OXIDIZED CARDIOLIPIN AS A PRO-INFLAMMATORY FACTOR

Abstract

Low levels of antibodies reactive with oxidised Cardiolipin (oxCL) in mammals are related to an increased risk of developing cardivacular diseases, auto-immune diseases or inflammatory conditions. High levels can have a protective function and in general there is a negative association between manifestations of these conditions and antibodies against oxCL. Thus, based on their relations methods of monitoring, determining and diagnosing as well as methods of immunisation and therapy of these diseases and conditions are provided.

Claims

1-14. (canceled)

15. A method of treating, or reducing the risk of developing, an acute inflammatory condition in a mammal, comprising administering to the mammal in need thereof a therapeutically effective amount of Annexin A5.

16. The method according to claim 15, wherein the mammal is selecting from the list consisting of: a mouse, a rat, a rabbit, a dog, a cat, cattle, a horse, and a human.

17. The method according to claim 16, wherein the mammal is a human.

Description

DRAWINGS

[0106] FIGS. 1A and 1B: Electrospray ionization mass MS spectrometer (Micromass, Beverly, Mass.) was used to demonstrate that bovine heart cardiolipin was oxidized.

[0107] FIGS. 2A and 2B: Induction of adhesion molecules. Oxidized CL but not CL induces adhesion molecules in endothelial cells, flow cytometry.

[0108] Endothelial cells were incubated with Oxidized CL (20 ug/ml) or CL (20 ug/ml) for 24 hours, oxidized CL induced ICAM-1 and VCAM-1 production but CL does not show same effect.

[0109] FIGS. 3A and 38: inhibition of oxCL-induced adhesion molecule expression by Annexin A5, detected by Flow cytometry.

[0110] Oxidized CL (20 ug/ml) was preincubated with Annexin A5 (10 ug/ml) before stimulating cells for 24 hours. Annexin A5 inhibited oxCL induced endothelial cell expression of ICAM-1 (CD54) and VCAM-1 (CD106).

[0111] FIG. 4: Induction of IL-6 by oxCL and inhibition by Annexin A5. determined by Luminex. Detected by intensity of fluorescence.

[0112] Endothelial cells were incubated with OxCL (20 ug/ml) with or without Annexin A5 (20 ug/ml) and CL (20 ug/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.

[0113] FIG. 5: Binding of oxCL by Annexin A5, ELISA.

[0114] OxCL, CL and reduced CL (5 ug/ml) were coated on ELISA plates overnight. Annexin A5 (0.32 ug/ml, 0.64 ug/ml, 1.28 ug/ml, 2.56 ug/ml, 5.12 ug/ml and 10.24 ug/ml) can bind to oxidized cardiolipin and air exposed CL but not reduced cardiolipin.

[0115] FIGS. 6A and 6B: Effect of oxCL on uptake of oxLDL in macrophages, detected by Flow cytometry.

[0116] OxCL competed macrophage uptake of OxLDL, but CL did not have such effect. (Human acute monocytic leukemia cell line) THP-1, cells differentiated MQ were studied uptake of dil-OxLDL, the uptake could be competed by OxCL but not non-oxidized CL.

[0117] FIGS. 7A, 7B, 7C and 7D: oxCL-induced calcium mobilization, Fluorometer.

[0118] Results shows that only oxidized cardiolipin can activate the neutrophils and induce the intracellular calcium mobilization. Furthermore, annexin can prevent the effects of oxCL on neutrophils.

[0119] FIG. 8: Effect of oxCL on leukotriene B4 release, EIA.

[0120] Oxidized cardiolipin promotes human neutrophils and macrophages to release leukotriene B.sub.4, but cardiolipin did not show the similar reaction.

[0121] FIG. 9: Effect of Annexin A5 on oxCL induced LTB.sub.4 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.

[0122] FIG. 10: Antibodies against oxCL were extracted from human Ig and incubated with oxCL. Experiment indicates that such extracted anti-OxCL decrease effects of oxCL, as seen by a shift to the left of the histogram.

[0123] FIGS. 11A, 11B, 11C, 11D, 11E and 11F: Effects of oxCL on activation of T-cells, flow cytometry.

[0124] FIG. 11A: Effects on CD4-positive T-cells, control

[0125] FIG. 11B: Effects on CD4-positive T-cells, 5 μg/ml of oxCL

[0126] FIG. 11C: Effects on CD4-positive T-cells, 5 μg/ml of CL (native, non-oxidized CL)

[0127] FIG. 11D: Effects on CD8-positive T-cells, control

[0128] FIG. 11E: Effects on CD8-positive T-cells, 5 μg/ml of oxCL

[0129] FIG. 11F: Effects on CD8-positive T-cells, 5 μg/ml of CL

[0130] Human PBMC (Peripheral blood mononuclear leukocytes) were incubated over night with 5 μg/ml of oxCL or CL. Both CD4 and CD8 positive T cells were studied. Quadrant Q2 represent (%) T-cells positive for CD69 expressing 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.

[0131] FIGS. 12A, 12B and 12C: A Neuroblastoma cell line was treated with amyloid peptide 1-42 which induced cell death (as determined by propidium iodide; PI). Extracted anti-oxCL inhibited the effect.

Example 1: Chemical Treatments of Cardiolipin

[0132] Native cardiolipin and oxidation product were further analyzed by mass spectrometry, FIGS. 1A and 1B. 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

[0133] 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 (FIGS. 2A and 2B). Both the increased expression of ICAM-1 and VCAM-1 (CD54 and CD106) induced by oxCL were significantly inhibited by Annexin A5 (FIGS. 3A and 3B).

Example 3: IL-6 Production

[0134] 10×6 HUVECs were seeded at 6 well plates. oxCL could stimulate endothelial cells produce 564.3±142.02 (pg/ml) IL-6 from the supernant. Annexin A5 significantly decreased Il-6 level to 276.4±28.62 (pg/ml). Comparing with the control group, both native CL and Annexin A5 themselves could not significantly increase endothelial cell Il-6 levels (FIG. 4). This supports the hypothesis that oxCL stimulates the IL-6, which is a known inflammatory marker, e.g. oxCL might be involved in inducing/mediating inflammatory responses.

Example 4: Uptake Experiment

[0135] To study potential mechanisms of oxCL bioactivity, 1×10®/well THP-1 differentiated macrophages (MQ) were used for uptake studies. oxLDL could inhibit dil-OxLDL uptake up to more than 60%, but LDL hade 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 (FIGS. 6A and 6B).

Example 5: Intracellular Calcium Mobilization

[0136] 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. 7A-7D).

Example 6: LTB4 Production

[0137] 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. 2×10.sup.6 cells/well were exposed to OxCL, CL or buffer solution. Results suggested OxCL could in a concentration dependent manner induce LTB4 production but not with native CL (FIG. 8). This increase was greatly inhibited by Annexin A5 (FIG. 9).

Example 7: ELISA for Annexin A5 Binding to Antigen

[0138] To test potential protective mechanisms of Annexin A5, oxCL, CL and reduced CL (5 ug/ml) were coated on ELISA plates overnight before different concentrations of Annexin A5 (0.32 ug/ml, 0.64 ug/ml, 1.28 ug/ml, 2.56 ug/ml, 5.12 ug/ml and 10.24 ug/ml) were added to plates. Results suggested that different concentration of Annexin A5 could bind to oxCL, which was also much stronger than overnights air exposed CL. Reduced CL could not bind to Annexin A5 (FIG. 5).

Example 8: The Role of IgM Antibodies Against Oxidized Cardiolipin (Anti-oxCL) in Prediction of Cardiovascular Disease (CVD) by Use of ELISA

[0139] Between Jul. 1, 1997 and Jun. 30, 1998, every third 60-year old man and woman, in the County of Stockholm, were invited to participate. The response rate was 78% and 4232 subjects (2039 men and 2193 women) were included. Information on sociodemography, lifestyle, medication and diseases was obtained by a self-administered questionnaire.

[0140] Physical examination (including blood pressure measurements, anthropometry and ECG) was performed and serum, plasma and whole blood were collected for storage (−80° C.). Routine tests were used for fasting levels of blood lipids (total cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides), apolipoproteins blood glucose, s-insulin, p-fibrinogen and hsCRP.

[0141] New events of coronary heart disease (CHD), myocardial infarction (MI), hospitalization for angina pectoris, ischemic stroke were registered. The study base of 4232 subjects was matched with that of the national cause of death registry (fatal events until Dec. 31, 2003) and the national in-hospital registry (non-fatal events until Dec. 31, 2005).

[0142] 211 incident cases of CVD were recorded. Only subjects without a history of CVD prior to recruitment were utilized for the matching procedures. To assess diagnoses of CVD events, the International Classification of Diseases (ICD-10) was used to register CHD-deaths (I 20, I 21, I 46), MI (I 21), angina pectoris including PCIs and CABGs (I 20, Z 95.5 and Z 95.1) and ischemic stroke (I 63-I 66). For each case three sex- and age-matched controls were randomly selected. Through this nested case-control design (211 cases and 633 controls) was applied to determine relative risks for future events.

[0143] The screening of 4232 subjects, 60-year-old (2039 men and 2193 women) with a follow up of 5-7 years was conducted and 211 incident cases of CVD (myocardial infarction, ischemic stroke, or hospitalized angina pectoris) and 633 age- and sex-matched controls were identified. Values were adjusted for smoking, BMI, type II diabetes, hypercholesterolaemia, and high blood pressure. An increased risk of CVD was determined as described in the tables.

[0144] Crude values include the matching factors age and sex. In addition adjustments have been made for smoking, BMI>29 kg/m.sup.2, type II diabetes, high s-cholesterol ≥25 mmol/l, high blood pressure >140/90 mmHg.

[0145] Descriptive analyses of were performed for cases and controls respectively with values expressed as medians (ranges) or proportions. Relative risks (RR) with 95% confidence intervals (CI) were calculated applying conditional logistic regressions. Analyses were run both crude and adjusted for traditional risk factors (as assessed by univariate analyses of significant differences between cases and controls). Statistical analyses were run with SAS®statistical software system version 9.1.

[0146] Thus, low anti-oxCL levels predicted a striking risk of stroke (CVD) and myocardial infarction (MI) in men, and also increased risk of myocardial infarction (MI) in women. It was not possible to study a similar effect of low anti-oxCL on the risk of stroke in woman due to a limited set of data.

[0147] Units were arbitrarily determined with sera two persons with moderately high-level anti-oxCL on each ELISA plate and these were used as controls and was the basis of Unit determinations. Quartile 4 represents the highest 25% of values in the whole cohort.

TABLE-US-00001 TABLE 1a Association between low levels of anti- OxCL and risk for CVD, men and women aOxCL Crude Adjust Quartiles RR 95% CI P-values RR 95% CI P-values Quartile 4 1 N/A N/A 1 N/A N/A Quartile 3 1.66 0.97-2.85 0.0653 1.84 1.08-3.33 0.0267 Quartile 2 1.67 0.97-2.88 0.0658 1.69 0.96-3.00 0.0699 Quartile 1 2.15 1.25-3.70 0.0036 2.15 1.23-3.78 0.0075

TABLE-US-00002 TABLE 3a Association between low levels of anti-OxCL and risk for MI, men aOxCL Crude Adjust Quartiles RR 95% CI P-values RR 95% CI P-values Quartile 4 1 N/A N/A 1 N/A N/A Quartile 3 1.39 0.73-2.66 1.6 0.81-3.17 Quartile 2 1.51 0.78-2.94 1.7 0.84-3.46 Quartile 1 2.16 1.14-4.09 2.30 1.17-4.51

TABLE-US-00003 TABLE 4 Association between low levels of anti-OxCL and risk for MI, women aOxCL Crude Adjust Quartiles RR 95% CI P-values RR 95% CI P-values Quartile 4 1 N/A N/A 1 N/A N/A Quartile 3 2.52 0.93-6.82 2.96 1.05-8.30 Quartile 2 2.13 0.82-5.52 1.97 0.73-5.31 Quartile 1 1.97 0.70-5.61 1.87 0.65-5.41

TABLE-US-00004 TABLE 5a Association between low levels of anti-OxCL and risk for stroke, men aOxCL Crude Adjust Quartiles RR 95% CI P-values RR 95% CI P-values Quartile 4 1 N/A N/A 1 N/A N/A Quartile 3 2.24 0.54-9.31  7.05 1.05-47.4  Quartile 2 2.7 0.54-13.48 8.03 0.93-69.56  Quartile 1 2.69 0.64-11.31 12.28 1.48-101.77

[0148] Relative risks (RR) using SAS were determined with 95% confidence intervals (CI) and quartiles of anti-oxCL levels where the 100% percentile was set as the reference value (RR=1.0). Thus, the percentile unit represent the indicated fraktile of the level of anti-oxCL of the analysed patients, respectively. Cut off levels are based on the total of men and women.

[0149] Thus, low anti-oxCL levels predicted a striking risk of stroke (CVD) and myocardial infarction, MI, in men, and also increased risk of myocardial infarction, MI, in women. It was not possible to have a similar effect of low anti-oxCL on the risk of stroke in woman confirmed due to a limited set of data].

[0150] In the following tables in this example, levels below the indicated were compared to those above.

TABLE-US-00005 TABLE 7a Association between low levels of anti-OxCL and risk for cvd men + women aOxCL percentile Crude Adjust unit RR 95% CI P-values RR 95% CI P-values 35 1.499 1.077 2.086 0.0163 1.520 1.076 2.146 0.0174 33 1.487 1.067 2.073 0.0191 1.484 1.049 2.099 0.0256 30 1.408 1.006 1.971 0.0460 1.386 0.976 1.968 0.0684 25 1.361 0.951 1.949 0.0919 1.366 0.941 1.982 0.1010 24 1.206 0.838 1.735 0.3136 1.219 0.836 1.776 0.3034 23 1.168 0.808 1.686 0.4086 1.185 0.810 1.733 0.3826 22 1.129 0.779 1.636 0.5220 1.145 0.781 1.677 0.4882 21 1.204 0.831 1.745 0.3253 1.219 0.831 1.787 0.3111 20 1.280 0.876 1.870 0.2018 1.282 0.867 1.895 0.2137 19 1.345 0.915 1.978 0.1315 1.333 0.896 1.983 0.1559 18 1.493 1.012 2.202 1.480 0.991 2.211 0.0556 17 1.400 0.938 2.090 0.0993 1.379 0.913 2.082 0.1265 16 1.385 0.915 2.097 0.1235 1.363 0.891 2.085 0.1531 15 1.410 0.924 2.151 0.1108 1.386 0.901 2.132 0.1377 14 1.527 0.988 2.360 0.0568 1.511 0.969 2.357 0.0688 13 1.620 1.040 2.524 0.0330 1.556 0.989 2.446 0.0558

TABLE-US-00006 TABLE 8a Association between low levels of anti-OxCL and risk for cvd men aOxCL percentile Crude Adjust unit RR 95% CI P-values RR 95% CI P-values 35 1.936 1.295 2.896 0.0013 2.050 1.342 3.133 0.0009 33 2.027 1.355 3.033 0.0006 2.142 1.401 3.274 0.0004 30 1.801 1.201 2.700 0.0044 1.885 1.232 2.884 0.0035 25 1.594 1.047 2.427 0.0296 1.655 1.067 2.566 0.0244 24 1.401 0.913 2.148 0.1224 1.440 0.924 2.246 0.1073 23 1.357 0.882 2.087 0.1648 1.412 0.903 2.208 0.1305 22 1.265 0.819 1.954 0.2895 1.311 0.837 2.053 0.2369 21 1.347 0.873 2.078 0.1777 1.389 0.887 2.174 0.1511 20 1.412 0.910 2.189 0.1235 1.460 0.927 2.299 0.1026 19 1.448 0.927 2.262 0.1037 1.479 0.932 2.346 0.0966 18 1.618 1.031 2.539 0.0362 1.638 1.026 2.613 0.0386 17 1.519 0.958 2.409 0.0754 1.507 0.937 2.424 0.0909 16 1.570 0.973 2.533 0.0646 1.560 0.955 2.549 0.0760 15 1.522 0.935 2.476 0.0910 1.485 0.903 2.442 0.1192 14 1.620 0.980 2.677 0.0598 1.591 0.952 2.660 0.0763 13 1.839 1.104 3.062 0.0193 1.731 1.028 2.915 0.0392

TABLE-US-00007 TABLE 9a Association between low levels of anti-OxCL and risk for MI men aOxCL percentile Crude Adjust unit RR 95% CI P-values RR 95% CI P-values 35 1.895 1.213 2.960 0.0050 1.912 1.191 3.069 0.0073 33 2.005 1.283 3.133 0.0023 2.023 1.259 3.250 0.0036 30 1.852 1.178 2.909 0.0075 1.829 1.135 2.947 0.0132 25 1.652 1.032 2.643 0.0363 1.609 0.980 2.642 0.0602 24 1.508 0.936 2.429 0.0912 1.460 0.884 2.413 0.1396 23 1.431 0.884 2.318 0.1449 1.409 0.846 2.347 0.1882 22 1.290 0.792 2.103 0.36 1.274 0.764 2.124 0.3525 21 1.397 0.859 2.270 0.1777 1.361 0.816 2.268 0.2376 20 1.484 0.906 2.431 0.1169 1.442 0.859 2.422 0.1661 19 1.477 0.895 2.438 0.1270 1.426 0.844 2.409 0.1851 18 1.614 0.978 2.663 0.0610 1.545 0.911 2.620 0.1062 17 1.561 0.937 2.599 0.0873 1.470 0.862 2.509 0.1575 16 1.589 0.935 2.701 0.0871 1.511 0.870 2.622 0.1426 15 1.529 0.889 2.630 0.1249 1.433 0.817 2.513 0.2097 14 1.658 0.954 2.882 0.0729 1.574 0.887 2.794 0.1212 13 1.895 1.080 3.327 0.0260 1.724 0.959 3.098 0.0685

TABLE-US-00008 TABLE 10a Association between low levels of anti-OxCL and risk for stroke men aOxCL percentile Crude Adjust unit RR 95% CI P-values RR 95% CI P-values 35 2.127 0.833 5.430 0.1143 4.031 1.107 14.674 0.0345 33 2.127 0.833 5.430 0.1143 4.031 1.107 14.674 0.0345 30 1.607 0.645 4.003 3.410 0.942 12.347 0.0617 25 1.385 0.543 3.531 0.4950 2.453 0.719 8.376 0.1521 24 1.042 0.397 2.735 0.9342 1.712 0.497 5.898 0.3941 23 1.104 0.426 2.866 0.8382 1.762 0.521 5.962 0.3622 22 1.174 0.452 3.051 0.7421 1.882 0.553 6.406 0.3118 21 1.174 0.452 3.051 0.7421 1.882 0.553 6.406 0.3118 20 1.174 0.452 3.051 0.7421 1.882 0.553 6.406 0.3118 19 1.344 0.506 3.565 0.5531 2.123 0.611 7.372 0.2358 18 1.637 0.584 4.588 0.3488 2.246 0.638 7.907 0.2078 17 1.352 0.465 3.933 0.5802 1.704 0.474 6.125 0.4145 16 1.491 0.495 4.490 0.4772 1.86 0.506 7.095 0.3423 15 1.491 0.495 4.490 0.4772 1.896 0.506 7.095 0.3423 14 1.454 0.441 4.792 0.5388 1.566 0.388 6.321 0.5290 13 1.605 0.486 5.306 0.4379 1.649 0.427 6.371 0.4679

Example 9

[0151] The same data material as described in example 8 was used herein.

[0152] Descriptive analyses were performed for cases and controls respectively with values expressed as medians (ranges) or proportions. Relative risks (RR) with 95% confidence intervals (CI) were calculated applying conditional logistic regressions. Analyses were run both crude and adjusted for traditional risk factors (as assessed by univariate analyses of significant differences between cases and controls). Statistical analyses were run with SAS®statistical software system version 9.1.

[0153] Relative risks (RR) using SAS were determined with 95% confidence intervals (CI) and quartiles of anti-oxCL levels where the highest quartile representing the highest values of anti-OxCL was set as the reference value (RR=1.0). Thus, percentile units represent the 76% and up to 98% fraktile of the level of anti-oxCL of the analysed patients, respectively. Cut off levels are based on the total of men and women.

[0154] Thus, low levels of anti-OxCL are associated with increased risk of development of CVD. Here it is demonstrated that high levels of anti-OxCL can be protective for CVD.

TABLE-US-00009 Association between high levels of anti-OxCL and risk for CVD (MI-stroke)men + women aOxCL percentile Crude Adjust unit RR 95% CI P-values RR 95% CI P-values 98 0.693 0.197 2.430 0.5662 0.566 0.154 2.078 0.3912 96 0.825 0.354 1.924 0.6564 0.716 0.297 1.722 0.4550 94 0.649 0.310 1.360 0.2522 0.606 0.283 1.296 0.1966 90 0.631 0.351 1.134 0.1239 0.600 0.331 1.089 0.0929 86 0.537 0.319 0.905 0.0196 0.485 0.283 0.829 0.0082 84 0.576 0.356 0.932 0.0246 0.534 0.326 0.874 0.0125 82 0.607 0.387 0.950 0.0291 0.577 0.365 0.913 0.0187 80 0.698 0.460 1.057 0.0892 0.653 0.427 1.001 0.0503 78 0.734 0.493 1.092 0.1272 0.706 0.469 1.062 0.0946 76 0.661 0.443 0.986 0.0425 0.643 0.427 0.969 0.0347

TABLE-US-00010 Association between high levels of anti-OxCL and risk for cvd (MI-stroke)men aOxCL percentile Crude Adjust unit RR 95% CI P-values RR 95% CI P-values 98 0.750 0.159 3.532 0.7161 0.522 0.104 2.625 0.4301 96 0.918 0.336 2.507 0.8672 0.705 0.245 2.024 0.5155 94 0.648 0.264 1.592 0.3438 0.539 0.211 1.375 0.1959 90 0.415 0.182 0.949 0.0371 0.364 0.157 0.843 0.0184 86 0.425 0.211 0.858 0.0170 0.359 0.174 0.743 0.0057 84 0.421 0.215 0.821 0.0112 0.358 0.179 0.712 0.0035 82 0.473 0.258 0.868 0.0156 0.421 0.225 0.787 0.0067 80 0.578 0.334 1.001 0.0505 0.518 0.293 0.914 0.0232 78 0.607 0.361 1.020 0.0594 0.565 0.330 0.968 0.0377 76 0.534 0.315 0.905 0.0197 0.496 0.286 0.858 0.0121

TABLE-US-00011 Association between high levels of anti-OxCL and risk for cvd (infakt-stroke)women aOxCL percentile Crude Adjust unit RR 95% CI P-values RR 95% CI P-values 98 0.600 0.070 5.136 0.6410 0.612 0.067 5.572 0.6628 96 0.652 0.136 3.133 0.5930 0.696 0.136 3.570 0.6641 94 0.653 0.178 2.391 0.5194 0.746 0.195 2.851 0.6688 90 1.121 0.475 2.643 0.7944 1.170 0.483 2.836 0.7280 86 0.756 0.343 1.663 0.4861 0.716 0.317 1.619 0.4222 84 0.878 0.430 1.793 0.7215 0.905 0.435 1.881 0.7889 82 0.865 0.439 1.702 0.6737 0.878 0.439 1.757 0.7136 80 0.924 0.486 1.755 0.8083 0.893 0.462 1.727 0.7371 78 0.992 0.530 1.856 0.9788 0.967 0.510 1.833 0.9179 76 0.919 0.491 1.719 0.7921 0.911 0.484 1.717 0.7739

Example 10

[0155] Patient sera from a women control population was used herein. Data was obtained by use of simple regression and Spearman Rank test was used in statistical analysis (Stat View).

[0156] The association between important parameters and determinants in relation to anti-oxCL is determined.

TABLE-US-00012 Controls from the general population (population controls; 26 women) Clinical role of antibodies against oxidized cardiolipin and R p Glucos in plasma −0.41 0.035 Systolic blood pressure −0.66 0.001 Diastolic blood pressure −0.428 0.015 Age no association 0.2 BMI −0.56 0.0053 No association with anticardiolipin antibodies or lupus antikoagulans (an indirect measure for anti-cardiolipin antibodies): p = 0.41

[0157] Thus, these data indicate strong and significant negative associations between anti-OxCL antibodies and important factors as blood pressure, diabetes and blood sugar, Body Mass Index 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.

[0158] The role of anti-oxCL in 52 women with a prototypic autoimmune disorder, systemic lupus erythematosus (SLE) is demonstrated.

TABLE-US-00013 SLE-patients: Association between anti- OxCL in women with SLE (n = 52) Clinical role of antibodies against oxidized cardiolipin and R P TNF-induction when leukocytes (PBMC) −0.32 0.02. are cultured with endotoxin 1 ng/ml and serum from patients Endothelial function (nitroinduced 0.43 0.023 dilatation of a Brachialis) VCAM (important inflammatory/vascular −0.40 0.0046 marker): TNF (Major inflammatory cytokine) −0.40 0.0043 Age no association 0.89 DIsease severity index SLICC: below index 4 (117 ± 48 compared to above index 4 (63 ± 14); p = 0.0085

[0159] 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, is 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 11

[0160] Both CD4 and CD8 positive T-cells from the cell line Human PBMC where studied in an incubation setup illustrated in FIG. 11A-F. Both T-cell types where incubated with, buffer (control), oxCL or CL to determine the effect on activation, interpreted by the expression of CD69 surface molecule after stimulation.

[0161] 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.

[0162] The control-stimulation (FIGS. 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 (FIGS. 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 (FIGS. 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.

[0163] 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.

[0164] FIG. 12A-C illustrates the effect of anti-oxCL on amyloid peptide 1-42 induced cell death.

[0165] 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.

[0166] The cells were treated with 5 uM 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.

[0167] In the control experiment the amount of dead cells was measured to 23.99%, whereas the amyloid peptide 1-42 induced cell death increased that number to 45.54%. When amyloid peptide 1-42 was incubated together with oxCL-IgG, 30 ug/ml, (anti-oxCL), the cell dead is reduced to 37.10%.

[0168] 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.

[0169] Thus, from the above it emerges, that oxCL has a T-cell activating properties, and is involved in cell death, which is supported by the cell death protective effect of anti-oxCL illustrated in FIG. 12A-C.

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[0206] All given references as listed above and discussed in the description are hereby incorporated by reference.