WO2011160845A2

WO2011160845A2 - Oxidized phospholipids and lipoproteins, and antibodies thereto, as biomarkers of inflammatory conditions and methods of treatment

Info

Publication number: WO2011160845A2
Application number: PCT/EP2011/003110
Authority: WO
Inventors: Johan FROSTEGÅRD
Original assignee: MEDIRISTA BIOTECHNOLOGIES AB
Current assignee: MEDIRISTA BIOTECHNOLOGIES AB
Priority date: 2010-06-24
Filing date: 2011-06-24
Publication date: 2011-12-29
Anticipated expiration: 2012-12-24
Also published as: WO2011160845A3

Abstract

A method is disclosed for assessing the risk of developing inflammatory conditions, and/or the risk of mortality related thereto, comprising monitoring and/or determining the levels of one or more oxidized phospholipids and lipoproteins, and antibodies thereto, and comparing said levels with predetermined cutoff values, as well as a kit for implementing the method. Also disclosed is a method for treating or decreasing the risk of developing inflammatory conditions, and/or the risk of mortality related thereto, by administering medicaments comprising annexin A5 and/or antibodies to one or more oxidized phospholipids and lipoproteins, as well as bioactive components and/or parts/fragments thereof. Compositions comprising one or more inhibitors of oxidized phospholipids and lipoproteins, as well as bioactive components and/or parts/fragments thereof, as well as compositions comprising one or more conjugates of oxidized phospholipids and lipoproteins, as well as bioactive components and/or parts/fragments thereof, are also disclosed.

Description

Oxidized phospholipids and lipoproteins, and antibodies thereto, as biomarkers of inflammatory conditions and methods of treatment Field of the invention

This invention relates to a method for assessing the risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto, by measuring the levels of one or more markers selected from the list consisting of oxCL, oxPS, oxLDL, MDA-LDL, anti-oxCL, anti-oxPS, anti-oxLDL, anti- MDA-LDL and anti-PC, as well as a kit for implementing the method. It also relates to a method for treating or decreasing the risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto, by administering medicaments comprising one or more compounds selected from the list consisting of annexin A5, anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC, as well as bioactive components and/or parts/fragments thereof. It further relates to compositions comprising one or more inhibitors selected from the list consisting of annexin A5, inhibitors of oxCL, inhibitors of oxPS, inhibitors of oxLDL, inhibitors of MDA-LDL and inhibitors of PC, as well as bioactive components and/or parts/fragments thereof, and also to compositions comprising one or more conjugates selected from the list consisting of oxCL conjugates, oxPS conjugates, oxLDL conjugates, MDA-LDL conjugates and PC conjugates, as well as bioactive components and/or parts/fragments thereof.

Levels of one or more markers selected from the list consisting of oxCL, oxPS, oxLDL, MDA-LDL, anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC may be used to indicate if and to which extent treatment according to the present invention, i.e. prophylactic, therapeutic and palliative treatment with one or more inhibitors selected from the list consisting of annexin A5, inhibitors of oxCL activity, inhibitors of oxPS activity, inhibitors of oxLDL activity, inhibitors of MDA-LDL activity and inhibitors of PC activity, as well as bioactive components and/or parts/fragments thereof, including, but not limited to, antibodies against oxCL (anti-oxCL), antibodies against oxPS (anti-oxPS), antibodies against oxLDL (anti-oxLDL), antibodies against MDA-LDL (anti-MDA-LDL) and antibodies against PC (anti-PC), may be beneficial or necessary to decrease the risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto. Moreover, such levels may be used by physicians to evaluate if, and to which extent, immunization according to this invention, i.e. treatment to elevate natural levels of anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC, and/or to decrease oxCL levels (or activity) and/or oxPS levels (or activity) and/or oxLDL levels (or activity) and/or MDA-LDL levels (or activity), should be the treatment of choice. Background of the invention

Auto-immune diseases and inflammatory conditions are in general major health problems in the Western World and increasing in developing countries. These diseases include, but are not limited to, rheumatic conditions like rheumatoid arthritis and systemic lupus erythematosus (examples of autoimmune diseases), the latter often described as a prototypic autoimmune disease⁶¹, and in text books more than 80 autoimmune rheumatic diseases are described, cf. e.g. Harrison's Principles of Internal Medicine, 17th Edition. Further diseases in the category of inflammatory conditions known to be challenging in the health systems of the Western World are cardiovascular diseases (CVDs), which include, but are not limited to, atherosclerosis, stroke, myocardial infarction, and heart failure (including, but not limited to, acute and chronic heart failure); these diseases are often correlated with or preceded by the metabolic syndrome. The metabolic syndrome has moreover been correlated to Type 2 diabetes mellitus, CVD and coronary heart disease, which are also categorized as inflammatory disorders, cf. e.g. Peter W.F. et al., Metabolic Syndrome as a Precursor of Cardiovascular Disease and Type 2 Diabetes Mellitus, Epidemiology, Circulation. 2005;112:3066-3072.

Atherosclerosis is an inflammatory disease and the dominating underlying cause of cardiovascular disease, including myocardial infarction (Ml) and heart failure, stroke and claudication. Leukocyte extravasation through the endothelial barrier is important in the pathogenesis of inflammatory disorders. Endothelial cells line the lumina of vessels, thus separating and also connecting the blood and the synovial tissue. It has become clear that, in inflammation, endothelial cells are not only passive bystanders but are actively responding to various stimuli, interacting with the immune system. Thus, endothelium is a target for inflammatory leukocytes and their mediators. Upon different stimuli, endothelial cells themselves produce a number of inflammatory mediators, express cellular adhesion molecules (CAMs) and therefore directly influence the immune system, for example by recruiting and/or activating white blood cells modulating the outcome of the inflammatory response. This recruitment is likely not only to be an early step in the immune response to infections, but also in atherogenesis and inflammatory processes in the development of atherosclerosis. (Pober, J.S. et al., 1990)³⁷.

Inflammatory conditions include not only conditions where an undesirable immune response is responsible for a pathological state or parts thereof including autoimmune diseases, infectious conditions, rheumatic conditions, and/or secondary complications, but also those conditions caused by side-effects of medicaments, surgery or other clinical therapy. In this regard, the term "condition" relates to systemic conditions, disease and inflammatory activity or those defined by a tissue and/or organ.

Early recognition and/or prophylactic, palliative and/or curative treatment of one or more infectious conditions, inflammatory conditions and/or pre-inflammatory states, inflammatory activity and/or secondary inflammatory conditions in pathological states are of great interest in clinical care or treatment.

One or more infectious conditions becoming inflammatory conditions, as well as the mortality related thereto, are known problems in clinical care of a broad range of patients with very different symptoms, conditions and diseases, including uremia and autoimmune conditions, such as severe Sjogren's syndrome and SLE, but also tuberculosis and HIV, as well as different status of morbidity and risk related to mortality, whether treated in their homes and/or in the hospital. The risk of being infected with bacteria or virus may be limited by isolation (e.g. avoiding or eliminating contact to people and animals and/or their surroundings) and a high level of hygiene. However, eliminating or even limiting the risk of viral and/or bacterial infections developing undesirably and/or uncontrollably is a greater challenge. A biological marker pointing out patients at special risk of developing one or more infectious conditions and/or determining the mortality related to such conditions, as well as a prophylactic, palliative and/or curative treatment for those patients may be of great use in the clinical care of patients at all levels of illness.

Patients of interest include, but are not limited to, patients with frequent bacterial and/or viral infections, patients with depressed immune systems, patients in poor physical conditions due to operations and/or patients in dialysis. One group of patients of great risk of developing inflammatory conditions are patients admitted into intensive care units (ICUs), which are at great risk of acquiring nosocomial infections. They are susceptible to infection because of the underlying diseases or conditions associated with impaired immunity and are at increased risk of infections during invasive monitoring as well as secondary infections after exposure to broad-spectrum antimicrobials (Eggimann, P. and Pittet, D., 2001 , and Cotran, R.S., 1993)^38,39. Patients on hemodialysis who develop an infection requiring hospitalization are at risk of substantial morbidity and mortality, especially if they are over the age of 60 (Allon, M. et al. 2005)⁴⁰. Chronic obstructive pulmonary disease (COPD) is and will remain a major cause of morbidity and mortality worldwide. The severity of airflow obstruction is known to relate to overall health status and mortality. However, even allowing for common etiological factors, a link has been identified between COPD and other systemic diseases such as cardiovascular disease, diabetes and osteoporosis. COPD is known to be an inflammatory condition and neutrophil elastase has long been considered a significant mediator of the disease. Pro-inflammatory cytokines, in particular TNF (Tumor Necrosis Factor), may be the driving force behind the disease process (Sevenoaks, M. J. and Stockley, R.A., 2006)⁴¹. The above reviewed groups of patients with different inflammatory conditions represent only a small section of patients which daily develop inflammatory conditions due to underlying systemic diseases or dysfunctions and/or diseases or dysfunctions defined by tissue and/or organs, or due to side-effects from surgery or clinical treatment, and do not limit the scope of the invention. However, the above- mentioned conditions do exemplify that inflammatory conditions are a great issue in health care. Thus, risk prediction, prophylactic treatment as well as monitoring options and palliative and/or curative treatment related to development of inflammations are of great interest in order to decrease the morbidity as well as mortality of such patients in need.

The recognition and use of the novel biomarkers, such as oxCL and anti-oxCL, have been shown to be favorable in predicting a potential development of an inflammatory state. Treatment with such agents in immunotherapy has also been revealed to be beneficial in prophylactic, palliative and/or curative treatment of developing inflammatory activity and/or autoimmune conditions and/or secondary inflammatory conditions in disease. The state of the art in prediction of disease in inflammatory conditions varies depending on the specific disease, but in general, there is a clear need of novel risk factors and risk markers. For example, in rheumatoid arthritis, a supposedly autoimmune disease, novel prediction (or risk) markers include anti-citrullin antibodies. Rheumatoid factor (RF) is also used, but is less effective. In dementia/Alzheimer^'s disease, which also has inflammatory properties, genetic markers can be used (apoE4). In SLE there is no established risk marker.

Palliative treatment of inflammatory conditions varies from condition to condition as well as among patients and their overall physical state. Common choices of treatment in inflammatory conditions are immunosuppressant drugs, non-steroidal- inflammatory drugs (NSAIDs), histamines, anti-rheumatic drugs, such as the disease-modifying anti-rheumatoid drugs (DMARDs) and different chemotherapies, as well as antibacterial, antifungal and antiviral drugs. In general, a specific combination of drugs has to be selected for each patient due to side-effects, disease, physical state and several other circumstances influencing the patient's course of disease. For example, most antibiotics are effective only against certain bacteria. In selecting an antibiotic to treat a person with an infection, doctors estimate which bacteria are likely to be the cause. Moreover, antibiotics that are effective in the laboratory do not necessarily work in an infected person. The effectiveness of the treatment depends on how well the drug is absorbed into the bloodstream, how much of the drug reaches the sites of infection in the body, and how quickly the body eliminates the drug. These factors may vary from person to person, depending on other drugs being taken, other disorders present, and the person's age. In selecting an antibiotic, doctors also consider the nature and seriousness of the infection, the drug's possible side effects, the possibility of allergies or other serious reactions to the drug, and the cost of the drug (Merck Manuals, online medical library)⁵⁷. Prediction and modulation of inflammation must take into account a complex biology not only including cytokines and other inflammatory factors and/or cells, but also several other biochemical cascades acting in parallel with the immune system. Such cascades include, but are not limited to, the complement system and coagulation. The recognition and use of biomarkers predicting one or more potential developing inflammatory condition may thus be as favorable as biomarkers indicating the patient's status in regard to risking inflammatory conditions.

Mortality risk is greatly enhanced in patients with end-stage renal disease (ESRD). In addition to being an important clinical problem, this increased mortality risk could also be of importance in mortality studies in general, where one or more infectious conditions play major roles.

Acute renal failure (ARF) is a frequent problem in the intensive care unit and is associated with a high mortality in critically ill patients. Patients on maintenance dialysis (hemodialysis and peritoneal dialysis¹) appear to experience an increased risk of developing ARF due to secondary infections (bacterial and/or virus). This risk is among others connected to the exposure of bodily fluids during dialysis procedure as well as the patient's general poor physical condition.

Peritoneal dialysis (PD) is a treatment for patients with severe chronic kidney failure. The process uses the patient's peritoneum in the abdomen as a membrane across which fluids and dissolved substances (electrolytes, urea, glucose, albumin and other small molecules) are exchanged from the blood. Acute renal failure (ARF) is defined as decreased glomerular filtration, detected by increased serum creatinine. ARF can be a consequence of three etiological scenarios; prerenal causes, intrarenal causes and postrenal causes, and is characterized in that the failure is preceded by an acute injury, which may be healed and regenerated in some degree, thus not resulting in failure of the kidney.

Prerenal causes include, but are not limited to, renal failure due to the ingestion of non-steroidal anti-inflammatory drugs (NSAIDs), angiotensin converting enzyme (ACE) inhibitor initiated acute renal failure in patients suffering from hypoperfusion of the kidneys due to, amongst others, renal artery stenosis, congestive heart failure or intrarenal small vessel disease, dependent on angiotensin ll-mediated vasoconstriction of the efferent arteriole to maintain renal perfusion.

Intrarenal causes for acute renal failure may be further divided into inflammatory diseases and acute tubular necrosis. Inflammatory diseases are included in the non- exhaustive list of conditions; vasculitis, glomerulonephritis and drug-induced injury. Acute tubular necrosis may be caused by a plurality of conditions, including, but not limited to, ischemia, poisons and hemolysis. Notable intrarenal diseases are among others the toxic effects of aminoglycoside antibiotics or rhabdomyolysis, after crush injury of muscle results in precipitation of myoglobin into the renal tubules. The former may be prevented by monitoring especially renal compromised patients and/or elderly patients during treatment with antibiotics. Furthermore, sepsis is a common cause of acute renal failure, including prerenal conditions in combination with intrarenal causes.

Postrenal causes for acute renal failure may be conditions, where the urinary tract is obstructed.

Chronic renal failure (CRF) may be caused by a non-exhaustive list of conditions, such as diabetes mellitus, hypertension and glomerulonephritis, as well as polycystic kidney disease, obstructions and/or infections.

The pathogenesis of acute renal disease is very different from that of chronic renal disease. Whereas an acute injury of the kidney results in death and sloughing of the tubular epithelial cells, often followed by regeneration with establishment of normal architecture in acute renal disease, chronic injuries in chronic renal disease result in irreversible loss of nephrotic mass. As a result, an increased functional burden is borne by fewer nephrons, leading to increased glomerular filtration pressure and hyperfiltration, subsequently leading to fibrosis and scarring (glomerular sclerosis), speeding up the progression to uremia, which indicates the stadium of renal disease when residual renal function is inadequate and renal failure is in progress.

About 50% of nephrons can be lost without any short term evidence of functional impairment. Thus, maintenance dialysis may be required for patients with loss of nephrotic mass over 50%, in periods between regeneration of renal tissue (e.g. during chronic renal disease/failure) or whenever glomerular filtration is severely impaired.

Cardiovascular complications are the leading cause of mortality in patients with end- stage renal disease (ESRD). The excess cardiovascular risk and mortality is already demonstrable in early renal disease and in patients with chronic renal failure, with the highest relative risk of mortality in the youngest patients. The high risk for cardiovascular disease (CVD) results from the additive effect of multiple factors, including but not limited to hemodynamic overload and several metabolic and endocrine abnormalities more or less specific to uremia. CVD includes disorders of the heart, including, but not limited to, left ventricular hypertrophy [LVH] and cardiomyopathy, as well as disorders of the vascular system, including, but not limited to, atherosclerosis and arteriosclerosis, these two disorders being usually associated and interrelated (Semin Dial. 2003 Mar-Apr; 16(2):85-94).

Early recognition and/or prophylactic, palliative and/or curative treatment of one or more infectious conditions in general and/or pre-inflammatory states, inflammatory activity and/or secondary inflammatory conditions in renal disease could help clinical management, but current indices lack sufficient predictive value for developing one or more infectious conditions, ARF and/or CRF (ARF and CRF are collectively referred to as renal failure). Therefore, there is a great need for biomarkers in detecting one or more infectious conditions, renal disease, renal failure, renal tubular damage, injury and/or dysfunction. Moreover, biomarkers related to one or more infectious conditions, inflammatory activity and and/or secondary inflammatory conditions related to renal disease, renal failure and/or renal tubular injury, damage and/or dysfunction at an early stage before a decline in glomerular filtration rate is noted by an increased serum creatinine are of great interest. Moreover, a method for evaluation of the mortality related to such conditions, activity or diseases may be a powerful tool for physicians in determining treatment and the status of the patient's overall physical condition.

Known biomarkers related to renal failure include tubular enzymes (alpha- and pi- glutathione S-transferase, N-acetyl-glucosaminidase, alkaline phosphatase, gamma-glutamyl transpeptidase, Ala-(Leu-Gly)-aminopeptidase, and fructose-1 ,6- biphosphatase), low-molecular weight urinary proteins (alphal- and beta2- microglobulin, retinol-binding protein, adenosine deaminase-binding protein, and cystatin C), Na+/H+ exchanger, neutrophil gelatinase-associated lipocalin, cysteine- rich protein 61 , kidney injury molecule 1 , urinary interleukins/adhesion molecules, and markers of glomerular filtration, such as proatrial natriuretic peptide (1-98) and cystatin C (Trof RJ et al. 2006, Schock Sep 26(3):245-53).

Studies comparing healthy subjects and end-stage renal disease patients on maintenance hemodialysis revealed that patients with ARF had significantly higher plasma levels of all measured cytokines. Proinflammatory cytokines IL-6 and IL-8 were significantly higher in nonsurvivors versus survivors, and anti-inflammatory cytokine IL-10 was also significantly higher in nonsurvivors. For each natural log unit increase in the levels of IL-6, IL-8, and IL-10, this study established correlation with odds of death to be increased by 65%, 54%, and 34%, respectively, corresponding to increases in relative risk of approximately 30%, 25%, and 15% (Simmons et al., Kidney Int. 2004 Apr; 65(4): 1357-65). This inflammatory activity indicates that ARF may be an inflammatory condition itself and that this inflammatory state affects the mortality of the condition. Chronic kidney injuries, such as seen in, for example, renal disease and/or failure, are commonly considered a chronic inflammation, supported by the elevated levels of TNF-alpha, IL-1beta and IL-6 in bodily fluids of patients.

Modulation of inflammation must therefore take into account the complex cytokine biology in patients with established renal failure. The recognition and use of biomarkers predicting a potential development into this inflammatory state of ARF and/or CRF may thus be as favorable as biomarkers indicating the patient's status in regard to risking secondary infectious diseases, CVD and the development of ARF. Low levels of anti-oxCL have previously been shown to be correlated with higher risk of developing inflammatory conditions. OxCL has been found to have proinflammatory properties, and it has also been shown that a low concentration of anti- oxCL is an effective indicator of inflammatory conditions. Surprisingly, it has been found that anti-oxCL is a suitable biomarker for predicting risk, early pathogenesis, progression of as well as mortality related to one or more infectious conditions, renal disease and/or conditions including renal damage, injury and/or dysfunction. Moreover, the risk of secondary inflammatory conditions in renal disease, such as CVD and secondary infectious conditions and/or inflammatory activity in renal disease and/or renal failure can be predicted when determining the anti-oxCL level in bodily fluids of patients.

Cardiolipin (CL) is a dimeric phospholipid which is known to be present in eukaryotic cells, bacteria and Archaebacteria but its functional role is only partly known, (Schlame, M., 2008)¹. It is more prevalent in cells with high metabolic activity, like heart and skeletal muscle, and especially in mitochondrial membranes. The presence of CL in mitochondria and bacteria is interesting from an evolutionary point of view since mitochondria are likely to have a bacterial origin, (Martin, W. et al., 2001 )². Also lipoproteins, including low density lipoprotein (LDL), contain CL, in contrast to what has previously has been thought, and two thirds of CL are present in low density lipoprotein (LDL), (Deguchi, H. et al., 2000)³.

CL has a unique dimeric structure, highly enriched in linoleic acid groups susceptible to oxidation (Schlame, M. et al., 2000, and Chicco, AJ., and Sparagna, GC, 2007)^4,5. It has been suggested to play a role in generation of an electrochemical potential for substrate transport and ATP synthesis both in bacteria and mitochondria, (Belikova, NA. et al., 2007, and Basova, LV. et al., 2007)⁶·⁷ CL that has undergone oxidation (oxCL) promotes delocalization and release of cytochrome c, predisposing to its release from mitochondria and the activation of the cell death programmes, (Chicco, AJ. and Sparagna, GC, 2007, Gonzalvez, F. et al., 2007, and Nakagawa, Y., 2004)⁵·⁸·⁹

Antibodies against CL are known to be thrombogenic both in arteries and veins (when present in exceedingly high levels typically above 2 standard deviations as compared to a normal healthy control group). This is especially the case in autoimmune diseases including SLE. However, little is known about a clinical role of antibodies against oxCL (anti-oxCL). PS (also named phosphatidylserine, 1 ,2-diacyl-sn-glycero-3-phospho-L-serine and Ptd-L-Ser) is distributed widely among animals, plants and microorganisms. PS usually represents less than 10% of the total phospholipids, the greatest concentration being in myelin from brain tissue. However, it may comprise 10 to 20 mol% of the total phospholipid in the plasma membrane and endoplasmic reticulum of the cell.

In animal cells, the fatty acid composition of PS varies from tissue to tissue, but does not appear to resemble the precursor phospholipids. In human plasma, 1- stearoyl-2-oleoyl and 1-stearoyl-2-arachidonoyl species predominate, but in brain (especially grey matter), retina and many other tissues 1 -stearoyl-2- docosahexaenoyl species are very abundant. Indeed, the ratio of n-3 to n-6 fatty acids in brain PS is very much higher than in most other lipids. (Wood, R. and Harlow, R.D., 1969, and Yabuuchi, H. and O'Brien, J.S., 1968)⁴²·⁴³

Apoptosis (also known as "controlled cell death") is a cell suicide program under the influence of hormones, growth factors and cytokines, which depending upon the receptors present on the target cells, may activate a genetically controlled cell elimination process. During apoptosis, the cell membrane remains intact and the cell breaks into apoptotic bodies, which are engulfed by macrophages and other phagocytic cells. Apoptosis, in contrast to necrosis, is not harmful to the host and does not induce any inflammatory reaction. The principal events that lead to inflammatory conditions are necrosis and/or cell damage induced by chemical/physical injury, anoxia or starvation. Cell damage means leakage of cell contents into the adjacent tissues, resulting in the capillary transmigration of granulocytes to the injured tissue. The accumulation of neutrophils and release of enzymes and oxygen radicals enhances the inflammatory reaction and form part of the factors controlling the accumulation and the tissue load of granulocytes and their histotoxic products in inflammatory processes. In chronic inflammation, oxidized phospholipids have been suggested to be part of the oxidative stress, triggering the chronic conditions; however, further evaluation or elaboration of which oxidized phospholipids may be involved have not been made (Kadi, A. et al., 2004)⁴⁴.

PS is known to have an essential role in the chain of events of apoptosis after a response to particular calcium-dependent stimuli. Also, PS is exposed on the surface of platelets upon their activation (Sun, J. et al., 1993)⁴⁵.

The normal distribution of PS on the inner leaflet of the membrane bilayer is affected when the enzyme scramblase is stimulated and/or aminophospholipid translocases are inhibited. The enzyme scramblase is able to move PS in both directions across the membrane, whereas aminophospholipid translocases return the lipid to the inner side of the membrane. After transfer to the outer leaflet of the cell, it is believed that a receptor on the surface of macrophages and related scavenger cells recognizes the PS and facilitates the removal of the apoptotic cells and their potentially toxic or immunogenic contents in a non-inflammatory manner. Binding of PS to specific proteins, such as apolipoprotein H ^-glycoprotein 1), enhances the recognition and clearance. This process is essential for the development of lung and brain, and it is also relevant to clinical situations where apoptosis plays an important part, such as cancer, chronic autoimmunity, and infections.

It has also been reported that PS is involved in blood coagulation by being represented on the surface of plasma membrane, binding blood coagulation proteins such as X, Xa and Va and forming stable prothrombinase complexes. Annexin A5 has been shown to inhibit this process (van Heerde, W.L. et al., 1994)⁴⁶. Moreover, the use of antibodies with high affinity for PS (at least 90% of the affinity of annexin A5 for PS) has been suggested as treatment for patients at risk of thrombosis (US patent US 2005/0222030)⁴⁷ and for treatment of patients with vascular dysfunction (International patent application WO 200907764)⁴⁸. Annexin A5 has been disclosed in the prevention and treatment of artherosclerosis and plaque rupture (International patent application WO 20Ό5099744)⁴⁹ and treatment of inflammatory disorders, SLE as well as sepsis (International patent application WO 2010043045⁵⁰ and Cederholm, A., and Frostegard, J., 2007⁵¹) and restenosis (International patent application WO 2009103977)⁵². Phospholipids (e.g. PS) as well as oxidized phospholipids (e.g. oxPS) have been correlated with inflammatory conditions in prior art. Especially PS and oxPS have been shown to be present on the surface of apoptotic cells (Kadi, A. et al., 2004, and Letter to editor, 2004)⁴⁴·⁵³.

It has also previously been shown that the class B cluster of differentiation 36, scavenger receptor, (CD36) plays an essential role in macrophage clearance of apoptotic cells in vivo. Further, macrophage recognition of apoptotic cells via CD36 has been shown to occur via interactions with membrane-associated oxidized PS (oxPS) and, to a lesser extent, oxidized phosphatidylcholine (oxPC), but not nonoxidized PS molecular species (Greenberg, M.E. et al., 2006)⁵⁴. Also lipoproteins including low density lipoproteins (LDLs) contain PS in contrast to what previously has been thought.

It has been hypothesized that oxidized phospholipids that emerge during apoptosis positively regulate the resolution of acute bacterial inflammation, on the one hand by inducing protective, anti-inflammatory genes, and on the other hand by representing a negative feedback by inhibiting LPS action and thus blunting innate immune response. Antibodies against CD36 and antibodies against the phosphatidylserine receptor have been shown to inhibit the uptake of oxPS coated cells by macrophages. This finding in combination with CD36 and the PS receptor recognition of oxPS indicates that antibodies against CD36 may inhibit phagocytosis by inhibiting oxPS normal activity, promoting engulfment and therefore being inflammation protective (Kadi A. et al., 2004)⁴⁴. Another hypothesis regarding the clearance of apoptotic hepatocytes in alcoholic liver disease confirms this understanding of oxPS function by suggesting that antibodies against oxPS hide phagocytic recognition sites on oxPS, impairing the anti-inflammatory response (e.g. engulfment of the apoptotic cell) (Albano, E., 2008)⁵⁵.

Further, antioxidants that are capable of inhibiting PS oxidation have been suggested to interfere with PS externalization and/or its recognition by macrophages and a mechanistic link between the oxidative stress in a cell during apoptosis, and the oxidation of PS stimulating a PS-dependent signaling pathway culminating in the disposal of cells by macrophages has previously been suggested (Kagan, V.E. et al., 2003)⁵⁶. Another interesting study showed that milk fat globule epidermal growth factor 8 (MFG-E8) serves as an opsonin for apoptotic cells and recognizes phosphatidylcholine (PC) and PS and preferably interacts with oxPS (Letter to the editor 2004)⁵³.

Oxidized low density lipoprotein (oxLDL) is taken up through specific scavenger receptors on endothelial cells, which are not down-regulated when exposed to increasing amounts of oxLDL (as opposed to the uptake of LDL), (Hansson, G.K., 2005)¹⁴. Inhibition of the scavenger function is generally believed to be atheroprotective, preventing foam cell formation in the vascular wall which is a key process in development of atherosclerosis. In line with this, mice defective in scavenger receptor function develop less atherosclerosis as compared to control mice, (Febraio, M. et al. 2000)²³. It should be noted, however, that recent findings indicate that different scavenger receptors may play different roles and the role of scavenger receptors may vary depending on disease stage and type, (Moore, K.J. et al., 2006)²⁴.

If oxPS is predominantly exposed on oxLDL, binding and uptake of oxLDL could through oxPS promote atherogenesis. On the other hand, if oxPS is present mainly on other compounds, e.g. apoptotic and/or necrotic cells, other proteins, or even bacteria, it is not clear how this would influence foam cell development, which in principle could be decreased. However, oxPS has been found to play an important role in regulation of apoptosis as well as blood coagulation, which are both bio- molecular systems interacting with the immune response as reviewed above. Further research is needed to clarify which parts of oxLDL play the major role in foam cell formation, as well as if and to what extent phospholipids and/or their oxidized variants influence such processes.

In conclusion, prior art associates oxPS with apoptosis and clearance of apoptotic cells preventing inflammation by acting as an "eat me" marker to phagocytic cells and/or opsonins attracting such engulfing cells, when presented on the membrane surface of apoptotic cell.

Surprisingly, it has now been found that oxPS (and not PS) and/or antibodies against oxPS (anti-oxPS) and not anti-PS have predictive abilities regarding the risk of developing inflammatory conditions when detected in bodily samples of animal origin. In contrast to the findings of the prior art, it has been found that increased levels of oxPS are correlated with increased risk of inflammation. Moreover, decreased levels of anti-oxPS are correlated with an increased risk for developing inflammatory conditions.

These findings have been made by analyzing a cohort of patients with SLE, establishing a first-time connection between oxPS and anti-oxPS levels as predictive markers for developing inflammatory conditions and the potential of anti- oxPS as inflammation-protective agents in prophylactic, palliative and/or curative treatment. Thus, the increased levels of oxPS (or increased activity thereof) detected in an animal is a potentially predictive value when assessing these individuals' risk of developing acute and/or chronic inflammatory conditions, when infected by microbes or other infectious agents, or experiencing other deficiencies related to systemic and/or organ and/or tissue related disease, injury and/or damage, which can be interpreted as latent stages of (or prior to) developing inflammatory conditions.

Potentially, the measurement of oxPS and/or anti-oxPS levels in animals prior to, or during any kind of clinical treatment may give confirmational information to a presumption of increased risk of inflammation upon infections at an early stage and most importantly and preferably before onset of such an inflammation. This could indicate that prophylactic and/or palliative treatment (at early stages, maybe even before symptoms are detected) by administering anti-oxPS could be beneficial, reducing the possibilities of inflammations due to infections for animals at such risk by increasing serum levels of anti-oxPS, thereby decreasing oxPS activity and/or levels of oxPS, under or after the onset of treatment related to the primary disease. In this way, the risk of developing inflammatory conditions or consequences of such inflammations may be constrained dramatically.

Moreover a prediction marker as well as one general prophylactic, palliative and/or curative treatment of inflammatory disease given by the present invention has a great potential of reducing the need and/or amount of additional and disease specific treatments and has the potential of saving time and decreasing mortality and morbidity related to such conditions.

Summary of the invention

Anti-PC has anti-inflammatory properties, inhibiting inflammatory phospholipids like PAF, and anti-PC may be atheroprotective in humans, since these antibodies are negatively associated with atherosclerosis development. Further, as previously published, low anti-PC levels are independently associated with increased risk of cardiovascular diseases (CVD). Moreover, it has surprisingly been found that oxidized phosphatidylserine (oxPS), and not phosphatidylserine (PS), increase the expression of vascular cell adhesion molecule-1 (VCAM-1) (also known as CD106) on the surface of endothelial cells and increase tumor necrosis factor (TNF) levels, suggesting a stimulatory role of oxPS in inflammatory events. Increased anti-oxPS levels have been revealed to have a strong negative correlation with both TNF serum levels and VCAM-1 expression on endothelial cells. It has further been shown that decreased levels of anti-oxCL are correlated with infectious conditions, and thus therapy involving an elevation of the patient's serum anti-oxCL levels could potentially decrease the risk of such patients developing infectious conditions into chronic inflammatory conditions. Furthermore, the combination of high anti-MDALDL with high anti-oxCL or high anti-oxPS levels or high anti-OxLDL levels, as well as the combination of high anti-PC levels with high anti-MDA-LDL levels and/or high anti-OxLDL levels in the ELSA study gave a strong protective effect against atherosclerosis, indicating that the combination of these markers is useful in assessing disease risk. These results also suggest that a combination of these antibodies, as well as antibodies against components, fragments or derivatives of MDA-LDL or OxLDL (such as apoBIOO or fragments thereof), would be more beneficial than single therapies. The antibodies could either be endogenously produced as a result of active immunization (vaccination), or exogenously administered (passive immunization). Likewise, our finding that a combination of low IgM anti-PC with low anti-oxCL or low anti-oxPS increases the excess risk of CVD would suggest that the combination of these markers is useful in assessing disease risk and that a combination of these antibodies could also improve therapy, if raised through active immunization or passive immunization.

These new findings relating to inflammatory-protective antibodies (anti-oxCL, anti- oxPS, anti-oxLDL, anti-MDA-LDL and/or anti-PC) mentioned above, all being protective against inflammatory conditions, are useful in assessing disease risk, as well as revealing a new combination-possibility in replacement-therapy relying on a synergic and/or potentiated effect through a combination, in patients at risk of developing inflammation.

Immunization in regard to stimulating the patient's own production of natural antibodies against oxCL, oxPS, oxLDL, MDA-LDL and/or PC may reduce the risk of patients with inflammatory conditions and provide a tool for prophylactic, palliative and/or curative treatment of developing inflammatory conditions, and/or inflammatory activity and/or secondary inflammatory conditions in disease and/or primary or secondary infectious conditions. Moreover treatment with such combinations may reduce the mortality risk of patients with low natural levels of the above antibodies and/or high levels of oxCL, oxPS, oxLDL, MDA-LDL and/or PC, especially during other medical treatment with increased risk of secondary infections and/or suppressive effect on the overall immune system, such as transfusion medicine, immunosuppressant medicine and/or chemotherapy.

Further, we hypothesize that a combination comprising inhibitors of oxCL and inhibitors of oxPS and optionally inhibitors of PC could be effective against infections as such. Atherosclerosis is an inflammatory disease where oxidized low-density lipoprotein may play an important role through oxCL. Both atherosclerosis and its clinical consequence cardiovascular disease (CVD) are highly prevalent in patients with end-stage renal disease (ESRD). The association between antibodies against oxCL and risk of death in patients suffering from renal diseases and/or renal failure, undergoing haemodialysis (HD) has surprisingly been found.

The surprising characteristics of oxCL are confirmed by the fact that low concentrations of antibodies to mammal oxCL are an effective indicator of development of and/or mortality related to developing infectious disease, in one embodiment especially related to renal disease and/or conditions, renal damage, injury and/or dysfunction. Moreover, the risk of secondary inflammatory conditions in renal disease, such as, but not limited to, CVD and/or development of ARF, as well as inflammatory activity in renal disease and/or related to renal damage and/or injury and/or dysfunction can be predicted when determining the anti-oxCL level in bodily fluids of patients.

It is within this present invention found that antibodies to this particular antigen (i.e. oxCL) do not develop sufficiently in patients before the clinical onset of inflammatory conditions due to one or more infectious conditions and/or inflammatory activity in renal disease related to renal damage, injury and/or dysfunction and/or secondary inflammatory conditions in renal disease and presently it is a preferred hypothesis that an insufficiently developed natural immunity against oxCL represents an underlying mechanism for development of such diseases.

Moreover, it is within this present invention found that patients found with insufficiently developed antibodies to this particular antigen (i.e. oxCL) are subject to increased mortality related to developing one or more infectious conditions, and/or inflammatory activity in renal disease related to renal damage, injury and/or dysfunction and/or secondary inflammatory conditions in renal disease. Potentially, replacement-therapy with anti-oxCL in patients with low natural anti- oxCL levels, or immunization in regard to stimulating the patient's own production of natural antibodies against anti-oxCL may reduce the risk of developing infectious disease and/or reduce the risk of patients with renal disease and/or conditions, renal damage, injury and/or dysfunction, to develop inflammatory activity and/or secondary inflammatory disease and/or to develop ARF and/or CRF. Moreover, treatment with anti-oxCL may reduce the mortality risk when administered during renal disease, especially during dialysis. Thus, the anti-oxCL levels in the patients' bodily fluids may indicate whether and to which extent an immunization and/or a replacement therapy with anti-oxCL may reduce the morbidity of the disease as well as the morbidity related to developing one or more infectious conditions, ARF and/or CRF.

It has been found that oxidized PS, in contrast to native PS, has pro-inflammatory properties and serves as a predictive risk marker for developing inflammatory conditions.

It has also surprisingly been found that oxPS, and not PS, increases the expression of vascular cell adhesion molecule-1 (VCAM-1) (also known as cluster of differentiation 106 receptor (CD106)) on the surface of endothelial cells. Thus, oxPS function is not restricted to the expression on apoptotic cells, as described by the prior art reviewed in "backgrounds of the invention". In contrast to the prior art, a stimulatory role of oxPS in inflammatory events has now been revealed. VCAM-1 is commonly used as a biological marker for vascular inflammation. Moreover, it has been found that annexin 5A (a calcium-phospholipid binding protein involved in the coagulation cascade) inhibits the inflammatory effect of oxPS. In patients with systemic lupus erythematosus (SLE), a negative correlation between anti-oxPS and tumor necrosis factor (TNF) levels has now been found, further supporting the proinflammatory role of oxPS.

The surprising characteristics of oxPS are confirmed by the fact that low concentrations of anti-oxPS and high levels of oxPS are an effective indicator of developing inflammatory conditions. Anti-oxPS, and not anti-PS, has now been found to have inflammation protective abilities and, when present in low levels relative to predetermined cutoff values, anti-oxPS predict an increased risk of developing inflammatory conditions. Thus, prediction of increased risk of developing inflammatory conditions can accordingly be based on using oxPS, anti-oxPS or both oxPS and anti-oxPS as markers.

A great potential lies in prophylactic, palliative and/or curative replacement-therapy with anti-oxPS in animals with low natural levels of anti-oxPS or high levels of oxPS to prevent, reduce or cure development of inflammation or reduce physical consequences of inflammatory conditions. Immunization by stimulation of an animal's own production of natural antibodies against anti-oxPS reduces the risk of developing infectious and/or inflammatory conditions, inflammatory activity and/or secondary inflammatory conditions related to damage of tissue and/or organs. Replacement therapy may be conducted by administering inhibitors of oxPS and/or anti-oxPS (or bioactive components and/or parts thereof) by injection and/or administered via infusion products enriched with inhibitors of oxPS and/or anti-oxPS (bioactive components and/or parts thereof) such as, but not limited to, plasma products with anti-oxPS and/or any other suitable means of supplying the patient's bodily fluid with anti-oxPS. Vaccines according to this invention that elevate the animals' natural synthesis of anti-oxPS may also be administered by injection and/or infusion.

Several methods of administration may be used, including, but not limited to, intradermal, subcutaneous, intramuscular, intravenous, intraosseous, and intraperitoneal injection or infusion. Long-acting forms of subcutaneous/intramuscular injections such as depot injections are available for various drugs, and may also be a choice of administration in connection with the present invention. Administration to mucosal tissues and lungs may be used, including, but not limited to, liquid and solid preparations for nebulisation and rectal application. Dermal application in the form of emulsions, creams, ointments, lotions, gels or other standard formulations for skin preparations may be used. OxPS conjugates, oxPS as such or bioactive components and/or parts thereof, optionally in combination with any suitable adjuvants, may be administered orally, nasally, rectally or through other mucosal tissues in order to achieve an alternative immune-specific reaction than achieved by injection.

Transfusion medicine (or transfusiology) is the branch of medicine that is concerned with the transfusion of blood and blood components. A blood product is any component of the blood which is collected from a donor for use in a blood transfusion. Whole blood is uncommonly used in transfusion medicine at present; most blood products consist of specific processed components such as red blood cells, blood plasma, or platelets. However, other blood substitutes such as, but not limited to, cryoprecipitate, plasma frozen within 24 Hours after phlebotomy (PF24), fresh frozen plasma (FFP) or cryosupernatant may also be used. Whenever blood products are needed or when other products used in transfusion medicine are administered, a beneficial effect in relation to developing inflammatory conditions due to infections during the process of injection and/or infusion can be achieved by enriching the product with a medicament containing anti-oxPS. Thus, according to the present invention, oxPS or anti-oxPS or both oxPS and anti- oxPS are used as biologic risk markers for inflammatory conditions of different underlying conditions, diseases and/or dysfunctions.

The invention also relates to use of inhibitors of oxPS activity and/or antibodies against oxPS in a method of replacement therapy, i.e. elevating the patients' serum levels of anti-oxPS in patients with low levels of functional anti-oxPS and/or high levels of oxPS and therefore experiencing an increased risk of developing inflammatory conditions. Means for replacement therapy may be anti-oxPS as such, administered by injection and/or administered through enrichment of infusion products, such as, but not limited to, plasma products with anti-oxPS and/or any other suitable means of supplying the patient's bodily fluid with anti-oxPS. The invention also relates to a vaccine (agent that is suitable for increasing the anti- oxPS immune response in an animal, in particular oxPS conjugates, oxPS as such or bioactive components and/or parts thereof optionally in combination with any suitable adjuvants). In patients with normal production and levels of oxPS and/or functional anti-oxPS, a vaccine with oxPS and/or fragments thereof may be a choice of prophylactic treatment prior to surgery, in order to elevate anti-oxPS levels and to decrease the risk of developing post-operative inflammatory conditions due to infections, or prevent inflammation due to treatment with immunosuppressant drugs, in patients with unstable immune systems or other clinical treatments known to increase risk of infection and/or inflammation.

The present invention further relates to a kit needed to perform the immunoassay comprising oxPS markers.

In one aspect of the present invention, anti-oxPS levels measured in a sample of animal origin (such as, but not limited to, bodily fluids and/or tissue) may indicate whether and to which extent an immunization using a vaccine against oxPS and/or a replacement therapy with anti-oxPS or bioactive components and/or parts thereof may reduce the risk and/or the morbidity related to such conditions.

In another aspect of the present invention, oxPS levels measured in a sample of animal origin (such as, but not limited to, bodily fluids and/or tissue) may indicate whether and to which extent an immunization using a vaccine against oxPS and/or a replacement therapy with anti-oxPS or bioactive components and/or parts thereof may reduce the risk and/or the morbidity related to such conditions.

In another aspect of the present invention, anti-oxPS and oxPS levels measured in a sample of animal origin (such as, but not limited to, bodily fluids and/or tissue) may indicate whether and to which extent an immunization using a vaccine against oxPS and/or a replacement therapy with anti-oxPS or bioactive components and/or parts thereof may reduce the risk and/or the morbidity related to such conditions. In connection with this invention, the term "infectious condition" is understood in the general meaning of the term, comprising microbial infections, provoking a normal immune response which will be cured in an untreated state (e.g. not develop into a prolonged inflammatory condition).

In connection with the present invention, the term "developing infectious condition" means a condition caused by an infectious agent of bacterial, protozoal, viral or parasitic type, developing into an inflammatory condition due to an abnormal increased or decreased immune response to an exogenous stimulus (e.g. virus and/or bacteria and/or toxins originating from infectious agents).

In connection with this invention, the term "developing" is understood in the general meaning of the word, which comprises both developing and progressing. In connection with this invention, "condition" means both disease and/or condition, systemic and/or defined by tissue and/or organs.

In connection with this invention, "renal disease" means both renal disease and/or renal conditions where glomerular filtration is acutely and/or chronically decreased and/or failing due to prerenal, intrarenal and/or postrenal causes. Such renal diseases include, but are not limited to, renal conditions and/or failure including ARF and/or CRF where prerenal causes include, but are not limited to, the ingestion of non-steroidal anti-inflammatory drugs (NSAIDs), angiotensin converting enzyme (ACE) inhibitor initiated acute renal failure in patients suffering from hypoperfusion of the kidneys due to, amongst others, renal artery stenosis, congestive heart failure or intrarenal small vessel disease, dependent on angiotensin ll-mediated vasoconstriction of the efferent arteriole to maintain renal perfusion. Intrarenal causes may be further divided into inflammatory diseases and acute tubular necrosis. Inflammatory diseases are included in the non-exhaustive list of conditions such as; vasculitis, glomerulonephritis and drug-induced injury. Acute tubular necrosis may be caused by a plurality of conditions, such as, but not limited to, ischemia, poisons and hemolysis. Notable intrarenal causes are among others the toxic effects of aminoglycoside antibiotics or rhabdomyolysis, after crush injury of muscle results in precipitation of myoglobin into the renal tubules. Postrenal causes for renal disease including acute renal failure may not be limited to conditions where the urinary tract is obstructed. Furthermore, CRF may also be caused by a non- exhaustive list of conditions, such as diabetes mellitus, hypertension and glomerulonephritis, as well as polycystic kidney disease, obstructions and/or infections.

In connection with the present invention, "inflammatory activity" means inflammatory activity in tissue originated in a diseased, damaged and/or injured organ not related to a systemic inflammatory condition. Inflammatory activity includes, but is not limited to, acute and chronic tissue and/or organ damages or injuries showing elevated levels of inflammatory biomarkers that include, but are not limited to, TNF, IL-1-beta, IL-6 and IL-8 in bodily fluids of patients. In connection with this invention, "secondary inflammatory condition in disease" means inflammatory activity, conditions and/or disease with an onset following the onset of a primary disease, condition and/or tissue or organ damage, injury and/or dysfunction. Thus, secondary inflammatory conditions in this invention include both systemic inflammatory conditions and inflammatory conditions defined by tissue and/or organ, and include, but are not limited to, any of the following diseases, conditions and/or activities; auto-immune diseases, type II diabetes, Alzheimer's disease, dementia in general, rheumatic diseases, atherosclerosis, acute and/or chronic inflammatory conditions, type I diabetes, rheumatoid arthritis, psoriasis, psoriatic arthritis, acne, ankylosing spondylitis, Reiter's Syndrome, systemic lupus erythematosus (SLE), dermatomyositis, Sjogren's syndrome, multiple sclerosis, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), arthritis, including osteoarthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, and/or an allergic disorder.

In connection with the present invention, "animal" means mammals and other vertebrates and invertebrates, in particular mammals such as mice, rats, rabbits, dogs, cats, cattle, horses and humans. In connection with the present invention, samples of animal origin means any natural sample from an animal, including but not limited to tissue and/or fluid, such as, but not limited to, plasma, serum, blood, urine, saliva or samples collected via bronchioalveolar lavage (BAL) or aspiration.

In connection with the present invention, monoclonal or polyclonal antibodies of isotype IgA, IgD, IgE, IgG, IgM, raised against oxCL, oxPS, oxLDL, MDA-LDL, PC, apoBIOO or bioactive components and/or parts/fragments/derivatives thereof refer to any monoclonal or polyclonal antibody produced by immunisation of a suitable mammal, including, but not limited to, mouse, rabbit, goat, sheep, or horse.

In connection with the present invention, antibodies against oxCL (anti-oxCL), antibodies against oxPS (anti-oxPS), antibodies against oxLDL (anti-oxLDL), antibodies against MDA-LDL (anti-MDA-LDL), antibodies against apoBIOO (anti- apoBIOO), antibodies against PC (anti-PC) and/or or antibodies against bioactive components and/or parts/fragments/derivatives thereof may be determined using any of the methods and techniques conventional in the art for such determination. Conveniently, such a method may comprise an immunoassay e.g. enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), immunofluorescence, Western blot, immunodiffusion, Immunoelectrophoresis, immunoprecipitation, magnetic immunoassay and flow cytometry. Conveniently, the components needed to perform the immunoassay may be supplied in a kit form. In connection with the present invention, a medicament may be intended for oral, rectal, parenteral and mucosal administration and may be formulated with excipients normally employed for such formulations. The medicament may be administered in a way so as to be compatible with the dosage formulation and in such amount as will be therapeutically effective and immunogenic.

In connection with the present invention, vaccine means any agent that is suitable for increasing the animal's natural anti-oxCL response, in particular oxCL conjugates, oxCL as such or bioactive components and/or parts thereof, and/or the animal's natural anti-oxPS response, in particular oxPS conjugates, oxPS as such or bioactive components and/or parts thereof, and/or the animal's natural anti-oxLDL response, in particular oxLDL conjugates, oxLDL as such or bioactive components and/or parts thereof and/or the animal's natural anti-MDA-LDL response, in particular MDA-LDL conjugates, MDA-LDL as such or bioactive components and/or parts thereof and/or the animal's natural anti-apoB100 response, in particular apoBIOO conjugates, apoBIOO as such or bioactive components and/or parts thereof and/or the animal's natural anti-PC response, in particular PC-containing bacteria, such as pneumococcae or parts of it, PC conjugates, PC as such or bioactive components and/or parts thereof, optionally in combination with any suitable adjuvants.

Detailed description of the invention Investigations into the relationships between oxCL, oxPS, anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and/or anti-PC on the one hand, and inflammatory conditions, infectious conditions, and/or mortality related thereto on the other hand, have revealed that the risk of developing one or more of the latter can be assessed by measuring the levels of one or more of the former. High levels of oxCL or oxPS are associated with increased risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto. Similarly, low levels of anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL or anti-PC are also associated with increased risk. Conversely, low levels of oxCL or oxPS, or high levels of anti- oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL or anti-PC are associated with decreased risk.

It has been found that oxidized CL, in contrast to native CL, has pro-inflammatory properties. OxCL, but not CL, induced endothelial cells to express ICAM-1 , intracellular adhesion molecule 1 , and VCAM-1 , vascular cell adhesion molecule 1. Adhesion molecules play an important role in recruiting monocytes into the intimal compartment of arteries, which is an early step in inflammatory processes. Further, it has been found and demonstrated that oxCL, but not CL, has the capacity to induce interleukin 6, IL-6, production. Several clinical studies have implicated three well-known markers of inflammation, C-reactive protein (CRP), fibrinogen (Fb) and IL-6, as CVD risk factors, and IL-6 has been identified as an independent risk factor for coronary artery disease (CAD).

Furthermore, it has previously been reported that oxCL, but not CL, could induce leukotriene B4 (LTB4) production in both neutrophils and macrophages. It is also found that oxCL, but not CL, can provoke intracellular calcium mobilization which is an initiation signal for LTB4 production and also in general a sign of cell activation. Leukotrienes are short-lived lipid mediators that have potent pro-inflammatory biological activities. Leukotriene B4 (LTB4) is one of the most potent chemotactic agents for other inflammatory cells and is biosynthesized from arachidonic acid by the sequential action of 5-lipoxygenase (5-LO) and leukotriene A4, LTA4, hydrolase, mainly in cells of myeloid lineage, such as neutrophil and macrophage, (Funk, CD., 2005)¹⁶. Two G-protein coupled LTB4 receptors have been identified, BLT1 and BLT2, with high and low affinity for LTB4, respectively, (Yokomizo, T. et al., 1997, and Yokomizo, T. et al., 2000)^17,18. LTB4 is known to exert broad pro-inflammatory effects, and evidence is accumulating regarding the antimicrobial functions of LTB4, (Serezani, CH. et al., 2005, and Wan, M. et al., 2007)¹⁹·²⁰ Furthermore, the LTB4- BLT1 pathway was found to be important for linking early immune responses and the multiple classes of effector cells associated with acquired immunity, (Goodarzi, K. et al., 2003)²¹. LTB4 may play an important role in atherosclerosis and CVD since mRNA levels for the three key proteins are significantly increased in human atherosclerotic plaque, and more pronounced in patients with ongoing CVD (Qui, H. et al., 2006)²².

Thus, by inducing LTB4 production, oxCL could play a role in initiating plaque rupture and CVD. Another finding was that oxCL but not CL (or reduced CL), inhibits uptake of oxLDL in macrophages.

If oxCL is predominantly exposed on oxLDL, binding and uptake of oxLDL through oxCL could promote atherogenesis. On the other hand, if oxCL is present mainly on other compounds, e.g. apoptotic cells, other proteins, or even bacteria, it is not clear how this would influence foam cell development, which in principle could be decreased. However, since apoptotic cells are known not to have any pronounced pro-inflammatory effects, this would suggest at least that oxCL is not an important factor exposed during apoptosis. Further research is needed to clarify which parts of oxLDL play the major role in foam cell formation.

It has recently been demonstrated that annexin A5 is abundant in atherosclerotic lesions and that anti-CL can interfere with its binding to endothelial cells, promoting CVD in SLE, (Cederholm, A. et al., 2005)¹². Rand et al. have demonstrated that annexin A5 can form a crystalline shield over cell surfaces, which could have a protective function. However, this can be disrupted by anti-CL, causing the antiphospholipid antibody syndrome, characterized by arterial and venous thrombosis and also miscarriage, (Rand, JH., and Wu, XX., 1999)²⁵. Annexin A5 has recently been implicated in CVD in general also as indicated by its function as a potent anti-atherothrombotic agent in a rabbit model of arterial thrombosis, by interfering with tissue factor expression and by recovery of hypercoagulability, (Cederholm, A. et al., 2005, Thiagarajan, P., and Benedict, CR., 1997, and Ishii, H. et al., 2007) ^26"28.

In connection with the previous invention, novel anti-inflammatory properties of annexin A5 with potential interest in atherosclerosis and CVD are reported. Annexin A5 inhibited the pro-inflammatory effects of oxCL, including induction of adhesion molecules, IL-6. The mechanism could be that annexin A5 binds to phosphatidylserine (PS) of endothelial cells, PS being a pro-thrombotic factor. It is also demonstrated that annexin A5 can bind to oxCL but not CL (Figure 5), though the exact mechanisms are not clear. Annexin A5 can thus in principle prevent these oxCL-induced effects by interacting with oxCL, though the exact mechanisms remain unknown. Compatible with this are the unusual properties of annexin A5, enabling it to form crystalline layers, which by themselves could be antiinflammatory, inhibiting pro-inflammatory effects of oxCL by a mechanistic barrier. CL is synthesized in cells de novo through the action of cardiolipin synthase, which is most active in high-metabolic tissue (where CL itself is most abundant). In mitochondria, CL modification and remodelling occurs, including substantial changes in the acyl composition¹. Accumulating evidence now also suggests that remodeling defects of CL could play a role in physiology and also pathology such as in diabetes, heart failure and Barth syndrome, (Han, X. et al., 2007, Sparagna, GC. et al., 2007, and Schlame, M., 2002)^29"31. In an interesting report, it was demonstrated that CL is quickly oxidized when coated on ELISA-plates for determination of anti-CL and many anti-CL in fact recognize oxidized CL (oxCL) (Horkko, S. et al., 1996)³². OxCL being an antigen for anti-CL could thus contribute to the antiphospholipid antibody syndrome, (Horkko, S. et al., 1996)³². The nature of these antibodies is still debated, almost three decades after their discovery, and it is likely that some anti-CL recognize plasma protein co-factors binding to CL, but that others recognize the phospholipid moiety (Frostegard, J., 2005)¹⁰.

Based on the findings herein, it is hypothesized that oxCL also could contribute to human chronic inflammatory disease in general, by its pro-inflammatory effects. CL binds easily to proteins, and it is possible that such complexes can become proinflammatory e.g. if exposed to the hypoxic and/or pro-oxidant environment in atherosclerotic plaques, but in principle also in other chronic inflammatory conditions like rheumatoid arthritis (RA) and SLE.

Interestingly, oxLDL and foam cells are present in rheumatoid arthritis, RA-synovia, (Winyard, PG. et al., 1993)³³, and oxLDL is raised and associated with disease activity in RA (unpublished observation). In systemic lupus erythematosus (SLE), the risk of CVD is very high due to a combination of traditional and non-traditional risk factors, (Frostegard, J., 2005)¹⁰. Both anti-CL and oxLDL are important examples of non-traditional risk factors. In addition to being raised in SLE per se, oxLDL is raised in CVD in SLE, (Frostegard, J., 2005, and Frostegard, J. et al., 2005)^10,34. Since CL is easily oxidized, it could play a role in these chronic inflammatory conditions. A prospective observational study was performed examining the relationship between anti-oxCL levels and mortality risk in a well-characterized cohort of 203 prevalent HD patients [56% men, median age 66 (interquartile range 51-74) years, vintage time 29 (15-58) months] with a mean follow-up period of 29 (14-58) months. The patients with an anti-oxCL value below the median had a higher mortality rate (ROC curve value 60%, p<0.05). These patients remained at higher risk of death even after adjustment for age, sex, smoking habits, cardiovascular (CVD) risk factors, albumin and inflammation. Thus, it is within this present invention found that antibodies to this particular antigen (i.e. oxCL) do not develop sufficiently in patients before the clinical onset of inflammatory conditions due to infectious conditions (e.g. developing infectious conditions), and/or inflammatory activity in renal disease related to renal damage, injury and/or dysfunction and/or secondary inflammatory conditions in renal disease and presently it is a preferred hypothesis that an insufficiently developed natural immunity against oxCL represents an underlying mechanism for development of such diseases and the mortality related thereto.

It has also recently been demonstrated that low levels of natural antibodies against phosphorylcholine (anti-PC), another exposed antigen in oxLDL, are risk factors for development of CVD in men, (Sjoberg, BG., 2008)³⁵. OxCL belongs to a novel class of pathogen-associated molecular patterns (PAMPs) and natural antibodies against oxCL bind oxLDL, (Tuominen, A. et al., 2006)³⁶. Thus, it is not known if there are common patterns of recognition in oxCL and PC, but the possibility that natural antibodies against oxCL like anti-PC, are risk factors for CVD at low levels deserves further study.

Taken together, the findings of the present invention indicate that oxCL can be a novel pro-inflammatory factor, causing development and progression of infectious diseases, also playing a role in mortality related to such one or more infectious conditions and/or secondary inflammatory diseases in renal diseases, activity and/or conditions including CVD, RA and SLE. Further, based on its capacity to inhibit oxCL-effects, it can be hypothesized that antibodies against oxCL could also be developed into a therapeutic agent for prophylactic, palliative and/or curative treatment in diseases where mortality and/or morbidity are increased due to oxCL activity.

Based on the material presented above under "Summary of the invention", it is established that oxPS contributes to human chronic inflammatory disease in general, by its pro-inflammatory effects. OxPS may easily bind proteins, and it is possible that such complexes can become pro-inflammatory e.g. if exposed to the hypoxic and/or pro-oxidant environment in atherosclerotic plaques, but in principle also in other chronic inflammatory conditions like rheumatoid arthritis (RA) and SLE.

The present invention thus confirms that oxPS has great potential as a novel proinflammatory factor causing development and progression of infectious and/or inflammatory conditions, including auto-immune as well as inflammatory conditions, such as, but not limited to, RA and SLE. Further, based on its capacity to inhibit oxPS-effects, annexin A5 and/or anti-oxPS and/or bioactive components and/or parts thereof could be developed into a therapeutic agent for prophylactic, palliative and/or curative treatment in diseases where morbidity is related to increased oxPS activity and/or decreased anti-oxPS activity. Further, the administration of vaccines against oxPS, such as oxPS conjugates, oxPS as such and/or bioactive components and/or parts thereof may be used in immune therapy for protection against developing inflammatory conditions and/or activity in animals at risk thereof, including scenarios that are not limited to preparation of a patient for surgery and/or chemotherapy.

Immunoassays

Detection of antibodies is a common form of medical diagnostics. For example, in different biochemical assays for disease diagnosis, a titre of antibodies indicative for a particular disease is estimated from the blood and if those antibodies are not present, the person does not have the disease in question. If however antibodies are found, a diagnosis is normally accepted when symptoms are correlating such findings. Several immunodiagnostic methods based on detection of antigen-antibody complex are used to diagnose infectious diseases, for example ELISA, immunofluorescence, Western blot, immunodiffusion, Immunoelectrophoresis, and magnetic immunoassay. Targeted monoclonal antibody therapy has been used to treat diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, and many forms of cancer including non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer.

An immunoassay is a test that measures the concentration of a substance in a sample of animal origin (such as, but not limited to, bodily fluid and/or tissue), using the reaction of an antibody or antibodies to its antigen. Both polyclonal and monoclonal antibodies can be used. Monoclonal antibodies usually bind only to one site of a particular molecule, and therefore provide a more specific and accurate test. Both the presence of antigen or antibodies can be measured.

In many applications, the response (often a fluorescence signal or dye intensity; however also other labelling options may be used) measured is compared to standards of a known concentration. This can be done through the plotting of a standard curve on a graph, the position of the curve at response of the unknown is then examined, and so the quantity of the unknown can be determined.

The most common method used for detecting the quantity of antibody or antigen is to label either the antigen or antibody. The label may consist of an enzyme (enzyme immunoassay (EIA, also called enzyme-linked immunosorbent assay or ELISA), colloidal gold (lateral flow assays), radioisotopes such as 1-125 radioimmunoassay (RIA), magnetic labels (magnetic immunoassay - MIA) or fluorescence. Other techniques include agglutination, nephelometry, turbidimetry and Western Blot.

Immunoassays are normally divided into those that involve labelled reagents and those which involve non-labelled reagents. Those which involve labelled reagents can be subdivided into homogenous and heterogeneous immunoassays. Heterogeneous immunoassays can furthermore be competitive or non-competitive. In simple terms, in ELISA or EIA a primary antigen or antibody is affixed to a surface in a dish, well or any other comparable laboratory means and then a sample of interest suspected to contain the corresponding antibody or antigen is added and incubated to let the corresponding antigen/antibody complex react. An additional (i.e. secondary) antibody linked to an enzyme or other form of labelling means is added to the well or dish after an intermediate wash. This antibody is targeted to bind the antigen/antibody complex and/or parts thereof establishing a possibility to detect a signal upon an activation reaction with suitable instruments measuring intensity of the signal chosen.

ELISA

Thus, any form of ELISA (it being direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA or reverse ELISA) can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, and is consequently a useful tool for determining serum antibody concentrations.

The ELISA, or the enzyme immunoassay (EIA), has a high sensitivity. Thus, in an ELISA of relevance for the present invention, a sample of bodily fluid, e.g. a person's serum, can, for example, be diluted 400-fold and applied to a plate to which one of the relevant compounds (for example oxCL, oxPS, oxLDL, or MDA-LDL) is attached. If antibodies to the compound are present in the serum, they may bind to the compound. The plate can then be washed to remove all other components of the serum. A specially prepared "secondary antibody"— an antibody that binds to the antigen/antibody complex or parts thereof— can then be applied to the plate, followed by another wash. If this secondary antibody is chemically linked in advance to an enzyme, the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme can then be applied, and catalysis by the enzyme leads to a change in colour or fluorescence. ELISA results can then be reported as a number, which can subsequently be compared to the relevant "cutoff point between a positive and negative result.

A cutoff value may be determined by comparing it with a known standard. This can be determined by applying a sample of known concentration to a surface and fixing it to the surface to render it immobile. Samples of known concentrations can then be used to generate a standard curve.

Immunomodulation therapy (immunotherapy)

Immunotherapy is normally defined within medicine as "Treatment of disease by inducing, enhancing, or suppressing an immune response".

Passive immunity (e.g. replacement therapy) can be achieved through the transfer of ready-made antibodies into the affected individual. Immunotherapies designed to elicit or amplify an immune response are normally termed "activation immunotherapies".

Immunotherapies designed to reduce, suppress or more appropriately direct an existing immune response, as in cases of autoimmunity or allergy, are normally termed "suppression immunotherapies". The active agents of immunotherapy are collectively called "immunomodulators".

Cutoff value

The average level of oxCL, oxPS, oxLDL, MDA-LDL, anti-oxCL, anti-oxPS anti- oxLDL, anti-MDA-LDL and/or anti-PC in animals depends on the type of bodily fluid sample, the specific species, and may vary between the different population groups.

A cutoff value may be chosen so that concentrations of oxCL, oxPS, oxLDL or MDA- LDL higher than said cutoff value are associated with an increased risk of developing a cardiovascular disease, an auto-immune disease or inflammatory condition.

A cutoff value may be chosen so that concentrations of oxCL, oxPS, oxLDL or MDA- LDL lower than said cutoff value are not associated with an increased risk of developing a cardiovascular disease, an auto-immune disease or inflammatory condition. A cutoff value may be chosen so that concentrations of anti-oxCL, anti-oxPS, anti- oxLDL, anti-MDA-LDL and/or anti-PC lower than said cutoff value are associated with an increased risk of developing a cardiovascular disease, an auto-immune disease or inflammatory condition.

A cutoff value may be chosen so that concentrations of anti-oxCL, anti-oxPS, anti- oxLDL, anti-MDA-LDL and/or anti-PC higher than said cutoff value are not associated with an increased risk of developing a cardiovascular disease, an autoimmune disease or inflammatory condition.

The cutoff value may also be determined from the ratio between the oxCL level and the anti-oxCL level, the ratio between the oxPS level and the anti-oxPS level, the ratio between the oxLDL level and the anti-oxLDL level, and the ratio between the MDA-LDL level and the anti-MDA-LDL level.

Embodiments of the invention

One embodiment of this invention relates to a method for assessing the risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto, comprising monitoring and/or determining the levels of one or more markers selected from the list consisting of oxCL, oxPS, oxLDL, MDA- LDL, anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC, and comparing said levels with predetermined cutoff values.

Another embodiment of this invention relates to a method for assessing the risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto, comprising monitoring and/or determining the levels of oxPS or anti-oxPS, and comparing said levels with predetermined cutoff values.

Another embodiment of this invention relates to a method for assessing the risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto, comprising monitoring and/or determining the levels of two or more markers selected from the list consisting of oxCL, oxPS, oxLDL, MDA- LDL, anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC, and comparing said levels with predetermined cutoff values.

In one embodiment of the present invention, the levels of one or more markers selected from the list consisting of oxCL, oxPS, oxLDL and MDA-LDL, are compared with predetermined cutoff values for a given population of individuals leading to a diagnosis.

In another embodiment of the present invention, the levels of two or more markers selected from the list consisting of oxCL, oxPS, oxLDL and MDA-LDL, are compared with predetermined cutoff values for a given population of individuals leading to a diagnosis.

One way of testing the levels of oxCL, oxPS, oxLDL and MDA-LDL in such samples may be immunoassays.

In another embodiment, the level of one or more markers selected from the list consisting of anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC is compared with a predetermined cutoff value for a given population of individuals leading to a diagnosis.

In another embodiment, the level of two or more markers selected from the list consisting of anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC is compared with a predetermined cutoff value for a given population of individuals leading to a diagnosis.

One way of testing the levels of anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and/or anti-PC in such samples may be immunoassays.

One preferred embodiment of the method relates to a predetermined cutoff value for a given population of individuals being chosen so that concentrations of oxCL, oxPS, oxLDL and/or MDA-LDL higher than said cutoff value is associated with an increased risk of developing inflammatory conditions. Another preferred embodiment of the method relates to a predetermined cutoff value for a given population of individuals being chosen so that concentrations of oxCL, oxPS, oxLDL and/or MDA-LDL lower than said cutoff value is not associated with an increased risk of developing inflammatory conditions.

Another preferred embodiment of the method relates to a predetermined cutoff value for a given population of individuals being chosen so that concentrations of anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and/or anti-PC lower than said cutoff value is associated with an increased risk of developing inflammatory conditions.

Another preferred embodiment of such method relates to a predetermined cutoff value for a given population of individuals being chosen so that concentrations of anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and/or anti-PC higher than said cutoff value is not associated with an increased risk of developing inflammatory conditions.

Referring to this invention, the inflammatory condition may be caused by infectious agents directly or indirectly (via e.g. toxins).

Referring to this invention, inflammatory conditions comprise one or more primary and/or secondary inflammatory diseases, inflammatory conditions and/or inflammatory activity.

Inflammatory condition may be selected from the non-exhaustive group comprising infections, auto-immune diseases, type II diabetes, Alzheimer's disease, dementia in general, rheumatic diseases, cardiovascular disease (CVD), such as atherosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris, high blood pressure, acute and/or chronic inflammatory conditions, type I diabetes, rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, asthma, acne, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), arthritis, including osteoarthritis, idiopathic inflammatory myopathies (MM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, vasculitis and/or an allergic disorder.

Referring to this invention, inflammatory conditions include conditions where an undesirable immune response is responsible for a pathological state or parts thereof and include autoimmune diseases, infectious conditions, rheumatic conditions, or secondary complications, also those caused by side-effects of medicaments. In this regard, the term condition relates to conditions, disease and activity defined by a tissue and/or organ. Referring to this invention, said inflammatory conditions are systemic or defined by the tissue and/or organ of origin, such as a renal condition, a renal disease and/or renal failure.

Referring to this invention, said renal condition, renal disease and/or renal failure is an autoimmune disease, a metabolic disease or a disease caused by toxic compounds.

Referring to this invention, said levels of oxCL, oxPS, anti-oxCL, anti-oxPS, anti- oxLDL, anti-MDA-LDL and/or anti-PC are monitored and/or determined in samples of animal origin, wherein said animal is selected from the group comprising mammals, vertebrates or invertebrates and wherein said mammal is selected from the group comprising human, dog, horse, cat mouse, rat, pig or cattle.

According to this invention, said samples of animal origin comprise tissue, plasma, serum, blood, urine, saliva or samples collected via bronchioalveolar lavage (BAL) or aspiration.

According to this invention, said method may be an immunoassay, making use of oxidized cardiolipin (oxCL) or fragments thereof, oxidized phosphatidylserine (oxPS) or fragments thereof, oxidized LDL (oxLDL) or fragments thereof, and a marker of antibody origin for binding to oxCL, oxPS, anti-oxCL, anti-oxPS, anti-oxLDL, anti- MDA-LDL, anti-PC, anti-oxCL/oxCL-antibody-antigen complex, anti-oxPS/oxPS- antibody-antigen complex, anti-oxLDL oxLDL-antibody-antigen complex, anti-MDA- LDUMDA-LDL-antibody-antigen complex and/or anti-PC/PC-antibody-antigen complex selected from ELISA, radioimmunoassay (RIA), flow cytometry or EIA. Another embodiment of this invention relates to a method for treating or decreasing the risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto, comprising administering to an animal in need thereof a therapeutically effective amount of one or more compounds selected from the list consisting of annexin A5, anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA- LDL and anti-PC, as well as antibodies against components, fragments or derivatives of MDA-LDL or OxLDL (such as apoBIOO or fragments thereof), and also bioactive components and/or parts/fragments of these compounds.

Another embodiment of this invention relates to a method for treating or decreasing the risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto, comprising administering to an animal in need thereof a therapeutically effective amount of two or more compounds selected from the list consisting of annexin A5, anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA- LDL and anti-PC, as well as antibodies against components, fragments or derivatives of MDA-LDL or OxLDL (such as apoBIOO or fragments thereof), and also bioactive components and/or parts/fragments of these compounds.

Referring to antibodies in this invention, antibodies can be monoclonal or polyclonal of isotype IgA, IgD, IgE, IgG, IgM against oxCL, oxPS or PC, or bioactive components and/or parts/fragments thereof.

In one embodiment of the method, the animal is suffering from a renal condition, a renal disease and/or renal failure. Another embodiment of this invention relates to a method for assessing and decreasing the risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto, comprising monitoring and/or determining the level of one or more markers selected from the list consisting of oxCL, oxPS, oxLDL, MDA-LDL, anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC, comparing said levels with predetermined cutoff values, and administering to an animal in need thereof a therapeutically effective amount of one or more compounds selected from the list consisting of annexin A5, anti-oxCL, anti- oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC, as well as antibodies against components, fragments or derivatives of MDA-LDL or OxLDL (such as apoBIOO or fragments thereof), and also bioactive components and/or parts/fragments of these compounds. Another embodiment of this invention relates to a method for assessing and decreasing the risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto, comprising monitoring and/or determining the level of two or more markers selected from the list consisting of oxCL, oxPS, oxLDL, MDA-LDL, anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC, comparing said levels with predetermined cutoff values, and administering to an animal in need thereof a therapeutically effective amount of two or more compounds selected from the list consisting of annexin A5, anti-oxCL, anti- oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC, as well as antibodies against components, fragments or derivatives of MDA-LDL or OxLDL (such as apoBIOO or fragments thereof), and also bioactive components and/or parts/fragments of these compounds.

Compositions comprising one or more inhibitors selected from the list consisting of annexin A5, inhibitors of oxCL, inhibitors of oxPS, inhibitors of oxLDL, inhibitors of MDA-LDL and inhibitors of PC, including antibodies, and/or bioactive components and/or fragments thereof, may, according to this invention, be used as a medicament, preferably used for treatment of inflammatory conditions, wherein said treatment comprises prophylactic, palliative and/or curative treatment. Compositions comprising two or more inhibitors selected from the list consisting of annexin A5, inhibitors of oxCL, inhibitors of oxPS, inhibitors of oxLDL, inhibitors of MDA-LDL and inhibitors of PC, including antibodies, and/or bioactive components and/or fragments thereof, may, according to this invention, be used as a medicament, preferably used for treatment of inflammatory conditions, wherein said treatment comprises prophylactic, palliative and/or curative treatment.

In one embodiment this medicament is in the form of a vaccine.

According to this invention, compositions comprising one or more inhibitors selected from the list consisting of annexin A5, inhibitors of oxCL, inhibitors of oxPS, inhibitors of oxLDL, inhibitors of MDA-LDL and inhibitors of PC, including antibodies, and/or bioactive components and/or fragments thereof are administered by injection and/or infusion, such as, but not limited to, transfusion. Another route of administration could be nasal, rectal or oral, in order to modify and raise anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC levels.

According to this invention, compositions comprising two or more inhibitors selected from the list consisting of annexin A5, inhibitors of oxCL, inhibitors of oxPS, inhibitors of oxLDL, inhibitors of MDA-LDL and inhibitors of PC, including antibodies, and/or bioactive components and/or fragments thereof are administered by injection and/or infusion, such as, but not limited to, transfusion. Another route of administration could be nasal, rectal or oral, in order to modify and raise anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC levels.

According to this invention, products used in transfusion medicine where the inhibitors of oxCL and/or inhibitors of oxPS and/or inhibitors of oxLDL and/or inhibitors of MDA-LDL and/or inhibitors of PC and/or bioactive components and/or fragments thereof is/are added comprise blood products selected from the group comprising whole blood products, platelet products, cryosupernatant or plasma products.

Another embodiment of this invention is the use of one or more conjugates selected from the list consisting of oxCL conjugates, oxPS conjugates, oxLDL conjugates, MDA-LDL conjugates and PC conjugates and/or bioactive components and/or fragments thereof for activation immunotherapy for treatment of inflammatory conditions, optionally adjuvants may be added. Another embodiment relates to the use of one or more inhibitors selected from the list consisting of inhibitors of oxCL, inhibitors of oxPS, inhibitors of oxLDL, inhibitors of MDA-LDL, anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and/or anti-PC and/or bioactive components and/or fragments thereof as a part of infusion products selected from the group comprising whole blood products, platelet products, cryosupernatant or plasma products.

Another embodiment of this invention is the use of two or more conjugates selected from the list consisting of oxCL conjugates, oxPS conjugates, oxLDL conjugates, MDA-LDL conjugates and PC conjugates and/or bioactive components and/or fragments thereof for activation immunotherapy for treatment of inflammatory conditions, optionally adjuvants may be added. Another embodiment relates to the use of two or more inhibitors selected from the list consisting of inhibitors of oxCL, inhibitors of oxPS, inhibitors of oxLDL, inhibitors of MDA-LDL, anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and/or anti-PC and/or bioactive components and/or fragments thereof as a part of infusion products selected from group comprising whole blood products, platelet products, cryosupernatant or plasma products.

Another embodiment of this invention relates to a kit for monitoring and/or determining the level of one or more markers selected from the list consisting of oxCL, oxPS, oxLDL, MDA-LDL, anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL, and anti-PC for assessing the risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto.

Another embodiment of this invention relates to a kit for monitoring and/or determining the level of two or more markers selected from the list consisting of oxCL, oxPS, oxLDL, MDA-LDL, anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL, and anti-PC for assessing the risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto. Another embodiment of this invention relates to a method of preventing or treating inflammation induced by radiation, for example in radiotherapy of cancer, comprising administering to an animal in need thereof a therapeutically effective amount of a) anti-oxCL and anti-oxPS, or

b) anti-oxCL and anti-oxPS and annexin A5.

Another embodiment of this invention relates to a composition comprising

a) anti-oxCL and anti-oxPS, or

b) anti-oxCL and anti-oxPS and annexin A5

for use in preventing or treating inflammation induced by radiation, for example in radiotherapy of cancer. Another embodiment of this invention relates to a topical skin care composition comprising

a) anti-oxCL and anti-oxPS, or

b) anti-oxCL and anti-oxPS and annexin A5. Another embodiment of this invention relates to a method of preventing or treating inflammation induced by UVA and/or UVB rays comprising administering to an animal in need thereof a therapeutically effective amount of a topical skin care composition comprising

a) anti-oxCL and anti-oxPS, or

b) anti-oxCL and anti-oxPS and annexin A5.

Experimental

The materials and methods and examples disclosed below are provided only for the purpose of illustrating the present invention and should not be considered as any limitation of the scope as outlined in the appended claims.

Antibodies binding oxidised cardiolipin in renal disease

Materials and methods

Chemical treatments of cardiolipin

Cardiolipin from bovine heart was purchased as ethanol solution from Sigma (Sigma product C 1649) and was stored at -20°C. Hydro heart cardiolipin (reduced CL) was purchased from Avanti Polar Lipids, Inc. To generate saturated molecular species, cardiolipin was oxidized in aqueous solutions containing 1 .5 mmol/L tert- butylhydroperoxide and 20 μιηοΙ/L CuSO4. Both cardiolipin and copper treated cardiolipin were measured with MS-spectrophotometer, and it was confirmed to have been oxidized by copper and tert-butylhydroperoxide.

Endothelial cells culture and adhesion molecules

Pooled human umbilical vascular endothelial cells (HUVECs) at passage 2 were purchased from Cascade Biologies, Inc (Portland, Ore). Cultures were maintained in EGM™ phenol red-free medium (Clonetics, San Diego, CA), containing 2% of fetal bovine serum and supplements, at 37°C under humidified 5% CO₂ conditions. All experiments were performed at passage 3 to 5. HUVECs were seeded at 6x10⁴ cells/2mL density on 6-well plates (NUNC Inc, Naperville, III). After allowing endothelial cells to attach overnight, cells were used for stimulation.

After washing with PBS, oxCL and reduced CL (20pg/ml) were added. For annexin A5 (from Bender MedSystems GmbH, Austria) inhibition study, oxCL was incubated with annexin A5 (10pg/ml) for a half hour before adding to cells. After 24 hours incubation, detached floating cells were washed away and cells were harvested into Falcon FACS tubes. After centrifuging at 1200 rpm for 5 minutes, cells were resuspended in 300μΙ FACS buffer (1 % FBS-PBS), incubated with 10μΙ PE- conjugated anti-CD54 (eBioscience) and 10μΙ FITC-conjugated anti-Human CD106 (Becton, Dickinson) for 30 minutes on ice. The intercellular adhesion molecule (ICAM-1 ) CD54 and the vascular cell adhesion molecule (VCAM-1 ) CD106 were studied with flow cytometry analysis equipped with CellQuest software. For each sample, 10,000 events were analyzed.

Macrophage differentiation and complement induction

The macrophages which transformed from human monocyte-derived THP-1 cells (American Type Culture Collection, Manassas, VA, U.S.A.) were plated in a 6-well plate at a density of 1 x 10⁶ cells/well in DMEM (Invitrogen, USA) containing 10% FBS overnight. Then the cells were washed three times with serum free medium before being incubated with oxCL (2 ig/m\). Thereafter, the cells were washed 4 times with ice-cold 0.2% BSA/PBS and once with PBS. The cells were harvested in PBS containing 0.1% BSA and 0.01% NaN3. 10μΙ monoclonal mouse anti-human C5b-9 (from Dakocytomation Demark) were added, and after incubation for 30 minutes on ice, cells were centrifuged at 4°C and supernatant was discarded, PBS containing 0.1% BSA and 0.01 % NaN3 was added and 10 μΙ polyclonal rabbit anti- mouse IgG-FITC (Fab ^'2) (From Dakocytomation Demark) were added. Complement production was measured by flow cytometry (BD Biosciences, San Jose, CA, USA). For each sample, a minimum of 10,000 events were analyzed. IL-6 production

HUVEC cells as above were seeded at density 10⁶/2ml into 6 well plates. Allowing cell attachment for 24 hours, oxCL (20pg/ml) with or without annexin A5 (20pg/ml) and reduced CL were added and incubated for 24 hours. Cell supernatants were collected. Production of IL-6 was measured with protein multiplex immunoassay kits (from Bioscource, USA) and Bio-Plei™ system (BioRad, USA).

Enzyme-Linked Immunosorbent Assay (ELISA) for annexin A5 Binding

F96 microtiter polysorb plates (Roskilde, Denmark) were coated with oxCL, CL or

Hydro Heart cardiolipin (Biosearch Technologies, Inc, Ca, USA) 10pg/ml incubated overnight at 4°C. After five washings with PBS, the plates were blocked with 2% PBS-BSA for 2h at room temperature. Annexin A5 was added and incubated for 1 hour. After washing, bound annexin A5 was detected by incubating subsequently with rabbit anti-human annexin A5 polyclonal antibodies (Hyphen Biomed, Andresy, France) 1 :2000 and polyclonal goat anti-rabbit Immunoglobulins/AP (DakoCytomation) 1 :3000. The reaction was developed with alkaline phosphatase substrate (Sigma), and optical density (OD) was read at 405nm with an ELISA Multiscan Plus spectrophotometer (Molecular Devices Emax, San Francisco). All samples were measured in duplicates and the coefficient of variation was below 15%.

Use of other suitable monoclonal or polyclonal antibodies for detecting bound annexin A5 is also possible. Labeling oxLDL with Dil

OxLDL was incubated with Dil (Molecular Probes Eugene, Oregon, USA) in lipoprotein-deficient serum (Sigma) at 37°C for 15 hours, and then dialyzed against saline-EDTA buffer for 6 hours.

Uptake of Dil-labeled oxLDL

Uptake of Dil-labelled oxLDL was studied either by flow cytometry or fluorescence/confocal microscopy. For microscopy, the macrophages (1 x 10⁶) were grown overnight on culture slides (Nunc, Naperville, New York), for flow cytometry, the macrophages were plated in a 6-well plate at density of 1 x 10⁶ cells/well in DMEM (Invitrogen, USA) containing 10% FBS overnight. Then the cells were incubated with Dil-oxLDL (5pg/ml), with oxCL (40, 80pg/ml), with CL control (40, 80pg/ml), with unlabelled oxLDL (40 g/ml) or with unlabelled LDL (40pg/ml) for 4 hours. For the annexin A5 inhibition of Dil-oxLDL experiment, macrophages were incubated with Dil-ox-LDL (5ug/ml), with annexin A5 (0.01 , 0.04, 0.16, 0.64, 1 , 10, 20, 40 Mg/ml). Thereafter, the cells from above were washed 4 times with 0.2% BSA/PBS and once with PBS. The cells were harvested in PBS containing 0.1% BSA and 0.01 % NaN3. Mean fluorescence intensity was measured by flow cytometry (BD Biosciences, San Jose, CA, USA). For each sample, fluorescence emission above 550nm was measured and a minimum of 10,000 cells were analyzed.

Cell culture

Human mononuclear cells were isolated from freshly prepared buffy coats (Karolinska Hospital blood bank, Stockholm, Sweden) by gradient centrifugation on Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden). The mononuclear cells were cultured at a density of 5 ^χ 10⁶/ml in RPMI-1640 medium with 25mM Hepes, 1% L-glutamine, 1% penicillin-streptomycin and 10% FBS. After 7 days, there were approximately 2 * 10⁶ macrophages per well.

Isolation of polymorphonuclear neutrophils (PMNs)

Human PMNs were isolated from freshly prepared buffy coats (Karolinska Hospital blood bank, Stockholm, Sweden) by dextran sedimentation, hypotonic lysis of erythrocytes and gradient centrifugation on Lymphoprep (Axis-Shield PoC AS, Oslo, Norway). PMNs were suspended at a density of 10 * 10⁶/ml in Dulbecco's PBS (Gibco (Invitrogen), Paisley, UK). PMN purity (> 95%) and viability (> 98%) was determined using Hemacolor (J.T. Baker, Utrecht, Holland) and Trypan Blue (Sigma Chemical Co.) staining, respectively.

Intracellular calcium mobilization

Neutrophils were added into black, 96-well plates with transparent bottom (Corning Costar; 5 χ 10⁴ cells/well), and the plate was spun down at 120 χ g for 3 min, afterwards changing the medium containing 4 μΜ FURA-2AM (Fura-2 acetoxymethyl ester), or buffer as appropriate, and the cells were incubated for 30 min at 37°C and 5% CO₂. Cells were washed four times with 50 μΙ of a buffer solution (135 mM NaCI, 4.6 mM KCI, 1.2 mM MgCI₂, 1.5 mM CaCI₂, 11 mM glucose, 11 mM Hepes, pH 7.4) before a final 45 μΙ volume of buffer was added to each well.

The plates were transferred to a fluorometer (Fluostar™, BMG Technologies), and 50 μΙ of different agonists according to experimental designs or buffer solution as control were injected into individual wells and the cells were monitored for the next 120 seconds. Control wells containing cells that had not been exposed to FURA- 2AM were used to subtract background auto-fluorescence. The results are given as the ratio of mean fluorescence intensity (MFI) between 340 and 380 nm, and normalized by control.

Ultrasound scan for assessing FMD and NTG induced vasodilatation

All ultrasound scans were made using a duplex scanner (Acuson Sequoia, Mountain View, California, USA) with an 8 MHz ART linear array transducer in a quiet semi- darkened room with the subject in the supine position. Scans were videotaped for off line analysis. Subjects were asked not to smoke, and not to drink coffee or tea for at least 2 h before the study. All studies were done by the same operator using the same equipment. All measures were done off-line from digitalised cine-loops by one measurer. The brachial artery was scanned longitudinally 2-10 cm above the elbow, with the vessel placed horizontally across the screen. Settings were made to optimise lumen-arterial wall interface, and thereafter not changed during the study. The transducer was held in the same position throughout the study by a mechanical arm. A resting B-mode scan was recorded, flow velocity was measured with a 5 MHz pulsed Doppler with a 1.5 mm gate width in the centre of the vessel at a 70 degree angle. Flow-increase was induced by the post ischemic response to deflation of a pneumatic tourniquet placed around the forearm of the patient to 250 mmHg for 4.5 min. A second scan was recorded from 30 s before to 90 s after cuff deflation. Flow velocity was recorded with a pulsed wave Doppler for 15 s before and 15 s after cuff deflation. This was followed by 10 minutes rest for vessel recovery. A third scan at rest was taken and 0.40 mg sublingual nitro-glycerine spray was administered. Three to four minutes after nitro-glycerine, the last scan was recorded. The vessel was measured at a fixed point in all scans. Measures were made on digitalised cine- loops from the anterior wall leading edge of the intima-media echo to the leading edge of the far wall intima-media vessel interface incidentally with the R-wave on the ECG for three consecutive cardiac cycles and the average measurements were used. After reactive hyperemia, measurements were made 50-70 s after cuff deflation. The increase in vessel diameter during hyperemia and after nitro-glycerine administration is expressed as percentages of the first control scan.

Determination of anti-oxCL antibody levels

The Immulon 1 B plate was coated with 50 μΙ/well of oxCL 10μg/ml, and allowed to dry overnight at 4°C. After washing with PBS, the plate was blocked with 2% BSA at room temperature for 2 hours. 1 :50 diluted sera were added in duplicates. The plate was incubated overnight at 4°C. The secondary antibody (anti-lg) was added 100 μΙ/well, then left overnight at 4°C. After washing five times with PBS, substrate was added (100 μΙ/well). The ELISA Multiscan Plus Spectrophotometer was used to determine optical density.

TNF production

The PBMC were cultured in 96-well culture plates at density of 2^*10⁵cells/1 ΟΟμΙ/well with stimulation of 1 pg/ml PHA in medium presence with different sera for 48 hours, and the supernatants were measured for TNF-a concentration by commercially available ELISA kits.

Extraction of anti-oxCL IgG from intravenous immunoglobulin (IVIG)

OxCL-MBSA and MBSA were coupled to a HiTrap NHS column (Amersham Biosciences) separately according to the manufacturer's instructions. Human pooled immunoglobulin (IVIG; Gammaguard, S/D) was diluted in binding buffer (20mM Na₂HPO₄) at 50mg/ml and filtered through 0.45 pm filter before passing through pre- coupled oxCL-MBSA and MBSA Sepharose gel column. Anti-oxCL IgG was eluted by 0.1 M Glycine-HCI buffer. The purified fractions were desalted using PD-10 columns (Amersham Pharmacia Biotech AB). Binding to oxCL (as described for determination of anti-oxCL antibodies) was confirmed.

Adhesion molecule expression by endothelial cells - inhibition by extracted anti- oxCL

Pooled human umbilical vascular endothelial cells (HUVECs) at passage 2 were purchased from Cascade Biologies, Inc (Portland, Ore). Cultures were maintained in EGMTM phenol red-free medium (Clonetics, San Diego, CA), containing 2% of fetal bovine serum and supplements, at 37°C under humidified 5% CO₂ conditions. All experiments were performed at passage 3 to 5. HUVECs were seeded at 6x10⁴ cells/2mL density on 6-well plates (NUNC Inc, Naperville, III).

After allowing 24 hours for cell attachment, the cells were incubated with oxidized cardiolipin (oxCL) 10pg/ml either with or without anti-oxCL-lgG 0.22 mg/ml. After 24 hours incubation, detached floating cells were washed away and cells were harvested into Falcon FACS tubes. After centrifuging at 1400 rpm for 5 minutes, cells were resuspended in 300μΙ FACS buffer (1% FBS-PBS), incubated with 10μΙ FITC-conjugated anti-Human CD106 (Becton, Dickinson) for 30 minutes on ice. The vascular cell adhesion molecule (VCAM-1) CD106 was studied with flow cytometry analysis equipped with CellQuest software. For each sample, 10,000 events were analyzed. Brief description of the drawings

Figure 1 : Electrospray ionization mass MS spectrometer (Micromass, Beverly, MA) was used to demonstrate that bovine heart cardiolipin was oxidized.

Figure 2: Induction of adhesion molecules. Oxidized CL but not CL induces adhesion molecules in endothelial cells, flow cytometry.

Endothelial cells were incubated with oxidized CL (20pg/ml) or CL (20pg/ml) for 24 hours, oxidized CL induced ICAM-1 and VCAM-1 production but CL did not show the same effect.

Figure 3: Inhibition of oxCL-induced adhesion molecule expression by annexin A5, detected by flow cytometry.

Oxidized CL (20pg/ml) was preincubated with annexin A5 (10pg/ml) before stimulating cells for 24 hours. Annexin A5 inhibited oxCL induced endothelial cell expression of ICAM-1 (CD54) and VCAM-1 (CD106). Figure 4: Induction of IL-6 secretion by endothelial cells by oxCL and inhibition by annexin A5.

Determined by Luminex. Detected by intensity of fluorescence.

Endothelial cells were incubated with oxCL (20pg/ml) with or without annexin A5 (20 g/ml) and CL (20pg/ml) for 24 hours. Oxidized CL can significantly induce IL-6 production and this effect can be inhibited by annexin A5. CL had no such effect.

Figure 5: Binding of oxCL by annexin A5, ELISA. OxCL, CL and reduced CL (5pg/ml) were coated on ELISA plates overnight. Annexin A5 (0.32pg/ml, 0.64pg/ml, 1.28pg/ml, 2.56pg/ml, 5.12pg/ml and 10.24pg/ml) can bind to oxidized cardiolipin and air exposed CL but not reduced cardiolipin. Figure 6: Effect of oxCL on uptake of oxLDL in macrophages, detected by flow cytometry. OxCL competed macrophage uptake of oxLDL, but CL did not have such an effect. (Human monocytic cell line) THP-1 cells (differentiated MQ), a model for monocyte/macrophage function, were studied for uptake of Dil-oxLDL, the uptake could be competed by oxCL but not non-oxidized CL. Figure 7: OxCL-induced calcium mobilization, fluorometer.

Results show that only oxidized cardiolipin can activate the neutrophils and induce the intracellular calcium mobilization. Furthermore, annexin A5 can inhibit the oxCL induced calcium mobilization in neutrophils.

Figure 8: Effect of oxCL on leukotriene B4 release, EIA.

Oxidized cardiolipin promotes human neutrophils and macrophages to release leukotriene B₄, but cardiolipin did not show the similar reaction.

Figure 9: Effect of annexin A5 on oxCL induced LTB₄ production from oxCL- stimulated human neutrophils and macrophages, which may give new insights into clinical novel targets for medical treatments of the associated inflammatory conditions, EIA.

Figure 10: Antibodies against oxCL were extracted by conjugating bovine serum albumin (BSA) with oxCL, whereupon the oxCL-BSA was loaded on a sepharose containing column and Ig was added. Anti-oxCL could then be extracted (IgM), and used in experiments, where 5 pg/ml of anti-oxCL was added 30 minutes before addition of oxCL. As indicated in Figure 10, anti-oxCL had the capacity to inhibit the proinflammatory effects of oxCL on expression of the adhesion molecule VCAM (CD106) as determined by flow cytometry, as seen by a shift to the left of the histogram. Figure 11 : Effects of oxCL on activation of T-cells, flow cytometry.

Human PBMC (Peripheral blood mononuclear leukocytes) were incubated overnight with 5 pg/ml of oxCL or CL. Both CD4 and CD8 positive T cells were studied. Quadrant Q2 represent (%) T-cells positive for CD69 expression and thus activated T-cells. Thus, it is demonstrated that oxCL but not CL can activate CD8-positive and CD4-positive T cells as determined by an increase in Q2 which is highly significant. Figure 12: Protective effect of anti-oxCL on amyloid peptide 1-42 induced cell death. A Neuroblastoma cell line was treated with amyloid peptide1-42 which induced cell death (as determined by propidium iodide; PI). Extracted anti-oxCL inhibited the effect. Figure 13: OxPS is positively correlated with endothelial cell expression of VCAM-1. HUVEC cells were stimulated with oxPS or oxPS that was preincubated with annexin A5 for 30 minutes. VCAM- 1 (CD106) expression was measured by flow cytometry (BD Biosciences, San Jose, CA, USA). Relative to the control (curve marked with "control"), the curve shifts to the right (curve marked with "oxPS"), confirming that oxPS does induce VCAM-1 expression. The shift of the curve (marked with "oxPS + annexin A5") to the left when HUVEC cells were stimulated with oxPS pre-incubated with annexin A5 indicates that annexin A5 inhibits oxPS induced VCAM-1 expression.

Figure 14: Effect of antibodies against phosphorylcholine (anti-PC) IgM antibodies on lysophosphatidylcholine (LPC) induced necrosis, expressed as fluorescent measure of leakage of lactate dehydrogenase from cells with a damaged membrane in PBMC. (300,000 cells/well, 96 well plates) were incubated with LPC for 2 h and LDH activity in supernatant was measured. Anti-PC or control antibodies which did not bind PC were preincubated with LPC 90 minutes before starting the treatment. The results were expressed as units per liter of LDH. Figure 15: Effect of human IgM antibodies on lysophosphatidylcholine (LPC) induced cytotoxicity, measured as decrease in mitochondrial dehydrogenase activity in PBMC. (3,000,000 cells/well) were incubated with LPC for 18 h and the mitochondrial dependent reduction of MTT to formazin was measured. Anti-PC, total IgM and IgM which did not bind PC (flow through) were preincubated with LPC 90 minutes before starting the treatment. The results were expressed as percentage of viable cells compared with non stimulated cells (control).

Figure 16: Antibodies against oxidized phosphatidylserine (anti-oxPS) negatively associated with disease activity in SLE.

One hundred fourteen patients with SLE were compared with one hundred twenty two age- and sex matched population-based controls. SLE activity was determined with the Systemic Lupus Activity Measure (SLAM) and also with Systemic Lupus Erythematosus diseases activity index (SLEDAI). Organ damage was determined with Systemic Lupus International Collaborating Clinics (SLICC) damage index. Antibodies were determined by ELISA. Anti-oxPS was negatively associated with SLAM >6; a sign of high disease activity. 0 = SLAM≤ 6, 1 = SLAM > 6. Example 1 : Chemical treatments of cardiolipin

Native cardiolipin and oxidation product were further analyzed by mass spectrometry, figure 1. Native cardiolipin from bovine heart yielded two major signals, corresponding to the double charged anion (m/z 724.0), the single charged anion (m/z 1447). The oxidized derivative peaks from both double charged and single charged ions in the oxidized fraction were 8 m/z units apart, suggesting progressively oxidized cardiolipins.

Example 2: Endothelial cells and adhesion molecules

To study whether oxCL can stimulate endothelial cells to express adhesion molecules, HUVECs from passage 3 to 5 were incubated for 24 h with oxCL or native CL. Results suggested oxCL can significantly increase both ICAM-1 and VCAM-1 expression, but native CL did not show the same effect (Fig. 2). Both the increased expression of ICAM-1 and VCAM-1 (CD54 and CD106) induced by oxCL were significantly inhibited by annexin A5 (Fig. 3).

Example 3: IL-6 production

1 x 10⁶ HUVECs were seeded in 6 well plates. OxCL could stimulate endothelial cells to produce 564.3±142.02 (pg/ml) IL-6 from the supernatant. Annexin A5 significantly decreased II-6 level to 276.4 ±28.62 (pg/ml). Compared to the control group, both native CL and annexin A5 themselves could not significantly increase endothelial cell II-6 levels (Fig. 4). This supports the hypothesis that oxCL stimulates IL-6, which is a known inflammatory marker, e.g. oxCL might be involved in inducing/mediating inflammatory responses.

Example 4: Uptake experiment

To study potential mechanisms of oxCL bioactivity, 1x10⁶/ well THP-1 differentiated macrophages (MQ) were used for uptake studies. OxLDL could inhibit Dil-oxLDL uptake up to more than 60%, but LDL had almost no effect on the uptake of Dil- oxLDL, which confirmed the specificity of uptake results. Further results suggested oxCL could, in a concentration dependent manner, inhibit Dil-oxLDL uptake while the native CL did not show the competition effects (Fig. 6).

Example 5: Intracellular calcium mobilization

To study the influence of oxCL on intracellular calcium mobilization, 5 * 10⁴ neutrophil cells/well were used for measuring calcium mobilization. After adding oxCL, MFI of calcium mobilization in the cells were significantly increased within 120 seconds. This increase induced by oxCL was greatly inhibited by incubation of annexin A5, but annexin A5 and native CL did not increase calcium mobilization (Fig 7). Example 6: LTB4 production

Human mononuclear cells isolated from freshly prepared buffy coats were differentiated into macrophages according to protocols which are well known to those skilled in the art. 2x10⁶ cells/well were exposed to oxCL, CL or buffer solution. Results suggested that oxCL could, in a concentration dependent manner, induce LTB4 production, but native CL could not (Fig 8). This increase was significantly inhibited by annexin A5 (Fig. 9).

Example 7: Negative association between major parameters in systemic lupus erythematosus (SLE), inflammation and anti-oxCL

Patient sera from a control population of women were used herein. Data was obtained by use of simple regression, and Spearman Rank test was used in statistical analysis (Stat View).

The association between important parameters and determinants in relation to anti- oxCL was determined. Table 1 : Controls from the general population (population controls; 26 women)

No association with anticardiolipin antibodies or lupus anticoagulans (an indirect measure for anti-cardiolipin antibodies): p=0.41

Thus, these data indicate strong and significant negative associations between anti- oxCL antibodies and important factors such as blood pressure, diabetes and blood sugar, body mass index (BMI), and a positive association with endothelial function. One mechanism can be oxCL-effects on endothelium, causing endothelial activation and dysfunction if protective anti-oxCL are low. The role of anti-oxCL in 52 women with a prototypic autoimmune disorder, system lupus erythematosus (SLE), was demonstrated.

Table 2: SLE-patients: Association between anti-oxCL in women with SLE (n=52)

Disease severity index SLICC: below index 4 (117 ±48 compared to above index 4 (63±14); p=0.0085

Thus, strong negative association between major SLE measures and inflammation and anti-oxCL exists. In the prototypic autoimmune disease SLE, a strong negative association between anti-oxCL on the one hand, and TNF-induction and other inflammatory markers on the other hand, was determined, indicating an anti- inflammatory role played by these antibodies. Further, there is a positive association with endothelial function, indicating a positive effect which also has implications for cardiovascular disease. A specific potential role in SLE-manifestations and disease severity is demonstrated by an association with SLICC, an index of disease damage, where higher anti-oxCL could ameliorate disease. Example 8: Activation of CD4 and CD8 positive T cells by oxCL

Both CD4 and CD8 positive T-cells from the cell line human PBMC were studied in an incubation setup illustrated in figure 11. Both T-cell types were incubated with buffer (control), oxCL or CL to determine the effect on activation, interpreted by the expression of CD69 surface molecule after stimulation.

In this experiment, the attention must be drawn to the quadrant Q2 of the flow cytometry data shown. This quadrant represents the percentage of T-cells in the population tested to be CD69 positive, e.g. activated by the stimuli prior to the data collection.

In the control-stimulation (figure 11 a and d), without CL or oxCL, no induction of CD69 expression was detected in CD4 or CD8 positive lymphocytes. The same is true for the population of T-cells treated with CL (figure 11 c and f) before measuring the CD69 expression, represented by very similar data plots. However, the expression of CD69 was notably elevated in oxCL stimulated T-cells positive for CD4 and CD8 (figure 11 b and e). A distinct shift from quadrant Q1 to Q2 is seen when comparing data from the control and CL-experiment to the oxCL-data.

This experiment shows that an immune response can be induced by oxCL, indicated by the oxCL, but not CL, mediated expression of CD69 on the surface of CD4 and CD8 positive T-cells. Example 9: Protective effect of anti-oxCL on amyloid peptide 1-42 induced ceil death.

The beta amyloid (Abeta) and especially Abeta peptide (1-42), is an important component of senile plaques in Alzheimer's disease, and is known to be directly responsible for the production of free radicals toxic to brain tissue. Abeta (1-42)- induced free radical oxidative stress in the neurodegeneration observed in AD brain may be one mechanism for neurotoxicity. Human SH-SY5Y neuroblastoma cells were used in cell culture systems. The cells were treated with 5μΜ of the peptide for 24 hours. Cell death was determined by the addition of 1 mg/ml_ propidium iodide (PI), which labels the nucleus in dying cells which lack an intact plasma membrane.

In the control experiment, the amount of dead cells was measured as 23.99%, whereas the amyloid peptide 1-42 induced cell death increased that number to 45.54%. When amyloid peptide1-42 was incubated together with oxCL-lgG, 30pg/ml, (anti-oxCL), the cell death was reduced to 37.10%.

This experiment confirms that anti-oxCL will have a protective effect on cell death induced by the amyloid peptide 1-42. Thus, this supports the notion that anti-oxCL can have a protective effect against Alzheimer's disease. Thus, from the above it emerges that oxCL has T-cell activating properties, and is involved in cell death, which is supported by the cell death protective effect of anti- oxCL illustrated in figure 12.

Example 10: Association between IgM anti-oxCL and anti-OxPS and risk of death in patients undergoing haemodialysis

The association between IgM antibodies against oxCL (IgM anti-oxCL) and oxPS (IgM anti-oxPS) and risk of death in patients undergoing haemodialysis (HD) was studied.

A prospective observational study examining the relationship between anti-oxCL and anti-oxPS levels and mortality risk in a well-characterized cohort of 203 prevalent HD patients [56% men, median age 66 (interquartile range 51-74) years, vintage time 29 (15-58) months] with a mean follow-up period of 29 (14-58) months, was performed.

RESULTS: The patients with an anti-oxCL value below the median had a higher mortality rate (ROC curve value 60%, p<0.05). The patients with anti-OxPS levels under median also had increased mortality rate (p<0.05). These patients remained at higher risk of death even after adjustment for age, sex, smoking habits, cardiovascular (CVD) risk factors, albumin and inflammation.

These data reveal that low levels of natural IgM antibodies against oxCL and oxPS are independent predictors of death among HD patients, where death from infections is a major factor. It furthermore shows that active immunization, with oxCL or oxPS as an antigen, or passive immunization, aimed at raising levels of protective anti-oxCL or anti-oxPS, should be expected to have a clinical effect. Further, anti- oxCL and anti-oxPS determination can be used for determination of risk.

OxPS and/or anti-oxPS in inflammatory conditions Materials and methods Chemical treatments of phosphatidylsehne

Commercially available PS is stored at -20°C. To generate saturated molecular species, PS is oxidized in aqueous solutions containing tert-butylhydroperoxide and CuSO₄. Both PS and copper treated PS (oxidized product) are then measured with mass spectrometry (MS)-spectrophotometer to confirm that the phosphatidylserine has been oxidized by the copper and tert-butylhydroperoxide.

Endothelial cells culture and adhesion molecules

Pooled human umbilical vascular endothelial cells (HUVECs) at passage 2 can be purchased from Cascade Biologies, Inc (Portland, Ore). Cultures are maintained in EGM™ phenol red-free medium (Clonetics, San Diego, CA), containing 2% of fetal bovine serum and supplements, at 37°C under humidified 5% CO₂ conditions. All experiments are performed at passage 3 to 5. HUVECs are seeded at 6x10⁴cells/2ml_ density on 6-well plates (NUNC Inc, Naperville, III). After allowing endothelial cells to settle and attach overnight, cells are ready for stimulation.

After washing with phosphate-buffered saline (PBS), oxPS is added. For annexin A5 (from Bender MedSystems GmbH, Austria) inhibition study, oxPS is incubated with annexin A5 (10pg/ml) for half an hour before adding to cells. For anti-oxPS inhibition study, oxPS is incubated with anti-oxPS (10, 15, 20, 25 or 30 pg/ml) for half an hour before adding to cells, or the cells are incubated with oxPS, PS or reduced PS for 24 hours, followed by stimulation with anti-oxPS (10, 15, 20, 25 or 30 pg/ml) for additional 12 hours. After 24 hours incubation, detached floating cells are washed away, cells are harvested into Falcon FACS tubes and optionally anti- oxPS are allowed to incubate for 12 hours. After centrifuging at 1200 rpm for 5 minutes, cells are resuspended in 300μΙ direct flow cytometry (FACS) buffer (1% FBS-PBS), incubated with 10μΙ PE-conjugated anti-CD54 (eBioscience) and 10μΙ fluorescein isothiocyanate (FITC)-conjugated anti-Human CD106 (Becton, Dickinson) for 30 minutes on ice. The intercellular adhesion molecule (ICAM-1) CD54 and the vascular cell adhesion molecule (VCAM-1) CD106 are studied with flow cytometry analysis equipped with CellQuest software. For each sample, 10,000 events are analyzed. Controls for anti-oxPS should be incubated additional 12 hours after the first 24 hours of incubation for proper comparison.

Macrophage differentiation and complement induction

The macrophages which may be transformed from Human monocyte-derived THP- 1 cells (American Type Culture Collection, Manassas, VA, U.S.A.) are seeded in a 6-well plate at a density of 1 x 10⁶ cells/well in DMEM (Invitrogen, USA) containing 10% fetal bovine serum (FBS) overnight. Then the cells are washed three times with serum free medium before being incubated with PS, reduced PS or oxPS (2pg/ml). Thereafter, the cells are washed 4 times with ice-cold 0.2% bovine serum albumin (BSA)/PBS and once with PBS. The cells are harvested in PBS containing 0.1% BSA and 0.01% NaN3. 10μΙ monoclonal mouse anti-human C5b-9 (from Dakocytomation Demark) are added after incubation for 30 minutes on ice, cells are centrifuged at 4°C and supernatant is discarded, PBS containing 0.1% BSA and 0.01% NaN₃ is added and 10 μΙ polyclonal rabbit anti-mouse Immunoglobulin G (IgG)-FITC (Fab'2) (from Dakocytomation Demark) are added. Complement production is measured by flow cytometry (BD Biosciences, San Jose, CA, USA). For each sample, a minimum of 10,000 events should be analyzed. IL-6 production

To evaluate the IL-6 production on stimulation with oxPS, human umbilical vein endothelial cells (HUVEC) from the above setup are seeded at density 10⁶/2ml into 6 well plates. Cells are allowed to attach for 24 hours. OxPS (20pg/ml), with or without annexin A5 (20pg/ml) and reduced PS, are then added in combination or to separate wells and incubated for 24 hours. A combination comprising anti-oxPS may also be added. Cell supernatants are then collected and IL-6 production can be measured with protein multiplex immunoassay kits (from Bioscource, USA) and Bio- Plei™ system (BioRad, USA).

Induction of IL-6 by oxPS and inhibition by annexin A5 are determined by Luminex and detected by intensity of fluorescence.

Cell culture

Human mononuclear cells are isolated from freshly prepared buffy coats (Karolinska Hospital blood bank, Stockholm, Sweden) by gradient centrifugation on Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden). The mononuclear cells are cultured at a density of 5 * 10⁶/ml in RPMI-1640 medium with 25mM Hepes, 1% L-glutamine, 1% penicillin-streptomycin and 10% FBS. After 7 days, approximately 2 χ 10⁶ macrophages per well may be expected.

Isolation of polymorphonuclear neutrophils (PMNs)

Human PMNs are isolated from freshly prepared buffy coats (Karolinska Hospital blood bank, Stockholm, Sweden) by dextran sedimentation, hypotonic lysis of erythrocytes and gradient centrifugation on Lymphoprep (Axis-Shield PoC AS, Oslo, Norway). The PMNs are then suspended at a density of 10 ^x 10⁶/ml in Dulbecco's PBS (Gibco (Invitrogen), Paisley, UK). PMN purity and viability is determined using Hemacolor (J.T. Baker, Utrecht, Holland) and Trypan Blue (Sigma Chemical Co.) staining, respectively. Intracellular calcium mobilization

For evaluation of the calcium mobilization, neutrophils are added into black, 96-well plates with transparent bottom (Corning Costar; 5 * 10⁴ cells/well), and the plate is spun down at 120 * g for 3 min, afterwards changing the medium to 4 μΜ Fura-2- acetoxymethyl ester (FURA-2AM) or buffer as appropriate. The cells are then incubated for 30 min at 37°C and 5% CO₂. Cells are washed four times with 50 μΙ of a buffer solution (135 mM NaCI, 4.6 mM KCI, 1.2 mM MgCI₂, 1.5 mM CaCI₂, 11 mM glucose, 11 mM Hepes, pH 7.4) before a final 45 pi volume of buffer is added to each well.

Following the above, the plates are transferred to a fluorometer (Fluostar™, BMG Technologies), and 50μΙ of different agonists according to experimental designs or buffer solution as control are injected into individual wells and the cells are monitored for the next 120 seconds. Control wells containing cells that have not been exposed to FURA-2AM are used to subtract background auto-fluorescence. The results are measured as the ratio of mean fluorescence intensity (MFI) between 340 and 380 nm, and normalized by control. Determination of oxPS levels

As an example of determining the levels of oxPS in any fluid and/or sample, oxPS may be conjugated with BSA, which acts as carrier protein. Anti-oxPS is extracted by use of columns loaded with oxPS-BSA. Such antibodies against oxPS are generated and another monoclonal antibody against low density lipoprotein (LDL) is coated on ELISA plates. Alternatively, monoclonal antibodies (e.g. anti-oxPS) are coated on ELISA plates, serum is added and monoclonal anti-oxPS is added again; thus, all oxPS containing particles are measured. Subsequently, a fluid or sample subject for the oxPS determination, such as serum, is added. After incubation and washing of the plate, bound oxPS is detected with an antibody against oxPS conjugated with a color detection marker.

Determination of anti-oxPS antibody levels

An Immulon 1 B plate is coated with 50 μΙ/well of oxPS 10μg/ml, and allowed to dry overnight at 4°C. After washing with PBS, the plate is blocked with 2% BSA at room temperature for 2 hours. 1 :50 diluted sera is added in duplicates. The plate is incubated overnight at 4°C. The secondary antibody (anti-lg) is added 100pl/well and incubated overnight at 4°C, followed by 5 rounds of washing with PBS before substrate is added (ΙΟΟμΙ/well). ELISA Multiscan Plus Spectrophotometer can be used to determine optical density.

TNF production

Peripheral blood mononuclear cells (PBMCs) are cultured in 96-well culture plates at a density of 2x10⁵cells/100pl/well with stimulation of 1pg/ml polyhydroxyalkanoates (PHA) in medium comprising different sera for 48 hours, and the supernatants are measured for TNF-a concentration by commercially available ELISA kits.

For stimulation studies, the cells are washed after the first 48 hours and stimulated an additional 12 hours with pure medium, anti-oxPS, annexin A5 or a combination thereof, before harvesting and evaluation by commercially available ELISA kits. Extraction of anti-oxPS IgG from intravenous immunoglobulin (IVIG)

OxPS-mannosylated BSA (mBSA) or PS-mBSA and mBSA are coupled to a HiTrap N-hydroxysuccinimide (NHS) column (Amersham Biosciences) separately according to the manufacturer's instructions. Human pooled immunoglobulin (IVIG; Gammaguard, S/D) is diluted in binding buffer (20mM Na₂HP0₄) at 50mg/ml and filtered through 0.45pm filter before passing through pre-coupled oxPS-mBSA and mBSA Sepharose gel column. Anti-oxPS IgG is eluted by 0.1 M Glycine-HCI buffer. The purified fractions are desalted using PD-10 columns (Amersham Pharmacia Biotech AB). Adhesion molecule expression by endothelial cells - inhibition by extracted anti- oxPS

Pooled human umbilical vascular endothelial cells (HUVECs) at passage 2 can be purchased from Cascade Biologies, Inc (Portland, Ore). Cultures are maintained in EGMTM phenol red-free medium (Clonetics, San Diego, CA), containing 2% of fetal bovine serum and supplements, at 37°C under humidified 5% CO₂ conditions. All experiments are performed at passage 3 to 5. HUVECs are seeded at 6x10⁴ cells/2mL density on 6-well plates (NUNC Inc, Naperville, III). After allowing 24 hours for cell attachment, the cells are incubated with oxidized phosphatidylserine (oxPS) 10 g/ml, either with or without anti-oxPS-lgG 0.22 mg/ml. After 24 hours incubation, detached floating cells are washed away and cells are harvested into Falcon FACS tubes. After centrifuging at 1400 rpm for 5 minutes, cells are resuspended in 300μΙ FACS buffer (1% FBS-PBS), incubated with 10μΙ FITC-conjugated anti-Human CD106 (Becton, Dickinson) for 30 minutes on ice. The vascular cell adhesion molecules VCAM-1 (CD106) and ICAM-1 (CD54) are then studied with flow cytometry analysis equipped with CellQuest software. For each sample, 10,000 cells should be analyzed.

Immunization of atherosclerotic mice

OxPS or conjugates containing epitopes of oxPS are used to immunize mouse models of atherosclerosis/chronic inflammation, such as, but not limited to, apoE knock out models, or other relevant models for chronic inflammatory diseases. Increased anti-oxPS levels after such immunization are then monitored.

Another method involving immunization of mouse models is, when monoclonal antibodies against oxPS are produced, and used in the abovementioned mouse models of atherosclerosis/chronic inflammation, such as, but not limited to, apoE knock out models, or other relevant models for chronic inflammatory diseases, to evaluate effects on inflammation. Different oxPS dependent changes may then be determined, including vascular inflammation or inflammation in tissues such as kidney, or inflammation-related changes in brain, such as amyloid measurements or other dementia markers.

Example 11: Chemical treatments of PS; Electrospray ionization mass MS spectrometer (Micromass, Beverly, MA) is used to demonstrate that commercially available phosphatidylserine is oxidized.

Native PS and oxidation product can be analyzed by standard methods in mass spectrometry. Example 12: Anti-oxPS binds oxPS (and not PS)

Binding to oxPS (as described for determination of anti-oxPS antibodies) can be confirmed when performing an extraction of anti-oxPS IgG from intravenous immunoglobulin (IVIG) as described above. It is expected that the results show that anti-oxPS does not bind PS.

Example 13: Endothelial cells and adhesion molecules

To study whether oxPS can stimulate endothelial cells to express adhesion molecules, HUVECs from passage 3 to 5 were incubated for 24 h with oxPS or native PS. Results showed that oxPS can significantly increase VCAM-1 expression. Native PS did not show the same effect. The increased expression of VCAM-1 (CD 06) induced by oxPS was significantly inhibited by annexin A5 (Figure 13).

Example 14: IL-6 production Endothelial cells can be incubated with oxPS (20pg/ml) with or without annexin A5, anti-oxPS (20pg/ml) and PS (20pg/ml) for 24 hours, oxidized PS is expected to significantly induce IL-6 production and this effect is moreover expected to be inhibited by annexin A5 and anti-oxPS. PS is not expected to show such effect.

Compared with the control group, both native PS and annexin A5 themselves should not be expected to significantly increase endothelial cell IL-6 levels.

Example 15: Uptake experiment

To study potential mechanisms of oxPS bioactivity, 1x10⁶/ well THP-1 differentiated macrophages (MQ) are used for uptake studies. OxLDL is expected to inhibit Dil- oxLDL uptake, but LDL should not have such an effect on the uptake of Dil-oxLDL then confirming the specificity of uptake results. Thus, oxPS could in a concentration dependent manner inhibit Dil-oxLDL uptake while the native PS is expected not to show the competition effects. OxPS is expected to compete with the macrophage uptake of oxLDL, but PS should not have such effect. Example 16: Intracellular calcium mobilization

To study the influence of oxPS on intracellular calcium mobilization, 5 χ 10⁴ neutrophil cells/well are used for measuring calcium mobilization. After adding oxPS, mean fluorescence intensities (MFI) of calcium mobilization in the cells are expected to be significantly increased within 120 seconds. This increase induced by oxPS is moreover expected to be greatly inhibited by incubation with annexin A5 or anti-oxPS. Annexin A5 and native PS as such should not increase calcium mobilization. OxPS-induced calcium mobilization is measured by a fluorometer.

Example 17: Anti-oxPS is negatively correlated with endothelial cell expression of VCAM-1

Patient sera from a control population were used herein. Data was obtained by use of simple regression and Spearman Rank test was used in statistical analysis (Stat View).

The association between important parameters and determinants in relation to anti- oxPS was determined. The role of anti-oxPS in 26 patients with a prototypic autoimmune disorder, systemic lupus erythematosus (SLE) but without CVD, was demonstrated.

Table 3: SLE-patients: Association between anti-oxPS in patients with SLE without

CVD (n=26)

Thus, strong negative association between major SLE measures and inflammation and anti-oxPS was shown to exist. In the prototypic autoimmune disease SLE, a strong negative association between anti-oxPS and levels of VCAM-1 in serum was determined, indicating an anti-inflammatory role played by these antibodies. PS has not been found to have the same effect as oxPS in this experimental setup. Example 18: OxPS but not PS is positively correlated with endothelial cell expression of VCAM-1 The association between important parameters and determinants in relation to oxPS was determined in a group of patients with a prototypic autoimmune disorder, systemic lupus erythematosus (SLE), without CVD. This was performed similar to the experiment above, by analysing patient sera from a control population. Data was obtained by use of simple regression and Spearman Rank test and analysed statistically (Stat View).

The correlation between oxPS in patients with SLE and without CVD was shown to have a strong (indirect) association between major SLE measures and inflammation and oxPS. In the prototypic autoimmune disease SLE, a strong negative association between anti-oxPS and the expression of VCAM-1 is determined, and thus this experiment confirms a pro-inflammatory role of oxPS, by measuring low levels of anti-oxPS correlated to increased VCAM-1 expression.

Example 19: Inhibitors of oxPS and their effect on oxPS induced VCAM-1 expression on endothelial cells

HUVECs are seeded, incubated for settlement and attachment and stimulated with oxPS or PS for 24 hours, followed by 12 hours of incubation before harvest and evaluation by flow cytometry, with pure medium, anti-oxPS, annexin A5 or a combination thereof. Comparative data is expected to reveal that the inhibitors of oxPS decrease oxPS induced VCAM-1 expression on the HUVECs.

Other inhibitors of oxPS (not PS)

Inhibitors that bind oxPS, but not PS, are obviously expected to be part of the invention. Such inhibitors should have an inhibitory effect on oxPS activity similar to anti-oxPS and annexin A5, however, should not (as annexin A5) inhibit PS. Example 20: Anti-oxPS inhibits TNF activation

The association between important parameters and determinants in relation to anti- oxPS was determined and the role of anti-oxPS in 26 patients with a prototypic autoimmune disorder, systemic lupus erythematosus (SLE), but without CVD, was demonstrated.

Table 4: SLE-patients: Association between anti-oxPS in patients with SLE without CVD (n=26)

Thus, strong negative association between major SLE measures and inflammation and anti-oxPS exists. In the prototypic autoimmune disease SLE, a strong negative association between anti-oxPS and the level of TNF was determined, indicating an anti-inflammatory role played by these antibodies.

Example 21 : Inhibitors of oxPS and their effect on oxPS induced TNF levels

The peripheral blood mononuclear cells (PBMCs) are cultured in 96-well culture plates at a density of 2x10⁵cells/100pl/well with stimulation of 1 pg/ml Polyhydroxyalkanoates (PHA) in medium comprising different sera for 48 hours, followed by 12 hours of stimulation with pure medium, anti-oxPS, annexin A5 or a combination thereof. TNF-a concentrations are measured by commercially available ELISA kits. Comparative data is expected to reveal that the inhibitors of oxPS decrease oxPS induced TNF levels in PBMCs. Example 22: OxPS effects on atherosclerosis

OxPS or conjugates containing epitopes of oxPS are used to immunize mouse models of atherosclerosis/chronic inflammation, such as, but not limited to, apoE knock out models, or other relevant models for chronic inflammatory diseases. Increased anti-oxPS levels after such immunization are monitored.

Immunization of the above mentioned mouse models of atherosclerosis/chronic inflammation, such as, but not limited to, apoE knock out models or other relevant models for chronic inflammatory diseases, with monoclonal antibodies, is performed and different oxPS dependent changes are determined.

Example 23: Inhibition of lysophosphatidylcholine induced cell death by anti- PC IgM

Peripheral blood mononuclear cells (PBMC) in 96 well plates (with 300,000 cells/well) were incubated with lysophosphatidylcholine (LPC) for 2 h, and cell death was assessed by measuring lactate dehydrogenase (LDH) activity in the supernatant. Antibodies against phosphorylcholine (anti-PC IgM) or control antibodies which did not bind PC were preincubated with LPC 90 minutes before starting the treatment. The results were expressed as units per liter of LDH. As can be seen from Figure 14, anti-PC IgM reduced cell death more than total IgM and flowthrough IgM.

PBMC (3,000,000 cells/well) were incubated with LPC for 18 h, and cell death was assessed by measuring the decrease in mitochondrial dependent reduction of MTT to formazin. Anti-PC, total IgM and IgM which did not bind PC (flow through) were preincubated with LPC 90 minutes before starting the treatment. The results were expressed as percentage of viable cells compared with non-stimulated cells (control). As can be seen from Figure 15, anti-PC IgM reduced cell death more than total IgM or flowthrough IgM. Example 24: Protective effect of combination of high anti-MDA-LDL, high anti- OxLDL, high anti-PC, high anti-oxCL or high anti-oxPS

ELSA-study

Materials and methods Subjects Serum samples were obtained from 226 subjects with established hypertension (diastolic pressure >95 mm Hg) prior to their entry into the Swedish component of the European Lacidipine Study on Atherosclerosis (ELSA)^{58, 59} Samples were collected following a 4-week washout period without medication to minimize the effects of treatment on the measured parameters. Blood pressure, cholesterol and triglyceride levels were determined as described previously^{58, 59}. One hundred and fifteen of the subjects were subsequently assigned to treatment with the β-blocker atenolol, and 111 of the subjects were assigned to treatment with the calcium antagonist lacidipine. Carotid ultrasound determinations were performed and analyzed as detailed elsewhere^58"60. A total of 226 patients had valid ultrasound measurements at baseline and after 4 years of follow up. Briefly, the right and left carotid arteries were examined with Biosound 2000 MA duplex scanner using a 8.0 MHz annular array transducer. The intima-media (l-M) thickness was determined in the far wall as the distance between the leading edge of the lumen-intima echo and the leading edge of the media-ad ventitia echo. The outcome measurement as a surrogate indicator for atherosclerosis was the change in mean maximum Intimal-Medial thickness (IMT) of the four far walls in the distal common carotids and carotid bifurcations bilaterally (CBM_max) at the 4-year follow-up. The associations between antibody levels at enrolment into the study with an increase or decrease in IMT at the 4-year follow-up were evaluated. IgM antibodies to PC-BSA were determined by enzyme-linked immunosorbent assay (ELISA). Pooled serum from the same donors was used as an internal standard and tested on every plate. The plateau of antibody binding was reached with the antigen concentration of 10pg/ml. F96 microtiter polysorb plate was therefore coated with PC-BSA(10pg/ml) 50pl/well in PBS. Coated plates were incubated overnight at 4°C. After five washings with PBS, the plates were blocked with 2% BSA-PBS for 2h at room temperature and washed as described above. Serum samples were diluted (1 :30) in 0.2% BSA-PBS and added at 50pl/well. IgM antibodies to anti-oxCL and anti-oxPS were determined by ELISA as described. Cardiolipin and phosphatidylserine were oxidized in aqueous solutions containing 1.5 mmol/L tert-butylhydroperoxide and 20 pmol/L CuSO4.

LDL was isolated from plasma of healthy donors by sequential preparative ultra- centrifugation and oxidized by use of copper ions (OxLDL) or derivatized with MDA (MDA-LDL) as described⁶¹. OxLDL and MDA-LDL were determined by ELISA essentially as described⁶¹. OxLDL or MDA-LDL was diluted to 2 pg/ml in coating buffer (carbonate-bicarbonate buffer 50 mM pH 9.7), and 100 μΙ/well was used to coat ELISA plates (Costar 2581). The plates were kept at 4°C overnight, washed 4 times with PBS, and then blocked with 20% adult bovine serum in PBS (20% ABS- PBS) for 2 hours in room temperature. They were then incubated with 100 μΙ serum, diluted 1 :30 in 20% ABS-PBS at 4°C overnight.

Plates were incubated overnight at 4°C and washed as described above. Alkaline phosphatase conjugated goat anti-human IgG (diluted 1 :9000 in the sample buffer) and alkaline phosphatase conjugated goat anti-human IgM (diluted 1 :7000 in the sample buffer) were added at 100ul/well and incubated at 4°C overnight. After five washings, color was developed by adding the alkaline phosphatase substrate (PNPP) at 100ul/well and incubating the plates for 60 min at room temperature in the dark. The plates were read in an ELISA Multiskan Plus spectrophotometer (Molecular Devices Emax, San Francisco) at 405nm. All samples were measured in a single assay and the coefficient of variation was below 10-15%.

Results Details of the study on Basic characteristics and anti-PC, anti-OxLDL and anti-MDALDL separately have been presented elsewhere⁶⁰.

When high anti-MDALDL and anti-oxCL or anti-oxPS (both IgM) were combined, surprisingly, the associations with decreased development of atherosclerosis (protection effect) were strongly and significantly raised (Table 6). Likewise, when high anti-MDA-LDL and high anti-PC (both IgM) or high anti-OxLDL and high anti- PC (both IgM) were combined, surprisingly, the associations with decreased development of atherosclerosis (protection effect) was also strongly and significantly raised (Table 7 & Table 8). Furthermore, high anti-OxLDL and high anti-MDALDL when combined provided strong protection (OR 0.09, p<0.01).

Conclusion

Anti-oxPS and anti-oxCL IgM as protection factors against chronic inflammatory diseases as atherosclerosis becomes stronger when combined with anti-MDALDL. Anti-PC IgM as a protection factor also becomes stronger when combined with anti- MDA-LDL or anti-OxLDL. These results suggest that a combination of these antibodies, raised through active immunization (vaccination) or passive immunization (where antibodies are administered), has advantages as compared to single therapies.

Table 5. Basic characteristics of the study group at enrolment. Results are presented as means (SD) or percentage (%) and mg/dL for lipids.

Total Atenolol Lacidipine

(N=226) (N=115) (N=111)

57.7 (7.8) 57.6 (7.6) 57.7 (7.9) 50 46 53

26.7 (3.7) 26.3 (3.3) 27.1 (3.9) 232.4 (37.8) 233.5 (38.1) 231 .4 (37.4) 55.6 (27.6) 56.5 (25.8) 54.7 (27.6) 149.4 (37.8) 149.7 (37.1) 149.2 (38.6) 131.6 (58.2) 128.6 (57.0) 134.7 (59.5)

Table 6.

Combination of high anti-oxCL or high anti-oxPS in combination with high anti- MDALDL as protective factors in atherosclerosis development.

Model 1 indicates exposure to high levels of any of the antibodies, Model 2 exposure to having high levels of both antibodies.

MODEL! MODEL2

Table 7: Prediction of changes in IMT with baseline levels of IgM

autoantibodies to phosphorylcholine (PC) or MDA-LDL.

Variable Odds Ratio (95% CI) P

Lower Upper

Anti-MDA-LDL .67 .36 1.2 .18 (highest 25^th

percentile

Anti-PC (highest 10^th .36 .15 0.87 .024 percentile)

Anti-MDA-LDL(lgM) .14 .038 0.512 .003 and anti-PC

(combined) Table 8: Prediction of changes in IMT with baseline levels of IgM autoantibodies to phosphorylcholine (PC) or Ox-LDL.

Example 25: Increased risk of CVD from combination of low IgM anti-PC with low anti-oxCL or low anti-oxPS

Study of 60-year olds

Objective. We here determine the role of IgM antibodies against phosphorylcholine (anti-PC) in combination with antibodies against oxidized cardiolipin (anti-oxCL) or antibodies against oxidized phosphatidylserine (anti-oxPS) in prediction of cardiovascular disease (CVD).

Methods. From a screening of 4232 subjects, 60-years old (2039 men and 2193 women), 21 1 incident cases of CVD (myocardial infarction, ischemic stroke, or hospitalized angina pectoris) and 633 age- and sex-matched controls were identified through a 5-7 year follow-up^62"65. Serum levels of antibodies were determined by ELISA. Cardiolipin and phosphatidylserine were oxidized in aqueous solutions containing 1.5 mmol/L tert-butylhydroperoxide and CuSO in 20 pmol/L.

Results. In women, there were no significant associations between anti-PC and CVD. Among men, individuals with anti-PC levels in lowest quartile 4 (lowest) as the reference value yielded a trendwise excess risk for CVD (p=0.069) which was not significant. However, when combined with low anti-oxCL or low anti-oxPS (both below 25th percentile), associations became significant with an increase from p=0.069 to p=0.042 (when combined with anti-oxCL) and 0.024 (when combined with anti-oxPS).

Conclusions. A combination of low IgM anti-PC with low anti-oxCL or low anti-oxPS increases the excess risk of CVD. A combination of these antibodies could improve therapy, if raised through active immunization or passive immunization.

Example 26: Association between low levels of IgM anti-oxPS and risk for myocardial infarction and stroke (CVD) among 60-year olds in Stockholm county, followed for 5 years. Study of 60-year olds

Objective. We here determine the role of IgM antibodies against oxidized phosphatidylserine (anti-oxPS) in prediction of cardiovascular disease (CVD). Methods. From a screening of 4232 subjects, 60-year old (2039 men and 2193 women), 211 incident cases of CVD (myocardial infarction, ischemic stroke, or hospitalized angina pectoris) and 633 age- and sex-matched controls were identified through a 5-7 year follow-up^62"65. Serum levels of antibodies were determined by ELISA. Phosphatidylserine and cardiolipin were oxidized in aqueous solutions containing 1.5 mmol/L tert-butylhydroperoxide and CuS0₄ in 20 pmol/L

Results. Anti-oxPS levels below the highest 25^th percentile had increased risk of CVD, (Odds ratio (OR) 1.74, p = 0.0232, and below the highest 75^th percentile, OR was 1.84, p = 0.0191). Among men, anti-oxPS below the 75^th percentile meant a high risk, OR = 2.56, P= 0.0043. In women, there were no significant associations between anti-oxPS and CVD, though OR below the highest 25^th percentile IgM anti- oxPS were raised. Conclusions. IgM anti-oxPS is a novel protection marker for CVD. Active immunization or passive immunization to raise anti-oxPS levels could be a novel therapy against chronic inflammation including CVD and atherosclerosis.

Table 9: Association between low levels of IgM anti-oxPS and risk for myocardial infarction and stroke (CVD) among 60-year olds in Stockholm county, followed for 5 years.

Men and women combined

anti-oxPS Crude Adjusted

Quartiles (U/ml) OR 95 % CI P-values OR 95 % CI P-values

Quartile 4 125.727 > 1 N/A NA 1 N/A N/A

Quartile 3 83.99< <=125.727 1.63 1.02 2.58 0.0397 1.74 1.08 2.80 0.0232

Quartile 2 64.6564< <=83.99 1.39 0.87 2.23 0.1718 1.48 0.91 2.41 0.1166

Quartile 1 <=64.6564 1.77 1.09 2.86 0.0212 1.84 1.10 2.99 0.0191

Table 10: Association between levels of anti-oxPS and risk for CVD men

anti-oxCL Crude Adjusted

Quartiles (U/ml) OR 95 % CI P-values OR 95 % CI P-values

Quartile 4 125.727 > 1 N/A NA 1 N/A N/A

Quartile 3 83.99< <=125.727 1.82 0.91 3.36 0.0557 2.04 1.08 3.85 0.0288

Quartile 2 64.6564< 1.34 0.72 2.47 0.3357 1.56 0.83-2.96 0.1 24

Quartile 1 <=64.6564 2.31 1.25 4.25 0.0074 2.56 1.34 4.88 0.0043

Table 11 : Association between levels of anti-oxPS and risk for CVD women anti-oxCL Crude Adjusted

Quartiles (U/ml) OR 95 % CI P-values OR 95 % CI P-values

Quartile 4 125.727 > ^{1 N/A NA 1 N/A N/A}

Quartile 3 83.99< <=125.727 1.42 0.70 2.92 0.0557 1.47 0.70 3.10 0.3096

Quartile 2 64.6564< <=83.99 1.61 0.75 3.45 0.2225 1.56 0.70 3.47 0.2753

Quartile 1 <=64.6564 0.94 0.39 2.27 0.89 0.95 0.39 2.34 0.9128 Example 27: Antibodies against oxidized phosphatidylserine (anti-oxPS) in atherosclerosis

ELSA-study

Materials and methods Subjects Serum samples were obtained from 226 subjects with established hypertension (diastolic pressure >95 mm Hg) prior to their entry into the Swedish component of the European Lacidipine Study on Atherosclerosis (ELSA)^{58, 59} Samples were collected following a 4-week washout period without medication to minimize the effects of treatment on the measured parameters. Blood pressure, cholesterol and triglyceride levels were determined as described previously^{58, 59} One hundred and fifteen of the subjects were subsequently assigned to treatment with the β-blocker atenolol, and 111 of the subjects were assigned to treatment with the calcium antagonist lacidipine. Carotid ultrasound determinations were performed and analysed as detailed elsewhere^58"60. A total of 226 patients had valid ultrasound measurements at baseline and after 4 years of follow up. Briefly, the right and left carotid arteries were examined with Biosound 2000 IIA duplex scanner using an 8.0 MHz annular array transducer. The intima-media (l-M) thickness was determined in the far wall as the distance between the leading edge of the lumen-intima echo and the leading edge of the media-adventitia echo. The outcome measurement as a surrogate indicator for atherosclerosis was the change in mean maximum Intimal-Medial thickness (IMT) of the four far walls in the distal common carotids and carotid bifurcations bilaterally (CBM_max) at the 4-year follow-up. The associations between antibody levels at enrolment into the study with an increase or decrease in IMT at the 4-year follow-up were evaluated. IgM antibodies to anti-oxCL and anti-oxPS were determined by ELISA as described. Cardiolipin and phosphatidylserine were oxidized in aqueous solutions containing 1.5 mmol/L tert-butylhydroperoxide and CuS0₄ in 20 μηηοΙ/L LDL was isolated from plasma of healthy donors by sequential preparative ultra-centrifugation and derivatized with MDA (MDA-LDL) as described⁶¹.

MDA-LDL was diluted to 2 g/ml in coating buffer (carbonate-bicarbonate buffer 50 mM pH 9.7), and 100 μΙ/well was used to coat ELISA plates (Costar 2581). The plates were kept at 4°C overnight, washed 4 times with PBS, and then blocked with 20% adult bovine serum in PBS (20% ABS-PBS) for 2 hours in room temperature. They were then incubated with 100 μΙ serum, diluted 1 :30 in 20% ABS-PBS at 4°C overnight.

Plates were incubated overnight at 4°C and washed as described above. Alkaline phosphatase conjugated goat anti-human IgG (diluted 1 :9000 in the sample buffer) and alkaline phosphatase conjugated goat anti-human IgM (diluted 1 :7000 in the sample buffer) were added at 100ul/well and incubated at 4°C overnight. After five washings, color was developed by adding the alkaline phosphatase substrate (PNPP) at 100ul/well and incubating the plates for 60 min at room temperature in the dark. The plates were read in an ELISA Multiskan Plus spectrophotometer at 405nm. All samples were measured in a single assay and the coefficient of variation was below 10-15%.

Results

Details of the study on Basic characteristics and have been presented elsewhere⁶⁰. Anti-oxPS is a protection marker for atherosclerosis development, where high levels, above 75^th, 80^th and 90^th percentile, were associated with a favourable atherosclerosis development (p<0.05). Conclusion.

Anti-oxPS is a protection factor against chronic inflammatory diseases such as atherosclerosis, suggesting that anti-oxPS can be raised through active immunization (vaccination) or passive immunization (where antibodies are administered), as a novel therapy.

Table 12: Basic characteristics of the study group at enrolment. Results are presented as means (SD) or percentage (%) and mg/dL for lipids.

Total Atenolol Lacidipine (N=226) (N=115) (N=111)

Age (years) 57.7 (7.8) 57.6 (7.6) 57.7 (7.9)

Sex (% males) 50 46 53

BMI 26.7 (3.7) 26.3 (3.3) 27.1 (3.9)

Total Cholesterol 232.4 (37.8) 233.5 (38.1) 231.4 (37.4)

HDL 55.6 (27.6) 56.5 (25.8) 54.7 (27.6)

LDL 149.4 (37.8) 149.7 (37.1) 149.2 (38.6)

Triglycerides 131.6 (58.2) 128.6 (57.0) 134.7 (59.5)

Table 13: Prediction of increase in atherosclerosis measurements during 5 years. High anti-oxPS levels predict decreased risk.

CRUDE

anti-oxPS IgM anti-oxPS IgG

Cutoff

>75 0.48 (0.26-0.89) 0.65(0.35-1.21)

ADJUSTED: anti-oxPS IgM anti-oxPS IgG

>75 0.49 (0.26-0.93) 0.66(0.35-1.25)

adjusted for: sex, cholesterol, treatment, age, smoking

CRUDE

anti-oxPS IgM anti-oxPS IgG

Cutoff

>80 0.45(0.23-0.90) 0.64(0.33-1.26)

>90 0.47(0.19-1.16) 0.47(0.19-1.16)

ADJUSTED

anti-oxPS IgM anti-oxPS IgG

Cutoff

>80 0.45(0.23-0.90) 0.63(0.32-1.25)

>90 0.38(0.15-0.98) 0.47(0.19-1.19)

adjusted for: sex, cholesterol, treatment, age, smoking

Example 28: Antibodies against oxidized phosphatidylserine (anti-oxPS) negatively associated with vulnerable plaque in SLE Introduction

The risk of cardiovascular disease (CVD) and atherosclerosis is reported to be increased in systemic lupus erythematosus (SLE). We recently reported a negative association between natural IgM-antibodies against phosphorylcholine (anti-PC) in the general population, high anti-PC levels leading to decreased atherosclerosis development and low levels to increased risk of CVD. Potential mechanisms include anti-inflammatory properties and inhibition of uptake of oxidized low density lipoprotein in macrophages. The objective herein was to study atherosclerosis in SLE in detail and in relation to traditional and non-traditional risk factors including novel factors antibodies against oxidized phosphatidylserine (anti-oxPS) and antibodies against oxidized cardiolipin (anti-oxCL).

Methods

One hundred fourteen patients with SLE were compared with one hundred twenty two age- and sex-matched population-based controls. Common carotid intima- media thickness (IMT), calculated intima-media area (cIMa) and plaque occurrence were determined by B-mode ultrasound as a surrogate measure of atherosclerosis.

Plaques were graded according to echogenicity and grouped as 1-4, with 1 being echolucent, and considered most vulnerable.

Antibodies were studied by ELISA.

Results

Hypertension, triglycerides and insulin resistance (determined by homeostasis model assessment of insulin resistance) and C-reactive protein (CRP) were increased in SLE (p<0.01) while smoking, LDL, HDL did not differ between groups. Low levels of anti-PC IgM (lowest tertile) were more common in SLE patients than in controls (p=0.0022) while anti-oxCL and anti-oxPS did not differ between groups. IMT and cIMa did not differ significantly between groups. However, plaques were more often found in SLE patients (p=0.029). Age, LDL and IgM anti-PC (lowest tertile) were independently associated with plaque occurrence in SLE. Further, in the left carotid arteries echolucent plaques (grade 1) were more prevalent in SLE as compared to controls p<0.016).

Anti-oxPS were lower in individuals with vulnerable plaque (grade 1 ; p=0.01), at left side where vulnerable plaque were raised significantly, the association was strong (p=0.001). When controlled for age, by use of logistic regression, this association remained significant (p=0.01). Likewise, anti-oxCL were lower in individuals with vulnerable plaque and at left side, the association was significant (p=0.01). Conclusion

Plaque occurrence in the carotid arteries is increased in SLE and independently associated with age, LDL and low anti-PC levels. Vulnerable plaques were more common in SLE and associated independent of age, negatively with anti-oxPS and anti-oxCL. Anti-oxPS could be a novel risk marker also with a therapeutic potential in SLE, by itself or in combination with anti-oxCL and anti-PC.

Example 29: Antibodies against oxidized phosphatidylserine (anti-oxPS) negatively associated with disease activity in SLE

Introduction

The risk of cardiovascular disease (CVD) and atherosclerosis is reported to be increased in systemic lupus erythematosus (SLE). The objective herein was to study disease manifestations SLE in detail and in relation to traditional and non-traditional risk factors including novel factors antibodies against oxidized phosphatidylserine (anti-oxPS) and antibodies against oxidized cardiolipin (anti-oxCL) and also antibodies against phosphorylcholine (anti-PC). Methods

One hundred fourteen patients with SLE were compared with one hundred twenty two age- and sex matched population-based controls. SLE activity was determined with the Systemic Lupus Activity Measure (SLAM) and also with Systemic Lupus Erythematosus diseases activity index (SLEDAI). Organ damage was determined with Systemic Lupus International Collaborating Clinics (SLICC) damage index. Antibodies were determined by ELISA.

Results

Anti-oxPS was negatively associated with SLAM >6; a sign of high disease activity. Figure 16: 0 = SLAM < 6, 1 = SLAM > 6. Conclusion

Anti-oxPS is associated with disease activity in SLE, negatively. Anti-oxPS could be a novel risk marker also with a therapeutic potential in SLE. Example 30: Anti-oxPS negatively associated with TNF in serum in SLE

Twenty six women with systemic lupus erythematosus were studied. We have previously demonstrated that TNF is an important proinflammatory factor in SLE and also in other rheumatic diseases like RA this is well known. The objective was to determine the role of the novel factor antibodies against oxidized phosphatidylserine (anti-oxPS) in SLE. Anti-oxPS and TNF levels were determined by ELISA (in house method and R&D systems respectively).

Results:

Anti-oxPS was significantly and negatively associated with TNF in serum (R= -0.47, P= 0.018).

Conclusion:

Anti-oxPS is negatively associated with the important proinflammatory cytokine TNF, implying it is anti-inflammatory.

Example 31 : Treatment with anti-oxCL and anti-oxPS to prevent radiation damage In cancer therapy, a major problem is radiation side effects, causing serious damage to tissues like lungs, Gl-tract and endothelium. Recently it was demonstrated that oxidized cardiolipin and oxidized phosphatidylserine (oxCL and oxPS) are major causes of radiation damage⁶⁶ and that inflammation is the mechanism. Our previous results indicate anti-inflammatory properties in antibodies against oxCL and oxPS (anti-oxCL and anti-oxPS) and also that annexin A5 inhibits the inflammatory effects of oxCL and oxPS. The following experiment can illustrate treatment with anti-oxCL and anti-oxPS to prevent radiation damage. Three groups of mice are subjected to total-body irradiation (5-15 Gy (or other relevant doses)) and euthanized after 24 h. One group is pre-treated with anti-oxCL and anti-oxPS, one group is pre-treated with anti-oxCL and anti-oxPS and annexin A5, and one group is untreated control. Mouse lung endothelial cells and GIT tissue can be analyzed 48 h after gamma radiation and apoptosis and inflammatory changes detected.

Example 32: Topical skin care composition

Effect of anti-oxCL and anti-oxPS and annexin A5 can be studied in cell cultures subjected to UVA and UVB irradiation. Cell death, both apoptosis and necrosis can be studied as described above in example 23. Further, inflammatory effects and cell activation are studied, including examples 2-6.

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Claims

1. A method for assessing the risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto, comprising monitoring and/or determining the levels of one or more markers selected from the list consisting of oxCL, oxPS, oxLDL, MDA-LDL, anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC, and comparing said levels with predetermined cutoff values.

2. A method according to claim 1 wherein the levels of one or more markers selected from the list consisting of oxCL, oxPS, oxLDL, and MDA-LDL are compared with predetermined cutoff values for a given population of individuals, said cutoff values being chosen so that concentrations of the one or more markers higher than said cutoff values are associated with an increased risk of developing inflammatory conditions.

3. A method according to claim 1 wherein the levels of one or more markers selected from the list consisting of oxCL, oxPS, oxLDL and MDA-LDL are compared with predetermined cutoff values for a given population of individuals, said cutoff values being chosen so that concentrations of the one or more markers lower than said cutoff values are not associated with an increased risk of developing inflammatory conditions.

4. A method according to claim 1 wherein the levels of one or more markers selected from the list consisting of anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC are compared with predetermined cutoff values for a given population of individuals, said cutoff values being chosen so that concentrations of the one or more markers lower than said cutoff value are associated with an increased risk of developing inflammatory conditions.

5. A method according to claim 1 wherein the levels of one or more markers selected from the list consisting of anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC is compared with predetermined cutoff values for a given population of individuals, said cutoff values being chosen so that concentrations of the one or more markers higher than said cutoff value are not associated with an increased risk of developing inflammatory conditions.

6. A method according to any of claims 1 to 5 wherein said inflammatory conditions comprise one or more primary and/or secondary inflammatory diseases, inflammatory conditions and/or inflammatory activity.

7. A method according to any of claims 1 to 6, wherein said inflammatory conditions are selected from the group comprising infections, auto-immune diseases, cardiovascular diseases, type II diabetes, Alzheimer's disease, dementia in general, rheumatic diseases, acute and/or chronic inflammatory conditions, type I diabetes, high blood pressure, arthrosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, ischemic attack, transient ischemic attack, claudication, angina, rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, asthma, acne, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease, arthritis including osteoarthritis, idiopathic inflammatory myopathies, dermatomyositis, polymyositis, inclusion body myositis, vasculitis, an allergic disorder and/or osteoarthritis.

8. A method according to any of claims 1 to 7, wherein said inflammatory condition is systemic or defined by the tissue and/or organ of origin.

9. A method according to any of claims 1 to 8 where an animal is suffering from a renal condition, a renal disease and/or renal failure.

10. A method according to claim 9 wherein said renal condition and/or renal failure is an autoimmune disease, a metabolic disease or a disease caused by toxic compounds.

11. A method according to any of claims 1 to 10, wherein the levels of said markers are monitored and/or determined in samples comprising plasma, serum, blood, urine, saliva, or samples collected via bronchioalveolar lavage (BAL) or aspiration.

12. A method according to any of claims 1 to 11 , wherein monitoring and/or determining the levels of said markers comprises an immunoassay.

13. A method for treating or decreasing the risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto, comprising administering to an animal in need thereof a therapeutically effective amount of one or more compounds selected from the list consisting of annexin A5, anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC, as well as antibodies against components, fragments or derivatives of MDA-LDL or OxLDL (such as apoBIOO or fragments thereof), and also bioactive components and/or parts/fragments of these compounds.

14. A method according to claim 13 wherein said anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC are monoclonal or polyclonal antibodies of isotype IgA, IgD, IgE, IgG or IgM.

15. A method according to either of claims 13 or 14 where an animal is suffering from a renal condition, a renal disease and/or renal failure.

16. A method for assessing and decreasing the risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto, comprising monitoring and/or determining the levels of one or more markers selected from the list consisting of oxCL, oxPS, oxLDL, MDA-LDL, anti-oxCL, anti- oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC, comparing said levels with predetermined cutoff values, and administering to an animal in need thereof a therapeutically effective amount of one or more compounds selected from the list consisting of annexin A5, anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti- PC, as well as antibodies against components, fragments or derivatives of MDA- LDL or OxLDL (such as apoBIOO or fragments thereof), and also bioactive components and/or parts/fragments of these compounds.

17. A method according to claim 16 wherein the levels of one or more markers selected from the list consisting of oxCL, oxPS, oxLDL, and MDA-LDL are compared with predetermined cutoff values for a given population of individuals, said cutoff values being chosen so that concentrations of the one or more markers higher than said cutoff values are associated with an increased risk of developing inflammatory conditions.

18. A method according to claim 16 wherein the levels of one or more markers selected from the list consisting of anti-oxCL, anti-oxPS, anti-oxLDL, anti-MDA-LDL and anti-PC are compared with predetermined cutoff values for a given population of individuals, said cutoff values being chosen so that concentrations of the one or more markers lower than said cutoff value are associated with an increased risk of developing inflammatory conditions.

19. A composition comprising one or more inhibitors selected from the list consisting of annexin A5, inhibitors of oxCL, inhibitors of oxPS, inhibitors of oxLDL, inhibitors of MDA-LDL, and inhibitors of PC, for use as a medicament.

20. A composition according to claim 19 wherein one or more of said inhibitors are antibodies.

21. Composition according to claim 20 wherein one or more of said antibodies are monoclonal or polyclonal antibodies of isotype IgA, IgD, IgE, IgG or IgM.

22. Composition according to any of claims 19-21 for use in treatment of one or more primary and/or secondary inflammatory diseases, inflammatory conditions and/or inflammatory activity.

23. Composition according to claim 22 wherein said primary and/or secondary inflammatory diseases, inflammatory conditions and/or inflammatory activity comprise infections, auto-immune diseases, cardiovascular diseases, type II diabetes, Alzheimer's disease, dementia in general, rheumatic diseases, acute and/or chronic inflammatory conditions, type I diabetes, high blood pressure, arthrosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, ischemic attack, transient ischemic attack, claudication, angina, rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, asthma, acne, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease, arthritis including osteoarthritis, idiopathic inflammatory myopathies, dermatomyositis, polymyositis, inclusion body myositis, vasculitis, an allergic disorder and/or osteoarthritis.

24. A composition comprising one or more conjugates selected from the list consisting of oxCL conjugates, oxPS conjugates, oxLDL conjugates, MDA-LDL conjugates or PC conjugates, for use in activation immunotherapy.

25. A composition according to claim 24 for treatment of one or more primary and/or secondary inflammatory diseases, inflammatory conditions and/or inflammatory activity.

26. Composition according to claim 25 wherein said primary and/or secondary inflammatory diseases, inflammatory conditions and/or inflammatory activity comprise infections, auto-immune diseases, cardiovascular diseases, type II diabetes, Alzheimer's disease, dementia in general, rheumatic diseases, acute and/or chronic inflammatory conditions, type I diabetes, high blood pressure, arthrosclerosis, atheromatous plaque rupture, myocardial infarction, acute coronary syndrome, stroke, ischemic attack, transient ischemic attack, claudication, angina, rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, asthma, acne, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease, arthritis including osteoarthritis, idiopathic inflammatory myopathies, dermatomyositis, polymyositis, inclusion body myositis, vasculitis, an allergic disorder and/or osteoarthritis.

27. A kit for monitoring and/or determining the level of one or more markers selected from the list consisting of oxCL, oxPS, oxLDL, MDA-LDL, anti-oxCL, anti-oxPS, anti- oxLDL, anti-MDA-LDL and anti-PC for assessing the risk of developing inflammatory conditions, including infectious conditions, and/or the risk of mortality related thereto.

28. A method of preventing or treating inflammation induced by radiation, for example in radiotherapy of cancer, comprising administering to an animal in need thereof a therapeutically effective amount of

a) anti-oxCL and anti-oxPS, or

b) anti-oxCL and anti-oxPS and annexin A5.

29. A composition comprising

a) anti-oxCL and anti-oxPS, or

b) anti-oxCL and anti-oxPS and annexin A5

for use in preventing or treating inflammation induced by radiation, for example in radiotherapy of cancer.

30. A topical skin care composition comprising

a) anti-oxCL and anti-oxPS, or

b) anti-oxCL and anti-oxPS and annexin A5.

31. A method of preventing or treating inflammation induced by UVA and/or UVB rays comprising administering to an animal in need thereof a therapeutically effective amount of a topical skin care composition comprising

a) anti-oxCL and anti-oxPS, or

b) anti-oxCL and anti-oxPS and annexin A5.