EP1747011A2 - Means and method for diagnosis, prophylaxis and therapy of connective tissue diseases - Google Patents

Abstract

The invention relates to compositions, comprising at least one substance capable of binding to a pathogen-associated molecular patterns (PAMP) receptor and at least one tri-helical fibrous protein, a fragment or a variant thereof. According to a preferred embodiment, the tri-helical fibrous protein is pro-collagen, fragments or variants thereof. The invention further relates to pharmaceutical compositions comprising the above for prophylaxis/therapy and test kits, comprising the above for the diagnosis of connective tissue diseases. Furthermore, specific binding partners and the use thereof in pharmaceutical compositions for prophylaxis/therapy and in test kits for diagnosis are disclosed.

Description

 Means and methods for the diagnosis, prophylaxis and therapy of connective tissue diseases

The present invention relates to compositions, pharmaceutical preparations containing them and test kits for the diagnosis of connective tissue diseases; this also includes the connective tissue in parenchymatous organs and vascular structures. Furthermore, the present invention relates to the diagnosis, prophylaxis and or treatment of connective tissue diseases using the composition. Suitable specific binding partners of the composition are also provided for diagnosis and prophylaxis / therapy.

Clinical laboratory diagnostics are an essential part of rheumatological diagnostics. The so-called rheumatoid factors play an important role, the term rheumatoid factor being a historical term. These rheumatoid factors are autoantibodies against gamma globulins, whereby the analysis of the respective rheumatoid factor IgG, IgM and Ig As is of different clinical relevance. They are predominantly anti-isotypic immunoglobulins, i.e. antibodies directed against the constant regions (Fc) of immunoglobulin molecules. Depending on the titer height, RF-IgM is relevant for rheumatoid arthritis, SLE, Sjögren's syndrome, MCTD and other clinical pictures. Accordingly, these parameters are also not specific for rheumatoid diseases. The RF-IgA value is important in clinically noticeable patients with negative RF-IgM. RF-IgG plays a pathophysiological role in later stages of the disease.

A direct derivation of a rheumatic disease from these laboratory values is not possible, as this can also be used in pregnancy, asthma, some inflammatory Chen diseases, Heφes or flu may be increased. 6-8% of healthy people and 10% in old age have a rheumatoid factor. This is also the reason why rheumatoid factors do not have the pathological importance, such as anti-ds-DNA-AK in the diagnosis of SLE. Nevertheless, the detection of these autoantibodies is an essential diagnostic criterion. They are also of prognostic importance, since rheumatoid factor-positive polyarthrides usually take a more progressive course than forms of rheumatoid factor. Apparently the rheumatoid factors play a role pathogenetically, especially in the expression of extracellular manifestations of rheumatoid arthritis. In any case, patients with rheumatoid vasculitis usually have higher rheumatoid factor titers than patients with only articular manifestations. Immune complexes consisting of rheumatoid factors and IgG molecules in the cell walls of pulmonary vessels as well as in the alveoli were also detected in lung manifestations.

In laboratory diagnostics for rheumatic diseases, inflammation values such as CRP, the blood count are used to assess disease activity and the determination of uric acid in connection with gout.

The pathogenetically triggering causes of a rheumatic disease cannot be ascertained using any of the previously known methods.

Accordingly, many rheumatic patients are not covered by the studies mentioned. On the other hand, many other sufferers, especially autoimmune sufferers, who are not rheumatic are also included.

Rheumatic diseases have a better chance of treatment if they are diagnosed early and clearly. A number of different treatment options for the therapy of rheumatoid arthritis are available. Not every joint inflammation is equally quick and the same aggressive. The therapy must therefore be tailored to the individual patient. Targeted and especially early treatment is of great importance for the success of treatment. Rheumatological research has shown that treatment of diseases with a severe course is particularly successful within the first few years and can stop irreparable joint disorders.

Diagnostics that provide indications of a possible cause of the diseases, which may can be avoided or reduced by metaphylaxis / secondary prevention, is therefore of primary importance. This is where the pathogenetic concept based on the requirements of the substances and processes to be protected comes into play.

For this reason, early and clear diagnosis is of particular interest for successful therapy. In earlier years, rheumatoid arthritis was often only recognized at an advanced stage when irreversible macroscopic damage to the structures had already occurred. It is now known that the disease progresses particularly quickly within the first two years after the first symptoms appear. The earlier an effective therapy begins, the greater the chance of influencing the inflammatory process and stopping the destruction of the bones and joints. That is why early and clear diagnosis is particularly important.

The collagenous tissue is generally built up in the following steps:

Fiber-forming cells, such as fibrocytes, chondrocytes, osteocytes, reticular cells and other cells specialized thereon, produce connective tissue structures through exocytosis of trihelical, rope-like fiber proteins or their precursors. The synthesis process is shown below using the example of chondrocytes and their specific products, but can be transferred to other fiber-forming cells, taking into account the respective variabilities of the various known collagens.

In the Golgi apparatus of these cells, three amino acid chains (hereinafter referred to as cardels) are first synthesized independently of one another and are characterized by the following characteristic sections: • A 15 to 50 amino acid (AS) long N-terminal section, which is used later to form the N -terminal propeptide. This section has a higher variability among the different collagens and is rich in the amino acids cysteine, resp. in the cystine form Cys-SS-Cys for loops and pocket-forming longitudinal crosslinks, especially for the collagen I - III, which make up a large part of the total mass of all Faseφroteeine. • This is followed by a section characterized by high regularity which repetitively contains the sequence (Gly, X, Y) _n and comprises approximately 1050 AS. The amino acid proline or leucine is found almost exclusively under X; in the course of further intracellular processing, these are optionally post-translational in hydroxproline. Hydroxylysine converted. These hydroxyamino acids subsequently serve to crosslink the cardels after stranding and further to crosslink the stranded fiber segments with one another. The position marked with Y is occupied by tissue-specific, variable amino acids. • This is followed by a propeptide which is analogous to the N-terminal end and of comparable length. In addition to the fact that this section again contains a high proportion of cysteine / cystine, there is a special motif of two resp. three cysteine AS close to each other (for example, the sequence GlyProCysCysGly) at the (N-terminal) end of the C-propeptide - i.e. close to the transition point between telopeptide and C-terminal propeptide - characteristic and of great importance: The cross-linking of the three AS chains begins here.

After the three AS chains have been aligned in the same direction in parallel (the N-terminal ends lying together on one side and likewise the C-terminal ends lying together on the other side), the three individual cardelas begin to strand at this point through the formation of covalent ones Cys-SS-Cys bindings of the cardels with each other, with double or triple cross-links. The formation and localization of this structure called "cystine knot" is precisely determined from the primary structure of the AS chains of the individual cardels.

The cystine node is the evolutionary solution for the task of fixing the relative displaceability of the three cardels in the longitudinal axis to one another. As a result, the three cardels can wrap around each other. This stranding runs spontaneously from the cystine node towards the N-terminal end (and to a small extent also into the C-terminal propeptide). It is only possible due to the uniform repetitiveness of the Gly-X-Y groups, which come together in a rigid, staggered grid of the cardels. This grid synchronization runs through the entire middle rope section, which thus forms the telopeptide, right down to the N-terminal section, which is initially stranded, but then presents itself as an untwisted end due to the dissolution of the repetitiveness. Even if the three arms of the section - now called the N-terminal propeptide - remain free, the spatial structure of the amino acids lying on these arms with the binding epitopes emanating from them is fixed.

The cystine knot realized on the C-terminal propeptide thus has a remote effect on the spatial structure of the opposite end, the free cardiac arms of the N-terminal propeptide. This raster synchronization of the free ends of the propeptides, which is forced by the cystine node, is retained even if the propeptides are cut off from procollagen to tropocollagen in the course of processing, at least as long as the cut of the responsible endopeptidases (metalloproteinases) occurs at the physiologically correct location.

Thus, not only the exact tertiary structure of the C-terminal propeptide on which the cysteine node is located is defined, but this applies in the same way to the N-terminal propeptide, even if the structure which triggers the grid synchronization - the cysteine node - on this fragment is no longer detectable.

After the intracellular, stranded procollagen fiber pieces with a length of approx. 300 nm are transported to the cell surface and further processed there.

To build up collagen tissue, matrix metalloproteinase enzymes (MMP), e.g. MMP3, the above-mentioned ends of the procollagen fibers, which are referred to as propeptides, are separated and the respective “cut” fiber piece is released into the matrix as tropocollagen. The fiber piece conditioned in this way is then specifically incorporated into the respective collagen tissue (linear, grid-shaped, net-like, felt-like, etc.).

Inflammatory or degenerative diseases of the connective tissue structures formed from Faseφroteins, whether they represent their own organs, such as ligaments, tendons, joints, fascia, etc., or that they serve as scaffolding structures in parenchymatous organs, can be divided into different groups.

With rheumatic diseases in the narrower sense, after an initially clinically often not very noticeable phase, autoantibodies are formed against the connective tissue structures themselves - or with them directly associated tissues. Inflammatory episodes follow, which maintain the further formation of autoantibodies.

In the second group, rheumatoid diseases, there are no autoantibodies typical of an autoimmune disease; Primarily inflammatory and painful symptoms with no recognizable background dominate the clinic. The primary cause of these diseases is unknown; After a recurrent course, you can move to the above Full frame of a rheumatic disease so that it is disputed in the literature whether the latter diseases may only be preliminary stages of the former. It is not known which additional complications must occur in order for a rheumatoid disease to develop into the full picture of a rheumatic autoimmune disease. In any case, a longer and progressive course is to be regarded as an unfavorable requirement. It is discussed that, as a result of the immune system's deficiency, the persistent inflammation ultimately leads to the formation of autoantibodies against its own, non-pathologically altered structures, because the antibodies expressed over time show their specific selectivity for distinguishing between self and not-self lose.

On the other hand, there is a group of diseases of connective tissue structures, in particular the joints and tendons, which is referred to as primary arthrosis and which neither begin with inflammatory flare-ups nor are characterized by the occurrence of the autoantibodies typical of an autoimmune disease. For these diseases, long-term loss of the fiber-forming cells, e.g. of the chondrocytes is secured without the cause of which is known.

These diseases are causally assigned to wear (due to unphysiological stress), but it can be assumed that the focus is less on excessive wear and tear on the structures, but rather on a reduced regeneration capacity. The clinical picture is dominated by a a slow, unexplained shrinkage of the connective tissue, especially the knot, which has been going on for years / decades.

The present invention was therefore based on the object of specifying new means and methods for the diagnosis, prophylaxis and therapy of connective tissue diseases and diseases of the musculoskeletal system.

The object on which the present invention is based is achieved by the provision of a composition comprising: i) at least one substance capable of binding to a PAMP receptor; and ii) at least one trihelical Faseφrotein, a fragment or a variant thereof.

Further solutions result from the subject matter of the claims.

Some terms are explained below in order to clarify how they are to be understood in the context of the present application.

PAMP receptors comprise receptors of the innate immune system that recognize typical molecular structures for parasites, especially microorganisms. The pathogen associated molecular patterns (= PAMP) are characteristic structures of microorganisms that show a high genetic and phenotypic stability within larger groups of microorganisms (= fungi, bacteria and protozoa). This indicates an essential structural unit for the affected microorganisms (= MO), which cannot be changed without serious evolutionary disadvantage. It is precisely for this reason that these special molecular patterns prove to be important for the host organism, which does not depend on these characteristics in its structural inventory, and therefore these molecular patterns are suitable as a reliable detection criterion for non-body MOs.

The expression “pathogen-associated molecular patterns (PAMP) receptor” is understood synonymously with the expression “pattern recognition receptor (PRR) receptor”. For an overview, reference is made to the article by Medzhitov and Janeway, Current Opinion Immunol (1997), 9, 4-9. According to the invention, PAMP receptors include toll-like receptors. The expression also includes non-toll-like, cell membrane-based PAMP receptors such as phagocytic receptors. The scavenger receptors, in particular SR-A and MARCO, the macrophage mannose receptor, the B-glucan receptor and peptidoglycan recognition proteins (PGRP) may be mentioned here as examples. The expression also includes intracellular PAMP receptors such as protein kinase R, oligoadenylate synthase and molecules of the NOD family. In addition, the term soluble PAMP receptors includes pentraxins such as e.g. CRP, serum amyloid A, collectine, lipid transferases such as the lipid-binding protein (LBP) and soluble variants of the aforementioned PGRP are mentioned. The PAMP receptors are preferably of human origin. The amino acid sequences are available from the generally accessible sequence databases such as Genbank and SwissProt.

There are cellular PAMP receptors that are dissolved in body fluids. Cellular receptors either serve for signal transduction into the cell, eg CD-14 receptor, or have no known intracellular signal connection for triggering a reaction. The same applies to soluble forms of PAMP ligands that have no direct contact with cells. The function of the receptors without intracellular signal transduction connection (cellular or soluble) consists in the binding of vagabonding MO or fragments of MO; accordingly, they are referred to as scavenger receptors. “Scavenger receptor” (= SR) here included SR-A including SR-A I and SR-A II, and MARCO. Furthermore, SR includes SR-A-like SR. These include SR-CLI (SR with C- Type lectin I), SR-CLII, CL-Pl (= collectin from placenta receptor I), LOX-I (= lectin-like oxidized LDL receptor I), dSR-CI (Drosophila SR-CI) Scavenger receptor SR-A, in particular SR-AI. Furthermore, with regard to the scavenger receptors, reference is made to the review article Peiser et al., In Current Opinion in Immunology 2002, 14: 123-128.

According to the invention, “substances capable of binding to a PAMP receptor” include all PAMPs known in the prior art. For an overview, see articles by Medzhitov and Janeway, Current Opinion Immunol. (1997), 9, 4-9. PAMPs include bacterial and mycobacterial PAMPs and PAMPs derived from fungi The PAMPs include, for example, lipopolysaccharides (= LPS, also called endotoxins), lipoteichoic acid (= LTA) and their variants, wall-specific proteoglycans (= PGN), non-methylated DNA segments (CpG) specific for microorganisms , Lipoproteins and N-formylated peptides such as F-Met-Leu-Phe and bacterial proteins. PAMP derived from fungi are, for example, fungus-specific peptidoglycans. PAMP derived from mycobacteria are mycolic acid and mycolic acid ester.

The binding of lipoteichoic acid, lipopolysaccharide (LPS), bacterial DNA, bacterial cell fragments to SR, which is a PAMP receptor, is known from Peiser et al., Above.

Further substances capable of binding to a PAMP receptor can be obtained by simply performing a binding assay, as described, for example, in Dünne et al., PNAS 91 (1994), 1863-1867, the PAMP receptor preferably SR for the binding assay -AI is. According to the invention, the term “trihelical phase phrotein” encompasses collagen, procollagen, tropocollagen, elastin, fibrillin, fibronectin, scavenger receptors and other collagen-like constituents. Furthermore, the expression includes the synthesis precursors of the aforementioned. As is generally known and described above, collagens are initially referred to as After the hydroxylation of L-proline and L-lysine residues and after glycosylation in the endoplasmic reticulum or in the Golgi apparatus, where they come together to form three chains, they are excreted into the extracellular space At the ends of the chains, so-called propeptides or those separated at a non-typical interface are split off, and the tropocollagen is formed, which assembles into fibrils by oxidation of amino groups in the L-lysine side chains to form aldehyde groups and their aldol and aldimine formation with aldehyde or amino groups of adjacent chains these are linked together. (Networking).

The term “fragments”, insofar as they are proteins, preferably means fragments with a minimum length of 10, particularly preferably 20, amino acids. If they are nucleic acids, the fragments have at least 20 nucleotides, preferably 50 nucleotides.

The term “variants” basically encompasses all modifications of a given sequence, muteins which differ at the amino acid level by addition, substitution, deletion, insertion or inversion of at least one amino acid from the given peptide sequence being preferred. The mutation particularly preferably comprises 1 to 5 amino acids , in particular 1 to 3 amino acids.

The term “conjugates” encompasses di-, oligo-, and polymerizations, whereby both homo- and hetero-di-, oligo-, and polymerizations are encompassed. The coupling here can be carried out on a carrier molecule, such as, for example, polyethylene glycol or others Carrier molecules known in the prior art are Furthermore, the term conjugates also includes naturally occurring conjugates, such as, for example, post-translational modifications, such as, for example, hydroxylations of the side chain.

Lipopolysaccharides (LPS) contain a very strictly preserved amino sugar-2-X-phosphate-lipid complex called Lipid (A), which is the actual membrane part. In the endotoxin molecule, the lipid (A) constituting the bacterial membrane is followed by an area essentially consisting of specific sugar with sometimes other phosphates as core antigen (Core A). The core antigen is followed by the very variable and very differently long sugar chains, called surface antigen (Surface-A), which can differ greatly from bacterial species, but also within one species from strain to strain. According to the invention, both the mono- and the bi-phosphorylated form of the lipid (A) are included.

The present invention is based on the following surprising findings by the inventors:

It has been observed in numerous cases that people who are in buildings where there is certain moisture damage that is associated with microbial infestation, suffer conspicuously often from pain in the area of the joints, so-called rheumatoid complaints. These complaints are usually related to your time in the building, i.e. when the patient is absent, the complaints subside and when they return to the relevant building, they recur in a relatively short time. The more often those affected were exposed, the faster the complaints come from exposure and the slower they resolve after the exposure has been removed.

More detailed investigations showed that initially in the initial stage it was usually not the joints that hurt in the narrower anatomical sense, but those close to the joints Soft tissue structures. The occurrence of the pain was strictly correlated with a previous biomechanical load. The observations showed that it was precisely the areas with the highest cross-sectional stresses that hurt the patient the next day.

When examining numerous such buildings, it could be shown that the connective tissue diseases correlated with a characteristic microbial infestation pattern in building materials.

Through in-tro experiments it could be demonstrated that bacterial endotoxins (= LPS), extracted from damp building materials, destroy human cartilage tissue in the area of moisture damage. With this destruction, the chondrocytes and the extracellular matrix are destroyed and a sharp increase in the amounts of MMP enzymes in the tissue culture can be observed. It was also demonstrated that the effect of the endotoxins on the tissue shows a clear dose-response relationship, i.e. the higher the concentration of endotoxins, the greater was the destruction of the chondrocytes that are responsible for collagen synthesis.

Thus, for the first time, LPS or endotoxins as noxae, i.e. as a pathogenic cause for connective tissue diseases of patients who were in buildings with microbial infestation. It is therefore possible for the first time to differentiate between a microbial-related connective tissue disease and a non-microbial-related connective tissue disease.

The diagnostic approach of the methods in question is the detection of a reduced repair and regeneration capacity for fibrillar structures by an exogenous noxa (PAMP, definition see below).

The task of PAMP receptors, especially scavenger receptors, is among other things the binding of endotoxins for removal from the bloodstream. Which he- Finders of the present invention have also found that the LPS-binding epitopes of the scavenger receptors have a high degree of homology to analog structures at the ends of the procollagen molecules. The spatial structures of SR-AI and SR-AII and procollagen are shown schematically in Figures 1 and 2. As a result, in particular the N- and / or C-terminal propeptides in various fragmentation lengths, possibly including some or all of the length of the telopeptide or modifications of these structures, are suitable as near-natural and thus low-antigenic ligands.

If the systemic cleaning system is overloaded with PAMP's, which is provided by scavenger receptors, e.g. in the case of septic infections, antibiotic therapy with bactericidal antibiotics, or as in the present case due to exogenous endotoxin contamination, the PAMPs, especially LPS, are circulated and these can bind to the next comparatively avoid structure. This is the freshly formed procollagen not yet processed to tropocollagen on stimulated fiber-forming cells, e.g. Chondrocytes.

Using the example of growth-induced chondrocytes, the inventors of the present invention were able to show that a complete blockage of the collagen synthesis by endotoxin through an excessive activation of the metalloproteinases involved in the processing leads to damage to already existing fiber structures and ultimately to a death of the fiber-forming cells. It can be assumed that the non-processing to procollagen causes the cell to continue to provide large amounts of procollagen on the surface and to provide metalloproteinases for processing. An excessive, no longer only local synthesis of MMPs leads to large-scale destruction of fibers and cells; with extensive destruction resulting in clinically apparent inflammation and pain. Fig. 1 shows the spatial structures of SR-AI and SR-AII. Fig. 2 shows the spatial structure of procollagen.

3 (a) to (c) show destroyed chondrocytes after addition of the eluate described in Example 1. 4 (a) to (b) show healthy chondrocytes.

The invention thus relates to a composition comprising: i) at least one substance capable of binding to a PAMP receptor; and ii) at least one trihelical Faseφrotein, a fragment or a variant thereof.

The substance capable of binding to a PAMP receptor is preferably a bacterial, mycobacterial or fungus-derived PAMP, preferably the PAMP is a lipopolysaccharide (= LPS, also called endotoxin), lipoteichoic acid (= LTA) and their variants, wall-specific proteoglycans (= PGN), non-methylated DNA segments (CpG) specific for microorganisms, lipoproteins and N-formylated peptides such as F-Met-Leu-Phe and bacterial proteins. It is further preferred that the PAMP derived from fungi is a mushroom-specific peptidoglycan. Also preferred is the PAMP mycolic acid or mycolic acid ester derived from mycobacteria. The PAMP lipopolysaccharide is particularly preferred.

Fragments, variants or conjugates of the substance capable of binding to a PAMP receptor are also preferred, the fragments, variants or conjugates thereof, at least 50% of the binding capacity of LPS from E. coli to the scavenger receptor SR-A I. Die Binding of the LPS to the scavenger receptor is determined as described above. The fragments, variants or conjugates particularly preferably have a binding capacity of at least 80%, in particular at least 90%, of the binding ability of LPS to the scavenger receptor.

The substance capable of binding to a PAMP receptor is particularly preferably a lipolysaccharide (LPS), fragments or variants thereof, the fragments or variants having a phosphorylated N-acetylglucosamine dimer, preferably containing a complete lipid A complex. It is known that lipid (A) mediates the binding of LPS to the macrophage receptor via this structural element to the CD14 receptor LBP complex. The lipopolysaccharide is preferably the lipopolysaccharide of the genera e.g. Salmonella, Shigella, E. coli, Pseudomonas, Neisseria, Klebsieila and Haemophilus.

It is also particularly preferred that the PAMPs derived from microorganisms originate from bacteria of the order Actinomycetales and fungi of the genera Aspergillus, Botrytis, Cladosporium, Eurotium, Penicillium, Wallemia, Chaetomium, Mucor, or Scopulariopsis.

According to a preferred embodiment, the trihelical Faseφrotein (component (ii) of the composition) is selected from collagen, procollagen, tropocollagen, elastin, fibrillin, fibronectin, scavenger receptors or other collagen components, synthesis precursors, fragments, variants or conjugates of the above components.

According to the invention, the collagen comprises types I to XIV, with types I, II, III, V and IX being preferred. Elastin is the main component of the elastic fibers of the connective tissue, especially in organs with high elasticity, such as blood vessels, lungs, skin, tendons and uterus. Elastin differs from similar collagens in its higher valine and leucine content, its lower arginine and hydroxyproline content and its lack of lysine residues. The above proteins are preferably of human origin, however animal proteins are also included in the invention. The trihelical phase phrotein is preferably procollagen, fragments or variants thereof. The fragments or variants of procollagen preferably have at least 50%, particularly preferably at least 80%, in particular at least 90% of the binding capacity of human procollagen to LPS from E. coli. The binding ability can be carried out using a generally known binding assay.

The variants of procollagen are preferably at least 60% homologous, preferably at least 80%, particularly preferably at least 90%, in particular at least 95% homologous at the amino acid level to human procollagen. The homology can be determined using standard programs known to those skilled in the art (e.g. FASTA, BLAST) using standard settings.

The procollagen fragment preferably has at least 10, particularly preferably 20, in particular 50, amino acids of procollagen. The variant of procollagen is preferably a mutein which differs from procollagen by the addition, substitution, deletion, insertion or inversion of at least one amino acid. The mutations preferably comprise 1 to 5 amino acids, in particular 1 to 3 amino acids. The substitution is particularly preferably a conservative substitution in which an amino acid is replaced by another amino acid which belongs to the same physico-chemical group.

In general, the fragments and variants can be produced by chemical methods or recombinantly.

Procollagen has a stranding of the three peptide chains about 10 to 30 amino acid residues before the end. The point of transition from the stranded structure to the free poor is of particular importance. At this transition point a very distinctive amino acid motif for covalent bonds is responsible. By double or triple incorporation of the amino acid cysteine at precisely defined intervals on each of the three peptide chains, a structure is formed in which each chain with each chain is bonded to one another via Cys-SS-Cys. This coordination structure made of cysteine building blocks is called a cysteine node.

The trihelical phase phrotein, the fragment or the variant thereof therefore preferably comprises a sequence suitable for forming a cysteine node, so as to provide binding sites for the binding of the substance which is capable of binding to a PAMP receptor. The sequence suitable for forming a cysteine node is preferably the sequence GlyProCysCysGly (SEQ ID NO: 1).

For the variants of procollagen, its secondary and cleavage products discussed in the context of the invention, it should be noted that the evolution of the cysteine node represents only one of the conceivable solutions to the task of grid synchronization. For relevant biosynthetic sections of the procollagen and variants of its propeptides, it should be noted that the task of grid synchronization, which is essential for maintaining the affinity of the propeptides to PAMPs used below, also by other space-coordinating measures between the three cardelas of the AS strands is to be solved.

This applies in particular to the use of the binding ability of an N-terminal fragment of the procollagen. In addition to the obvious variant of the total or partial shortening of the telopeptide section in a biotechnologically expressed product, as a result of which the cystine node would be shifted further N-terminally, bonds formed differently (as by covalent Cys-SS-Cys) can also be used between the AS of the three cardels as long as the spatial coordination of the free, together the binding epitope for PAMP's ends of the three cardels is maintained. This technique, without using the natural Chen cysteine node or a functional equivalent, is also applicable to the C-terminal side.

According to a preferred embodiment, the fragment or variant of the trihelical Faseφroteins therefore has the N- and / or C-terminal end of a trihelical Faseφroteins and optionally a part of the telomer.

According to a particularly preferred embodiment, the substance capable of binding to a PAMP receptor is a lipopolysaccharide and the trihelical fiber protein is collagen, procollagen or tropocollagen, preferably procollagen, fragments or variants thereof.

According to a preferred embodiment of the present invention, the composition further contains chondrocytes, fibrocytes, osteocytes, reticulo-endothelial cells, endothelial cells and / or components thereof. The aforementioned cells are capable of producing collagen. Chondrocytes and their components are preferred. Preferred components are the cell membrane or fragments thereof. According to the invention, the cell nucleus, Golgi apparatus, endoplasmic reticulum and mitochondria can be components of the cells.

In the composition according to the invention, components (i) and (ii) can be linked to one another non-covalently or covalently. If not already present, components (i) and (ii) of the composition can be covalently bound by crosslinking using known methods, such as e.g. can be achieved by carbodiimides.

According to a further aspect of the present invention, a method for

Preparation of the composition according to the invention specified, the method comprising contacting component (i) with component (ii) and optionally cleaning and isolating the composition. The compo Component (i) and component (ii) can be used both in the naturally occurring form and can also be produced synthetically. Synthetic methods for this are known to the person skilled in the art. The components can be brought into contact in liquid form or one of the two components can be bound to a carrier.

Methods for purifying or isolating the composition include centrifugation, gradient fractionation, chromatographic methods, one- or two-dimensional gel electrophoresis.

According to a preferred embodiment of the method, the method is carried out as in vz ^' tro method, which comprises the steps: a) providing a bacterial or fungal eluate containing a component (i); b) providing a growth-stimulated chondrocyte culture; c) bringing the bacterial or fungal eluate into contact with the chondrocyte culture; and d) optionally purifying the composition.

The mushroom eluate is preferably an eluate obtained from Aspergillus, Botrytis, Cladosporium, Eurotium, Penicillium, Wallemia, Chaetomium, Mucor or Scopulariopsis. The bacterial eluate is preferably an eluate obtained from Actonomycetales, Salmonella, Shigella, E. coli, Pseudomonas, Neisseria, Klebsieila, and Haemophilus. A growth-stimulated chondrocyte culture is preferably as described by Shakibaei et al., Biochem. J. (1999), 342, 615-623. The knot tissue is mechanically crushed, incubated and cleaned in Ham's F-12 medium. The chondrocytes are then suspended in growth medium. The growth medium consists of Ham's F-12 and Dulbecco's modified medium according to Eagle (50%: 50%). 10% fetal calf slurry, 25 μg / ml ascorbic acid and 50 μg / ml gentamicin are added to this medium. As stated above, the purification can include methods known to those skilled in the art, chromatographic methods, centrifugation, ultrafiltration and gel electrophoresis. A preferred embodiment for the production consists in coupling the component (i) to a column matrix and subsequently passing a detergent-lysed chrondrocyte lysate over this column prepared in this way. After incubation of the chondrocyte lysate with the coupled component (i), the composition can subsequently be eluted after washing the column. The elution conditions should preferably be chosen so that the composition does not denature.

According to a further aspect of the present invention, a pharmaceutical composition is provided which contains the composition set out above and at least one pharmacologically acceptable carrier. Pharmacologically acceptable carriers are known to the person skilled in the field of galenics. The pharmaceutical compositions are preferably suitable as a vaccine in order to elicit an immune reaction of the person against at least one component of the composition for prophylaxis against the development of an autoimmune reaction. In a preferred embodiment, the vaccine formulation may further contain suitable carrier molecules in the prior art in order to increase the immunogenicity of the composition. The vaccine formulation can preferably be administered subcutaneously, intravenously or intramuscularly. The pharmaceutical composition may generally be in liquid form or in the form of an aerosol with the appropriate use of aerosol stabilizing compounds.

The use of the pharmaceutical composition, in particular as a vaccine for the prophylaxis and / or treatment of connective tissue diseases, is thus provided according to the invention. According to the invention, connective tissue diseases include diseases of the musculoskeletal system, but also of other diseases known to be associated with connective tissue, such as, for example, vasculitis. den, glomerulonephritis, endocarditis (with and without valve involvement), dermatitis and dermatomyositis. The connective tissue diseases are preferably selected from arthrosis, soft tissue rheumatism, fibromyalgia, rheumatic diseases or rheumatic autoimmune reactions.

According to a further embodiment of the present invention, a test kit for the diagnosis of connective tissue diseases is provided, which contains the composition according to the invention. The test kit is particularly suitable for the detection of antibodies which the body produces in particular a complex of endotoxin and procollagen in response to the composition formed under natural conditions. The antibody titer against the composition formed under natural conditions is an indicator of whether the complaints of the connective tissue and the musculoskeletal system are due to exposure to the substance capable of binding to a PAMP receptor, in particular endotoxins. A comparison with the titer on autoantibodies against normal collagen shows whether there is a preliminary stage of the incipient rheumatic disease or whether the rheumatic disease is already fully developed. The former would be indicated by a clear titer on the naturally formed composition, with an additional weak titer on the body's own collagen possibly being present. In the further case there is a titer on the naturally formed composition and a high titer on the body's own collagen. The test kits according to the invention are accordingly suitable for detecting an antibody. The test kit can furthermore contain the buffer substances known in the prior art which are suitable for use of the test kit in the determination and diagnostic method. The body fluid for examination by the test kit is preferably selected from blood or blood products including serum. Methods which are advantageous for determination purposes are immunoassays, ELISA, RIA, membrane-bound test strips, receptor binding tests or biosensory determinations, the implementation of which is known to the person skilled in the art. In an ELISA detection, for example, the composition would be immobilized on a microtiter plate. in the Test bind the specific antibodies and are then converted into a signal by correspondingly labeled autoantibodies using known methods.

The present invention furthermore provides reagents which bind to the composition according to the invention, preferably are specific for this. An example of such specific reagents are antibodies, antibody fragments, for example Fv, F (ab) or F (ab) ₂ fragments or antibody derivatives. The Antiköφer, Antiköφerfragmente, for example Fv, F (ab) or F (ab) ₂ fragments or Antiköφerderivate can be of monoclonal or polyclonal origin. In general, specific antibodies are available in which experimental animals, such as mice or rabbits, are immunized with the composition according to the invention or at least component (i) or component (ii), which are preferably coupled to suitable high-molecular carrier molecules. Immunization can be facilitated by adding suitable adjuvants known in the art. Monoclonal antibodies are usually obtainable by fusing spleen cells, which have been removed from an immunized mouse, with tumor cells and selecting the resulting hybridomas. Those hybridomas that efficiently secrete specific antibodies can be determined by searching for the supernatant. Alternatively, antibodies can be produced recombinantly; in the production of recombinant antibodies, the mRNA is isolated from hybridoma cells or B lymphocytes, which acts as the basis for the synthesis of the corresponding cDNA and is amplified by PCR. After ligation into a suitable vector and the introduction of a suitable host cell culture, the antibody can be obtained from the cell culture supernatants or the cell lysates. Recombinant antibodies allow a "humanization" of the antibody and are therefore less immunogenic. The methods in this regard are known in the prior art. Antibody derivatives comprise conjugates of antibodies with markers suitable for detection, for example for use in scintigraphy. The present invention furthermore provides a composition comprising a first binding partner which binds to component (i), preferably specific for this, and / or a second binding partner which binds to component (ii), preferably specific for this , The composition is preferably a pharmaceutical composition. The first and second binding partner is preferably an antibody. Another example of the embodiment in which component (i) is an endotoxin, preferably LPS, is the binding partner LAL. Further binding partners of endotoxin, preferably LPS, are albumin, transferrin, HDL, LDL, apolipoproteins, C-reactive protein, CRl, CR3, CD14, scavenger receptors, CD18, gangliosides, lectin-like receptors, polysaccharide receptors, bactericidal / permeability increasing protein (BPI), cationic antimicrobial proteins (CAP), and LPS-binding protein (LBP), as well as soluble variants of the factors mentioned above. According to the invention, component (ii) is specified as a further binding partner for component (i). Component (ii) is preferably procollagen, tropocollagen, collagen, synthetic precursors, fragments or variants thereof. In particular, the component is procollagen, fragments and variants thereof. The fragments and variants of procollagen are defined as above. In addition to the first binding partner and / or second binding partner, the pharmaceutical composition can contain a pharmacologically acceptable carrier. Suitable carriers are known in the prior art. The pharmaceutical compositions are preferably suitable for intravenous, subcutaneous or intramuscular administration. As stated above, the substance capable of binding to a PAMP receptor, in particular endotoxin, is the noxa for the death of the collagen-producing chondrocytes, the substance binding to the chondrocytes via the trihelical phase phrotein. The pharmaceutical composition can therefore on the one hand directly neutralize the substance capable of binding to a PAMP receptor before it binds to the chondrocytes. On the other hand, the pharmaceutical composition can remove already formed toxic complexes from the bloodstream. Suitable concentrations of binding partner in the pharmaceutical composition can preferably be in the range from 1 μg / ml to 10 mg / ml. The pharmaceutical composition is suitable for the treatment of connective tissue diseases, including diseases of the musculoskeletal system. The connective tissue disease is preferably osteoarthritis, soft tissue rheumatism, fibromyalgia or rheumatic disease.

According to the invention, a test kit for diagnosing connective tissue diseases is also provided, containing the antibody according to the invention and / or the above-mentioned composition. The test kit can also contain buffer substances known from the prior art which are suitable for use of the test kit in determination and diagnostic methods. The test kit can be used to determine the naturally formed composition, in particular a complex containing endotoxin and procol layers and / or the substance in the body fluid capable of binding to a PAMP receptor, in particular an endotoxin. The body fluid is preferably selected from blood or blood products including serum. The detection can be carried out using immunoassays, ELISA, RIA, membrane-bound test strips, receptor binding tests or biosensory determinations, the implementation of which is known to the person skilled in the art.

Accordingly, according to the invention, a method for the diagnosis of connective tissue diseases comprising detecting the composition by bringing it into contact with the antibody or with the composition containing a first binding partner which binds component (i), is preferably specific to it, and / or a second binding partner which binds to component (ii), is preferably specific for this.

Furthermore, the use of component (ii) for neutralizing endotoxins such as LPS for the prophylaxis of an autoimmune reaction is also proposed according to the invention. given. The administration of component (ii) leads to binding and thus neutralization of the endotoxins present in the patient's body fluid. Component (ii) is preferably selected from procollagen, tropocollagen, collagen, synthetic precursors, fragments or variants thereof, particularly preferably procollagen, fragments or variants. The fragments or variants are defined as above.

The following examples are intended to illustrate the invention, but in no way limit it. Based on the description of the examples, the person skilled in the art can access further embodiments, which are also included.

Examples

example 1

Determination of the effective noxa in bacterial eluates from microbially populated building material.

Microbially populated building materials were taken from the basement of a residential building. To produce the eluate, 150 g of this material and 60 ml of PBS were suspended and stirred at 200 rpm for 30 minutes. The sample was then switched off for 5 minutes so that the solid components could sediment. The liquid part was then centrifuged (at 4000 g) and then filtered with a 0.2 μm cellulose acetate filter. The liquid obtained in this way was tested for sterility by cultivation on nutrient media. This sterile eluate was added at a dilution of 1: 100 chondrocyte cultures.

The primary chondrocyte cultures used here were prepared in accordance with Shakibaei et al., Biochem. J. (1999), 342, 615-623; Shakibaei et al., J. Biol. Chem (2001), 276, 13289-13294. Knoφelgewebe was mechanically crushed, incubated and cleaned in Ham's F-12 medium. The pieces of cartilage were then dissolved with 1% pronase (2 hours at 37 ° C.) and then with 0.2% collagenase (4 hours at 37 ° C.). Here, the extracellular matrix is dissolved while the chondrocytes have not been attacked. Both enzymes were dissolved in Hank's solution with 5% (v / v) fetal calf suspension.

The chondrocytes were then suspended in growth medium. The growth medium consisted of Ham's F-12 and DMEM (3: 1) (v / v)). 10% (v / v) fetal calf semen, 25 μg / ml ascorbic acid and 50 μg / ml gentamicin were added to this medium. The chondrocytes were separated by repeated pipetting. The cells (cell density: 2xl0 ⁶ / 10μl) were cultivated in alginate beads (Shakibaei and de Souza, Cell Biol. Int. (1997), 21, 75-86). After two weeks, some cells had continuously grown out of the alginate beads and adhered to the petri dishes. After three days, when the cells had grown confluently, the cells of the first passage were removed with 0.05% (v / v) trypsin / 1.0 mM EDTA and sown again in culture bottles. The confluent monolayer was passaged every three days. The cells from the monolayer passage were introduced into high density cultures. High density cultures were prepared as detailed in Zimmermann et al., Cell Differ. Dev. (1990), 11-22. Briefly, the cells were washed twice in growth medium and sedimented by centrifugation (600 rpm for 10 minutes). One or two drops of a dense cell suspension (1.5 x 10 ⁶ cells / 10 μl) were applied to a membrane filter (pore diameter 0.2 μm, Sartorius, Göttingen, Germany) on the surface of a stainless steel grid in the intermediate area between medium and air in one Petri dish. The medium was changed every three days. The cultures were cultured at 37 ° C in a humidified atmosphere with 5% CO ₂ . The eluate was added to this medium. After three days and seven days, the cartilage was examined microscopically. Cuts were made for this. 3 (a) to (c) show the destruction of the chondrocytes after addition of the eluate. Figures 4 (a) and (b) show healthy chondrocytes for comparison. It was clear to see that both the extracellular matrix and the chondrocytes themselves were largely dissolved. Further investigations using immunofluorescence microscopy and Western blot showed that at the same time as the tissue destruction, there was a significant increase in enzymes of the matrix of the matrix metalloproteinases.

1 x 1 mg / ml, 1 x 10 mg / ml or 1 x 100 mg / ml polymyxin B (Sigma, P1004; polymyxin B sulfate salt,> 6000 USP units / mg) was also added to the sample eluate. Polymyxin B specifically binds endotoxins. Various amounts of polymyxin B were added until only very small amounts of endotoxins were finally detectable using LAL tests (LAL test, Pyrogent, BioWhittaker, Inc., MD, USA). The chondrocyte exposure experiments were then repeated. It was shown that the chondrocytes and the extracellular matrix were damaged in correlation to the LPS concentration in the eluate. The enzyme production of collagenase and matrix metalloproteinase was inversely proportional to the endotoxin content.

Example 2:

Applications of detection methods for the detection of antibodies which are directed against the composition according to the invention for diagnostics.

The composition according to the invention can be used as a marker for the medical diagnosis of connective tissue diseases in humans and animals. In this function it can also be used for therapy control. Also hygienic tests, for example of the living and working environment with regard to bacterial or mold colonization and in In this context, contamination with endotoxins and, if necessary, measures to eliminate them play an important role in the context of therapy concepts.

The antigens described are preferably used as markers in customary diagnostic methods, such as immunoassays, blotting methods, biosensor methods or comparable methods. The fact that the antigenic marker molecule or its modifications (for example modifications to improve the coating efficiency) were used in corresponding binding tests, for example immunoassays. It can be directly adsorbed to the surfaces of suitable highly adsorbing microtiter plates. The assay is carried out using appropriate blocking and coating protection methods, as are well known in the art, using dilute sera or plasma samples in the assay. Autoantibodies that are present in the appropriate titer in specifically affected rheumatic patients bind to the immobilized antigen. The signal is generated according to methods known in the art with enzyme-labeled (often horseradish peroxidase), anti-human IgG (possibly also IgM or IgA) antibodies from rabbits, sheep or goats. The final signal generation takes place via the enzymatic formation of a chromogen. A commonly used substrate is the tetramethylbenzidine substrate. The evaluation is carried out at 450 nm using an ELISA photometer after stopping the reaction with dilute sulfuric acid.

For calibration, specific and high-titer autoantibodies of a patient term (specifically ill patient) or a pool of several patient sera are used. The concentration or the titer of the specifically directed autoantibodies in the individual calibration sera is to be set by producing suitable dilutions in such a way that a suitable standard curve is achieved. The cut-off value for discrimination between normal and pathological values is set by measuring and evaluating sera or plasma collectives from specifically ill patients and control subjects. The cut-off value is calculated on the basis of these measurements by statistical evaluation. The diagnostic method described is indicated for rheumatic diseases which are associated with inco-endotoxins. Increased titers of specific autoantibodies indicate rheumatic diseases which can often not be determined by laboratory tests used to date, such as measuring the known rheumatoid factor. The diagnosis therefore records other specifically ill patients and thereby improves and supplements the established laboratory diagnosis alone. Furthermore, after a positive diagnosis has been made, a hygienic analysis of the living and working environment is possible, which eliminates eliciting factors, in particular endotoxins or the causes for the existence of these endotoxins.

In this context, the test can be used to monitor therapy. On the one hand to check a treatment, on the other hand to check the elimination of the possible causes discussed, ie endotoxin pollution of the environment.

Example 3:

Applications of detection methods of the marker and its modifications for diagnostics.

In addition to the autoantibodies, the composition itself, in particular the endotoxin or the endotoxin-procollagen complex, can also be detected in the serum. However, detection of the autoantibodies is preferred. The antigen itself can be detected in a conventional method such as in an immunoassay. The antigen is preferably detected in a sandwich ELISA. The basis is two, preferably different, antibodies which are obtained by immunization with the composition, preferably on endotoxin-procollagen complex, by methods which are known in the prior art. In an ELISA, which can be used as a preferred method, one of the two antibodies is used in a preferred method for coating, while the other is coupled to a marker enzyme. The coupling methods are known from the prior art.

This method can be used as an alternative or in addition to the autoantibody determination. As in Example 3, veterinary tests are also possible. Relevant structures or antigens from cell cultures can also be detected using the invention described here.

Claims

claims

1. A composition comprising: (i) at least one substance capable of binding to a pathogen associated molecular pattern (PAMP) receptor; and (ii) at least one trihelical Faseφrotein, a fragment or a variant thereof.

2. Composition according to claim 1, characterized in that the substance capable of binding to a PAMP receptor is a bacterial, mycobacterial or fungus-derived PAMP.

3. Composition according to claim 1 or 2, characterized in that the substance capable of binding to a PAMP receptor is selected from lipopolysaccharides (LPS), lipoteichoic acid, wall-specific proteoglycans, bacterial cell fragments, non-methylated DNA sections specific for microorganisms , Lipoproteins, N-formylated peptides, mushroom-specific peptidoglycans, mycolic acid or mycolic acid esters, fragments, variants or conjugates thereof, wherein the fragments, variants or conjugates thereof, at least 50% of the binding capacity of LPS from E. coli to the Scavenger receptor SR-AI exhibit.

4. Composition according to one of claims 1 to 3, characterized in that the substance capable of binding to a PAMP receptor is a lipopolysaccharide (LPS), a fragment or a variant thereof, the fragment or the variant being a phosphorylated N-acetylglucosamine - Dimer, preferably contains a lipid A complex.

5. Composition according to one of claims 2 to 4, characterized in that the PAMP the lipopolysaccharide (LPS) from the bacteria Salmonella, Shigella, E. coli, Pseudomonas, Neisseria, Klebsieila or Haemophilus.

6. Composition according to one of claims 2 to 4, characterized in that the PAMP originates from Actinomycetales, Aspergillus, Botrytis, Claosporium, Eurotium, Penicillum, Wallemia, Chaetomium, Mucor, or Scopulariopsis.

7. Composition according to one of claims 1 to 6, characterized in that the trihelical Faseφrotein is selected from collagen, procollagen, tropocollagen, elastin, fibrillin, fibronectin, scavenger receptors or other collagen components, synthetic precursors, fragments or conjugates of the above components.

8. Composition according to Anspmch 7, characterized in that the collagen is selected from collagen types I, II, III, V and IX.

9. Composition according to one of claims 1 to 7, characterized in that the trihelical Faseφrotein means procollagen, fragments or variants thereof.

10. The composition according to claim 9, characterized in that the fragment or variant is at least 50% of the binding ability of human procollagen to LPS from E. coli and / or at least 60% homologous to the amino acid sequence of human procollagen.

11. Composition according to Claim 9 or 10, characterized in that the variant is a mutein which differs from the amino acid sequence of human procollagen by the addition, substitution, deletion, insertion or inversion of at least one amino acid, preferably at most 5 amino acids ,

12. Composition according to one of claims 1 to 11, characterized in that the fragment or the variant of the trihelical Faseφroteins have a sequence suitable for forming a cysteine node, preferably the sequence GlyProCysCysGly (SEQ ID NO: 1).

13. The composition according to any one of claims 1 to 11, characterized in that the fragment or variant of the trihelical Faseφroteins contain the N- and / or C-terminal end and optionally a part of the telomer of the trihelical Faseφroteins.

14. The composition according to any one of claims 1 to 13, characterized in that the substance capable of binding to a PAMP receptor is a bacterial endotoxin, preferably LPS and the trihelical Faseφrotein tropocollagen, procollagen or collagen I, II, III, V or IX ,

15. The composition according to any one of claims 1 to 14, characterized in that the composition further contains chondrocytes, fibrocytes, osteocytes, reticulo-endothelial cells, endothelial cells, preferably chondrocytes, or components thereof.

16. The composition according to any one of claims 1 to 15, characterized in that the components (i) and (ii) are non-covalently linked to one another.

17. A method for producing the composition according to any one of claims 1 to 16 comprising contacting component (i) with that of component (ii) and optionally cleaning or isolating the composition.

18. In vitro method according to claim 17, characterized in that the method comprises the following steps: a) providing a bacterial or fungal eluate containing a component (i) according to one of claims 1 to 16; b) providing a growth-stimulated chondrocyte culture; c) bringing the bacterial or fungal eluate into contact with the chondrocyte culture; and d) optionally purifying the composition.

19. In vitro method according to Anspmch 18, characterized in that the fungal eluate comes from Aspergillus, Botrytis, Cladosporium, Eurotium, Penicillum, Wallemia, Chaetomium, Mucor, or Scopulariopsis.

20. In vitro method according to Anspmch 18, characterized in that the bacterial eluate comes from Actinomycetales, Salmonella, Shigella, E. coli, Pseudomonas, Neisseria, Klebsiella or Haemophilus.

21. The method according to any one of claims 17 to 20, characterized in that the purification comprises performing a chromatographic method, centrifugation, ultrafiltration or gel electrophoresis.

22. Pharmaceutical composition containing the composition according to one of claims 1 to 16 and at least one pharmacologically questionable carrier, preferably as a vaccine.

23. Use of the composition according to one of claims 1 to 16 for the prophylaxis and / or treatment of connective tissue diseases or diseases of the musculoskeletal system, preferably arthrosis, soft tissue rheumatism, fibromyalgia, rheumatic diseases or rheumatic autoimmune reaction.

24. Test kit for the diagnosis of connective tissue diseases containing the composition according to one of claims 1 to 16.

25. A method for the diagnosis of connective tissue diseases comprising the detection of autoantibodies by contacting a composition according to any one of claims 1 to 16 and detection of the binding.

26. Reagent that binds to at least one component of the composition according to any one of claims 1 to 16, preferably specific for the composition.

27. Reagent according to Anspmch 26, characterized in that the reagent is an antibody, preferably a monoclonal or polyclonal antibody.

28. Reagent according to Anspmch 27, characterized in that the antibody is conjugated with a marker suitable for detection.

29. Composition comprising a first binding partner that binds to component (i) and / or a second binding partner that binds to component (ii).

30. Composition according to Anspmch 29, characterized in that the first binding partner for component (i) and the second binding partner for component (ii) is specific, preferably the first and the second binding partner is an antibody.

31. Composition according to Claim 29 or 30, characterized in that the first binding partner is selected from LAL, transferrin, HDL, LDL, apolipoproteins, C-reactive protein, CRl, CR3, CD14, scavenger Receptors, CD 18, gangliosides, lectin-like receptors, polysaccharide receptors, bactericidal / permeability increasing protein (BPI), cationic anti-microbial proteins (CAP), and LPS-binding protein (LBP), as well as soluble variants of the factors mentioned.

32. Composition according to claim 30 or 31, characterized in that the first binding partner is selected from component (ii) according to one of claims 1 to 16, preferably procollagen, tropocollagen, collagen, synthetic precursors, fragments or variants thereof.

33. Pharmaceutical composition containing the composition according to any one of claims 29 to 32 and a pharmacologically acceptable carrier.

34. Use of the composition according to any one of claims 29 to 32 for the prophylaxis and / or treatment of connective tissue diseases or diseases of the musculoskeletal system, preferably arthrosis, soft tissue rheumatism, fibromyalgia, rheumatic diseases and rheumatic autoimmune reaction.

35. Test kit for the diagnosis of connective tissue diseases containing the reagent according to one of claims 26 to 28 and / or the composition according to one of claims 29 to 32.

36. A method for diagnosing connective tissue diseases comprising detecting the composition according to any one of claims 1 to 16 by contacting it with a composition according to any one of claims 29 to 32, preferably in blood or serum, and detecting the binding.

37. Use of component (ii) according to one of claims 1 to 16 for the neutralization of endotoxins in individuals, preferably for the prophylaxis of an autoimmune reaction.

38. Use according to Anspmch 37, characterized in that component (ii) is selected from collagen, procollagen, tropocollagen, synthetic precursors, fragments or variants thereof.

39. Use according to spoke 37 or 38, characterized in that the use for the prophylaxis of connective tissue diseases or diseases of the musculoskeletal system, preferably arthrosis, soft tissue rheumatism, fibromyalgia, rheumatic diseases and rheumatic autoimmune reaction is carried out.