composition of claim 29, where in the disease is selected from the group consisting of the immune system, infectious disease, autoimmune disease, neoplastic disease, hematological disease, and an underlying or related disease, having impaired complement-mediated lipid metabolism.

36. The composition according to claim 35, further comprising a second drug for the treatment of an immune disease.

37. The composition according to claim 36, wherein said second drug is selected from the group consisting of Tumor Necrosis Factor (TNF), blockers of said TNF, such as Humicade (CDP 571), suppressors of said TNF, such as thalomide (Thalidomide), interleukins, such as IL-1, IL-12 and IL-15, blockers of said interleukins, suppressors of said interleukins, interferons, such as IFNg, blockers of said interferons, suppressors of said interferons, integrin, blockers of said integrin, suppressors of said integrin, Intercellular Adhesion Molecules, such as ICAM-1, blockers of said Intercellular Adhesion Molecules, and suppressors of said Intercellular Adhesion Molecules.

38. The composition according to any of the claims 29-37, wherein at least one modulator capable of modulating the activity of one or more elements in said pathway modulates elements of a pathway selected from the group consisting of the lectin pathway and the alternative pathway for complement activation.

39. The composition according to any of the claims 29-38, wherein said modulator is selected from the group consisting of MBL, MBL-replacement factors, C4A and functional equivalents thereof, C4B and functional equivalents thereof, C2 and functional equivalents thereof, C3 and functional equivalents thereof, IgG- and IgM-antibodies raised against triglyceride-rich particles and LDL or parts thereof, C3adesArg, factor B and functional equivalents thereof, factor D and functional equivalents thereof, factor P and functional equivalents thereof, serum carboxypeptidases, such as sCP-N, and functional equivalents thereof, erythrocyte-bound CR1 and functional equivalents thereof, free CR1 and functional equivalents thereof, CR1 mimetics such as C3b antibodies, vitronectin and functional equivalents thereof, clusterin and functional equivalents thereof, apo B (48 and 100) and functional equivalents thereof, apo B replacement factors, and esterases, such as one of the MASP-proteins, and functional equivalents thereof.

40. The composition according to any of the claims 29-39, wherein said modulator is selected from the group consisting of MBL-replacement factors and apo B replacement factors.

41. The compositions according to any of the claims 29-40, wherein said modulator comprises molecules selected from the group consisting of metabolic precursors of modulators, biochemically functional analogues, functional equivalents of modulators and derivatives of such modulators.

42. The composition according to any of the claims 29-41, further comprising a pharmaceutically acceptable carrier chosen from the group consisting of natural lipids, artificial lipids, synthetic lipids, mineral oil, natural oil, and processed oil, wherein said processed oil is purified or modified.

43. The composition according to any of the claims 29-42, comprising a food product or pharmaceutical product.

44. A kit for diagnosing a diseases of the immune system or an infectious, autoimmune, neoplastic, hematological disease, or an underlying or related disease, related to impaired complement-dependent lipid metabolism, by a method according to any of the claims 24 to 28, further comprising means for carrying out an assay for the detection of at least one modulator and/or element of the complement/lipid pathway.