DE102004024674A1 - Means and methods for the diagnosis, prophylaxis and therapy of connective tissue diseases - Google Patents

Abstract

The present invention provides compositions comprising at least one substance capable of binding to a pathogen associated molecular pattern (PAMP) receptor; and at least one tri-helical fiber protein, fragment or variant thereof. According to a preferred embodiment, the trihelical fiber protein is procollagen, fragments or variants thereof. Also provided are pharmaceutical compositions for prophylaxis / therapy containing them and test kits for diagnosing connective tissue diseases containing them. Furthermore, specific binding partners and their use in pharmaceutical compositions for prophylaxis / therapy and in test kits for the diagnosis of connective tissue diseases are provided.

Description

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

The clinical laboratory diagnostics is an essential part of rheumatological diagnosis. Play an important role the so-called rheumatoid factors, where the term rheumatoid factor a historical term is. In these rheumatoid factors acts it is autoantibodies against gamma globulins, whereby the analysis of the respective rheumatoid factor IgG, IgM and IgAs is of varying clinical relevance. It are predominantly anti-isotypic immunoglobulins, i. to antibodies that directed against the constant regions (Fc) of immunoglobulin molecules are. RF IgM is, dependent on to the titer height relevant for Rheumatoid arthritis, SLE, Sjögren Syndrome, MCTD and other diseases. Accordingly are these parameters also not specific for rheumatoid diseases. The RF-IgA value is important in clinically conspicuous patients with negative RF IgM. RF-IgG plays a pathophysiological role in later Disease states.

A direct derivation of a rheumatic disease from these laboratory values can not, since these also in pregnancy, asthma, some inflammatory Diseases, herpes or flu can be increased. 6-8% of the healthy and 10% in old age have a rheumatoid factor. This is also the reason why Rheumatoid factors do not have the pathological significance, such as Anti-ds-DNA-AK in the diagnosis of SLE. Nevertheless, the evidence provides this autoantibody an essential diagnostic criterion. Moreover, they come also a prognostic significance, since rheumatoid factor-positive polyarthrids usually a more progressive course than rheumatoid factor negative Take forms of disease. Apparently the rheumatoid factors are playing Pathogenetic, especially in the expression of extracellular manifestations of rheumatic arthritis. Patients with also rheumatoid vasculitis In any case, they are mostly higher Rheumatoid factor titers on, as patients with only articular manifestations. In pulmonary manifestations, immune complexes of rheumatoid factors were also found and IgG molecules in the cell walls pulmonary vessels as well also be detected in the alveoli.

In Laboratory diagnostics for Rheumatic diseases are still inflammatory, such as CRP, the blood picture related to the assessment of disease activity and the determination of uric acid used with gout.

The pathogenetically triggering Causes of a rheumatic disease are with all previously known Methods not to capture.

Accordingly Many rheumatics are affected by these studies not recorded. On the other hand, many other sick people, especially autoimmune patients who are not rheumatic are ill.

Rheumatic Illnesses have an improved chance of treatment, though you early and be clearly diagnosed. A range of different treatment options for the treatment of rheumatoid arthritis are available. Not every arthritis extends equally fast and equally aggressive. Therapy is therefore individual to the individual patient. Of great importance for the success of the treatment is a targeted and especially an early treatment. The rheumatological Research has shown that in diseases with severe history the treatment within the first years particularly successful is and irreparable joint disorders can stop.

A Diagnostics, indications of a possible cause of the diseases supplies that u.U. through metaphylaxis / secondary prevention avoid or lessen is therefore of paramount importance. Here is the according to the claims of to be protected Substances and methods underlying pathogenetic concept.

For this reason, an early and clear diagnosis for a therapeutic success of special their interest. In earlier years, rheumatoid arthritis was often recognized only at an advanced stage, in which irreversible macroscopic damage of the structures had already occurred. It is now known that the disease progresses very fast within the first two years after the onset of the first symptoms. The sooner an effective therapy begins, the greater the chance of influencing the inflammatory process and stopping the destruction of cartilage and joints. Therefore, an early and clear diagnosis is particularly important.

Of the Construction of the collagenous tissue generally takes place in the following steps: Fiber-forming cells, such as fibrocytes, chondrocytes, osteocytes, reticular Cells and other specialized cells produce connective tissue Structures by exocytosis of trihelical, rope-like fiber proteins or their precursors.

Of the Synthesis process is described below using the example of chondrocytes and whose specific products are shown, but is under consideration the respective variabilities of the various known collagens to other fiber-forming cells.

• • Einem 15 bis 50 Aminosäuren (AS) langen N-terminalen Abschnitt, welcher später zur Bildung des N-terminalen Propeptids beiträgt. Dieser Abschnitt weist unter den verschiedenen Kollagenen eine höhere Variabilität auf und ist reich an den Aminosäuren Cystein, bezw. in der Cystin Form Cys-S-S-Cys bei Schleifen und Taschen bildenden Längsvernetzungen, insbesondere bei den einen großen Teil der Gesamtmasse aller Faserproteine ausmachenden Kollagenen I – III.
• • Im weiteren folgt ein durch hohe Regelmäßigkeit geprägter Abschnitt der repetetiv die Sequenz (Gly, X, Y)_n enthält und ca. 1050 AS umfaßt. Unter X wird fast ausschließlich die Aminosäure Prolin oder Leucin gefunden; im Zuge der weiteren intrazellulären Prozessierung werden diese fakultativ posttranslational in Hydroxprolin bezw. Hydroxylysin umgewandelt. Diese Hydroxyaminosäuren dienen im Folgenden der Quervernetzung der Kardele nach Verseilung und weiter der Vernetzung der verseilten Fasersegmente untereinander. Die mit Y gekennzeichnete Position ist durch gewebespezifische, variable Aminosäuren besetzt.
• • Anschließend folgt ein dem N-terminalen Ende analoges und vergleichbar langes Propeptid. Abgesehen davon, dass auch dieser Abschnitt insgesamt wieder einen hohen Anteil an Cystein/Cystin einhält, ist ein spezielles Motiv von zwei bezw. drei Cystein AS dicht nebeneinander (zum Beispiel die Sequenz GlyProCysCysGly) am (N-terminalen) Ende des C-Propeptids – also dicht an der Über gangsstelle zwischen Telopeptid und C-terminalen Propeptid – characteristisch und von großer Bedeutung: Hier beginnt die Vernetzung der drei AS Ketten.

In the Golgi apparatus of these cells, initially three amino acid chains (hereinafter referred to as Kardele) are synthesized independently of one another, which are characterized by the following characteristic sections:

• A 15 to 50 amino acids (AA) long N-terminal portion, which later contributes to the formation of the N-terminal propeptide. This section has a higher variability among the different collagens and is rich in the amino acids cysteine, bezw. in the cystine form Cys-SS-Cys in loop and pocket-forming longitudinal crosslinks, especially in the collagen I - III, which accounts for a large part of the total mass of all fiber proteins.
• In the following there follows a section marked by high regularity which repetitively contains the sequence (Gly, X, Y) _n and comprises approximately 1050 AA. Under X almost exclusively the amino acid proline or leucine is found; in the course of further intracellular processing these are optionally posttranslational in hydroxproline BEZW. Converted to hydroxylysine. In the following, these hydroxyamino acids serve to cross-link the cardels after stranding and further to crosslink the stranded fiber segments with one another. The Y position is occupied by tissue-specific, variable amino acids.
• • This is followed by a N-terminal end analogous and comparably long propeptide. Apart from the fact that this section also holds a high proportion of cysteine / cystine again, a special motive of two bezw. three cysteine AS close to each other (for example, the sequence GlyProCysCysGly) at the (N-terminal) end of the C-propeptide - close to the junction between telopeptide and C-terminal propeptide - characteristic and of great importance: Here begins the networking of the three AS chains.

To same direction parallel alignment of the three AS chains (where the N-terminal ends are in common on one side and as well the C-terminal ends together are on the other side) begins At this point, the stranding of the three individual Kardele by the formation of covalent Cys-S-S-Cys bonds of Kardele with each other, with double or triple cross-linking. The formation and localization of this "cystine node" called structure is from the primary structure the AS chains of the individual Kardele exactly specified.

Of the Cystine knot is the evolutionary one solution for the Task, the relative mobility of the three Kardele in the longitudinal axis to fix each other. As a result, the three Kardele wrap around each other. This stranding proceeds spontaneously from the cystine knot proceeding towards the N-terminal end (and to a lesser extent also in the C-terminal propeptide). It is only possible through the uniform repetitiveness of Gly-X-Y Groups, thus in a rigid, each offset grid of the Kardele come to each other. This grid synchronization runs through the whole middle section of rope, which thus forms the telopeptide, right down to the N-terminal section, which initially still is stranded, but then because of the resolution of the repetition as unseparated End represents. Even if the three arms of - now called N-terminal propeptide - Section remain free, so is the spatial Structure of the amino acids on these arms with their origin Bond epitopes fixed to each other.

The cystine node realized on the C-terminal propeptide thus has a long-term effect on the spatial structure of the opposite end, the free cardiac arms of the N-terminal propeptide. This through The crosstalk enforced grid synchronization of the free ends of the propeptides is retained, even if the propeptides are cut off during processing from procollagen to tropocollagen, at least as long as the respective endopeptidases (metalloproteinases) intersect at the physiologically correct location.

Consequently is not just the exact tertiary structure of the C-terminal propeptide on which the cysteine node is located is defined, but this applies equally to the N-terminal propeptide, even if at this fragment the raster synchronization triggering structure - just the Cysteine node - not is more detectable.

To Providing the intracellular, Stranded Prokollagen - fiber pieces with a length of about 300 nm, these are transported to the cell surface and there further processed.

To the Build up of collagen tissue using matrix metalloproteinase enzymes (MMP), e.g. MMP3, mentioned above, separated as Propeptide ends of Prokollagenfasern and the respective "cut" fiber piece as Tropokollagen delivered into the matrix. The fiber piece conditioned in this way becomes furthermore specifically incorporated into the respective collagenous tissue (linear, grid-shaped, net-like, felt-like etc).

inflammatory or degenerative diseases of fiber proteins Connective tissue structures, whether they represent their own organs, like ribbons, Tendons, joints, fascia, etc., or that they are in parenchymatous organs as framework structures serve, are divided into different groups.

at The rheumatic diseases in the narrower sense, it comes after one initially clinically common little noticeable Phase for the formation of autoantibodies against the connective tissue structures yourself - or with these directly associated tissues. It is followed by inflammatory drawers, which promote the further formation of autoantibodies.

at the second group, rheumatoid diseases, lack the typical autoimmune disease Autoantibodies; primary inflammatory and master painful symptoms with no apparent background the clinic. The primary Cause of these diseases is unknown; they can be recurrent in the o.g. Full picture of a rheumatic disease, so that in The literature is debatable, though the latter may only be diseases Represent precursors of the former. It is not known what additional Complications must occur Thus, from a rheumatoid disease, the full picture of a Rheumatoid autoimmune disease is definitely developing a longer one and progressive course as an unfavorable condition. It is discussed that it by the persistent inflammation as a result of dysfunction of the immune system last to education of autoantibodies against own, not pathologically changed structures comes, because the antibodies expressed over time have their specific selectivity for differentiation lose between yourself and not-yourself.

on the other hand there is one as primary Osteoarthritis designated group of diseases of connective tissue structures, especially the joints and tendons that are neither inflammatory bouts still start by the appearance of an autoimmune disease typical autoantibodies Marked are. For this Disease is a long-term demise of the fiber-forming Cells, e.g. the chondrocytes secured, without their cause is known.

These Diseases become the cause the wear (by unphysiological burden), but it can be assumed that less the excessive wear structures, but rather a reduced one regenerative capacity prevails. The clinical picture is dominated by a slow, over years / decades ongoing unexplained Shrinkage of connective tissue functional tissues, especially cartilage.

Of the The present invention therefore an object of the invention, new means and methods for the diagnosis, prophylaxis and therapy of connective tissue diseases and diseases of the musculoskeletal system.

• i) wenigstens eine zur Bindung an einen PAMP-Rezeptor fähige Substanz; und
• ii) wenigstens ein trihelikales Faserprotein, ein Fragment oder eine Variante davon.

The object underlying the present invention is achieved by providing a composition comprising:

• i) at least one substance capable of binding to a PAMP receptor; and
• ii) at least one tri-helical fiber protein, fragment or variant thereof.

Further solutions result from the subject of the claims.

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

PAMP receptors include receptors of the innate immune system responsible for parasites, specifically recognize microorganisms, typical molecular structures. The pathogen associated molecular patterns (= PAMP) are characteristic Structures of microorganisms that are high in genetic and phenotypic stability within larger groups of microorganisms (= fungi, bacteria and protozoa) show. This points to a for the affected microorganisms (= MO) indispensable structural unit which are not changed without serious evolutionary disadvantage can. These specific molecular patterns prove to be just that as for the host organism, which he himself is not in his structural inventory relies on these features, and therefore these molecular Patterns as a safe recognition criterion for foreign body MO are.

Of the Expression "pathogen-associated molecular patterns (PAMP) receptor "becomes synonymous with the term" pattern Recognition Receptor (PRR) receptor. "To the overview will be on the article by Medzhitov and Janeway, Current Opinion Immunol (1997), 9, 4-9. According to the invention include PAMP receptors Toll-like receptors. Furthermore, the term non-toll-like, cell-membrane-bound PAMP receptors such as phagocytic Receptors. Here are exemplary the scavenger receptors, in particular SR-A and MARCO, the macrophage mannose receptor, the B-glucan receptor and peptidoglycan-recognition proteins (PGRP). Further includes the term intracellular PAMP receptors such as protein kinase R, oligoadenylate synthase and molecules the NOD family. Furthermore the term includes soluble PAMP receptors pentraxins such as e.g. CRP, Serum Amyloid A, Collectine, Lipid transferases such as the lipid-binding protein (LBP) and soluble variants of the above mentioned PGRP mentioned. Preferably, the PAMP receptors are of human origin. The amino acid sequences are from the generally accessible Sequence databases such as Genbank and SwissProt available.

It are cell-based and in body fluids dissolved PAMP receptors. Permanent cell Receptors serve either to signal transduction into the cell, e.g. CD-14 receptor, or have, no known intracellular signal terminal for triggering a Reaction. The same applies to soluble Forms of PAMP ligands that are not in direct contact with cells to have. The function of the receptors without intracellular signal transduction connection (cellular or soluble) consists in the binding of vague MO or fragments of NOT A WORD; accordingly, they are referred to as scavenger receptors (scavengers).

"Scavenger receptor" (= SR) included here SR-A including SR-A I and SR-A II, and MARCO. Further, SR includes SR-A-like ones SR. These include SR-CLI (SR with C-type lectin I), SR-CLII, CL-P1 (= Colectin from placenta receptor I), LOX-I (= lectin-like oxidized LDL receptor I), dSR-CI (Drosophila SR-CI). Preferably, the Scavenger receptor SR-A, especially SR-AI. Furthermore, in relation to the Sca venger receptors on the review article Peiser et al., in Current Opinion in Immunology 2002, 14: 123-128 taken.

"To bond to a PAMP receptor capable Substances " according to the invention all PAMPs known in the art. For overview see article from Medzhitov and Janeway, Current Opinion Immunol. (1997), 9, 4-9. PAMPs include bacterial and mycobacterial PAMPs and fungi PAMPs. Belong to the PAMPs e.g. Lipopolysaccharides (= LPS, also called endotoxins), lipoteichoic acid (= LTA) and their variants, wall-specific proteoglycans (= PGN), for microorganisms specific non-methylated DNA sections (CpG), lipoproteins and N-formylated Peptides such as F-Met-Leu-Phe and bacterial proteins. From mushrooms PAMPs are e.g. Fungus-specific peptidoglycans. Of mycobacteria derived PAMP are mycolic acid and mycolic acid ester.

The Binding of lipoteichoic acid, Lipopolysaccharide (LPS), bacterial DNA, bacterial cell fragments SR, which is a PAMP receptor, is from Peiser et al., known above.

Other substances capable of binding to a PAMP receptor can be obtained by simply performing a binding assay, as described, for example, in Dunne et al., PNAS 91 (1994), 1863-1867 wherein for the binding assay the PAMP receptor is preferably SR-AI.

Of the Term "trihelical Fiber protein " collagen according to the invention, Procollagen, tropocollagen, elastin, fibrillin, fibronectin, scavenger receptors and other collagenous components. Further, the term includes the synthesis precursors of the aforementioned. As is well known and As described above, collagens are initially called procollagen alpha chains synthesized. These become after hydroxylation of L-proline and L-lysine residues as well as after glycosylation in the endoplasmic reticulum or in the Golgi apparatus, where they meet in three chains, in the extracellular Room excreted. There are so-called propeptides from the ends of the chains or those that have been separated at a non-typical interface, split off, and there is the tropocollagen, which turns into fibrils assembles. By oxidation of amino groups of the L-lysine side chains to aldehyde groups and their aldol and aldimine formation with aldehyde or amino groups adjacent chains are linked together. (Networking).

Of the Term "fragments" means, provided they are proteins, preferably fragments with a minimum length of 10, more preferably 20 amino acids. If it concerns nucleic acids, have the fragments at least 20 nucleotides, preferably 50 nucleotides.

Of the Expression "Variants" basically includes all Modifications of a given sequence, with muteins preferred are at the amino acid level by addition, substitution, deletion, insertion or inversion at least one amino acid differ from the given peptide sequence. Especially preferred the mutation comprises 1 to 5 amino acids, especially 1 to 3 Amino acids.

Of the The term "conjugates" includes di-, oligo-, and polymerizations wherein both homo- and hetero di-, oligo-, and polymerizations. The coupling can here to a carrier molecule, such as Polyethylene glycol or other known in the art carrier molecules. Further the term conjugates also includes naturally occurring conjugates, such as post-translational modifications, such as Hydroxylations of the side chain comprises.

lipopolysaccharide (LPS) contain a very strictly conserved amino sugar 2-X-phosphate-lipid complex the lipid (A) is called, and the actual membrane part is. In the endotoxin molecule follows this - the Bacterial membrane constituting lipid (A) a substantially from specific sugars with sometimes other phosphates as the core antigen (Core-A) designated area. The nucleus antigen is followed by the Surface antigen (Surface-A), very variable and very different long sugar chains, ranging from bacterial species, but also within of a species can vary greatly from tribe to tribe. According to the invention both the mono- and the bi-phosphorylated form of the lipid (A) includes.

The present invention is based on the following surprising findings of the inventors:
It has been observed in numerous cases that people who are in buildings, in which there are certain moisture damage associated with a microbial attack, conspicuously often suffer from pain in the joints, so-called rheumatoid complaints. As a rule, these complaints are related to the time spent in the building, that is to say, in the absence of symptoms, the symptoms recur, and when they reside in the respective building, they reappear in a relatively short time. The more often those affected are exposed, the quicker the symptoms of exposure come and the slower they will go off after elimination of the exposure.

more accurate Investigations showed that initially in the initial stage usually not the joints in the narrower anatomical Senses ached, but joint-soft tissue structures. The appearance The pain was strictly correlated with a previous one biomechanical load. The observations showed that it was accurate the highest places cross-sectional stresses that affect the patient at next Days hurt.

at the investigation of numerous buildings has been proven that the connective tissue diseases with a characteristic microbial Infestation patterns in building materials correlated.

In vitro experiments have shown that bacterial endotoxins (= LPS), extracted from moist building materials in the area of moisture damage, destroy human cartilage tissue. This destruction destroys chondrocytes and the extracellular matrix and a large increase in the levels of MMP enzymes in tissue culture is detectable. It was also detectable that the effect of endotoxins on the tissue shows a clear dose-response relationship, ie the higher the concentration of endotoxins, the greater the destruction of the chondrocytes responsible for collagen synthesis.

Consequently For the first time, LPS, or endotoxins as Noxes, i. as pathogenic Cause for Connective tissue diseases are noted by patients who are in buildings with microbial attack. It is therefore for the first time a distinction between a microbial-related connective tissue disease and a Non-microbial connective tissue disease possible.

diagnostic Approach of the proceedings in question is the proof of a reduced repair and regeneration capacity for fibrillar structures by an exogenous Noxe (PAMP, definition below).

The role of PAMP receptors, particularly scavenger receptors, is, inter alia, the binding of endotoxins for removal from the bloodstream. The inventors of the present invention have also found that the scavenger receptor LPS-binding epitopes have high homology to analogous structures at the ends of the procollagen molecules. The spatial structures of SR-AI and SR-AII and procollagen are in the 1 and 2 shown schematically. As a result, in particular the N- and / or C-terminal propeptides in different fragmentation lengths, possibly including a part or the entire length of the telopeptide or modifications of these structures, offer themselves as near-natural and therefore low-antigenic ligands.

at an overload of the systemic purification system with PAMPs provided by scavenger receptors is such. in septic infections, antibiotic therapy with bactericidal antibiotics, or as in the present case by exogenous endotoxin load, there is a transport of PAMPs, especially LPS, in the circulation and these can to the next bind comparatively avide structure. This is the freshly formed, procollagen not yet processed into tropocollagen on stimulated fiber protein-forming cells, e.g. Chondrocytes.

The Inventors of the present invention were able to use the example of the growth-stimulated Chondrocytes show complete blockage of collagen synthesis by endotoxin through excessive activation the involved in the processing metalloproteinases to a Damage to existing fiber structures and ultimately to one Death of the fiber-forming cells leads. It is to be assumed that the failed processing to procollagen causes the cell to in the large Scope to continue to provide procollagen on the surface and metalloproteinases available for processing to deliver. An excessive, not more only local-acting synthesis of MMP's leads to a large-scale destruction of Fibers and cells; at extensive destruction in the consequence to the clinical apparent inflammation and pain.

1 shows the spatial structures of SR-AI and SR-AII.

2 shows the spatial structure of procollagen.

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

4 (a) bis (b) show healthy chondrocytes.

• i) wenigstens eine zur Bindung an einen PAMP-Rezeptor fähige Substanz; und
• ii) wenigstens ein trihelikales Faserprotein, ein Fragment oder eine Variante davon.

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 tri-helical fiber protein, fragment or variant thereof.

Preferably is the substance capable of binding to a PAMP receptor a bacterial, mycobacterial or fungal PAMP, preferably the PAMP a lipopolysaccharide (= LPS, also called endotoxin), lipoteichoic acid (= LTA) and their variants, wall-specific proteoglycans (= PGN), for microorganisms specific non-methylated DNA sections (CpG), lipoproteins and N-formylated peptides such as F-Met-Leu-Phe and bacterial proteins. Furthermore, it is preferred that the fungi-derived PAMP is a fungus-specific Peptidoglycan is. Further preferred is the PAMP of mycobacteria derived mycolic acid or mycolic acid ester. Particularly preferred is the PAMP lipopolysaccharide.

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

Especially preferred is the substance capable of binding to a PAMP receptor a lipolysaccharide (LPS), fragments or variants thereof, wherein the fragments or variants a phosphorylated N-acetylglucosamine dimer preferably contain a complete lipid A complex. It is known that lipid (A) over this structural element binds to the CD14 receptor LBP complex mediated by LPS to the macrophage receptor. Preferably lipopolysaccharide lipopolysaccharide of genera e.g. Salmonella, Shigella, E. coli, Pseudomonas, Neisseria, Klebsiella and Haemophilus.

Especially it is further preferred that the microorganisms derived PAMPs from bacteria of the Order Actinomycetales and fungi of the genera Aspergillus, Botrytis, Cladosporium, Eurotium, Penicillium, Wallemia, Chaetomium, Mucor, or Scopulariopsis.

According to one preferred embodiment is the trihelical fiber protein (component (ii) of the composition) selected from collagen, procollagen, tropocollagen, elastin, fibrillin, fibronectin, Scavenger receptors or other collagenous components, synthesis precursors, Fragments, variants or conjugates of the above components.

The Collagen according to the invention comprises the types I to XIV, with the types I, II, III, V and IX are 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 is different from the similar ones Collagen through the higher Content of valine and leucine, due to the lower arginine and hydroxyproline content and the absence of lysine residues. The aforementioned proteins are preferably of human origin, but are also animal Proteins of the invention also included. Preferably, this is tri-helical Fiber protein procollagen, fragments or variants thereof. The fragments or variants of procollagen preferably have at least 50%, more preferably at least 80%, especially at least 90% of the binding ability from human procollagen to LPS from E. coli. The binding ability can be carried out here with a well-known binding assay.

The Variants of procollagen are preferably at least 60% homologous, preferably at least 80%, more preferably at least 90%, especially at least 95% homologous at the amino acid level to human procollagen. The determination of homology can hereby using the Professional known standard Progarammen (e.g., FASTA, BLAST) using standard settings respectively.

The Fragment of procollagen preferably has at least 10, especially preferably 20, in particular 50 amino acids of procollagen. The Variant of procollagen is preferably a mutein that is characterized by the addition, substitution, deletion, insertion or inversion at least an amino acid different from procollagen. Preferably, the mutations include 1 to 5 amino acids, in particular 1 to 3 amino acids. Particularly preferably, the substitution is a conservative substitution, at the one amino acid by another amino acid exchanged, which belongs to the same physicochemical group.

Generally can the fragments and variants by chemical or recombinant methods getting produced.

procollagen has a stranding of the three peptide chains about 10 to 30 amino acid residues in front of the respective end. The place of transition from the stranded Structure to the free poor is of particular importance. At this transition point is a very distinctive amino acid motif for covalent Responsible bonds. Due to the double or triple installation the amino acid cysteine at precisely defined intervals a structure is formed on each of the three peptide chains, where each chain is rigid with each chain via Cys-S-S-Cys is connected to each other. This coordination structure is made of cysteine building blocks Called cysteine node.

Preferably therefore includes the trihelical fiber protein, the fragment or the Variant thereof, one suitable for forming a cysteine knot Sequence, so binding sites for the binding of the substance that binds to a PAMP receptor is capable ready to deliver. Preference is given to the formation of a Cysteine-Knotens suitable sequence the sequence GlyProCysCysGly (SEQ ID NO: 1).

For the im Under the invention discussing variants of the procollagen, of its derivatives and fission products, it should be noted that evolution of the cysteine knot is just one of the conceivable solutions to the task of grid synchronization represents. For biosynthetically relevant sections of the procollagen and Variants of its propeptides it should be noted that the task of grid synchronization, the for the preservation of the subsequently used affinity of the propeptides for PAMPs is essential, also by other space-coordinating measures between the three Kardelen the AS strands to solve is.

The applies to especially for Use of the binding ability an N-terminal fragment of the procollagen. In addition to the obvious Variant of total or partial truncation of telopeptidic Section in a biotechnologically expressed product, thereby The cystine knot could be shifted further N-terminally, also differently (as by covalent Cys-S-S-Cys) formed bonds between the AS of the three Kardele be used as long as the spatial coordination of the free, together the binding epitope for PAMP's forming Ends of the three Kardele is preserved. This technique, without use of the natural cysteine knot or a functional equivalent, is also on the C-terminal Page applicable.

According to one preferred embodiment therefore has the fragment or variant of the tri-helical fiber protein the N- and / or C-terminal end of a tri-helical fiber protein and optionally a part of the telomer.

According to one particularly preferred embodiment For example, 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 from that.

According to one preferred embodiment of the present invention the composition also comprises chondrocytes, fibrocytes, osteocytes, reticulo-endothelial cells, endothelial cells and / or components from that. The aforementioned cells are for the production of collagen capable. Chondrocytes and their constituents are preferred here. preferred Ingredients here are the cell membrane or fragments thereof. Components of the cells can According to the invention, the nucleus, Golgi apparatus, endoplasmic reticulum, and mitochondria.

In the composition of the invention can components (i) and (ii) are non-covalently or covalently linked together be. A covalent bond of components (i) and (ii) of the composition If this is not already available, it can be crosslinked by known methods, e.g. achieved by carbodiimides become.

According to one Another aspect of the present invention is a method for Preparation of the composition of the invention wherein the method comprises contacting the component (i) with the component (ii) and optionally the cleaning and isolation of the composition. Component (i) as well as the component (ii) can both naturally occurring form, as well as synthetically produced become. Synthetic processes for this purpose are known to the person skilled in the art. The Contacting the components can in this case in liquid form done or one of the two components can in this case to a carrier be bound.

method to purify or isolate the composition herein Centrifugation, gradient fractionation, chromatographic methods, one- or two-dimensional gel electrophoresis.

• a) Bereitstellen eines eine Komponente (i) enthaltenden Bakterien- oder Pilz-Eluates;
• b) Bereitstellen einer wachstumsangeregten Chondrozytenkultur;
• c) Inkontaktbringen des Bakterien- oder Pilz-Eluates mit der Chondrozytenkultur; und
• d) gegebenenfalls Aufreinigen der Zusammensetzung.

According to a preferred embodiment of the method, the method is carried out as an in vitro method comprising the steps:

• a) providing a bacterial or fungal eluate containing a component (i);
• b) providing a growth-enhanced chondrocyte culture;
• c) contacting the bacterial or fungal eluate with the chondrocyte culture; and
• d) optionally purifying the composition.

The fungal eluate is preferably an eluate derived from Aspergillus, Botrytis, Cladosporium, Eurotium, Penicillium, Wallemia, Chaetomium, Mucor or Scopulariopsis. The bacterial eluate is preferably an eluate derived from Actinomycetales, Salmonella, Shigella, E. coli, Pseudomonas, Neisseria, Klebsiella, and Haemophilus. A growth-enhanced chondrocyte culture is preferably as described by Shakibaei et al., Biochem. J. (1999), 342, 615-623. Here, the cartilage tissue is mechanically minced, incubated and purified 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 to Eagle (50%: 50%). 10% fetal calf serum, 25 μg / ml ascorbic acid and 50 μg / ml gentamicin are added to this medium.

As stated above, For example, purification may be by methods known to those skilled in the art, chromatographic Procedures, centrifugation, ultrafiltration and gel electrophoresis include. A preferred embodiment for preparation, component (i) is attached to a column matrix and subsequently a detergent-lysed chondrocyte lysate via them so prepared Column too give. After incubation of the chondrocyte lysate with the coupled Component (i) may subsequently, after washing the column Composition eluted. The elution conditions are here preferably so to choose that the composition does not denature.

According to one Another aspect of the present invention is a pharmaceutical Composition provided, the composition described above and at least one pharmacologically acceptable carrier. Pharmacologically acceptable carriers are known to those skilled in the field of galenics. Preferably, the pharmaceutical compositions suitable as a vaccine to an immune response the person against at least one component of the composition to prevent prophylaxis against the formation of an autoimmune reaction. According to one preferred embodiment For example, the vaccine formulation may also be useful in the art Carrier molecules contain immunogenicity to increase the composition. The vaccine formulation may preferably be administered subcutaneously, intravenously or intramuscularly become. The pharmaceutical composition may generally be in liquid form or in the form of an aerosol with suitable use of aerosol-stabilizing Compounds are present.

Consequently is the use according to the invention the pharmaceutical composition, in particular as a vaccine for Prophylaxis and / or treatment of connective tissue diseases provided. Connective tissue diseases according to the invention include diseases of the musculoskeletal system, but also known to others connective tissue mediated diseases such as vasculitis, Glomerulonephritides, endocarditis (with and without valve involvement), Dermatitis and dermatomyositis. Preferably, the connective tissue diseases selected from arthrosis, soft tissue rheumatism, fibromyalgia, rheumatic diseases or rheumatic autoimmune reaction.

According to one another embodiment The present invention is a test kit for the diagnosis of Connective tissue diseases providing the composition of the invention contains. The test kit is particularly suitable for detecting antibodies that the body in response to, among natural Conditions formed composition in particular a complex produced from endotoxin and procollagen. The antibody titer against the under natural Conditions formed composition is an indicator here for this, whether the discomfort of the connective tissue and the musculoskeletal system the exposure to the substance capable of binding to a PAMP receptor, especially endotoxins. A comparison with the titer on autoantibodies to normal collagen shows if a precursor of incipient rheumatic disease or the rheumatic disease is already fully developed. The former would indicated by a significant titer on the naturally formed Composition, where appropriate, an additional weak titer endogenous Collagen is present. In the further case, a titer is natural formed composition and a high titer on the body's own To determine collagen. The test kits according to the invention are accordingly detectable an antibody suitable. The test kit may further include those known in the art Buffer substances that are required for use of the test kit in Determination and diagnostic methods are suitable. Preferably is the body fluid for examination by the test kit of blood or blood products including Serum selected. For identification purposes advantageous methods here are immunoassays, ELISA, RIA, membrane-bound Test strips, receptor binding tests or biosensory determinations, their implementation the expert is known. For example, in ELISA detection the composition is immobilized on a microtiter plate. In the test, the specific antibodies bind and then go through correspondingly marked autoantibodies converted into a signal by known methods.

The present invention further provides reagents that bind to, preferably are specific to, the composition of the invention. An example of such specific reagents are antibodies, antibody fragments, eg Fv, F (ab) or F (ab) ₂ fragments or antibody derivatives. The antibodies, antibody fragments, eg Fv, F (ab) or F (ab) ₂ fragments or antibody derivatives may be of monoclonal or polyclonal origin. In general, specific antibodies are obtainable in which experimental animals, such as mice or rabbits, are coupled with the composition according to the invention or at least component (i) or component (ii), which are preferably coupled to suitable high molecular weight carrier molecules are to be immunized. Immunization can be facilitated by the addition of suitable adjuvants known in the art. Monoclonal antibodies are commonly available by fusing spleen cells taken from an immunized mouse with tumor cells and selecting the resulting hybridomas. Those hybridomas which efficiently secrete specific antibodies can be determined by screening the supernatant. Alternatively, antibodies can be recombinantly produced; In the production of recombinant antibodies, the mRNA is isolated from hybridoma cells or B lymphocytes, which acts as a basis for the synthesis of the corresponding cDNA and amplified by PCR. After ligation into a suitable vector and introduction of a suitable host cell culture, the antibody can be recovered from the cell culture supernatants or the cell lysates. Recombinant antibodies allow for "humanization" of the antibody and are thus less immunogenic, and the methods of this are well known in the art Antibody derivatives include conjugates of antibodies with markers suitable for detection, for example, for use in scintigraphy.

The The present invention further provides a composition containing a first binding partner which is attached to component (i) binds, preferably for this is specific, and / or a second binding partner, the binds to component (ii), preferably specific for them. The Composition is preferably a pharmaceutical composition. The first and second binding partner is preferably an antibody. One another example of the embodiment, that component (i) is an endotoxin, preferably LPS, is the binding partner LAL. Other binding partners of endotoxin, preferably LPS, are albumin, transferrin, HDL, LDL, apolipoproteins, C-reactive Protein, CR1, CR3, CD14, scavenger receptors, CD18, gangliosides, lectin-like Receptors, polysaccharide receptors, bactericidal / permeability increasing protein (BPI), cationic antimicrobial protein (CAP), and LPS-binding protein (LBP), as well as soluble variants of the above mentioned factors. As another binding partner for component (i) becomes component according to the invention (ii) indicated. Component (ii) is preferably procollagen, Tropocollagen, collagen, synthesis precursors, fragments or variants from that. In particular, the component procollagen, fragments and Variants of it. The fragments and variants of procollagen are here as defined above. The pharmaceutical composition can in addition to the first binding partner and / or second binding partner a pharmacologically acceptable carrier contain. Suitable carriers are known in the art. Preferred are the pharmaceutical Compositions for intravenous, subcutaneous or intramuscular Suitable administration. As stated above, that provides for bonding to a PAMP receptor capable Substance, especially endotoxin, the Noxe for the death of collagen-producing Chondrocytes, wherein the substance on the trihelical fiber protein binds to the chondrocytes. The pharmaceutical composition Therefore, on the one hand directly to the binding to a PAMP receptor able Neutralize substance before it binds to the chondrocytes. On the other hand, the pharmaceutical composition may already have arisen eliminate toxic complexes from the bloodstream.

suitable Concentrations of binding partner in the pharmaceutical composition may be preferred here in the range of 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. Preferably, the connective tissue disease Arthrosis, soft tissue rheumatism, fibromyalgia or rheumatic disease.

According to the invention is further a test kit for the diagnosis of connective tissue diseases containing the Antibodies according to the invention and / or the above composition is provided. The test kit may further contain buffers known in the art, the use of the test kit in diagnostic and diagnostic Methods are suitable. The test kit can be used for Determination of the natural formed composition, in particular a complex containing endotoxin and procollagen and / or the body fluid for binding capable of a PAMP receptor Substance, in particular an endotoxin. Preferably, the body fluid selected from blood or blood products including serum. Of the Detection may be performed using immunoassays, ELISA, RIA, membrane-bound Test strips, receptor binding tests or biosensory determinations, their accomplishments the person skilled in the art are known to be executed.

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

Further is the use according to the invention component (ii) for neutralizing endotoxins such as LPS for the prophylaxis of an autoimmune reaction. The administration the component (ii) leads for binding and thus for neutralization of the in the body fluid the patient's endotoxins. Component (ii) is preferred selected from procollagen, tropocollagen, collagen, synthesis precursors, fragments or variants thereof, more preferably procollagen, fragments or variants. The fragments or variants are here as above Are defined.

It is intended, with the following examples, the invention to explain but in no way limit these. The skilled person is due the description of the examples further embodiments accessible, which are also included.

Examples

Example 1:

determination the effective Noxe in bacterial eluates from microbial populated Building material.

It From the basement of a residential building, microbially populated building material became taken. For eluate preparation, 150 g of this material and 60 ml of PBS and stirred for 30 minutes at 200 rev / min. After that the sample was turned off for 5 minutes to allow the solid components could sediment. Then the liquid part was centrifuged (at 4000 g) and then with a 0.2 μm Cellulose acetate filter filtered. The liquid obtained in this way was by cultivation on nutrient media on sterility tested. This sterile eluate was diluted with a 1: 100 chondrocyte cultures added.

The primary chondrocyte cultures used were prepared according to Shakibaei et al., Biochem. J. (1999), 342, 615-623; Shakibaei et al., J. Biol. Chem. 2001 ), 276, 13289-13294.

cartilage was mechanically minced, incubated and purified in Ham's F-12 medium. The cartilaginous tissue pieces were subsequently with 1% pronase (2 hours at 37 ° C) and then dissolved with 0.2% collagenase (4 hours at 37 ° C). This is where the extracellular matrix becomes resolved while the chondrocytes were not attacked. Both enzymes were in Hank's solution with 5% (v / v) fetal calf serum solved.

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 serum, 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: 2 × 10 ^6/10 ul) were dissolved in alginate beads (Shakibaei and de Souza, Cell Biol Int (1997), 21, 75-86.). Cultured. After two weeks, some cells had grown out of the alginate beads continuously and adhered to the petri dishes. After three days, when the cells were grown confluent, the cells of the first passage were removed with 0.05% (v / v) trypsin / 1.0 mM EDTA and reseeded in culture flasks. Every three days, the confluent monolayer was passaged. The cells from the monolayer passage were introduced into high density cultures. The high density cultures were prepared as detailed by Zimmerman 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 × 10 ⁶ cells / 10 μl) were applied to a membrane filter (pore diameter 0.2 μm, Sartorius, Göttin gen, Germany) on the surface of a stainless steel grid in the intermediate region between medium and air in a petri dish. Every three days, the medium was changed. 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 tissue was examined microscopically. For this purpose, cuts were made. 3 (a) bis (c) show the destruction of the chondrocytes after addition of the eluate. 4 (a) and (b) show healthy chondocytes for comparison. It was clear that both the extracellular matrix and the chondrocytes themselves were mostly resolved. Further investigations by immunofluorescence microscopy and Western Blot showed that at the same time as the tissue destruction a significant increase of enzymes of the group of matrix metalloproteinases is associated.

Further, 1 × 1 mg / ml, 1 × 10 mg / ml and 1 × 100 mg / ml polymyxin B (Sigma, P1004, polymyxin B sulfate salt,> 6,000 USP units / mg, respectively) were added to the sample eluate. Polymyxin B specifically binds Endoto xine. Various amounts of polymyxin B were added until finally only very small amounts of endotoxins were detectable by LAL tests (LAL-Test, Pyrogent, BioWhittaker, Inc., MD, USA). Then the chondrocyte exposure experiments were repeated. It was found that in correlation to the LPS concentration in the eluate the chondrocytes and the extracellular matrix were damaged. 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 contrary to the composition according to the invention are directed to diagnostics.

The Composition according to the invention can be used as a marker for medical diagnosis of connective tissue diseases be applied to humans and animals. He can in this capacity also be used for therapy control. Also hygienic examinations for example, the living and working environment with regard to bacterial Colonization or mold colonization and in this context the Contamination with endotoxins and, if necessary, measures For their elimination play in the context of therapy concepts a important role.

Of the Use of the described antigens as markers is preferably carried out in the usual diagnostic procedures, such as immunoassays, blotting procedures, biosensory methods or comparable methods. Thereby, that the antigenic marker molecule or its modifications (for example, modifications for improvement the coating efficiency) in corresponding binding tests, for example Immunoassays were used. It can be directly adsorptive to the surfaces suitable highly adsorbent microtiter plates are bound. Following appropriate blocking and coating protection procedures, as they are well known in the art takes place the assay, being dilute Sera or plasma samples are used in the assay. Autoantibodies that in specifically affected rheumatoid patients in appropriate Titers are present, bind to the immobilized antigen. The Signal generation is accomplished by methods known in the art labeled with enzyme (common Horseradish peroxidase), anti-human IgG (possibly also IgM or IgA) antibodies Rabbit, sheep or goat. The final signal generation takes place over the enzymatic formation of a chromogen instead. A frequently used one Substrate is the tetramethylbenzidine substrate. The evaluation takes place at 450 nm an ELISA photometer after stopping the reaction with diluted Sulfuric acid.

to Calibration will become specific and high titre autoantibodies Patient's serum (specific patient) or a pool used by several patient sera. The concentration or the Titers of the specific autoantibodies in the individual calibration serums should be adjusted by making suitable dilutions such that: a suitable standard curve is achieved.

The Setting the cut-off value to discriminate between normal and pathological values is done by measuring and evaluating Sera or plasma pools of specifically ill patients and control subjects. The cut-off value will be based on this Measurements calculated by statistical evaluation. The described diagnostic procedure is used in rheumatic diseases associated with incorporated endotoxins. Increased Indicate titers of specific autoantibodies rheumatic diseases that are common by previously used laboratory tests, such as the Measurement of the known rheumatoid factor, can not be detected. The Diagnostics therefore detects more specifically ill patients and improved and supplemented solely by the established laboratory diagnostics. Furthermore, after successful positive diagnosis a hygienic analysis of the living and working environment possible, the elimination triggering Factors, in particular endotoxins or the causes of existence eliminates these endotoxins.

Of the Test can be used in this context for therapy control become. On the one hand to control a treatment, on the other hand to control the discussed Eliminate the possible Causes, ie endotoxin pollution of the environment.

Example 3:

applications of detection methods of the marker and its modifications for diagnosis.

In addition to the autoantibodies, the composition itself, in particular the endotoxin, can also be used or the endotoxin-procollagen complex can be detected in the serum. However, the detection of autoantibodies is preferred.

The Antigen itself can be in a usual Method as demonstrated in an immunoassay. About it will the antigen is preferably detected in a sandwich ELISA. basis are two, preferably different, antibodies produced by immunization with the composition, preferably to endotoxin-procollagen complex by method, which are known in the art, are obtained. In one ELISA, which can be used as a preferred method, becomes one of the two antibodies used in a preferred method of coating, while the other is coupled with a marker enzyme. The coupling methods are known from the prior art.

This Method may alternatively or additionally to autoantibody determination be used. Likewise, as in Example 3, are also veterinary Examinations possible. Also, let relevant structures or antigens from cell cultures prove with the invention described here.

SEQUENCE LISTING

Claims (39)

Composition comprising: (i) at least a linked to a pathogen associated molecular patterns (PAMP) receptor-capable substance; and (ii) at least one tri-helical fiber protein, a fragment or a variant of it. Composition according to Claim 1, characterized that the substance capable of binding to a PAMP receptor is a bacterial, mycobacterial or fungal PAMP. A 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, microorganism-specific, non-methylated DNA segments, lipoproteins, N -formylated peptides, fungal-specific peptidoglycans, mycolic acid or mycolic acid esters, fragments, variants or conjugates thereof, wherein the fragments, variants or conjugates are there of, at least 50% of the binding capacity of LPS from E. coli to the scavenger receptor SR-AI. A composition according to any one of claims 1 to 3, characterized in that for binding to a PAMP receptor able Substance a lipopolysaccharide (LPS), a fragment or a variant of which, wherein the fragment or variant is a phosphorylated N-acetylglucosamine dimer, preferably containing a lipid A complex. A composition according to any one of claims 2 to 4, characterized in that the PAMP is the lipopolysaccharide (LPS) from the bacteria Salmonella, Shigella, E. coli, Pseudomonas, Neisseria, Klebsiella or Haemophilus is. A composition according to any one of claims 2 to 4, characterized in that the PAMP from Actinomycetales, Aspergillus, Botrytis, Cladosporium, Eurotium, Penicillum, Wallemia, Chaetomium, Mucor, or Scopulariopsis. A composition according to any one of claims 1 to 6, characterized in that the trihelical fiber protein is selected from collagen, procollagen, tropocollagen, elastin, fibrillin, fibronectin, Scavenger receptors or other collagenous components, synthesis precursors, Fragments or conjugates of the above components. Composition according to Claim 7, characterized the collagen is selected from collagen type I, II, III, V and IX. A composition according to any one of claims 1 to 7, characterized in that the trihelical fiber protein procollagen, Fragments or variants thereof means. Composition according to Claim 9, characterized that the fragment or variant 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. A composition according to claim 9 or 10, characterized characterized in that the variant is a mutein that is characterized by the addition, substitution, deletion, insertion or inversion at least an amino acid, preferably from at most 5 amino acids from the amino acid sequence different from human procollagen. A composition according to any one of claims 1 to 11, characterized in that the fragment or variant of the tri-helical fiber protein one to form a cysteine knot suitable sequence, preferably the sequence GlyProCysCysGly (SEQ ID NO: 1). A composition according to any one of claims 1 to 11, characterized gekennnzeichnet that the fragment or variant of the tri-helical fiber protein, the N- and / or C-terminal end and optionally one Part of the telomere of the trihelical fiber protein. A composition according to any one of claims 1 to 13, characterized in that for binding to a PAMP receptor able Substance a bacterial endotoxin, preferably LPS and the trihelical Fiber protein tropocollagen, procollagen or collagen I, II, III, V or IX is. A composition according to any one of claims 1 to 14, characterized in that the composition further comprises chondrocytes, Fibrocytes, osteocytes, reticuloendothelial cells, endothelial cells, preferably chondrocytes, or contains components thereof. A composition according to any one of claims 1 to 15, characterized in that the components (i) and (ii) non-covalently connected to each other. Process for the preparation of the composition according to one of the claims 1 to 16 comprising contacting the component (i) with the component (ii) and optionally purifying or isolating the composition. In vitro method according to claim 17, characterized in that the method comprises the following Steps comprising: a) providing a bacterial or fungal eluate containing a component (i) according to any one of claims 1 to 16; b) providing a growth-enhanced chondrocyte culture; c) contacting the bacterial or fungal eluate with the chondrocyte culture; and d) optionally, purifying the composition. In vitro method according to claim 18, characterized in that that the fungal eluate from Aspergillus, Botrytis, Cladosporium, Eurotium, Penicillum, Wallemia, Chaetomium, Mucor, or Scopulariopsis. In vitro method according to claim 18, characterized in that that the bacterial eluate from Actinomycetales, Salmonella, Shigella, E. coli, Pseudomonas, Neisseria, Klebsiella or Haemophilus. Method according to one the claims 17 to 20, characterized in that the purifying performing a chromatographic method, centrifugation, ultrafiltration or gel electrophoresis. Pharmaceutical composition containing the composition according to one of the claims 1 to 16 and at least one pharmacologically acceptable Carrier, preferably as a vaccine. Use of the composition according to any one of claims 1 to 16 for the prophylaxis and / or treatment of connective tissue diseases or diseases of the musculoskeletal system, preferably osteoarthritis, Soft tissue rheumatism, fibromyalgia, rheumatic diseases or rheumatic Autoimmune reaction. Test kit for the diagnosis of connective tissue diseases containing the composition according to any one of claims 1 to 16th Method for the diagnosis of connective tissue diseases comprising detecting autoantibodies by contacting with a composition according to one the claims 1 to 16 and evidence of binding. Reagent attached to at least one component of the composition according to one of the claims 1 to 16 binds, preferably for the composition is specific. Reagent according to claim 26, characterized in that the reagent is an antibody, preferably a monoclonal or polyclonal antibody. Reagent according to claim 27, characterized in that the antibody with a suitable for detection Marker is conjugated. Composition containing a first binding partner, which binds to component (i), and / or a second binding partner, which binds to component (ii). Composition according to Claim 29, characterized that the first binding partner for the Component (i) and the second binding partner for component (ii) specifically is preferably, the first and the second binding partner is a Antibody. A composition according to claim 29 or 30, characterized characterized in that the first binding partner is selected from LAL, transferrin, HDL, LDL, apolipoproteins, C-reactive protein, CR1, CR3, CD14, scavenger receptors, CD18, gangliosides, Lec tin-like Receptors, polysaccharide receptors, bactericidal / permeability increasing protein (BPI), cationic antimicrobial protein (CAP), and LPS binding protein (LBP), as well as soluble variants of said Factors. A composition according to claim 30 or 31, characterized characterized in that the first binding partner is selected from component (ii) according to one of claims 1 to 16, preferably Procollagen, tropocollagen, collagen, synthesis precursors, fragments or variants thereof. Pharmaceutical composition containing the composition according to one of the claims 29 to 32 and a pharmacologically acceptable carrier. 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 osteoarthritis, Soft tissue rheumatism, fibromyalgia, rheumatic diseases and rheumatic Autoimmune reaction. 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 any one of claims 29 to 32. Method for the diagnosis of connective tissue diseases comprising detecting the composition according to any one of claims 1 to 16 by contacting with a composition according to any one of claims 29 to 32, preferably in blood or serum and detection of binding. Use of component (ii) according to a the claims 1 to 16 for the neutralization of endotoxins in individuals, preferably for the prophylaxis of an autoimmune reaction. Use according to claim 37, characterized in that the component (ii) is selected from Collagen, procollagen, tropocollagen, synthesis precursors, fragments or variants thereof. Use according to claim 37 or 38, characterized in that the use for prophylaxis connective tissue disorders or disorders of the musculoskeletal system, preferably osteoarthritis, soft tissue rheumatism, fibromyalgia, rheumatic Diseases and rheumatic autoimmune reaction is performed.