WO2015011073A1

WO2015011073A1 - Autoimmune derived antibodies to dermcidin as cardiovascular risk markers

Info

Publication number: WO2015011073A1
Application number: PCT/EP2014/065582
Authority: WO
Inventors: Christian Apfel; Paul Cutler; Philippe FERBER; Thomas Peter KRAEHENBUEHL; Daniel Roeder; Nicolas VUILLEUMIER; Gaby WALKER
Original assignee: F Hoffmann La Roche AG; Hopitaux Universitaires De Geneve; Hoffmann La Roche Inc
Current assignee: F Hoffmann La Roche AG; Hopitaux Universitaires De Geneve; Hoffmann La Roche Inc
Priority date: 2013-07-25
Filing date: 2014-07-21
Publication date: 2015-01-29
Anticipated expiration: 2016-01-25

Abstract

This invention relates to the detection of endogenous antibodies recognizing human Dermcidin (DCD) or its fragment peptide DCD-1L in human plasma and sera, and its use for the prognosis of cardiovascular events.

Description

AUTOIMMUNE DERIVED ANTIBODIES TO DERMCIDIN AS CARDIOVASCULAR

RISK MARKERS

FIELD OF THE INVENTION

The present invention relates to the detection of autoimmune derived (endogenous) antibodies to dermcidin (DCD) or DCD-derived peptide DCD-1L in biological fluids, in particular human plasma or human sera, and its use for the prognosis of cardiovascular diseases, especially for the prognosis of future cardiovascular events.

BACKGROUND

Cardiovascular diseases (CVDs) are the number one cause of death globally. An estimated 17.3 million people died from CVDs in 2008, representing 30 % of all deaths, and it is expected that the number of people who die from CVDs will increase to 23.3 million by 2030 (WHO Fact Sheet No. 317, March 2013). It is increasingly recognized that autoimmunity directed against cardiac antigens plays a crucial role for the pathogenesis of cardiovascular diseases, such as myocarditis, dilated cardiomyopathy (DCM), ischemic myocardiopathy, rheumatic fever and atherosclerosis. Autoimmunity may be caused by the superimposition of genetic, immune, hormonal as well as environmental factors. Furthermore, upon myocardial tissue damage normally sequestered cellular constituents (cryptic antigens) may be released or exposed to the immune system and thus trigger autoimmune responses.

Acute chest pain is a frequent reason to attend hospital emergency rooms (ER). Timely identification of patients with acute coronary syndrome (ACS) is of key relevance not only for patient management but also to optimize patient flow through the ER. Based on the clinical presentation, electrocardiographic (ECG) features and elevation in cardiac troponin I (cTnl) I or cardiac troponin T (cTnT). ACS is divided into ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation ACS (NST-ACS). Whereas diagnosis of STEMI is straightforward, NST-ACS diagnosis is more difficult and relies mostly on ECG changes and cTn elevation to discriminate between non-ST-segment elevation myocardial infarction

(NSTEMI) and unstable angina or chest pain of non-cardiac origin. However, it is well known that the sensitivity of cTn within the first 6 h following the onset of symptoms is low. Because of this, prolonged monitoring and repeated blood sampling over a period of 6 h are often required before NSTEMI can be diagnosed. It has been suggested that postponing diagnosis not only increases the risk of complications associated with this condition, but also contributes to ER overcrowding. Simultaneous assessment of multiple emergent cardiac biomarkers including new markers reflecting different underlying pathophysiological processes may therefore of high relevance for improving the NSTEMI diagnosis.

Autoantibodies are not only found to be present in serum from patients, but also in healthy individuals at low concentrations. Autoantibodies identified so far are directed against

mitochondrial proteins such as antimitochondrial M7, sarcolemmal proteins such as troponins and myosins as well as cardiac membrane receptors such as adrenergic and muscarinic receptors (for review: Kaya et al., 2013). Moreover, anti-phospholipid, anti-heat shock protein and anti- high-density lipoprotein/apolipoprotein A-l antibodies have been described (for review see Roux-Lombard et al, 2010).

DCD is a protein with 110 amino acids in length (SEQ ID NO: 1) and was first detected as expressed and secreted from the sweat glands where it is transported to the epidermal surface. Biological functions have been assigned to the entire DCD protein or proteolytic peptide fragments, designated DCD-IL (SEQ ID NO:2), YP-30 (SEQ ID NO:3) and PIF (SEQ ID NO:4), and are described in various tissues including the skin (Schittek et al, 2001), nervous system (Cunningham et al, 1998, Landgraf et al, 2005; 2008), cancer cells (Stewart et al, 2007), in development and embryogenesis (Landgraf et al, 2005).

EP 1397384 Bl refers to an antimicrobial peptide that comprises a fragment of a maximum of 50 amino acid residues from the C-terminal region of the DCD protein, in particular the fragment comprising the amino acid residues 63-110 of DCD that is named DCD-IL. The use of the peptide for the manufacture of medicaments for the treatment of diseases which are caused by microbial pathogens, in particular for skin diseases is also described.

In the cardiovascular system, DCD protein was first identified as an oxidative stress protein in plasma from MI patients (Ghosh et al. 2011). Injection of DCD protein into rabbits increased systolic blood pressure by 77% and diastolic pressure by 45% within 2 hours via the inhibition of endothelial NO-Synthase activity. A further effect of DCD-injection was a significant increase in platelet aggregation by stimulation of cyclooxygenase synthesis.

Moreover, pancreatic insulin secretion was reduced by about 50% of normal levels. All effects could be reversed by application of aspirin (Ghosh et al, 2012a; 2012b). In a proteomic study comparing protein compositions of the electropositive, native low- density lipoprotein LDL(+) and the electronegative, minor atherogenic LDL(-) by LC-ESI MS / MS analyses DCD was found predominantly in LDL(+) (Bancells et al, 2010).

In a study on the comparison of the mRNA and miRNA transcriptomes in cardiomyocytes derived from human differentiating-induced pluripotent stem cells (hiPSC) constitutive DCD expression over the entire period up to 120 days after differentiation initiation was observed (Babiarz et al, 2012).

The family of antimicrobial peptides (AMPs) further comprises cathelicidines, histatines and defensins (for review De Smet & Contreras, 2005). AMPs were first described for their antimicrobial activities against a wide range of pathogens as an important part in the innate host defense. However, recent reports suggest different functions including cell proliferation (Murphy et al, 1993), production of extracellular matrix (Gallo et al, 1994), angiogenesis (Li et al, 2000) and cellular immunity (Huang et al, 1997).

Histatins comprise a family of small, cationic, histidine-rich peptides of 3 - 4 kDa present in human saliva. The histatin family consists of several members HIS1 through HIS5. Human a- defensins are synthesized as prepropeptides with a 19-amino acid signal peptide and a 41- 51 amino acid anionic pro segment. Up to now, six different human a-defensin molecules have been described including HNP-1, HNP-3, HNP-4, HD-5, HD-6 and a truncated version HP-2. Human β-defensins HB-1 through HB-4 contain around 35 amino acid residues, including six cysteine residues with a spacing pattern forming a disulfide that differs from that of the a- defensins. Cathelicidins are a family of antimicrobial peptide. In humans, only one cathelicidin LL37 has been characterized. LL37 is composed of 37 amino acid residues, and has a linear structure because it does not contain cysteine residues. The peptide adapts its conformation to the local chemical environment: In a hydrophilic environment a largely random coil conformation, in a hydrophobic environment a-helical structure is formed (for review Schneider et al, 2005). DCD-1L is also a member of the AMP family, however, seems to be unique in terms of the structure and function.

So far, only few reports suggest a role of defensins in the cardiovascular system. The peptides are released into the blood during infectious and non-infectious (sterile) inflammation such as chronic (CHF) and acute heart failure (AHF) as well as diabetes mellitus. Plasma defensins may have the potential as clinical risk markers for patients with CHF and AHF.

Pleiotrophin (PTN), also called heparin binding growth- associated molecule (HB-GAM), is a protein of 18 kDa with a closely related structure to Midkine (MK). PTN and MK are mainly composed of two domains linked by disulfide bridges, and there are three antiparallel p-sheets in each domain. MK and PTN promote growth, survival, and migration of various cells, and play important roles in neurogenesis and epithelial mesenchymal interactions during organogenesis (Muramatsu, 2002). In the cardiovascular system, PTN was reported to have neovasculogenic effects in the damaged heart. During in vitro differentiation of cardiomyocytes from pluripotent stem cells PTN gene expression is upregulated. Gene expression profiling studies revealed PTN mRNA induction in myocard tissue from patients with heart failure (Asakura & Kitakaze, 2009). A recent report suggests a role for PTN as apoptotic factor induced in stressed heart cells.

Syndecans (SDCs) are a family of cell surface heparin sulfate proteoglycans. Four SDC genes (SDC-1, -2, -3, and -4) are characterized (for review; Tkachenko et al, 2005). SDCs may act in dual fashion, as adhesion and docking receptors. As cell adhesion receptor, the

extracellular domain of SDC can transduce extracellular signals to the cytoplasm thereby regulating focal adhesion, stress fiber formation, cell motility, cellular migration and

proliferation. Binding of extracellular ligands to SDC may cause a variety of processes inside and outside of the cell: (i) Activation of extracellular enzymes such as matrix metalloproteinases (MMPs). (ii) Binding and local concentration of diluted soluble ligands. (iii) Promotion of high affinity binding of growth factors such as FGF and VEDF to their specific receptors FGFR and VEGFR, respectively (for review Kwon et al, 2012).

So far, little is known about physiological or pathophysiological effects of SDCs in the cardiovascular system. Increased SDC-4 mRNA expression and protein shedding during heart failure was reported. In mice, pressure overload induced by aortic banding (AB) increased SDC- 4 mRNA. Moreover, in cardiac myocytes and fibroblasts, TNF-a, IL-Ιβ, and lipopolysaccharide (LPS) induced SDC-4 mRNA correlating with mRNA expression. In failing human hearts, SDC- 4 mRNA and shedding were found to be up-regulated. The authors suggest that SDC-4 may play an important role in the immune response of the heart to increased pressure, influencing cardiac remodelling and failure progression (Strand et al, 2013).

Creatine kinase (CK), Creatine kinase-MB (CKMB), Myoglobin and cardiac troponins T and I are the preferred biomarkers for the diagnosis of acute myocardial infarction (J. Adams, Circulation 2004; 109: el2-el4). It has also been recognized that elevated troponin levels may be detected in several non-acute chronic disease states, including coronary artery disease, heart failure, and chronic kidney disease (see e.g. Omland et al., N Engl J Med. 2009;361(26):2538- 2547). Troponins T and I have also been shown to be detectable in individuals from the general population (see e.g. Wallace et al, Prevalence and determinants of troponin T elevation in the general population. Circulation. 2006; 113(16): 1958-1965). Measuring the level of high sensitive C-reactive protein (hsCRP) in blood is a further test to improve risk prediction for myocardial infarction, stroke and sudden death. For diagnosis and treatment follow-up of congestive heart failure the level of NT-pro-BNP (N-terminal pro-B-type natriuretic peptide) in blood is the preferred biomarker.

However, in spite of all efforts made to estimate the cardiovascular risk in acute and chronic situations there is still an unmet medical need for further improvement in the accuracy of the risk prediction. Furthermore, a considerable number of cardiovascular events occur in asymptomatic patients with low risk scores according to current risk assessment tools.

Accordingly, there is a need for novel markers and new tests that could successfully and advantageously predict who is at higher risk of developing cardiovascular events.

SUMMARY OF THE INVENTION The invention provides a method for the detection of a cardiovascular disease in a human subject comprising the steps of

(a) measuring in a sample of serum or plasma from a human subject the level of endogenous antibodies recognizing Dermcidin (DCD), and

(b) comparing the level measured in (a) with the level of said autoantibodies representative for a human subject of a healthy population, wherein the presence or an increased level of said endogenous antibodies recognizing DCD in the sample is indicative for a cardiovascular disease.

In one embodiment, the invention provides a method for the detection of a cardiovascular disease, wherein said measuring of endogeneous antibodies recognizing DCD comprises the steps of: (a) providing a sample of serum or plasma from a human subject;

(b) bringing said sample into contact with a solid matrix where at least one peptide selected from DCD comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of said DCD comprising the amino acid sequence of SEQ ID NO: 2 is coupled to, wherein the contacting is under conditions sufficient for binding endogenous antibodies recognizing DCD present in said sample to said peptide through antigen-antibody interactions;

(c) removing any unbound antibody from the surface of said solid matrix; and

(d) detecting the level of an antigen-antibody complex bound to said solid matrix.

In a certain embodiment, the fragment peptide used in the method above is DCD-1L comprising the amino acid sequence of SEQ ID NO:2.

In certain embodiments, the solid matrix used in the methods above is a microtitre plate (microwell plate). The Microplate can be made from a variety of materials such as glass, polystyrene, polypropylene, polycarbonate or cycloolefines. Typically, the microplate has 6, 24, 96, 384 or even 1536 sample wells. In particular, the solid matrix is a 96-well polystyrene microplate. In a further embodiment, a method for the detection of a cardiovascular disease is provided, wherein the cardiovascular disease is acute coronary syndrome.

In another embodiment, a method for detecting endogenous antibodies recognizing human DCD in a sample of serum or plasma from a human subject is provided, said method comprising the steps of:

(a) providing a sample of serum or plasma from a human subject;

(b) bringing said sample into contact with a solid matrix where at least one peptide selected from DCD comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of said DCD comprising the amino acid sequence of SEQ ID NO:2 is coupled to, wherein the contacting is under conditions sufficient for binding an endogenous antibody recognizing

DCD present in said sample to said peptide through antigen-antibody interactions;

(c) removing any unbound antibody from the surface of said solid matrix; and

(d) detecting the presence of an antigen-antibody complex bound to said solid matrix; wherein the presence of said complex is indicative that the sample of serum or plasma from the human subject contains endogenous antibodies recognizing DCD.

In a further embodiment, a kit comprising a peptide of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 for detecting in a sample of serum or plasma from a human subject the presence or the level of endogenous antibodies recognizing human DCD is provided, wherein the presence or an increased level said endogenous antibodies recognizing DCD in the sample of serum or plasma from the human subject compared to a level of said antibodies representative for a human subject of a healthy population is indicative for a cardiovascular disease.

In another embodiment, the invention provides for the use of a peptide selected from DCD comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of said DCD comprising the amino acid sequence of SEQ ID NO: 2 for determining endogenous antibodies recognizing human DCD in a sample of serum or plasma from a human subject, wherein the presence or an increased level of said endogenous antibodies recognizing DCD in the biological fluid sample is indicative for a cardiovascular disease.

In a particular embodiment, the use of the peptide fragment DCD-1L comprising the amino acid sequence of SEQ ID NO: 2 is provided for determining endogenous antibodies recognizing human DCD in a sample of serum or plasma from a human subject, wherein the presence or an increased level of said endogenous antibodies recognizing DCD in the biological fluid sample is indicative for a cardiovascular disease. BRIEF DESCRIPTION OF THE FIGURES Fig. 1 shows DCD and its proteolytic cleavage products.

Fig 2A: Western-blot analyses of an ApoA-1 -enriched human plasma preparation using directly labeled IgG purified from serum of patient versus healthy donor for immunostaining. Fig 2B: Western-blot analyses of SDS-PAGE under reducing versus non-reducing conditions of an ApoA-1 -enriched human plasma preparation using directly labeled IgG purified from serum of patient for immunostaining.

Fig. 2C: Western-blot analyses of ApoA-1 -enriched human plasma preparation using an anti-DCD-antibody and competition experiments with recombinant ApoA-1 and a batch of ApoA- 1 -enriched plasma preparation.

Fig. 2D: Western-blot analyses of an immunoprecipitation experiment using ApoA-1- enriched human plasma preparations and an anti-DCD-antibody.

Fig. 3A: Amino acid sequences of DCD-related peptides DCD-IL and YP-30 applied to solid phase ELISA Fig. 3B: Outline of the solid phase ELISA using synthetic DCD-IL peptide for coating and

AHA for immunostaining

Fig. 4: Titration of serum from MI patients (A) versus healthy donors (B) and different amounts of DCD-IL peptide on the DCD-IL solid phase ELISA

Fig. 5: Titration of serum from MI patients versus healthy donors on DCD-IL- (A, B) and YP-30 (C, D) solid phase ELISAs

Fig. 6: Titration of purified serum IgG from MI patients versus healthy donors on DCD- IL- (A, B) and YP-30 (C, D) solid phase ELISAs

Fig. 7: Competition for the binding of anti-DCD and anti-ApoA-1 antibodies and autoantibody to solid phase DCD-IL peptide Fig. 8: A small cohort study of sera from 59 CVD patients and healthy donors was tested for autoimmunity to DCD-IL versus YP-30. A correlation with established markers for CVD such as NT-proBNP, CKMB and Troponin I was performed. Fig. 9: Size exclusion chromatography from total patient and healthy donor plasma and western blot analyses of SEC fractions using anti-DCD antibody in parallel to directly labeled patient serum IgG.

Fig. 10: DCD-1L solid phase ELISA on fractions 21 to 27 in sera from healthy donor (left) and patient sera (right). Each fraction 38 accounts for a negative control.

Fig. 11: Treatment of SEC fractions 21 to 27 with formic acid.

Fig. 12: Western blot analyses of SEC fractions 21 through 27 and 38 for

immunoreactivities to DCD, PTN and SDCs 1-3.

Fig. 13: Competition experiments of DCD-1L peptides coated on solid phase using recombinant PTN (A), SDC-1 (B) and ApoB (C) to autoimmune reactivity in patient serum.

Fig. 14: Respective diagnostic accuracies for NSTEMI prediction based on anti-DCD- 1L IgG and anti-YP-30 IgG (as measured by ELISA).

Fig. 15: Respective predictive accuracies for subsequent cardiac troponin I elevation based on anti-DCD- 1L IgG and anti-YP-30 IgG. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a test method for the detection of a cardiovascular disease, in particular for the prognosis of cardiovascular events. Said method is based on autoimmunity- derived antibodies directed against DCD, or more particularly a peptide fragment of DCD, in particular DCD-1L, alone or in a complex comprised with pleiotrophin and/or syndecan. These autoimmunity-derived antibodies are present in human body fluids, particularly in plasma or serum of the human subject.

Thus, the invention provides a method for the detection of a cardiovascular disease in a human subject comprising the steps of

(a) measuring in a sample of serum or plasma from a human subject the level of endogenous antibodies recognizing DCD, and

(b) comparing the level measured in (a) with the level of said autoantibodies in a sample representative for a human subject of a healthy population, wherein the presence or an increased level of said endogenous antibodies recognizing DCD in the biological fluid sample is indicative for a cardiovascular disease. The term "cardiovascular disease or disorder", as used herein, includes diseases affecting the heart or blood vessels or both or associated with the cardiopulmonary and circulatory systems including but not limited to artherosclerosis, LDL oxidation, adhesion of monocytes to endothelial cells, foam-cell formation, fatty-streak development, platelet adherence, and aggregation, smooth muscle cell proliferation, coronary heart disease, ischemia, myocardial ischemia, angina, acute coronary syndrome, myocardial infarction, stroke, thromboembolic diseases, arterial hypertension, reperfusion injury of the brain, heart, kidney or other organ or tissue; edematous conditions, arrhythmia (atrial or ventricular or both), cardiac or vascular aneurysm, vasculitis, peripheral obstructive arteriopathy of a limb, an organ, or a tissue, valvular heart disease, congestive heart failure, acute decompensated heart failure, congenital heart failure, traumatic, surgical or endotoxic shock, vascular abnormality, inflammation, and insufficiency limited to a single organ or tissue.

The terms "cardiovascular disease risk" or "cardiovascular risk" are defined herewith as the probability of developing or dying from a cardiovascular disease or event for subjects who have not already developed major cardiovascular symptoms or for patients already diagnosed with a cardiovascular disease. This probability is typically evaluated based on the observation of different traditional cardiovascular risk factors, such as age, gender, BMI, obesity, family history, tobacco use, diabetes, hypertension, dyslipidemia, physical inactivity, and atherogenic diets. Subjects without patent cardiovascular diseases are qualified as having a "high cardiovascular risk" when their 10-year risk of developing a cardiovascular disease is estimated to be higher than 10 -20 depending on the geographical region based on the 10-year global Framingham risk score. For patients with acute cardiovascular events risk prediction can be estimated by the TIMI Risk Score (NSTEMI and STEMI Risk Scores), which assess the risk of death and subsequent ischemic events. The term "endogenous autoantibody" is a type of protein produced by the immune system of a human subject that is directed against one or more of the human subject's own proteins. In the context of the present invention, the endogenous autoantibodies are autoimmunity-derived antibodies directed against dermcidin (DCD), or more particularly a peptide fragment of DCD, in particular DCD-1L, alone or directed against a confirmation epitope comprising DCD-1L in a macromolecular complex that potentially contains pleiotrophin and/or syndecans.

The term "human subject" in particular includes patients. A "patient" is a human subject for which a treatment is indicated or desired. More particularly, the patient is a subject suffering from a cardiovascular disease or disorder.

As used herein, "therapy" or "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to medical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms or diminishment of any direct or indirect pathological consequences of the disease. Dermcidin (DCD) is a protein of 110 amino acids in length (SEQ ID NO: 1) and was first detected as expressed and secreted from the sweat glands were it is transported to the epidermal surface. Proteolytic processing of DCD gives rise to several major fragments including the N- terminal YDP-42 (amino acids 20-62, SEQ ID NO:5) and DCD-1L, a 47 amino acid peptide derived from the C-terminal part (SEQ ID NO:2, Fig. 1; Stewart et al, 2008). From YDP-42 truncated peptides were identified as YP-30 (amino acids 20-49, SEQ ID NO:3) and proteolysis- inducing factor PIF (amino acids 20-39, SEQ ID NO:4). From DCD-1L, a total of 13 peptides with different lengths were identified by MS analyses (Steffen H, Doctoral Thesis 2006).

Unprocessed DCD protein is strongly hydrophobic. Among the major fragments, YP-30 and PIF are hydrophobic whereas DCD-1L displays an amphipathic character. DCD-1L has a helical structure spanning the entire 48 amino acids. Remarkably, the primary sequence of DCD- 1L contains only 9 different amino acids, with no cysteins and disulfide bridges, no aromatic or proline amino acids. It is active over a broad pH range and in high salt concentration. The DCD- 1L derived peptides display different net charges at neutral pH: nine peptides were found to be anionic, two peptides with neutral charge and two peptides that are cationic (Steffen et al, 2006). Under certain conditions DCD has been reported to oligomerise, and DCD related peptides can form heterocomplexes with other proteins. More specifically, YP-30 has been reported to form complexes with both the 18kDa growth factor pleiotrophin and syndecan (Landgraf et al 2008).

In a certain embodiment, the invention provides a method for the detection of a

cardiovascular disease, wherein said measuring of endogeneous antibodies recognizing DCD comprises the steps of:

(a) providing a sample of serum or plasma from a human subject;

(b) bringing said sample into contact with a solid matrix where at least one peptide selected from DCD comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of said DCD, in particular the fragment comprising the amino acid sequence of SEQ ID NO:2 is coupled to, wherein the contacting is under conditions sufficient for binding endogenous antibodies recognizing DCD present in said sample to said peptide through antigen- antibody interactions;

(c) removing any unbound antibody from the surface of said solid matrix; and (d) detecting the level of an antigen-antibody complex bound to said solid matrix.

The term "solid matrix" includes any solid phase support suitable for carrying out an immunoassay or a method according to the invention. It includes beads, microparticles, nanoparticles, tubes, fabrics or plates, films, slides, wells, formed from or coated with glass, polystyrene, polypropylene, polycarbonate, cyclo-olefines, nitrocellulose, quartz, ceramic, dextran or other materials. In particular, the solid matrix is in a form of microtitre plates having 6, 24, 96, 384 or even 1536 sample wells, such as in particular a 96-well microtitre plate. In certain embodiments, the solid matrix used in the methods above is 96-well polystyrene microtitre-plate as for example a Nunc Maxisorp™ plate.

In a certain embodiment, a method for the detection of a cardiovascular disease is provided, wherein the cardiovascular disease is selected from the group consisting of acute coronary syndrome, severe carotid stenosis and myocardial infarction. In a further certain embodiment, a method for the detection of a cardiovascular disease is provided, wherein the method is a method of predicting a cardiovascular event in a patient having acute chest pain. The cardiovascular event is for instance myocardial infarction (MI).

In a further embodiment, provided is a method for the detection of a cardiovascular disease, wherein the method comprises the combination of measuring of endogeneous antibodies recognizing DCD as defined above with the measuring of further markers indicating

cardiovascular events.

In another certain embodiment, a method for detecting endogenous antibodies recognizing human DCD in a sample of serum or plasma from a human subject is provided, said method comprising the steps of: (a) providing a sample of serum or plasma from a human subject;

(b) bringing said sample into contact with a solid matrix where at least one peptide selected from DCD comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of said DCD comprising the amino acid sequence of SEQ ID NO: 2 is coupled to, wherein the contacting is under conditions sufficient for binding an endogenous antibody recognizing DCD present in said sample to said peptide selected from DCD or the fragment of said

DCD through antigen-antibody interactions; (c) removing any unbound antibody from the surface of said solid matrix; and

(d) detecting the presence of an antigen-antibody complex bound to said solid matrix; wherein the presence of said complex is indicative that the sample of serum or plasma from the human subject contains endogenous antibodies recognizing DCD. In a certain further embodiment, provided is a method, wherein the method further comprises a step of comparing the signal obtained under the detection step (d) with the same signal obtained for at least one control sample, wherein the signal obtained for said at least one control sample is collected previously, simultaneously or posteriorly to the detection step (d) for said sample. Detection of the captured/bound antibodies under step (d) can be carried out by any suitable method known in the art for detecting captured antibodies or proteins on surfaces such as optical detection (e.g. ELISA), mass variation detection (e.g. surface Plasmon resonance, mass spectrometry), electrical detection (e.g. impedance spectroscopy, electrochemical) techniques. Results of the assay may be qualitative or quantitative. The amount of captured/bound antibodies associated with the solid matrix can be compared with positive and negative controls. The controls are typically run concomitantly with the sample to be tested. A positive control can be a serum or a solution containing antibodies that are immunoreactive with DCD. A negative control can be a serum or solution which does not contain antibodies that are immunoreactive with DCD. For quantization, a calibration curve using known quantities of anti-DCD antibodies can be generated and/or used. Antibodies for use as positive controls may be produced using all, or fragments of, the amino acid sequence of DCD.

The comparison with sample of serum or plasma from a normal healthy human subject may be achieved with different methods. According to one embodiment, it may be carried out by including a control reaction with a non-diseased blood sample. According to another

embodiment, it may be carried out by employing a value for the concentration of the

endogeneous anti-DCD antibody for a typical sample from a healthy subject. Typically, the comparison of the level of endogeneous anti-DCD antibody present in a sample under investigation may be performed with respect to a value determined in each single testing procedure or to a predetermined value. The predetermined value may be determined for the testing procedure in general, or alternatively, the value may be valid only for a certain batch of testing reagents. For example, the reference value may be valid for a defined calibration period only and may be redefined upon calibration of the testing process. In a certain embodiment, a kit for detecting in a sample of serum or plasma from a human subject the presence or the level of endogenous antibodies recognizing human DCD is provided, wherein the presence or an increased level said endogenous antibodies recognizing DCD in the sample of the human subject compared to a level of said antibodies representative for a human subject of a healthy population is indicative for a cardiovascular disease.

In a particular embodiment, a kit is provided, that comprises at least one peptide selected from DCD comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of said DCD comprising the amino acid sequence of SEQ ID NO: 2 according to the invention, a variant thereof, or a combination thereof for coupling, or already coupled to a solid matrix as solid phase support as referred herein.

Various solid matrices can be used, including but not limited to glass, polystyrene, polypropylene, nitrocellulose, quartz, ceramic, dextran or other materials. Suitable forms of the solid matrix include beads, microparticles, nanoparticles, tubes, fabrics or plates, films, slides, wells, formed from or coated with these materials. Typically, the solid matrix comprises microtiter wells, such as a 96-well microtiter plate.

The coupling, or fixation, of the peptide to the solid matrix in a kit according to the invention may be carried out by adsorption or chemical coupling to a solid phase support. Any means known in the art for immobilizing a protein or peptide to a solid support can be used. The peptides according to the invention can be either covalently or non-covalently bound to the solid matrix by techniques such as covalent bonding via an amide or ester linkage or adsorption.

Peptides can be bound using binding pairs such as biotin and avidin or antibody and antigen. After the peptides are affixed to the solid matrix, the solid matrix can be incubated with a blocking solution (containing a blocking protein such as bovine serum albumin) to reduce nonspecific adsorption of antibodies in a test sample to the support surface. The kit according to the invention optionally further comprises coupling reagents and/or a solid matrix for performing an immunoassay.

According to another further embodiment, the kit according to the invention further comprises at least one rinsing reagent for washing unbound material before detection in order to avoid background noise detection. Typically rinsing reagents comprise standard buffers known in the art. According to another further embodiment, the kit according to the invention further comprises at least one control sample optionally together with calibration information for quantification of detected anti-DCD antibodies. According to another embodiment, the invention provides an immunoassay plate comprising at least one DCD peptide, a variant thereof, or a combination thereof, which are coupled to a solid matrix as solid phase support.

The invention also provides for the use of a peptide selected from DCD comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of said DCD comprising the amino acid sequence of SEQ ID NO: 2 for determining endogenous antibodies recognizing human DCD in a sample of serum or plasma from a human subject, wherein the presence or an increased level of said endogenous antibodies recognizing DCD in the sample is indicative for a cardiovascular disease. In a particular embodiment, the use of the peptide fragment DCD-1L comprising the amino acid sequence of SEQ ID NO: 2 is provided for determining endogenous antibodies recognizing human DCD in a sample of serum or plasma from a human subject, wherein the presence or an increased level of said endogenous antibodies recognizing DCD in the sample is indicative for a cardiovascular disease. The methods, the kits and uses according to the invention may be suited for screening purposes as well as for diagnostic purposes and may be applied in primary diagnosis as well as in monitoring of disease course during or after treatment.

In particular, the methods, the kits and uses according to the invention may be suited for: i) diagnosis purposes in patients with acute chest pain to rule out or rule-in a myocardial ischemia and, thus, for the diagnosis of acute coronary syndrome, ii) prognosis and, possibly, therapeutic purposes in patients with acute chest pain, acute coronary syndrome, rheumatoid arthritis, systemic lupus erythematosus, severe carotid stenosis, end-stage renal disease or periodontitis, since the methods, the kits and uses according to the invention could identify a sub- set of patients having a high cardiovascular risk. The aforementioned diagnostic, prognostic and therapeutic purposes may be applied in primary as well as in secondary prevention.

The methods, the kits and uses according to the invention may, in particular, also be suited for determining whether a patient suffering from acute chest pain, acute coronary syndrome, rheumatoid arthritis, systemic lupus erythematosus, severe carotid stenosis, end-stage renal disease or periodontitis, for whom the usual cardiovascular risk factors including but not restricted to age, gender, BMI, obesity, tobacco use, alcohol use, hypertension, dyslipidemia, physical inactivity, unhealthy diets are not observed or insufficiently predictive, could nevertheless benefit from an appropriate therapeutic intervention to prevent subsequent cardiovascular events.

The prognostic and/or diagnostic assays described herein can therefore be used to determine whether a subject could benefit from the administration of a therapeutic agent (e.g. a drug) for treating or preventing cardiovascular diseases.

Another aspect of the invention is a method of monitoring the course of a cardiovascular disease in a subject during or after treatment. For this purpose, the detection of anti-DCD antibodies in a biological fluid sample of a human subject can be determined on biological samples from the human subject before, during, or after undergoing a treatment. Information obtained from practice of the above assays is useful in prognostication, identifying progression of, and clinical management of diseases and other deleterious conditions affecting an individual's health status. The information more specifically assists the clinician in designing therapy or other treatment regimen to treat cardiovascular disease, in particular in sub- populations of patients suffering from acute chest pain, acute coronary syndrome, rheumatoid arthritis, systemic lupus erythematosus, severe carotid stenosis, end-stage renal disease or periodontitis.

EXPERIMENTAL DATA

1. Identification of DCD as a component of a 66kDa autoantibody-positive

immunoreactivity band present in an ApoAl -enriched human plasma preparation

Protein detection with Western blot analysis For Western-blot analyses, samples (25μg/lane) were separated using 4-12% SDS-PAGE

(NuPAGE, Invitrogen) and transferred to Nitrocellulose membranes (Amersham Pharmacia Biotech). Membranes were blocked in SuperBlock (Pierce), followed by 5% non-fat-dry milk in 0.1% Tween/PBS for 30 min. at room temperature. The primary antibodies were diluted 1: 1000 in 0.5% non-fat-dry milk in 0.1% Tween/PBS solution and incubated overnight at 4°C.

Membranes were washed with 0.1% Tween / PBS and subsequently incubated with the secondary antibody in a dilution 1:2000 in 0.5% non-fat-dry milk in 0.1% Tween/PBS solution for 1 hour at room temperature. Immunodetection was performed using the Lumi-Light Western Blotting Substrate (Roche Biochemicals).

IgG purification using HiTrap protein G HP 1ml column. GE Healthcare code No 17-0404-01 High density lipoprotein (HDL) fractions were isolated by ultracentrifugation from human plasma samples and delipidized using a chloroform: methanol extraction of the lipid. The delipidized HDL samples were applied to Superose 12 gel filtration (Weisweiler P. 1987). The Apo A-l fractions were pooled. The samples were obtained from N. Vuilleumier/ R. James, Geneva University Hospitals. Sera from healthy donors and MI patients were ordered from SeraCare Life Sciences.

Serum from an ACS patient was obtained from N. Vuilleumier, Geneva University Hospitals.

Sample preparation: The samples were centrifugated for 10 minutes at lOOOg. Samples were adjusted to the composition of the binding buffer (20mM Sodium Phosphate, pH 7.0) by diluting the serum with binding buffer 1: 1. Purification: The column was washed with 10 column volumes of binding buffer using a syringe, followed by adding 2 ml of the sample. The column was then washed with 5 column volumes of binding buffer. Elution was performed using 4 column volumes of elution buffer (0.1 M Glycine-HCl, pH 2.7). To every collected fraction 600 μΐ Tris-HCl, pH 9.0 was added to neutralize pH of the elution products. The sample was dried and then resuspended in sterile endotoxin-free water at desired concentration.

In western blot analyses of a preparation of delipidized HDL from human plasma which was further enriched for Apolipoprotein A-l (Apo A-l) by size exclusion chromatography (herein expressed as "ApoA-1 -enriched plasma preparation") an autoimmune reactive band at an approximate molecular weight of about 66 kDa which co-stained with anti-ApoA-1 antibody was found.

In a first experimental approach to identify the exact identity of the protein(s)

immunostaining using directly labeled IgG from patient versus healthy donor serum revealed the 66kDa band only in patients whereas IgG from healthy donor showed no signal (Fig.2A).

Under conditions of non-reducing SDS-PAGE, immunostaining using directly labeled IgG from patient serum revealed a signal shift to a molecular weight of 200 kDa (Fig. 2B).

As ApoA-1 does not contain any Cys-residues, a shift cannot be achieved under non- reducing SDS-PAGE conditions. Thus it may be concluded that autoimmune reactivity is most likely not directed towards ApoA- 1.

In a further approach, MS analyses of the gel slice harbouring the region of the 66 kDa band were performed. Among other proteins, DCD was identified as a component of the 66 KDa autoantibody-positive immunoreactive band.

In western blot analyses immunostaining with a commercially available anti-DCD antibody detects the 66 kDa band (Fig. 2C). DCD immunodetection could not be competed by application of recombinant Apo A-l protein, but by another batch of ApoA 1 -enriched plasma preparation (Fig. 2C).

Finally, the 66 kDa immunostaining could also been shown using a commercially available anti-DCD antibody for immunoprecipitation and subsequent western blot analyses (Fig. 2D).

The major conclusions from these experiments are that (i) DCD is a component of the autoantibody immunoreactivity at 66 kDa present in the ApoA- 1 -enriched plasma preparation and (ii) serum from CVD patients contains an autoimmunity which is directed against DCD and is clearly different from ApoA-1. 2. Establishment of solid phase ELISA assays for detection of DCD peptide fragments DCD-1L and YP-30 in serum samples from CVD patient versus healthy donor

ELISA tests using synthetic peptides for DCD-1L and YP-30 as solid phase were established (Fig. 3A and 3B). Immunostainings were performed using either serum or IgG from CVD patients as compared to healthy donors. The outline of the solid phase ELISA is shown in Fig. 3B.

DCD-1L solid phase ELISA

Maxisorp plates (Nunc™, 456529) were coated with synthetic human DCD-1L peptide obtained from Abgent (Abgent SP2420a; amino acids 63-110 of dermcidin, Swiss-Prot P81605), at 40 μg/mL or as indicated in 50 μΐ_^ per well for 1 h at 37°C. After washing four times with 2% BSA/PBS, wells were blocked for 1 h with 50 2% BSA/PBS at 37°C. Thereafter, 50 triplicate serum samples diluted 1/50 (or as indicated) were added to the wells and incubated for 1 h. After washing six times with 2% BSA/PBS, 50 μΐ_^ per well alkaline phosphatase-conjugated anti-human IgG (Sigma- Aldrich, A3150) diluted 1/1000 in 2% BSA/PBS solution was added to wells and incubated for 1 h at 37 °C. After washing again six times with 2% BSA/PBS, wells were developed by adding 100 μΐ_^ alkaline phosphatase substrate disodium p-nitrophenyl- phosphate (Sigma-Aldrich S0942), dissolved in diethanolamine buffer (pH 9.8). After 30 min incubation at 37°C, ELISA signals (absorbance OD405 nm) were determined using a plate reader (Molecular Devices Versa Max™; Molecular Device, Sunnyvale, CA, USA). YP-30 solid phase ELISA

The same method was used as described above using synthetic human Y-P30 peptide purchased from Phoenix Pharmaceuticals Inc. (Catalog No. 075-32; amino acids 20-49 of dermicidin, Swiss-Prot P81605) at 40 μg/mL or as indicated, in 50 μΐ_^ per well.

Several experiments were performed using the solid phase ELISA. Experiment 2.1:

For the ELISA, DCD-1L peptide amounts applied to solid phase coating were in the range from 40 μg/ml, 20 μg/ml, 10 μg/ml and 5 μg/ml. As a total of 50 μΐ of peptide solution was spotted for coating, this equals to 2 μg, 1 μg, 0.25 μg and 0.125 μg of peptide, respectively. Patient and healthy donor sera were applied at dilutions of 1:50, 1: 100 and 1:200. As shown in Fig. 4, the ELISA allows to titrate the anti-DCD-lL autoantibodies in patient serum ranging from 40 μg/ml to 5 μg/ml DCD-1L peptide on the solid phase (Fig. 4A) whereas in serum from healthy controls no titration was detectable (Fig. 4B). Moreover, application of different amounts of DCD-1L peptide to solid phase revealed a dose-dependent titration but only by the use of patient serum (Fig. 4A and 4B).

Experiment 2.2:

In this experiment, a fixed concentration of 40 μg/ml of DCD-1L peptide was applied to solid phase coating and increasing dilutions of serum from MI patients versus healthy donors ranging from 1: 10, 1:50, 1: 100 and 1:200 were used for immunodetection.

In addition, YP-30 peptide in a concentration of 40 μg/ml was applied to solid phase ELISA and immunostaining was performed as described for DCD-1L peptide.

As shown in Fig. 5A and B, a titration can be observed using patient serum. Using healthy serum for immunostaining, only a 3 fold increase in OD levels over blank could be detected at the highest concentration.

No significant change in optical density could be observed using patient versus healthy donor serum using YP-30 peptide as shown in Fig. 5C and 5D.

These results indicate that the autoimmunity is directed against the DCD-1L moiety of DCD protein.

Experiment 2.3:

In a further experiment, IgGs were purified from MI patient and healthy donor sera and applied to solid phase ELISA containing 40 μg/ml immobilized DCD-1L in concentrations varying from 100 μg/ml, 50 μg/ml, 25 μg/ml, 12.5 μg/ml and 6.25 μg/ml. As shown in Fig. 6, application of purified, directly labeled IgG display the same pattern as already observed for sera in Fig. 5.

Comparison of the optical density values obtained for patient serum (1: 10 dilution; OD = 1.748; Fig. 5A, left) and for patient IgG (100 μg/ml; OD = 1.784; Fig. 6A, left) allows a calculation of enhancement factor. A reasonable assumption is that the total protein concentration in plasma is about 50 mg/ml; a 1: 10 serum dilution reveals a total amount of proteins equal to 5 mg/ml.

In light that a IgG solution at 100 μg/ml generates a comparable OD value, an

enhancement of about 50-fold can be assumed. Experiment 2.4:

In this experiment, a DCD-IL solid phase ELISA (each 40 μg/ml DCD-IL peptide for coating) was used for competition experiments using anti-DCD and anti-Apol antibodies.

Competition solid phase ELISA experiments using untagged recombinant protein and

commercial antibodies:

Maxisorp plates were coated and blocked as described herein before. Recombinant proteins (SDC1 Abnova H00006382-G01; PTN R&D 252-PL-250; ApoB;) dissolved in 2% BSA/PBS were added at different concentrations to plasma prior to the application to the wells coated with synthetic human DCD-IL peptide (competition of synthetic DCD- 1L and Rec protein for the binding to the autoantibody in the plasma). Commercial antibodies (anti-DCD; anti-ApoA-1) were added at different concentrations to plasma prior to the application to the wells coated with synthetic human DCD-IL peptide (competition of the commercial Ab and autoantibody in the plasma for the binding to the synthetic DCD-IL on the solid phase). Blocking, conjugation with ant-human IgG and alkaline phosphatase substrate were applied as described above. Antibodies and serum were mixed and subsequently applied to solid phase ELISA. Thus, in these experiments a competition of antibodies and autoantibody for the binding to DCD-IL solid phase was performed.

As shown in Fig. 7, anti-DCD antibody can compete the binding of the autoantibody to solid phase DCD-IL peptide, whereas anti-ApoA-1 antibody is not effective. This clearly demonstrates that DCD-IL is the antigen or a component of the antigen of the autoimmune antibody.

3. Correlation of DCD-1L levels to CVD markers NT-proBNP, CKMB and Troponin I

In a small cohort of 59 CVD patients and healthy donors anti DCD-1L autoimmunity levels were detected using the solid phase ELISA. A correlation with established markers for CVD such as NT-proBNP, CKMB and Troponin I was performed. As can be seen in Fig. 8, elevated autoantibody positive signals can be correlated best with

NT-proBNP positive sera over Troponin I, and no significant correlation with CKMB. No discrimination can be obtained using the YP-30 solid phase ELISA (data not shown).

4. Characterization of additional components of the autoimmune-positive 66 kDa complex present in plasma from patient and healthy donors Experiment 4.1:

Size exclusion chromatography from total patient and healthy donor plasma was performed followed by Western blot analyses using anti-DCD antibody in parallel to directly labeled IgG from patient serum.

As shown in Fig. 9, DCD immunoreactivity is observed at 66 kDa as already in the ApoA- 1 -enriched plasma preparation, whereas the molecular weight of DCD is 11 kDa, in fractions 23 to 27 in both, patient and healthy plasma fractions. Moreover, autoimmunity is pronounced detectable in patient serum fractions and to a much lesser extent in healthy serum. A similar immuno staining pattern as also obtained using directly labeled patient IgG.

Using MS analyses from gel slices excised at the apparent immunoreactive band around 66 kDa derived from healthy and patient SEC fractions 21 through 27 DCD peptides were identified. However, in slices excised around 66 kDa from DCD immune-negative SEC fractions 17 to 19 and 35 to 38 did not reveal DCD sequences.

This result suggests that size exclusion chromatography from total plasma is sufficient and specific for this particular antigen. HDL delipidization followed by enrichment for ApoA-1 clearly reveals lower antigen levels (as can be seen in Fig. 1C).

Experiment 4.2:

Each fraction from the size exclusion chromatography contained a total volume of 500 μΐ. In a further experiment, 1:5 dilutions (each 100 μΐ) of fractions 23 to 27 were applied to the DCD-1L solid phase ELISA using 40 μg/ml for coating. In accordance to the western blot analyses, strong autoimmune activity is present in fractions 24 to 26 in patient serum and no significant OD levels over background were detected from healthy donors as shown in Fig. 10. SEC fraction 38 served as negative control.

It should be noted that the use of long term frozen (-20°C) stored size exclusion fractions generated years ago revealed the same DCD immunostaining results and that different freeze and thaw cycle did not affect the outcome of the results. Moreover, size exclusion chromatography performed in different laboratories and by different operators revealed the same results (data not shown).

Experiment 4.3: In this experiment it was tried to chemically characterize the 66 kDa complex. For this purpose, SEC fractions were treated with formic acid in order to destruct potential oligomers which might be stable even under conditions of SDS-PAGE.

As shown in Fig. 11, the rather harsh treatment does not alter the protein pattern, neither on Coomassie-stained gels nor on the western blot analyses using the commercially available anti- DCD antibodies.

These results strongly suggest that the complex for autoimmunity is present in patient serum, is chemically very stable, and does not result from simple oligomerization of components.

Experiment 4.4:

As fractions 23 -27 represent plasma fractions where most of the HDL particles are eluted in size exclusion chromatography, western blot analyses using 12 different anti-lipoprotein antibodies were performed in order to identify potentially present lipoproteins in the complex. Antibodies used are listed below in Table 1.

Table 1

As a result, none of the antibodies revealed any immunostaining at 66 kDa.

5. Identification of PTN and SDC as components of the 66 kDa autoimmune complex

Recently, Landgraf and colleagues reported of a complex comprising YP-30 with its binding partners PTN and SDC-2 and 3 (Landgraf et al, 2008). PTN is known to promote neurite outgrowth in thalamic neurons. For this activity, an association with SDC-3 is an essential prerequisite. During pregnancy YP-30 peptide, maternally generated by peripheral blood mononuclear cells, is capable of penetrating the blood-placenta barrier and accumulating in the developing fetal brain thereby enhancing thalamic neuron survival and neurogenesis. YP-30 causes oligomerization and generation of large macromolecular complexes containing PTN and SDCs.

Experiment 5.1:

In order to investigate whether such complex could be within the 66 kDa

autoimmunoreactive band, western blot analyses were performed on SEC fractions 21 through 27 using antibodies directed against DCD, a-PTN and SDCs 1-3. As shown in Fig. 12, SEC fractions display immunoreactivity at a molecular weight of about 66 kDa for DCD, PTN and SDCs 1-3.

These data suggest a 66 kDa, SDS-stable complex comprising DCD, PTN and SDC. This is of significance as the apparent molecular weights of all three proteins are different. Moreover, it may be of particular interest that the patient sera do not display any SDC-3 immunoreactivity. Experiment 5.2:

In this experiment, a DCD-1L solid phase ELISA (each 40 μg/ml DCD-1L peptide for coating) was used for competition experiments with recombinant proteins for PTN, SDC- 1 and ApoB proteins.

Recombinant protein and serum were mixed and subsequently applied to solid phase ELISA. Thus, in these experiments a competition of recombinant proteins and solid phase DCD- 1L peptide for the binding of the autoantibody from patient serum was performed.

As shown in Fig. 13, recombinant PTN (Fig. 13A) and recombinant SDC (Fig. 13B) can compete in a dose-dependent fashion for the autoimmune binding to DCD-1L solid phase. ApoB served as a negative control and no competition for the autoimmunoreactivity was observed (Fig. 13C). These data suggest that a complex comprising DCD-1L, PTN and SDC comparable to that of YP-30, PTN and SDCs in the nervous system provides antigenic material for autoimmunity in the cardiovascular system.

6. Screening sera from acute chest pain patients with or without DCD autoantibodies Based on the clinical presentation, electrocardiographic (ECG) features and elevation in cardiac troponin (cTn) acute coronary syndrome (ACS) is divided into ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation ACS (NST-ACS). For the current study, serum samples of 132 patients with acute chest pain were screened for DCD autoantibody expression using the DCD solid phase ELISA as described herein before. Details of sample collection and processing, endpoint definitions and inclusion/exclusion criteria were performed as described (Keller P.-F. 2012). The primary endpoint was a discharge diagnosis of NSTEMI versus chest pain related to unstable angina or to Other diagnoses'. Chest pain aetiology was adjudicated by two senior cardiologists blinded to the participants' biochemical data. Diagnosis of NSTEMI was established using the universal criteria of type 1 acute myocardial infarction (AMI) based on dynamic changes in cTnl levels in the appropriate clinical context, excluding persistent STEMI. Patients were considered to have diagnoses other than NSTEMI when cTnl values were negative. The secondary endpoint was subsequent cTnl elevation (< 0.09 ng/ml) when the first cTnl result was negative (< 0.09 ng/ml).

ROC curve analyses indicate that the anti-DCD IgG is a potential predictor in NSTEMI diagnosis at discharge with an AUC of 0.69 (p=0.0003) (see Figure 14) and for subsequent cTn positivity with an AUC of 0.71 (p=0.0008) (see Figure 15).

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Claims

1. A method for the detection of a cardiovascular disease in a human subject comprising the steps of (a) measuring in a sample of serum or plasma from a human subject the level of endogenous antibodies recognizing Dermcidin (DCD), and

(b) comparing the level measured in (a) with the level of said autoantibodies representative for a human subject of a healthy population, wherein the presence or an increased level of said endogenous antibodies recognizing DCD in the sample of serum or plasma is indicative for a cardiovascular disease.

2. The method for the detection of a cardiovascular disease, wherein said measuring of endogeneous antibodies recognizing DCD comprises the steps of:

(a) providing a sample of serum or plasma from a human subject;

(b) bringing said sample of serum or plasma into contact with a solid matrix where at least one peptide selected from DCD comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of said DCD comprising the amino acid sequence of SEQ ID NO:2 is coupled to, wherein the contacting is under conditions sufficient for binding endogenous antibodies recognizing DCD present in said serum or plasma to said peptide through antigen-antibody interactions; (c) removing any unbound antibody from the surface of said solid matrix; and

3. The method according to claim 2, wherein the fragment peptide is DCD-IL comprising the amino acid sequence of SEQ ID NO:2.

4. The method according to claims 2 or 3, wherein the solid matrix is a microtiter plate.

5. The method according to any one of claims 1 to 4, wherein the cardiovascular disease is acute coronary syndrome.

6. A method for detecting endogenous antibodies recognizing human Dermcidin (DCD) in serum or plasma from a human subject comprising the steps of:

(a) providing a sample of serum or plasma from a human subject;

(b) bringing said sample into contact with a solid matrix where at least one peptide selected from DCD comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of said

DCD comprising the amino acid sequence of SEQ ID NO: 2 is coupled to, wherein the contacting is under conditions sufficient for binding an endogenous antibody recognizing DCD present in said sample of serum or plasma to said peptide through antigen-antibody interactions; (c) removing any unbound antibody from the surface of said solid matrix; and

7. A kit comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2 for detecting in a sample of serum or plasma from a human subject the presence or the level of endogenous antibodies recognizing human Dermcidin (DCD), wherein the presence or an increased level said endogenous antibodies recognizing DCD in the sample of serum or plasma from the human subject compared to a level of said antibodies representative for a human subject of a healthy population is indicative for a cardiovascular disease.

8. Use of a peptide selected from DCD comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of said DCD comprising the amino acid sequence of SEQ ID NO: 2 for determining endogenous antibodies recognizing human Dermcidin (DCD) in a sample of serum or plasma from a human subject, wherein the presence or an increased level of said endogenous antibodies recognizing DCD in the sample is indicative for a cardiovascular disease.

9. Use according to claim 8, wherein the peptide is DCD-1L comprising the amino acid sequence of SEQ ID NO: 2.