Phosphorylcholine conjugates and corresponding antibodies

Abstract

In subjects with hypertension, increases in intima-media thickness (IMT) at four years were less in subjects also having high autoantibodies particularly IgM, to phosphorylcholine. The presence or absence of autoantibodies, particularly IgM, against phosphrylcholine is thus related to an increased or decreased risk of developing ischemic cardiovascular diseases. A method to determining antibodies, particularly IgM antibodies, toward phosphorylcholine is proposed in this invention to identify subjects at risk of developing ischemic cardiovascular diseases. Animal experiments show that medium to high levels of antibodies, particularly IgM antibodies, can be detected in plasma after active immunization with a keyhole limpet hemocyanin (KLH)-phosphorylcholine conjugate. Pharmaceutical compositions comprising a phosphorylcholine conjugate or antibody preparations, for example a monoclonal antibody, with specificity to a phosphorylcholine conjugate is proposed as is use of these compositions as active or passive immunogens in the treatment or prevention of atherosclerosis.

Claims

1. A method for immunization and treatment of a human against an atherosclerotic related disease selected from the group consisting of cardiovascular disease, stroke myocardial infarction and/or peripheral artery disease, the method comprising the step of administering to the human a pharmaceutical composition comprising a monoclonal antibody preparation with specificity to a phosphorylcholine conjugate, wherein said monoclonal antibody preparation comprises monoclonal antibodies with specificity for a phosphorylcholine conjugate.

2. The method of claim 1 wherein the composition is administered by injection.

3. The method of claim 1, wherein the phosphorylcholine conjugate comprises phosphorylcholine linked to a carrier via a spacer.

4. The method according to claim 1, wherein the phosphorylcholine conjugate comprises phosphorylcholine linked to a protein carrier, optionally via a spacer.

5. The method according to claim 4, wherein the protein carrier is KLH (keyhole limpet hemocyanin) or human serum albumin (HSA).

6. The method according to claim 3, wherein the phosphorylcholine conjugate comprises phosphorylcholine linked to a latex bead, optionally via a spacer.

7. A method of prophylactic or therapeutic treatment of a human being suffering from or facing the risk of developing an atherosclerotic related disease selected from the group consisting of cardiovascular disease, stroke, myocardial infarction and/or peripheral artery disease, whereby a therapeutically effective amount of monoclonal antibody preparation with specificity to a phosphorylcholine conjugate is administered, wherein said monoclonal antibody preparation comprises monoclonal antibodies with specificity for a phosphorylcholine conjugate.

8. The method of claim 7, wherein the human that is immunized and treated is a human patient that has been determined to be at risk of developing or progression of cardiovascular disease, myocardial infarction, stroke and/or peripheral artery disease by a method comprising assessing the human patient's level of antibodies reactive with a phosphorylcholine conjugate, wherein the level of antibodies reactive with a phosphorylcholine conjugate correlates negatively with the risk of developing or progression of cardiovascular disease in a healthy human patient.

9. The method of claim 8, wherein the patient has been determined to be at risk of developing or progression of cardiovascular disease.

10. The method of claim 9 wherein the cardiovascular disease is atherosclerosis.

11. The method of claim 8, wherein the human patient has been determined to be at risk of developing or progression of cardiovascular disease by a method comprising assessing the human patient's level of IgM antibodies reactive with a phosphorylcholine conjugate.

12. The method of claim 8, wherein the human patient has been determined to be at risk of developing or progression of cardiovascular disease by a method comprising assessing the human patient's level of IgG antibodies reactive with a phosphorylcholine conjugate.

13. The method of claim 11 wherein the cardiovascular disease is atherosclerosis.

14. The method of claim 12 wherein the cardiovascular disease is atherosclerosis.

15. The method of claim 8, wherein the patient has been determined to be at risk of developing or progression of myocardial infarction.

16. The method of claim 15, wherein the human patient has been determined to be at risk of developing or progression of myocardial infarction by a method comprising assessing the human patient's level of IgM antibodies reactive with a phosphorylcholine conjugate.

17. The method of claim 8, wherein the human patient has been determined to be at risk of developing or progression of myocardial infarction by a method comprising assessing the human patient's level of IgG antibodies reactive with a phosphorylcholine conjugate.

18. The method of claim 8, wherein the patient has been determined to be at risk of developing or progression of peripheral artery disease.

19. The method of claim 15, wherein the human patient has been determined to be at risk of developing or progression of peripheral artery disease by a method comprising assessing the human patient's level of IgM antibodies reactive with a phosphorylcholine conjugate.

20. The method of claim 8, wherein the human patient has been determined to be at risk of developing or progression of peripheral artery disease by a method comprising assessing the human patient's level of IgG antibodies reactive with a phosphorylcholine conjugate.

Description

FIGURES

(1) FIG. 1a: Inhibition of antibody (IgM) binding to ELISA-plates coated with PC albumin by 2GPI, PS and CL. Inhibition by different antigens of binding to PC albumin-coated plates. In order to investigate the specificity of aPC, competition assays were performed as described in the Experimental section. Results are presented as meanSD.

(2) FIG. 1b: Inhibition of antibody (IgG) binding to ELISA-pates coated with PC albumin by 2GPI, PS and CL. Inhibition by different antigens of binding to PC albumin-coated plates. In order to investigate the specificity of aPC, competition assays were performed as described in the Experimental section. Results are presented as meanSD.

(3) FIG. 2a: Inhibition of antibody (IgM) binding to ELISA-pates coated with PC albumin by oxLDL and MDA-LDL. Inhibition by different antigens of binding to PC albumin-coated plates. In order to investigate the specificity of aPC, competition assays were performed as described in the Experimental section. Results are presented as meanSD.

(4) FIG. 2b: Inhibition of antibody (IgG) binding to ELISA-pates coated with PC albumin by oxLDL and MDA-LDL. Inhibition by different antigens of binding to PC albumin-coated plates. In order to investigate the specificity of aPC, competition assays were performed as described in the Experimental section. Results are presented as meanSD.

(5) FIGS. 3a-3b: effect on oxLDL uptake in macrophages by aPC extracted from IGIV

(6) We tested two groups: macrophages with oxLDL and macrophages with oxLDL+preincubation with aPC extracted from IVIG.

(7) macrophage+-oxLDL (total 107 cells):

(8) Weak staining 37/107=34.58%

(9) Strong Staining 10/107=9.35%

(10) total staining positive 47/107=43.93%

(11) macrophage+Dil-oxLDL+aPC-group (checked 156 cells):

(12) Weak staining 37/156=23.72%

(13) Strong Staining 2/156=1.28%

(14) total staining positive 39/156=25%.

(15) FIG. 3A shows staining with Dil-labelled oxLDL. FIG. 3B shows staining with unlabeled oxLDL.

(16) FIG. 4: Effect of pre-incubation of high antiphospholipid antibodies (aPLs) titer serum with human pooled immunoglobulin Gammagard on Annexin V binding to human umbilical endothelia cells (HUVECs): flow cytometry analysis after 24 hrs culture.

(17) TABLE-US-00001 IVIG pre-incubated Median fluorescence intensity with serum at (MFI) of Annexin V binding 0 mg/ml 649 2.5 mg/ml 913 5 mg/ml 1269 10 mg/ml 1382

(18) FIG. 5: effect of aPC on ICAM-induction in endothelial cells

(19) 1 ug/ml of PAF was added to cultures of endothelial cells, with or without preincubation with aPC IgM. Expression of ICAM-1 was tested by FACScan. Green line represents PAF effect, while red is PAF+aPC IgM and black control. The data clearly indicate a shift to the left of histogram when aPC IgM are added.

EXPERIMENTAL

(20) Subjects

(21) 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).sup.25,26. Samples were collected following a 4-week washout period with no medication to minimize the effects of treatment on the measured parameters. Blood pressure, cholesterol and triglyceride levels were determined as described previously.sup.25,26. One hundred and fifteen of the subjects were subsequently assigned to treatment with the -blocker atenolol, and 111 of the subjects were assigned to treatment with the calcium antagonist lacidipine. The study was approved by the Ethics Committee of the Karolinska Hospital and was conducted in accordance with the Helsinki Declaration. All subjects gave informed consent.

(22) Carotid Ultrasound

(23) Carotid ultrasound determinations were performed and analysed as detailed elsewhere25,26. A total of 218 patients had valid ultrasound measurements at baseline and 4-year 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 (I-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-adventitious 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 (CBMmax) at the 4-year follow-up. The associations between antibody levels to PC at enrolment into the study with an increase or decrease in IMT at the 4-year follow-up were evaluated.

(24) Reagents

(25) Polysorp F96 microtiter immuno-plates were purchased from Nunc (Roskilde Denmark), PC-BSA (Phosphorylcholine-Bovine Serum Albumin) was purchased from Biosearch Technologies, INC (USA).

(26) Bovine serum albumin (BSA), Alkaline phosphatase conjugated goat anti-human IgG (r-chain specific), Alkaline phosphatase conjugated goat anti-human IgM (u-chain specific), PNPP (Alkaline phosphatase substrate), were obtained from Sigma (St. Louis, Mo., USA). Cardiolipin (CL) was purchased from AVANTT (US, .sub.2glycoprotein (.sub.2GP1) was obtained from Calbiochem (US).

(27) Total IgG and IgM levels were determined by routine techniques as previously described.sup.6.

(28) CRP was analyzed in erum by highly sensitive methods using particle-enhanced immunonephelometry (Behring Nephelometer Analyzer, BN II (Dade Behring GmBH, Marburg, Germany)) with an antir-assay variation <4%.

(29) Determination of Autoantibodies Against PC, oxLDL and MDA-LDL

(30) IgG and IgM antibodies to PC-BSA were determined by enzyme-linked immunsorbent assay (ELISA). Pooled serum from 17 antiphospholipid syndrome patients was used as an internal standard and tested on every plate. The plateau of antibody binding was reached with the antigen concentration of 10 g/ml. F96 microtiter polysorp pate was therefore coated with PC-BSA (10 g/ml) 50 g.Math.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 2 h at room temperature and washed as described above. Serum saples were diluted (1:30) in 0.2% BSA-PBS and added at 50 l/well.

(31) 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 described.sup.6.

(32) OxLDL and MDA-LDL were determined by ELISA essentially as described.sup.6. OxLDL or MDA-LDL was diluted to 2 g/ml in coating buffer (carbonate-bicarboatne buffer 50 mM pH9.7), and 100 l/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 horus in room temperature. They were then incubated with 100 l serum, diluted 1:30 in 20% ABS-PBS at 4 C. overnight.

(33) 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 1001/well and incubated at 4 C. overnight. After five washing, color was developed by adding the alkaline phosphatase substrate (PNPP) at 100 l/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 405 nm. All samples were measured in a single assay and the coefficient of variation was below 10-15%.

(34) Specificity of Anti-Phosphorylcholine-BSA Antibodies

(35) In order to investigate the specificity of anti-phosphorylcholine-BSA, absorption assays were performed by use of pooled high titer sera. At a dilution giving 50% of maximal binding to PC-BSA, high titer pooled sera were preincubated with different concentration of PC-BSA. After vortexing, the tubes were incubated at 40 C. overnight and centrifuged at 13000 r.p.m. for 30 min (40 C.) The supernatants were tested for antibody binding to PC-BSA as described. The percentage of inhibition was calculated as follows:
Percent inhibition=(OD without competitorOD with competitor)100/OD without competitor.
Statistical Analysis

(36) anti-phosphorylcholine levels were dichotomized at the 75th and 90th percentile. The association between anti-phosphorylcholine (or other antibodies) and the progression of atherosclerosis over a 4-year period were determined by estimating increases in IMT (yes or no) using logistic regression analysis and the calculation of odds ratios (ORs) and 95% confidence intervals (CI), or comparison using Spearman correlation as indicated. Adjustments were made for possible confounders including age, smoking habits, serum cholesterol, serum triglycerides and mode of anti-hypertensive treatment (lacidipine, atenolol). A two-tailed p-value <0.05 was considered as significant.

(37) Results

(38) Basic characteristics of the subjects at the time of enrolment into the study have been detailed elswhere (Pockley et al (2003) Hypertension 42, 235-238) and are presented in Table I.

(39) Competition studies reveal that aPC of IgM and IgG subclass was competed out by preincubation with PC-BSA, while cardiolipin had a weak and phosphatidylserine no competitive capacity (FIG. 1a, 1b). 2-glycoprotein I competed to some extent with IgG binding to PC-BSA but not so much with IgM (FIG. 1a, 1b). PC-BSA had a low capacity to compete out binding to the other antigens tested (data not shown). OxLDL and MDA-LDL could compete out binding of IgM aPC to PC-BSA, and also of IGG aPC, though not to the same extent (FIG. 2a, 2b).

(40) Increases in IMT at follow-up were less prevalent in subjects having high serum levels of IgM to PC (75th or 90th percentile), oxLDL and MDA-LDL (90th percentile) at the time of enrolment, while CRP was not associated with IMT-changes (Table 2).

(41) Logistic regression analysis revealed that the relationships between IgM autoantibodies to PC, oxLDL and MDA-LDL and changes in IMT were independent of age, smoking habits, treatment with atenolol or lacidipine and blood lipids. aPC IgM were significantly associated with changes in IMT at both 75th and 90th percentile, while aOxLDL and aMDA-LDL of IgM subclass only showed significance at the 90th (table 3a-d). IgM autoantibodies were also independent of IgG values (data not shown). Furthermore, total IgG and IgM levels were not associated with IMT-measurements or changes (data not shown).

(42) IgG autoantibodies to PC were trendwise lower in subjects with increases in IMT but this difference did not reach statistical significance (Table 2).

(43) There were striking differences between men and women. aPC, aMDA-LDL, and aOxLDL of IgM subclass, were significantly higher in women than in men (p's<0.05). In contrast, there were no differences between men and women in IgG levels of these autoantibodies. In addition, women had a significantly lower occurrence of plaque at baseline and follow-up (p<0.05).

(44) aPC IgM levels correlated negatively with increase in IMT (Rho 0.18, p=0.006) in contrast to two other protection factors, HDL and HSP70 which did not correlate with IMT changes as continuous measurements (data not shown). Unlike aPC IgM, aOXLDL and aMDA-LDL did not reach significance in these determinations (data not shown).

(45) There were significant associations between aPC IgM levels and aOCLDL IgM (Rho 0.74, p<0.001) and aMDA-LDL IgM (rho 0.51, p<0.001). Likewise aPC correlated with HSP60 (Rho 0.28, p<0.001), HSP70 (Rho 0.35, p<0.001), which we recently described as a novel protective afactor for human athersclerosis in this cohort (Pockley et al (2003) supra) and also with HDL (Rho 0.23, p<0.01). There were not associations between aPC IgM, aOxLDL IgM or aOxLDL, MDA-LDL and LDL, CRP or triglycerides (data not shown).

(46) When separate logistic regression analyses were made for men and women, controlling for age, total cholesterol, triglycerides, smoking and treatment IgM aPC showed significant protective effects in women only when 90th percentile was studied (EXP (B)=0.17, 95% CI=0.05-0.68; p=0.01 and in men only when 75th percentile was studied EXP (B)=0.18, 95% CI=0.04-0.74; p=0.01, respectively).

(47) IgM to MDA-LDL and oxLDL differend in this respect, since only values for women reached statistical significance independently. Thus, when separate logistic regression analyses were to made for men and women, controlling for age, total cholesterol, triglycerides, smoking and treatment both IgM to MDA-LDL and IgM to OxLDL showed significant protective effects in women (EXP (B)=0.17, 95% CI=0.05-0.68, p=0.01 and EXP (B)=0.18, 95% CI=0.04-0.74, p=0.01, respectively), whereas the effect did not reach significance among men (EXP (B)=0.60, 95% CI=0.15-2.2, p=0.44, and EXP (B)=0.39, 95% CI=0.10-1.5, p=0.17 respectively), indicating that high IgM titers to OxLDL and to MDA-LDL may be specifically protective among women.

(48) TABLE-US-00002 TABLE 1 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 57.6 57.7 (7.8) (7.6) (7.9) Sex (% males) 50 46 53 BMI 26.7 26.3 27.1 (3.7) (3.3) (3.9) Total cholesterol 232.4 233.5 231.4 (37.8) (38.1) (37.4) HDL 55.6 56.5 54.7 (27.6) (25.8) (27.6) LDL 149.4 149.7 149.2 (37.8) (37.1) (38.6) Trigyclerides 131.6 128.6 134.7 (58.2) (57.0) (59.5)

(49) TABLE-US-00003 TABLE 2 Unadjusted prediction of changes in IMT with baseline levels of IgG and IgM autoantibodies to phosphorylcholine (PC). Odds (95% CI) Variable Ratio Lower Upper P 75th percentile aPC (IgG) .60 .32 1.1 .10 aPC (IgM) .46 .25 .85 .01 aOxLDL 1.2 .64 2.3 .57 (IgG) aOxLDL .77 .41 1.4 .40 (IgM) aMDA-LDL .80 .43 1.5 .48 (IgG) aMDA-LDL .67 .36 1.2 .18 (IgM) C-reactive .80 .43 1.5 .46 protein 90th percentile aPC (IgG) .60 .25 1.4 .24 aPC (IgM) .36 .15 0.87 .024 aOxLDL .94 .38 2.31 .90 (IgG) aOxLDL .27 .11 .69 .006 (IgM) aMDA-LDL .63 .26 1.5 .30 (IgG) aMDA-LDL .27 .11 .69 .006 (IgM) C-reactive .60 .24 1.4 .24 protein

(50) TABLE-US-00004 TABLE 3a prediction of changes in MT over a 4-year period using the 75th percentile of aPC IgM autoantibodies at baseline in subjects with established hypertension. Estimated Variable Co- odds in the efficient ratio 95% CI model (B) Exp(B) P Lower Upper Smoking .01 .99 .95 .66 1.5 Sex .05 .95 .87 .54 1.4 Total .003 1.0 .45 .99 1.0 choles- terol Plasma .001 .99 .63 .99 1.0 triglyc- erides Age .01 1.0 .59 .97 1.0 (years) Treat- .23 .79 .40 .45 1.4 ment (A/L) APC 1.0 .37 .0027 .15 .89 IgM

(51) TABLE-US-00005 TABLE 3b prediction of changes in IMT over a 4-hyear period using the 90th precentile of aPC IgM autoantibodies in subjects with established hypertension. Variable Co- Estimated in the efficient odds ratio 95% CI model (B) Exp(B) P Lower Upper Smoking .02 .97 .90 .65 1.5 Sex .005 1.0 .98 .56 1.8 Total .003 1.0 .42 .99 1.0 choles- terol Plasma .001 0.99 .67 .99 1.0 triglyc- erides Age .003 1.0 .87 .97 1.0 (years) Treat- .22 .80 .43 .46 1.4 ment (A/L) aPC .77 .46 .017 .24 .87 IgM

(52) TABLE-US-00006 TABLE 3c Prediction of changes in IMT over a 4-year period using the 90th percentile with IgM autoantibodies to OxLDL and other risk factors in subjects with established hypertension Variable in Coefficient Estimated odds 95% CI the model (B) ratio Exp(B) P Lower Upper Smoking .01 .99 .95 .66 1.5 Sex .001 1.1 .98 .56 1.8 Total .001 1.0 .72 .99 1.1 cholesterol Plasma .001 1.0 .71 .99 1.0 triglycerides Age (years) .01 1.0 .60 .97 1.1 Treatment .28 .77 .35 .44 1.3 (A/L) aOxLDL IgM 1.3 .26 .008 .11 .72

(53) TABLE-US-00007 TABLE 3d Predicition of changes in IMT over a 4-year period using the 90th percentile with IgM autoantibodies to MDA-LDL and other risk factors in subjects with established hypertension Variable in Coefficient Estimated odds 95% CI the model (B) ratio Exp(B) P Lower Upper Smoking .07 0.93 .73 .62 1.4 Sex .001 .99 .99 .56 1.7 Total 0.001 1.0 .78 .99 1.0 cholesterol Plasma .001 .99 .74 .99 1.1 triglycerides Age (years) 0.01 1.0 .54 .97 1.0 Treatment .27 .76 .34 .44 1.3 (A/L) aMDM-LDL 1.1 .31 .01 .12 .79 IgM

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Study Showing Protective Effect of aPC

(55) In an observational study from Malm (the Malm Diet and Cancer Study), about 6000 out of 30000 subjects from the cohort were recruited for extensive cardiovascular investigations, including non-invasive assessment of subclinical atherosclerosis through ultrasound measurements of the carotids. In addition, additional cardiovascular risk factors were measured at baseline. These subjects have been followed for over 10 years with regard to the occurrence of new events of cardiovascular diseases (myocardial infarction, chronic coronary heart disease, atherotrombotic stroke). In order to assess the relative risks (calculated as relative hazards) with 95% confidence intervals, nested-case-control analyses (3 controls per case) were undertaken for low levels of antibodies against phosphorylcholine (aPC-IgM). There were in total 145 CVD cases (mainly myocardial infarction (MI) and ischemic stroke) and 400 age and sex-matched controls. The cutoff level for aPC was 307 for the tenth percentile of aPC levels. There were in total 20 CVD cases with aPC levels below the tenth percentile (14%), and 34 controls (9%), corresponding to a relative hazard of 1.9 (95% CI 1.1-4.3). The corresponding number of male CVD cases below the tenth percentile of aPC was 16 (19%), and 25 control patients below this level (11%), corresponding to a relative hazard of 1.9 (95% CI 1.1-3.5). The number of female cases was too low to yield robust information on relative risks (see Tables 1 and 2). The results suggest that low aPC levels are predictive for the occurrence of cardiovascular disease in healthy subjects, and could act as markers for cardiovascular diseases.

(56) TABLE-US-00008 TABLE 1 Descriptive statistics for aPC (<10.sup.th percentile) SEX Males Females All Below 10.sup.th pct Case Control Case Control Case Control No n 68 206 57 160 125 366 % 81 89 93 95 86 92 Yes N 16 25 4 9 20 34 % 19 11 7 5 14 9

(57) TABLE-US-00009 TABLE 2 Univariate analysis of the influence of aPC (<10.sup.th percentile) on CVD by conditional logistic regression for all patients, males and females, respectively. 95% Hazard Ratio Variable p-value Hazard ratio Confidence Limits All patients aPC 0.0308 1.939 1.063 3.536 Males aPC 0.0262 2.181 1.097 4.338 Females aPC 0.6556 1.331 0.379 4.676
Effects of aPC
Introduction

(58) By use of columns which are preabsorbed with PC-BSA, PC-KLH or Pneumococcal vaccine (Statens Serum Institute, Denmark), we extract antibodies with reactivity against these compounds. Levels of aPC IgG are raised in at least the two first of these. Small amounts of IgM may be extracted out of IVIG and then also run through the columns preabsorbed with the abovementioned antigens. Through this method we can obtain polyclonal human aPC of IgG and IgM subclass. Protein measurement indicates that aPC IgM levels of 0.5 mg/ml could be extracted. Using these antibodies we can test their functional properties using in vitro models: 1. Can preincubation of increasing concentrations of aPC IgM with oxidized LDL decrease binding and uptake in monocyte/macrophage cell line, THP1? Test systems with confocal microscopy and/or FACS can be used. 2. Can preincubation of increasing concentrations of aPC IgM with normal IgM as control, with PAF, lysophosphatidylcholine (LPC) inhibit induction of adhesion molecules ICAM on endothelial cells by these lipids? Also other cytokines can be tested using a commercial kit (several different cytokines; BioSource). Tests can make use of FACScan.
Cell Culture

(59) Cryopreserved pooled HUVECs at passage 2 were purchased from Cascade Biologics, Inc. (Portland, Oreg., USA). Cultures were maintained in EGM phenol red-free medium (Clonetics, San Diego, Calif., USA), containing 2% of fetal bovine serum and supplements. The cells were incubated in 75 cm.sup.2 flasks (TPP, AG, Trasadingen, Switzerland) at 37 C. under humidified 5% CO.sub.2 conditions.

(60) All experiments were performed at passage 3 to 4. Cells were seeded at 210.sup.4 cells/ml density in 12-well plates (NUNC, Inc, Naperville, Ill., USA) for flow cytometry analysis. After allowing 12-24 hours for attachment the cells were made quiescent in SFM for at least 12 hrs prior to treatment.

(61) Monocytic cell line THP-1 was from AT&T (USA). Cells were maintained in RPMI with 10% FCS.

(62) Preparation of aPC

(63) Total IgM or IgG fraction was separated from commercially available pooled human immunoglobulin (Gammagard) at 50 mg/ml using HiTrap IgM or IgG columns (Amersham Biosciences). Antibodies against phosphorylcholine (PC) were eluted after loading IgM or IgG fraction on NHS-Sepharose columns coupled to PC conjugated either to keyhole limpet protein (KLH) (1 or 5 mg/ml) or to bovine serum albumin (BSA) (1 mg/ml), followed by BSA-only column. PC-BSA (Phosphorylcholine-Bovine Serum Albumin) and PC-KLH was purchased from Biosearch Technologies, INC (Ca, USA). Eluted fractions were buffer-exchanged on PD-10 columns and concentrated with Millipore Centricone devices. Procedures were performed according to instructions given by manufacturers. The concentration of IgM aPC prepared was typically 50 g/ml, and the concentration of IgG aPC was typically 30 g/ml.

(64) Scavenger Binding and Uptake of oxLDL by THP-1 Derived Macrophages

(65) Oxidized LDL (oxLDL) is prepared as described by incubation with copper ions. First, oxLDL is labeled with Dil (Dil-(1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate; Molecular Probes, Inc)) and diluted in Saline-EDTA buffer to 1 mg/ml. After that, 2 ml lipoprotein-deficient serum is added for 1 mg of oxLDL and then filtered (0.45 um). 50 ul Dil (3 mg/ml) in DMSO is added for 1 mg oxLDL and the mixture is incubated 15 h, 37 C. and then dialyzed against several changes of saline-EDTA for 6 h. After this the mixture is 0.45 um filtered again.

(66) Uptake of the oxLDL is studied with fluorescence/confocal microscopy. THP-1 cells as models for monocytes/macrophages are grown overnight on slide chamber. (medium: DMEM/10% FBS/Glu/PEST)

(67) 3 wash with DMEM medium without FBS

(68) Incubated with oxLDL-DiO 5 ug/ml (SFM medium) 6 h.

(69) The cells washed with 0.2% BSA-PBS 5, PBS 1

(70) Macrophage nucleus staining: the cells were incubated with 1 ug/ml bisbenzimide 10 and wash with PBS 3.

(71) Fix and mount: the cells were then fixed with 4% paraformaldehyde in PBS for 30, PBS 3, to finally, after 1 drop of mounting gel. The slides were covered with cover slip.

(72) Annexin V binding to endothelial cells

(73) Heparin-preserved plasma with high capacity to inhibit Annexin V binding was added to HUVECs monolayer at concentration of 10% in SFM. After 24 hrs cells were harvested with Cell Dissociation Solution (CDS; Sigma-Aldrich, St. Louis, Mo., USA) and carefully pooled with supernatants, to exclude selective loss of detached floating cells, centrifugation at 1200 rpm for 7 min followed. After resuspension in 100 l of annexin V-binding buffer (Molecular Probes Inc, Eugene, Oreg., USA) samples were stained with 2 l 5 mg/ml annexin V-FITC (Molecular Probes) and incubated for 15 min on ice. Shortly before acquisition 1 mg/ml of propidium iodide (PI; a vital dye; R&DSystems Europe Ltd, Abingdon, UK) was added. Analysis was performed as described above.

(74) Statistical Analysis

(75) The statistics were computed using Stat View software, SAS Institute AB, Gteborg, Sweden. Skewed continuous variables were logarithmically transforme. Study groups were compared using ANOVA for continuous variables and Chi square for categorical variables. Fischer's PLSD was used as post hoc test. Correlation coefficients were calculated using Simple regression or for not normally distributed variables Spearman's rank correlation. The significance level was set at p<0.05.

(76) Results

(77) Measurements of Annexin V Binding to Endothelial Cells

(78) The frequency of HUVECs positive for annexin V staining was determined either as percentage of annexin V.sup.+/PI.sup. cells on a bivariate dot plot or percentage of annexin V.sup.+ cells based on a histogram. Annexin V-binding to HUVECs in the presence of serum known to decreased binding and preincubated with IVIG was determined. Preincubation with IVIG could restore binding of Annexin, indicating that antibodies present in IVIG could neutralize binding (FIG. 4).

(79) APC-BSA and aPC-KHL were both associated significantly in SLE-patients with a history of CVD with Annexin V binding to EC (r=0.45; p=0.02 and r=0.42 and p=0.03 respectively). aPC were determined as described above.

(80) Effect on oxLDL Uptake in Macrophages by aPC

(81) aPC of IgM and IgG subclass, extracted from IVIG as indicated were preincubated with oxLDL indicated (fig 3). We used total IgM as control for aPC IgM (macrophage+Dil-oxLDL+IgM) and effect on macrophage uptake. The total percentage of positive staining cells is 46.62%, indicating that IgM per se does not have the inhibitory effects that aPC has. IgM was bought from SIGMA, and is purified human IgM is produced by precipitation and gel filtration techniques using normal human serum as the starting material. The immunoglobulin is determined to be at least 95% pure.

(82) Effect of aPC on ICAM-Induction in Endothelial Cells

(83) PAF was incubated with EC at the indicated concentrations. As demonstrated in FIG. 5 this lipid could induce a significant increase in ICAM-expression. aPC of IgM subclass, extracted from IVIG as indicated were preincubated with these lipids as indicated (FIG. 5).

(84) Correlations Between aPC and Other Risk Markers in ELSA Study (226 Individuals with Hypertension as Described Previously.

(85) aPC IgM was associated with two other protection factors, HSP 70 and HDL, as indicated in Table 4. There was also a weak albeit significant association with TNF, a marker of inflammation and a proatherogenic cytokine.

(86) TNF is an important pro-inflammatory cytokine and TNF levels negatively associated with aPC IgM levels. The association is weak, but significant.

(87) HSP 70 is a novel protection factor recently described by us and others. There is a clearly positive association. Also HSP60, which is a weaker protection factor, is associated.

(88) HDL is a well known good cholesterol, with anti-inflammatory properties. It is associated significantly with aPC IgM.

(89) TABLE-US-00010 ANTPCIGG ANTPCIGM Spearman's rho ANTPCIGG 1,000 ,245 Correlation Coefficient Sig. (2-tailed) , ,000 N 220 220 ANTPCIGM ,245 1,000 Correlation Coefficient Sig. (2-tailed) ,000 , N 220 220 HDL ,008 ,233 Correlation Coefficient Sig. (2-tailed) ,906 ,001 N 206 206 TNFA ,012 ,136 Correlation Coefficient Sig. (2-tailed) ,863 ,044 N 220 220 HSP60 ,138 ,279 Correlation Coefficient Sig. (2-tailed) ,047 ,000 N 209 209 HSP70 ,157 ,356 Correlation Coefficient Sig. (2-tailed) ,022 ,000 N 213 213 **Correlation is significant at the .01 level (2-tailed). *Correlation is significant at the .05 level (2-tailed).