Identification of IgG1 isotype phosphorylcholine antibodies for the treatment of inflammatory cardiovascular diseases.

BACKGROUND: Phosphorylcholine (PC) is an important pro-inflammatory damage-associated molecular pattern. Previous data have shown that natural IgM anti-PC protects against cardiovascular disease. We aimed to develop a monoclonal PC IgG antibody with anti-inflammatory and anti-atherosclerotic properties. METHODS: Using various techniques PC antibodies were validated and optimized. In vivo testing was performed in a femoral artery cuff model in ApoE3*Leiden mice. Safety studies are performed in rats and cynomolgus monkeys. RESULTS: A chimeric anti-PC (PC-mAb(T15), consisting of a human IgG1 Fc and a mouse T15/E06 Fab) was produced, and this was shown to bind specifically to epitopes in human atherosclerotic tissues. The cuff model results in rapid induction of inflammatory genes and altered expression of genes associated with ER stress and choline metabolism in the lesions. Treatment with PC-mAb(T15) reduced accelerated atherosclerosis via reduced expression of endoplasmic reticulum stress markers and CCL2 production. Recombinant anti-PC Fab fragments were identified by phage display and cloned into fully human IgG1 backbones creating a human monoclonal IgG1 anti-PC (PC-mAbs) that specifically bind PC, apoptotic cells and oxLDL. Based on preventing macrophage oxLDL uptake and CCL2 production, four monoclonal PC-mAbs were selected, which to various extent reduced vascular inflammation and lesion development. Additional optimization and validation of two PC-mAb antibodies resulted in selection of PC-mAb X19-A05, which inhibited accelerated atherosclerosis. Clinical grade production of this antibody (ATH3G10) significantly attenuated vascular inflammation and accelerated atherosclerosis and was tolerated in safety studies in rats and cynomolgus monkeys. CONCLUSIONS: Chimeric anti-PCs can prevent accelerated atherosclerosis by inhibiting vascular inflammation directly and through reduced macrophage oxLDL uptake resulting in decreased lesions. PC-mAb represents a novel strategy for cardiovascular disease prevention.

Immunoglobulin G1 Antibodies Against Phosphorylcholine Are Associated With Protection in Systemic Lupus Erythematosus and Atherosclerosis: Potential Underlying Mechanisms.

OBJECTIVE: Immunoglobulin M antibodies against phosphorylcholine (anti-PCs) may be protective in atherosclerosis, cardiovascular disease (CVD), and systemic lupus erythematosus (SLE). We study immunoglobulin G1 (IgG1) and immunoglobulin G2 (IgG2) anti-PCs, with a focus on atherosclerosis and SLE. METHODS: We determined anti-PCs by using the enzyme-linked immunosorbent assay in 116 patients with SLE and 110 age- and sex-matched controls. For functional studies, we used three in-house-generated, fully human monoclonal IgG1 anti-PCs (A01, D05, and E01). Apoptosis was induced in Jurkat T cells and preincubated with A01, D05, E01, or IgG1 isotype control, and effects on efferocytosis by human macrophages were studied. Anti-PC peptide/protein characterization was determined using a proteomics de novo sequencing approach. RESULTS: IgG1, but not IgG2, anti-PC levels were higher among patients with SLE (P = 0.02). IgG1 anti-PCs were negatively associated with Systemic Lupus International Collaborating Clinics (SLICC) damage index and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores (odds ratio [OR]: 2.978 [confidence interval (CI): 0.876-10.098] and OR: 5.108 [CI 1.3-20.067], respectively) and negatively associated with CVD, atherosclerotic plaques, and echolucent plaques (potentially vulnerable plaques), but the association for the two former was not significant after controlling for confounders. D05 had a maximum effect on macrophage efferocytosis efficiency, followed by A01 and E01. The monoclonal antibodies showed differential binding specificity to PC and PC-associated neoepitopes. A peptide analysis showed a difference in the complementarity-determining region 3 of the three IgG1 anti-PC clones that are crucial for recognition of PC on apoptotic cell surfaces and other neoepitopes. CONCLUSION: IgG1 anti-PCs are negatively associated with disease activity and disease damage in SLE, but the negative association with CVD is also dependent on confounding risk factors. One potential underlying mechanism could be increased clearance of dead cells.

Different subclasses and isotypes of antibodies against phosphorylcholine in haemodialysis patients: association with mortality.

The risk of premature death is high among patients on haemodialysis (HD patients). We previously determined that immunoglobulin (Ig)M antibodies against phosphorylcholine (anti-PC) are negatively associated with increased risk of cardiovascular disease (CVD), atherosclerosis, some autoimmune diseases and mortality among HD patients in this cohort. Here, we also study other subclasses and isotypes of anti-PC in HD patients in relation to mortality, inflammation and gender. The study group is a cohort of 209 prevalent HD patients [median age = 66 years, interquartile range (IQR) = 51-74], vintage time = 29 months (IQR = 15-58; 56% men) with a mean follow-up period of 41 months (IQR = 20-60). Fifty-six per cent were men. We also divided patients into inflamed C-reactive protein (CRP) > 5·6 mg/ml and non-inflamed CRP. Antibody levels were determined by in-house enzyme-linked immunosorbent assay. IgG1 anti-PC below median was significantly associated with increased all-cause mortality (after adjustment for confounders: P = 0·02), while IgG, IgA and IgG2 anti-PC were not associated with this outcome. Among non-inflamed patients, IgM and IgG1 anti-PC were significantly associated with mortality (P = 0·047 and 0·02). IgG1 anti-PC was significantly associated with mortality among men (P = 0·03) and trending among women (P = 0·26). IgM (as previously reported) and IgG1 anti-PC are negatively associated with survival among HD patients and non-inflamed HD patients, but among inflamed patients there were no associations. IgG, IgA or IgG2 anti-PC were not associated with survival in these groups and subgroups. Further studies are needed to determine if raising anti-PC levels, especially IgM and IgG1 anti-PC, through immunization is beneficial.

Phenotypic features of vascular calcification in chronic kidney disease.

BACKGROUND: Patients with chronic kidney disease stage 5 (CKD5) are predisposed to vascular calcification (VC), but the combined effect of factors associated with VC was sparsely investigated. We applied the relaxed linear separability (RLS) feature selection model to identify features that concomitantly associate with VC in CKD5 patients. METHODS: Epigastric arteries collected during surgery from living donor kidney transplant recipients were examined to score the histological extent of medial VC. Sixty-two phenotypic features in 152 patients were entered into RLS model to differentiate between no-minimal VC (n = 93; score 0-1) and moderate-extensive VC (n = 59; score 2-3). The subset of features associated with VC was selected on the basis of cross-validation procedure. The strength of association of the selected features with VC was expressed by the absolute value of 'RLS factor'. RESULTS: Among 62 features, a subset of 17 features provided optimal prediction of VC with 89% of patients correctly classified into their groups. The 17 features included traditional risk factors (diabetes, age, cholesterol, BMI and male sex) and markers of bone metabolism, endothelial function, metabolites, serum antibodies and mitochondrial-derived peptide. Positive RLS factors range from 1.26 to 4.05 indicating features associated with increased risk of VC, and negative RLS factors range from -0.95 to -1.83 indicating features associated with reduced risk of VC. CONCLUSION: The RLS model identified 17 features including novel biomarkers and traditional risk factors that together concomitantly associated with medial VC. These results may inform further investigations of factors promoting VC in CKD5 patients.

Autoantibody profiling reveals four protein candidate autoantigens associated with systemic lupus erythematosus.

Objectives In systemic lupus erythematosus (SLE) there are typically many autoantibodies. The disease heterogeneity could be better understood with discovery of phenotype-specific antigens targeted by autoantibodies. We here aimed to identify novel autoantigens potentially related to SLE disease and a major complication, atherosclerosis. Methods Antigen microarrays were used to profile IgG autoantibody reactivity against 77 protein fragments (20-140 amino acids (aa) long, median 89 aa) produced within the Human Protein Atlas project, in serum samples from SLE patients ( n = 107) and age- and sex-matched population-based controls ( n = 107). Common carotid intima-media thickness, plaque occurrence and echogenicity were determined by B-mode ultrasound. Results We determined significant differences between patients and controls in IgG reactivity against four proteins. In patients compared to controls, there was an increase of IgG reactivity against zinc finger protein 688 (ZNF688), early B cell factor 2 (EBF2), crystallin, alpha B (CRYAB) and tumor necrosis factor receptor superfamily member 13C (TNFRSF13C). Of these four antigens, only anti-ZNF688 was associated with carotid atherosclerosis (plaque occurrence) and vulnerable plaques in SLE. There was a weak association between anti-EBF2 and SLE disease activity but no significant associations were determined for other measured IgG reactivity. Conclusions In this discovery screening we here demonstrate new candidate autoantigens with differential reactivity (reflecting autoantibody levels) in SLE patients and in controls and in relation to atherosclerosis in SLE.

PCSK9 plays a novel immunological role in oxidized LDL-induced dendritic cell maturation and activation of T cells from human blood and atherosclerotic plaque.

BACKGROUND: Activated T cells and dendritic cells (DCs) occur in atherosclerotic plaques. Proprotein convertase subtilisin kexin 9 (PCSK9) targets the LDL-receptor (LDLR) and results in increased LDL levels. We here investigate immune effects of PCSK9 on OxLDL induced DC maturation and T-cell activation. METHODS: T cells were isolated from carotid specimens of patients undergoing carotid endarterectomy or from peripheral blood of healthy individuals. Human peripheral blood monocytes were differentiated into DCs. Naïve T cells were cocultured with pretreated DCs. The effects of PCSK9 and its inhibition by silencing were studied. RESULTS: OxLDL induced PCSK9 in DCs and promoted DC maturation with increased expressions of CD80, CD83, CD86 and HLA-DR and the scavenger receptors LOX-1 and CD36. T cells exposed to OxLDL-treated DCs proliferated and produced IFN-γ and IL-17, thus with polarization to Th1 and/or Th17 subsets. Silencing of PCSK9 reversed the OxLDL effects on DCs and T cells. DC maturation was repressed, and the production of TNF-α, IL-1ß and IL-6 was limited, while TGF-ß and IL-10 secretion and T regulatory cells were induced. OxLDL induced miRNA let-7c, miR-27a, miR-27b, miR-185. Silencing PCSK9 repressed miR-27a and to a lesser extent let-7c. PCSK9 silencing enhanced SOCS1 expression induced by OxLDL. Experiments on T cells from carotid atherosclerotic plaques or healthy individuals showed similar results. CONCLUSIONS: We demonstrate immunological effects of PCSK9 in relation to activation and maturation of DCs and plaque T cells by OxLDL, a central player in atherosclerosis. This may directly influence atherosclerosis and cardiovascular disease, independent of LDL lowering.

Dietary micronutrient intake and atherosclerosis in systemic lupus erythematosus.

OBJECTIVE: The aim of this study was to investigate the role of dietary micronutrient intake in systemic lupus erythematosus (SLE). METHODS: This study included 111 SLE patients and 118 age and gender-matched controls. Data on diet (food frequency questionnaires) were linked with data on Systemic Lupus Activity Measure, Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) and carotid atherosclerotic/echolucent plaque (B-mode ultrasound). Dietary micronutrient intake were compared between SLE patients and controls and in relation to lupus activity and atherosclerosis in SLE. Associations between micronutrient intake and plaque were analyzed through logistic regression, adjusted for potential confounders. RESULTS: Micronutrient intake did not differ between patients and controls, and between lower and higher lupus activity, apart from the fact that phosphorus was associated with SLEDAI > 6. In SLE patients, some micronutrients were associated with atherosclerotic plaque, left side. Lower intake of riboflavin and phosphorus was associated with atherosclerotic plaque, left side (odds ratio (OR) 3.06, 95% confidence interval (CI) 1.12-8.40 and OR 4.36, 95% CI 1.53-12.39, respectively). Higher intake of selenium and thiamin was inversely associated with atherosclerotic plaque, left side (OR 0.28, 95% CI 0.09-0.89 and OR 0.26, 95% CI 0.08-0.80, respectively). In addition, higher intake of thiamin was inversely associated with echolucent plaque, left side (OR 0.22, 95% CI 0.06-0.84). Lower intake of folate was inversely associated with bilateral echolucent plaque (OR 0.36, 95% CI 0.13-0.99). CONCLUSIONS: SLE patients did not have different dietary micronutrient intake compared to controls. Phosphorus was associated with lupus activity. Riboflavin, phosphorus, selenium and thiamin were inversely associated with atherosclerotic plaque, left side in SLE patients, but not in controls. Dietary micronutrients may play a role in atherosclerosis in SLE.

Health-related quality of life, fatigue and mood in patients with SLE and high levels of pain compared to controls and patients with low levels of pain.

Objective The objective of this paper is to investigate health-related quality of life (HRQoL), fatigue, anxiety and depression in patients with systemic lupus erythematosus (SLE) and higher levels of pain and to compare them to patients with lower levels of pain and controls. Method Patients were dichotomized into two groups based on SLE-related pain score on the visual analog scale (VAS): low-pain group (76%, n=64, VAS 0-39 mm) and high-pain group (24%, n=20, VAS 40-100 mm). Sex- and age-matched controls were randomly selected from the general population. Participants were asked to complete questionnaires regarding self-reported pain, HRQoL, fatigue, anxiety and depression. Medical assessments also were recorded. Result Fatigue score in the high-pain group (median, 36.5; interquartile range (IQR), 32.5-39.7) was significantly higher (p<0.001) compared to the low-pain group (median, 23; IQR, 14.6-34.1), as well as scores for anxiety (median, 9; IQR, 6.5-11.5) and depression (median, 7.5; IQR, 5.5-9) (p<0.001). The high-pain group had significantly lower scores compared to the low-pain group in all dimensions in the SF-36 (p ≤ 0.001-0.007). No statistical differences were detected between the low-pain group and controls in any measurement except for the dimensions physical function, general health, vitality and social function in SF-36. Conclusion Patients with SLE scoring higher degrees of pain were burdened with more fatigue, anxiety and depression and lower levels of HRQoL compared to patients with lower levels of pain who did not differ significantly from the general population in most dimensions. These results elucidate the importance of identifying patients with higher degrees of pain who are probably in need of more extensive multidimensional interventions to decrease symptom burden.

Immunoglobulin (Ig)M antibodies against oxidized cardiolipin but not native cardiolipin are novel biomarkers in haemodialysis patients, associated negatively with mortality.

The risk of premature death is high in haemodialysis (HD) patients. Antibodies against cardiolipin (anti-CL) are thrombogenic in diseases such as systemic lupus erythematosus (SLE). CL is easily oxidized (Ox) and plays a role in apoptosis. In this work we studied immunoglobulin (Ig)M anti-CL and anti-OxCL in HD-patients. We conducted an observational study with a prospective follow-up examining the relationship between anti-CL, anti-OxCL and mortality risk in a well-characterized cohort of 221 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 41 (20-48 months). According to the receiver operator characteristic (ROC) analysis, anti-OxCL [area under the curve (AUC) 0·62, P < 0·01], but not anti-CL (AUC 0·52, P = 0·2), is associated with mortality. In crude and adjusted Cox analysis, every log increase in anti-OxCL inversely predicted all-cause [adjusted hazard ratios (HR) 0·62 (0·43-0·89)] and CVD-related [adjusted HR 0·56 (0·32-0·98)] mortality. Patients with anti-OxCL levels below median also had increased all-cause and cardiovascular disease (CVD)-related mortality. Although anti-OxCL and anti-phosphorylcholine (PC) were related positively to each other (ρ = 0·57, P < 0·01), patients with one or two of these autoantibody levels below the median were associated with an incrementally increased death risk. Anti-OxCL were co-factor ß2-GPI-independent; anti-CL from patients with anti-phospholipid antibody syndrome were ß2-GPI-dependent, while sera from HD-patients less so. Sera from healthy donors was not ß2-GPI-dependent. Anti-OxCL IgM is ß2-glycoprotein 1 (GPI)-independent and a novel biomarker; low levels are associated with death among HD patients (and high levels with decreased risk). Combination with anti-PC increases this association. Putative therapeutic implications warrant further investigation.

Extent and characteristics of self-reported pain in patients with systemic lupus erythematosus.

OBJECTIVE: Patients' own experiences of subjective symptoms are scarcely covered, and the objective of this study was to investigate the extent and characteristics of self-reported pain in patients with systemic lupus erythematosus (SLE). METHODS: This study comprised a cross-sectional design where 84 patients with SLE were asked to complete self-assessments: visual analogue scale of pain and the Short-Form McGill Pain Questionnaire. Medical assessments, including ESR, SLAM, SLEDAI, and SLICC, were also performed. RESULTS: Of the study population, 24% reported higher levels of SLE-related pain (≥40 mm on VAS). This group had a significantly shorter disease duration, higher ESR, and higher disease activity, according to the SLAM and SLEDAI, compared to the rest of the study population. This group mainly used the words "tender," "aching," and "burning" to describe moderate and severe pain, and they used a greater number of words to describe their pain. Of the patients with higher levels of pain, 70% reported their present pain as "distressing." The most common pain location for the whole patient population was the joints. Patients rated their disease activity significantly higher than physicians did. CONCLUSION: These findings expand the current knowledge of the extent of SLE-related pain and how patients perceive this pain. The results can contribute to affirmative, supportive and caring communication and especially highlight SLE-related pain in patients with a short disease duration and high disease activity.

Diet and fatty acid pattern among patients with SLE: associations with disease activity, blood lipids and atherosclerosis.

OBJECTIVE: As atherosclerosis is increased in systemic lupus erythematosus (SLE) we compared dietary habits in patients with SLE with controls, and in the patients studied associations of diet components, especially fatty acids (FAs), with disease activity, serum lipids and carotid plaque presence. METHODS: In all 114 patients with SLE and 122 age- and sex-matched population-based controls answered a food frequency questionnaire (FFQ). Subcutaneous abdominal fat cell aspiration was analysed as to FA content and plaque occurrence was determined by B-mode ultrasound. RESULTS: The total diet energy intake did not differ between patients and controls. However, the patients with SLE reported a higher intake of carbohydrate, lower fibre intake and lower intake of omega-3 and omega-6, than controls (p < 0.05). In the patients, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in adipose tissue (AT) correlated negatively with disease activity (SLEDAI), r = -0.36, p = < 0.001 and r = -0.33, p = < 0.001, respectively. AT omega-3 was further positively associated with serum apoA1, r = 0.29, p = 0.004, whereas AT omega-6 showed a negative association, r = -0.21, p = 0.040. These FAs also had opposite associations with plaque presence, EPA and were DHA negative, r = -0.32, p = 0.002 and r = -0.33, p = 0.001, respectively, and omega-6 positive, r = 0.22, p = 0.027. The carbohydrate intake was positively correlated to AT omega-6, r = 0.38, p < 0.001, and negatively with serum apoA1, r = -0.27, p = 0.005. CONCLUSION: The macronutrient dietary pattern is different in SLE as compared with controls. The low intake of omega-3 and high intake of carbohydrate among patients with SLE appear to be associated with worse disease activity, adverse serum lipids and plaque presence.

Microcirculation as determined by iontophoresis in SLE-patients and controls.

BACKGROUND: The risk of cardiovascular disease (CVD), microangiopathy and prevalence of atherosclerotic plaques are increased in Systemic Lupus Erythematosus (SLE). As systemic endothelial dysfunction is one of the earliest signs of these vascular outcomes in the general population we assessed skin microvascular endothelial function in SLE patients. METHODS: Endothelial function in skin was tested with local application of acetylcholine (inducing endothelium-dependent vasodilatation) and any concomitant increase in skin perfusion was measured with Laser Doppler Fluxmetry (LDF) in 84 SLE-patients (83% women, mean age 47 years) and 81 age and sex matched controls. Common carotid intima-media thickness (cIMT) and plaque occurrence were also determined using B-mode ultrasound. RESULTS: There were no significant differences in skin microvascular endothelial function between SLE-patients and controls. In the SLE group, endothelial function did not vary in relation to skin manifestations, Raynaud's phenomenon, nephritis or plaque occurrence. In SLE patients with CVD, however, endothelial function was impaired. CONCLUSION: Skin microvascular endothelial function is associated with CVD but not with early signs of atherosclerosis in SLE-patients. The endothelial function is not different in SLE-patients as compared to controls.

Genetic association of miRNA-146a with systemic lupus erythematosus in Europeans through decreased expression of the gene.

A recent genome-wide association study revealed a variant (rs2431697) in an intergenic region, between the pituitary tumor-transforming 1 (PTTG1) and microRNA (miR-146a) genes, associated with systemic lupus erythematosus (SLE) susceptibility. Here, we analyzed with a case-control design this variant and other candidate polymorphisms in this region together with expression analysis in order to clarify to which gene this association is related. The single-nucleotide polymorphisms (SNPs) rs2431697, rs2910164 and rs2277920 were genotyped by TaqMan assays in 1324 SLE patients and 1453 healthy controls of European ancestry. Genetic association was statistically analyzed using Unphased. Gene expression of PTTG1, the miRNAs miR-3142 and primary and mature forms of miR-146a in peripheral blood mononuclear cells (PBMCs) were assessed by quantitative real-time PCR. Of the three variants analyzed, only rs2431697 was genetically associated with SLE in Europeans. Gene expression analysis revealed that this SNP was not associated with PTTG1 expression levels, but with the microRNA-146a, where the risk allele correlates with lower expression of the miRNA. We replicated the genetic association of rs2341697 with SLE in a case-control study in Europeans and demonstrated that the risk allele of this SNP correlates with a downregulation of the miRNA 146a, potentially important in SLE etiology.

Anti-apoA-1 IgG and oxidized LDL are raised in rheumatoid arthritis (RA): potential associations with cardiovascular disease and RA disease activity.

OBJECTIVE: To determine whether emerging cardiovascular risk factors such as anti-apolipoprotein A-1 (anti-apoA-1) immunoglobulin (Ig)G and oxidized low density lipoprotein (oxLDL) are associated with cardiovascular disease (CVD), carotid intima-media thickness (IMT), and disease activity in rheumatoid arthritis (RA). METHOD: We determined the aforementioned associations in 69 RA patients with disease duration of 5 years and 46 controls matched by age, sex, and smoking status. Anti-apoA-1 IgG and oxLDL were measured by enzyme-linked immunosorbent assay (ELISA). Carotid arteries were examined by ultrasound. Disease Activity Score calculated on 28 joints (DAS28) was used to assess disease activity. RESULTS: CVD prevalence was higher among RA patients than controls (17% vs. 2%, p = 0.01) but there was no difference in IMT (median: 0.67 vs. 0.66, p = 0.33). RA patients had a higher anti-apoA-1 IgG prevalence than controls (20% vs. 0%, p = 0.001). Anti-apoA-1 IgG and oxLDL levels were higher in cases than controls [median: 0.33 vs. 0.175 optical density (OD), p = 0.03; and 121 vs. 37.2 U/L, p = 0.0001, respectively]. Anti-apoA-1 IgG-positive patients had higher levels of oxLDL (median: 140.5 vs. 112 U/L, p = 0.01) than those tested negative. Receiver operating characteristic (ROC) curve analysis showed that only anti-apoA-1 IgG was a modest but significant predictor of CVD [area under the curve (AUC) = 0.65, p = 0.03] in RA patients. oxLDL was significantly associated with RA disease activity, whereas anti-apoA-1 IgG was not. CONCLUSIONS: Anti-apoA-1 IgG could be a marker of CVD in RA, whereas oxLDL levels seem to reflect RA disease activity. Other causes of CVD than a general increase in atherosclerosis (as determined by IMT measurements) including plaque stability may therefore be of importance to explain the increased incidence of CVD in RA.

Effects of anti-cardiolipin antibodies and IVIg on annexin A5 binding to endothelial cells: implications for cardiovascular disease.

OBJECTIVES: Anti-phospholipid antibodies (aPL), including anti-cardiolipin antibodies (aCL), are risk factors for cardiovascular disease (CVD) in the general population and in patients with the anti-phospholipid syndrome (APS; Hughes syndrome). APS may be primary but is also common in patients with systemic lupus erythematosus (SLE). The anti-coagulant protein annexin A5 (ANXA5) is implicated in CVD by interfering with phospholipids and aPL. METHODS: ANXA5 binding to human umbilical venous endothelial cells (HUVECs) was determined by flow cytometry. RESULTS: When cells were cultured in serum from APS patients with a high aPL titre (aPL-S), binding of ANXA5 to HUVECs was reduced. Monoclonal immunoglobulin (Ig)G aPL against cardiolipin (mAb-CL) dose-dependently reduced ANXA5 binding to endothelium. Preincubation of intravenous (IV)Ig at therapeutically relevant doses with aPL-S and mAb-aCL restored ANXA5 binding to comparable levels when normal healthy serum (NHS) was used. By contrast, IVIg per se had the capacity to reduce ANXA5 binding to endothelium when added to NHS (but not to aPL-S). CONCLUSIONS: Decreased ANXA5 binding to endothelium, mediated by aPL, is a novel mechanism of atherothrombosis that can be countered by IVIg in vitro. IVIg per se could, to a lesser degree, cause decreased ANXA5 binding in NHS, which raises the possibility that some antibodies in IVIg can be involved in a side-effect reported in IVIg treatment, namely atherothrombosis and CVD. Increasing ANXA5 binding, either by addition of ANXA5 or by use of neutralizing antibodies in IVIg, represents a possible therapeutic strategy that deserves further study.

Dyslipidaemia and lipoprotein pattern in systemic lupus erythematosus (SLE) and SLE-related cardiovascular disease.

OBJECTIVE: This study focused on lipoprotein composition and properties in systemic lupus erythematosus (SLE). METHODS: The size distribution of plasma lipoproteins was studied by nuclear magnetic resonance (NMR). Cholesteryl ester transfer protein (CETP) activity was determined by enzyme-linked immunosorbent assay (ELISA). The affinity of low density lipoprotein (LDL) for proteoglycans was assayed. Twenty-six women (aged 52+/-8.2 years) with SLE and a history of cardiovascular disease (CVD) (SLE cases) were compared with 26 age-matched women with SLE and without CVD (SLE controls) and 26 age-matched population-based control women (controls). RESULTS: Very low density lipoprotein (VLDL) particles (nmol/L) were more prevalent among SLE cases than SLE controls (0.039) and tended to be more common in SLE cases than in controls (p = 0.073). By contrast, high density lipoprotein (HDL) particles (nmol/L) were more prevalent among controls than SLE cases (p = 0.024) whereas the number of LDL particles (nmol/L) did not differ significantly. Small dense (sd)LDL (nmol/L) were more common in controls and tended to be more common in SLE cases than in SLE controls (p = 0.036 and 0.086, respectively). Small high density lipoproteins (sHDL) (nmol/L) were more prevalent in controls than in SLE controls and SLE cases (p = 0.002 and p<0.001, respectively). VLDL or LDL size (nm) did not differ significantly between groups (data not shown) whereas HDL size (nm) was increased among SLE controls as compared to controls (p = 0.024) and tended to be increased among SLE cases as compared to controls (p = 0.070). The affinity of LDL for proteoglycans or CETP activity did not differ between groups (data not shown). CONCLUSIONS: sdLDL was not increased and SLE cases and SLE controls had decreased levels of sHDL. VLDL differentiates between SLE cases and SLE controls. The lipid pattern in SLE-related CVD was thus not similar to the pattern seen in diabetes or in CVD in general.

Endothelial function and markers of endothelial activation in relation to cardiovascular disease in systemic lupus erythematosus.

OBJECTIVE: Cardiovascular disease (CVD) is common in patients with systemic lupus erythematosus (SLE) although it is not clear whether an increased risk of CVD is a general feature of SLE or whether it applies only to a subgroup of patients. Our objective was to evaluate endothelial function and markers of endothelial activation in relation to CVD in SLE. METHODS: Twenty-six women with SLE and previous CVD (SLE/CVD cases, defined as objectively verified angina pectoris, myocardial infarction, cerebral infarction, or intermittent claudication; 52+/-8.2 years) were compared with age-matched SLE women without CVD (SLE controls) and population control women. Flow-mediated dilatation (FMD) of the brachial artery after reactive hyperaemia and nitroglycerin-mediated dilatation (NMD) after sublingual nitroglycerin administration were determined by ultrasound. Soluble thrombomodulin (sTM) and soluble vascular cellular adhesion molecule-1 (sVCAM-1) were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: FMD and NMD levels did not differ between SLE controls and population controls. In SLE cases FMD and NMD were not assessed because of interference with nitro-related medication. sVCAM-1 discriminated between SLE cases, SLE controls, and population controls (ng/mL; 814+/-221 vs. 545+/-214 vs. 401+/-189, p<0.01), whereas sTM (ng/mL; 5.2+/-2.8 vs. 4.2+/-1.9 vs. 3.0+/-0.5) differed between both SLE groups and controls (p<0.05). CONCLUSION: In this study SLE women free of CVD had good endothelial function (FMD), a possible marker of protection from lupus-related CVD. In addition, high levels of sVCAM-1, associated with systemic tumour necrosis factor-alpha (TNFalpha) activity, were identified as a novel discriminator for SLE-related CVD. This supports our hypothesis that SLE patients with enhanced systemic TNFalpha activity are at high risk of developing CVD.

Natural antibodies against phosphorylcholine as potential protective factors in SLE.

OBJECTIVE: We have recently reported that natural antibodies against phosphorylcholine (anti-PC) have atheroprotective properties. Here we compare anti-PC with other autoantibodies in SLE patients with and without cardiovascular disease (CVD). METHODS: Twenty-six women (52 +/- 8.2 yrs) with SLE and a history of CVD (SLE cases) were compared with 26 age-matched women with SLE without CVD (SLE controls) and 26 age-matched population-based control women (controls). PC was conjugated with BSA (PC-BSA) or keyhole-limpet haemocyanin (PC-KLH). Anti-PC and antibodies against phosphatidylserine (anti-PS) and BSA (anti-BSA) were studied by ELISA. Anti-PC-IgG were extracted from intravenous immunoglobulin (IVIG). Activation of endothelial cells by platelet-activating factor (PAF) was studied with FACScan. RESULTS: IgG anti-PC-BSA and anti-PC-KLH were decreased among SLE-cases and SLE-controls as compared with controls (P < 0.005 and P < 0.05), respectively. SLE cases were more prevalent in the lowest 25th percentile of anti-PC-IgM (and IgG) as compared with controls (P < 0.05) but anti-PC-IgM levels did not differ significantly between groups. Among SLE controls, anti-PC-BSA were associated negatively with organ damage (SLICC) and disease activity (SLEDAI) (P < 0.05). Among SLE cases, anti-PC-BSA and anti-PC-KLH were associated negatively with SLICC (P = 0.021; P = 0.010) and anti-PC-BSA was negatively associated with SLEDAI (P < 0.039). Anti-PS-IgG and anti-BSA-IgG were raised among SLE cases as compared with other groups (P < 0.05) and did not cross-react with anti-PC. Anti-PC-IgG could be extracted from IVIG and inhibited PAF-induced expression of adhesion molecules. CONCLUSION: Low levels of anti-PC could be of importance in SLE. Anti-BSA and anti-PS and low levels of anti-PC could contribute to development of CVD in SLE.

Systemic lupus erythematosus and cardiovascular disease.

Atherosclerosis is an inflammatory disease characterised by presence of activated immune competent cells in middle-sized and large arteries and is the major cause of cardiovascular disease (CVD). The risk of CVD is very high in systemic lupus erythematosus (SLE). SLE-related CVD and atherosclerosis are, therefore, important clinical problems but may in addition also have implications for the role of immune reactions in CVD in general. Others and we have recently demonstrated that risk factors for CVD in SLE are both traditional and non-traditional acting in concert. Traditional risk factors implicated in SLE include, for example, dyslipidemia (especially high triglycerides), hypertension, renal disease, non-traditional as inflammation, antiphospholipid antibodies (aPL) and low-density lipoprotein (LDL) oxidation are also associated with CVD in SLE. Atherothrombosis is likely to be a major underlying mechanism and is not only an increased risk of thrombosis per se. It is possible that factors like proinflammatory reactions or prothrombotic factors, such as aPL, make atherosclerotic lesions in SLE more prone to rupture than in 'normal' atherosclerosis. Whether premature atherosclerosis is a general feature of SLE or only affects a subgroup of patients is presently not clear. Treatment of patients with SLE should include a close monitoring of traditional risk factors and also the above-mentioned non-traditional for CVD.

Treatment with combined oral contraceptives induces a rise in serum C-reactive protein in the absence of a general inflammatory response.

BACKGROUND: The role of inflammation in the pathogenesis of cardiovascular disease is well established. C-reactive protein (CRP) is the strongest independent predictor of myocardial infarction and stroke in women. Recent studies have indicated that CRP levels are raised during use of combined oral contraceptives (COCs). OBJECTIVES: The aim of the study was to investigate the effect of COCs on serum CRP levels and to indicate the underlying mechanisms of an expected increase. METHOD: In a prospective randomized cross over-study 35 women used two different preparations of COC, one second and one third generation. Serum levels of CRP, serum amyloid A (SAA), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), antibodies against oxidized LDL, insulin and insulin-like growth factor-I (IGF-I) along with insulin-like growth factor binding protein-1 (IGFBP-1) and IGFBP-3 were analyzed before and during the two treatments. E-selectin, von Willebrand factor and factor VIII concentrations in plasma were also measured. RESULTS: A rise in serum CRP was observed during both treatments; the median level increased from 0.45 mg L(-1) at baseline to 1.48 mg L(-1) with second generation and to 2.02 mg L(-1) with third generation COC. The serum levels of SAA increased slightly during treatment with the third generation COC. IL-6 and TNFalpha were unaffected by treatment. Both preparations lowered IGF-I and raised IGFBP-1 and IGFBP-3 concentrations. CONCLUSION: The raised serum CRP concentration during treatment with COCs appears to be related to a direct effect on hepatocyte CRP synthesis and does not reflect IL-6 mediated inflammation, endothelial activation or induction of insulin resistance.