High Levels of (Un)Switched Memory B Cells Are Associated With Better Outcome in Patients With Advanced Atherosclerotic Disease.

BACKGROUND: Atherosclerosis is an inflammatory lipid disorder and the main underlying pathology of acute ischemic events. Despite a vast amount of data from murine atherosclerosis models, evidence of B-cell involvement in human atherosclerotic disease is limited. We therefore investigated the association of circulating B-cell subtypes with the occurrence of secondary cardiovascular events in advanced atherosclerotic disease. METHODS AND RESULTS: This cohort study consists of 168 patients who were included in the Athero-Express biobank between 2009 and 2011. Before surgery, peripheral blood mononuclear cells were isolated and stored in liquid nitrogen. After gentle thawing of the peripheral blood mononuclear cells, different B-cell subtypes including naïve, (un)switched memory, and CD27+CD43+ B1-like B cells, were analyzed by flow cytometry. Univariable and multivariable Cox proportional hazard models were used to analyze associations between B-cell subtypes, circulating antibodies and secondary cardiovascular manifestations during the 3-year follow-up period. Mean age was 70.1±9.6 years, males represented 62.8% of the population, and 54 patients had secondary manifestations during follow-up. High numbers of unswitched memory cells were protective against secondary outcome (hazard ratio, 0.30 [95% CI, 0.13-0.69]; P<0.01). Similar results were obtained for the switched memory cells that also showed to be protective against secondary outcome (hazard ratio, 0.33 [95% CI, 0.14-0.77]; P=0.01). CONCLUSIONS: A high number of (un)switched memory B cells is associated with better outcome following carotid artery endarterectomy. These findings suggest a potential role for B-cell subsets in prediction and prevention of secondary cardiovascular events in patients with atherosclerosis.

Low-Grade Inflammation, Oxidative Stress and Risk of Invasive Post-Menopausal Breast Cancer - A Nested Case-Control Study from the Malmö Diet and Cancer Cohort.

OBJECTIVE: Although cancer promotes inflammation, the role of inflammation in tumor-genesis is less well established. The aim was to examine if low-grade inflammation is related to post-menopausal breast cancer risk, and if obesity modifies this association. METHODS: In the Malmö Diet and Cancer cohort, a nested case-control study was defined among 8,513 women free of cancer and aged 55-73 years at baseline (1991-96); 459 were diagnosed with invasive breast cancer during follow-up (until December 31st, 2010). In laboratory analyses of blood from 446 cases, and 885 controls (matched on age and date of blood sampling) we examined systemic inflammation markers: oxidized (ox)-LDL, interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor (TNF)-α, white blood cells, lymphocytes and neutrophils. Odds ratios (OR) and 95% confidence intervals (CI) for breast cancer risk was calculated using multivariable conditional logistic regression. RESULTS: Inverse associations with breast cancer were seen in fully-adjusted models, for 2nd and 3rd tertiles of ox-LDL, OR (95% CI): 0.65 (0.47-0.90), 0.63 (0.45-0.89) respectively, p-trend = 0.01; and for the 3rd tertile of TNF-α, 0.65 (0.43-0.99), p-trend = 0.04. In contrast, those in the highest IL-1ß category had higher risk, 1.71 (1.05-2.79), p-trend = 0.01. Obesity did not modify associations between inflammation biomarkers and breast cancer. CONCLUSION: Our study does not suggest that low-grade inflammation increase the risk of post-menopausal breast cancer.

Low levels of IgG autoantibodies against the apolipoprotein B antigen p210 increases the risk of cardiovascular death after carotid endarterectomy.

OBJECTIVES: Autoimmune responses against oxidized-LDL (oxLDL) have been suggested to modulate inflammation in atherosclerosis. Previous studies showed an association between autoantibodies against the apolipoprotein B (apoB) p210 antigen and a lower risk of cardiovascular (CV) events. In the present study we investigated if autoantibodies against p210 at the time of carotid endarterectomy (CEA) predict risk for future CV events. METHODS: Native (nat) and malondialdehyde (MDA)-modified apoB p210 autoantibodies (IgM-p210nat, IgG-p210nat, IgM-p210MDA and IgG-p210MDA) were analyzed by ELISA from plasma samples of 351 patients at the time they underwent CEA. The incidence of postoperative CV events was assessed using national registers. RESULTS: A total of 52 non-fatal and 15 fatal CV events were registered during the follow-up period (35.1 ± 16.7 months). Patients who suffered from a fatal CV event had significantly lower plasma levels of IgG-p210nat and IgG-p210MDA. Kaplan-Meier curves of event-free survival showed increased CV mortality in patients with levels of IgG-p210nat and IgG-p210MDA below the median (Log Rank 7.813, p .005 and 9.105, p .003 respectively). The association between low levels of p210 IgG and fatal post-operative CV events remained significant when adjusting for age, sex, total cholesterol, HDL cholesterol, smoking habits and hypertension in a Cox Proportional Hazard model (hazard ratios (HR) IgG-p210nat below median: HR 6.7 (95% C.I. 1.5-30.6, p .013) and IgG-p210MDA below median: HR 7.8 (95% C.I. 1.7-35.5, p .008). CONCLUSIONS: The present findings support the notion that autoantibodies against LDL antigens are involved in the atherosclerotic disease process and suggest that CEA patients with low levels of IgG-p210nat and IgG-p210MDA have an increased risk of post-operative CV death.

Plasma autoantibodies against apolipoprotein B-100 peptide 210 in subclinical atherosclerosis.

OBJECTIVE: Experimental studies have suggested that autoimmunity is involved in atherosclerosis and provided evidence that both protective and pro-atherogenic immune responses exist. This concept has received support from small clinical studies implicating autoantibodies directed against apolipoprotein B-100 (apoB-100) in human atherosclerosis. We examined circulating autoantibodies directed against native and malondialdehyde (MDA)-modified epitope p210 of apoB-100 (IgG-p210nat and IgM-p210MDA) in relation to early atherosclerosis in a large, European longitudinal cohort study of healthy high-risk individuals. APPROACH AND RESULTS: IgG-p210nat and IgM-p210MDA were quantified in baseline plasma samples of 3430 participants in the IMPROVE study and related to composite and segment-specific measures of severity and rate of progression of carotid intima-media thickness (cIMT) determined at baseline and after 30 months. IgM-p210MDA autoantibody levels were independently related to several cIMT measures both in the common carotid artery and in the carotid bulb, including measures of cIMT progression, higher levels being associated with lower cIMT or slower cIMT progression. Consistent inverse relationships were also found between plasma levels of IgG-p210nat and baseline composite measures of cIMT. These associations disappeared when adjusting for established and emerging risk factors, and there were no associations with rate of cIMT progression besides in certain secondary stratified analyses. CONCLUSIONS: The present study provides further evidence of involvement of autoantibodies against native and MDA-modified apoB-100 peptide 210 in cardiovascular disease in humans and demonstrates that these associations are present already at a subclinical stage of the disease.

Elevated CD14++CD16- monocytes predict cardiovascular events.

BACKGROUND: Although monocytes in peripheral blood are no longer considered to be a homogeneous population, associations between distinct monocyte subsets and cardiovascular disease have not been highlighted in large epidemiological studies. METHODS AND RESULTS: The study included 700 randomly selected subjects from the cardiovascular arm of the Malmö Diet and Cancer study. Among these, 123 subjects experienced ischemic cardiovascular events during the follow-up until December 2008. Mononuclear leukocytes frozen at the baseline investigation in 1991 to 1994 were thawed and analyzed with flow cytometry to enumerate monocyte subsets, based on CD14 and CD16 expression. The percentage and number of classical CD14(++)CD16(-) monocytes were increased in the cardiovascular-event group compared with the event-free subjects (median, 69% [interquartile range, 62% to 76%] versus 67% [59% to 72%], P=0.017; 344 [251 to 419] cells/µL versus 297 [212 to 384] cells/µL, P=0.003). The hazard ratio was 1.66 for suffering a cardiovascular event in the highest tertile of the number of CD14(++)CD16(-) monocytes compared with the lowest tertile, even after adjustment for common risk factors (HR, 1.66; 95% CI: 1.02 to 2.72). CD14(++)CD16(-) monocytes did not, however, associate with the extent of atherosclerosis at baseline. In contrast, the percentage of monocytes expressing CD16 was negatively associated to the extent of carotid atherosclerosis measured as intima-media thickness at baseline. The chemokine receptors CCR2, CX3CR1, and CCR5 were not differentially expressed between cases and controls on any of the monocyte subsets, but CCR5 expression on CD14(+)CD16(++) monocytes was negatively associated to carotid intima-media thickness. CONCLUSIONS: This study shows that classical CD14(++)CD16(-) monocytes can predict future cardiovascular risk independently of other risk factors in a randomly selected population.

Identification of a danger-associated peptide from apolipoprotein B100 (ApoBDS-1) that triggers innate proatherogenic responses.

BACKGROUND: Subendothelial deposited low-density lipoprotein particles are a known inflammatory factor in atherosclerosis. However, the causal components derived from low-density lipoprotein are still poorly defined. Apolipoprotein B100 (ApoB100) is the unexchangeable protein component of low-density lipoprotein, and the progression of atherosclerosis is associated with immune responses to ApoB100-derived peptides. In this study, we analyzed the proinflammatory activity of ApoB100 peptides in atherosclerosis. METHODS AND RESULTS: By screening a peptide library of ApoB100, we identified a distinct native peptide referred to as ApoB100 danger-associated signal 1 (ApoBDS-1), which shows sequence-specific bioactivity in stimulation of interleukin-8, CCL2, and interleukin-6. ApoBDS-1 activates mitogen-activated protein kinase and calcium signaling, thereby effecting the expression of interleukin-8 in innate immune cells. Ex vivo stimulation of carotid plaques with ApoBDS-1 enhances interleukin-8 and prostaglandin E2 release. Furthermore, we demonstrated that ApoBDS-1-positive peptide fragments are present in atherosclerotic lesions using immunoassays and that low-molecular-weight fractions isolated from plaque show ApoBDS-1 activity inducing interleukin-8 production. CONCLUSIONS: Our data show that ApoBDS-1 is a previously unrecognized peptide with robust proinflammatory activity, contributing to the disease-promoting effects of low-density lipoprotein in the pathogenesis of atherosclerosis.

Immunization with cationized BSA inhibits progression of disease in ApoBec-1/LDL receptor deficient mice with manifest atherosclerosis.

Immune responses against modified self-antigens generated by hypercholesterolemia play an important role in atherosclerosis identifying the immune system as a possible novel target for prevention and treatment of cardiovascular disease. It has recently been shown that these immune responses can be modulated by subcutaneous injection of adjuvant. In the present study we immunized 25-week old ApoBec-1/LDL receptor deficient mice with manifest atherosclerosis with adjuvant and two different concentrations of the carrier molecule cationized BSA (cBSA). Plasma levels of Th2-induced apolipoprotein B (apoB)/IgG1 immune complexes were increased in the cBSA immunized groups verifying induction of immunity against a self-antigen. Mice were sacrificed at 36 weeks of age and atherosclerosis was monitored by en face Oil red O staining of the aorta. Immunization with 100 µg cBSA inhibited plaque progression, whereas the lower dose (50 µg) did not. In addition, the higher dose induced a more stable plaque phenotype, indicated by a higher content of collagen and less macrophages and T cells in the plaques. Moreover, there was an increased ratio of Foxp3+/Foxp3⁻ T cells in the circulation suggesting activation of a regulatory T cell response. In conclusion, we show that immunization with cBSA induces an immune response against apoB as well as an activation of Treg cells. This was associated with development of a more stable plaque phenotype and reduced atherosclerosis progression.

The pro-inflammatory effect of uraemia overrules the anti-atherogenic potential of immunization with oxidized LDL in apoE-/- mice.

BACKGROUND: Uraemia increases oxidative stress, plasma titres of antibodies recognizing oxidized low-density lipoprotein (oxLDL) and development of atherosclerosis. Immunization with oxLDL prevents classical, non-uraemic atherosclerosis. We have investigated whether immunization with oxLDL might also prevent uraemia-induced atherosclerosis in apolipoprotein E knockout (apoE-/-) mice. METHODS: ApoE-/- mice were immunized with either native LDL (n = 25), Cu(2+)-oxidized LDL (n = 25), PBS (n = 25), the apolipoprotein B-derived peptide P45 (apoB-peptide P45) conjugated to bovine serum albumin (BSA) (n = 25) or BSA (n = 25) prior to induction of uraemia by 5/6 nephrectomy (NX). RESULTS: Immunization with oxLDL increased plasma titres of immunoglobulin G (IgG) recognizing Cu(2+)-oxLDL and malondialdehyde-modified LDL (MDA-LDL). However, 5/6 NX induced a marked increase in plasma concentrations of anti-oxLDL antibodies as well as pro-atherogenic cytokines [interleukin (IL)-2 (IL-2), IL-4, IL-6 and IL-12)] in native mouse LDL (nLDL)-, oxLDL- and PBS-immunized mice. Even though nLDL- and oxLDL-immunized mice displayed higher anti-MDA-LDL IgG titres than the PBS group, aortic atherosclerosis lesion size was not affected by immunization. Immunization with the apoB-peptide P45, which consistently reduces classical atherosclerosis in non-uraemic mice, also did not reduce lesion size in uraemic apoE-/- mice. CONCLUSION: The results suggest that the pro-inflammatory and pro-atherogenic effect of uraemia overrules the anti-atherogenic potential of oxLDL immunization in apoE-/- mice.

High plasma concentrations of autoantibodies against native peptide 210 of apoB-100 are related to less coronary atherosclerosis and lower risk of myocardial infarction.

AIMS: We examined whether antibodies against peptides 45 and 210 of apoB-100 are related to myocardial infarction (MI) and severity of coronary atherosclerosis. METHODS AND RESULTS: Three hundred and eighty-seven survivors of a first MI (aged <60 years) and 387 sex- and age-matched controls were characterized in detail. IgG and IgM autoantibodies against native and malondialdehyde (MDA)-modified peptides 45 and 210 of apoB-100 (amino acids 661-680 and 3136-3155) were quantified in plasma and quantitative coronary angiography was performed in 243 patients. Post-infarction patients had significantly lower IgG against the native peptide 210 (IgG-p210(nat)) and higher IgM against the MDA-modified peptide 210 (IgM-p210(MDA)) compared with controls, whereas no differences were found for other antibodies. Plasma concentrations of IgG-p210(nat), but not IgM-p210(MDA), were independently and inversely related to the degree of coronary atherosclerosis in patients. In multiple logistic regression analysis (including established risk indicators), MI risk was 0.55 (95%CI: 0.37-0.81) for individuals in the IgG-p210(nat) upper quartile compared with the remaining individuals. CONCLUSION: Circulating IgG antibodies against the native peptide 210 of apoB-100 are inversely related to the severity of coronary atherosclerosis and associated with lower risk of MI. Epitope 210 of apoB-100 emerges as a target for immunization against atherosclerosis in humans.

Autoantibodies against modified apolipoprotein B-100 in relation to low-density lipoprotein size and the metabolic syndrome in otherwise healthy men.

The role of inflammation in atherosclerotic disease is well established, but the role of autoantibodies against modified apolipoprotein (apo) B-100 remains unclear. The metabolic syndrome is associated with a proinflammatory state, a predominance of small dense low-density lipoprotein (LDL) particles, and an increased risk for atherosclerotic diseases. Previous studies have shown specific autoantibodies against modified apo B-100 (within LDL) to be related to human atherosclerotic disease. The objective of the present study was to investigate whether autoantibodies against modified apo B-100 are related to parameters of the metabolic syndrome, such as small dense LDL. Two hundred ninety-one healthy men were investigated for different metabolic, anthropometric, and inflammatory variables; LDL peak particle size; and distribution of LDL in 4 subfractions. Subjects were grouped according to LDL peak size > or = 23.5 nm (pattern A, n = 230) or <23.5 nm (pattern B, n = 61). Immunoglobulin (Ig) G and IgM antibodies against 2 aldehyde-modified peptide sequences, denoted as 45 and 210, within apo B-100 were quantified. Levels of IgG(45), but not the other autoantibodies, were significantly higher in pattern B individuals (with a predominance of small dense LDL particles) compared with pattern A (P < .01). Relationships for both IgG(45) and IgG(210) with parameters typically associated with the metabolic syndrome were found. Only IgG(45) tended to be higher in individuals with the metabolic syndrome compared with those without (P = .07). We conclude that subjects with a predominance of small dense LDL particles have elevated concentrations of IgG(45) in the circulation, which reflect an activated immune response to a specific epitope of modified apo B-100.

Immunomodulation of atherosclerosis with a vaccine.

Experimental observations have established that the innate and adaptive immune mechanisms both have roles in the modulation of atherosclerosis. The complex function that the immune system has in the pathophysiology of atherosclerosis is highlighted by the fact that both proatherogenic and atheroprotective effects of immune activation can be demonstrated. An immune response to the protein and lipid components of oxidized LDL cholesterol has been observed in experimental models, and immunization with these antigens has generally reduced atherosclerosis. The findings suggest the tantalizing possibility that an atheroprotective vaccine can be developed. Our laboratories have identified several antigenic epitopes in the human apolipoprotein B100 component of LDL cholesterol. Active immunization with some of these epitopes has reduced atherosclerosis in hyperlipidemic mice. We believe, therefore, that a vaccine based on apolipoprotein B100-related peptide could have a role in reducing atherosclerosis. In this review, we discuss the possible immunologic mechanisms by which vaccines against atherosclerosis might work and the ways in which such treatment might be most effectively administered.

Vaccination for atherosclerosis: a novel therapeutic paradigm.

Numerous studies have identified a role for the innate and adaptive immune response in atherosclerosis; both pro- and antiatherogenic roles for the immune responses have been demonstrated. Common autoantigens against which an immune response has been identified in experimental and human models of atherosclerosis include oxidized low-density lipopoteins, beta2 glycoprotein 1 and heat shock protein 60. Activation of atheroprotective adaptive immune responses have been demonstrated for oxidized low-density lipoprotein-related antigens. Conversely, atheroprotection has been demonstrated with the induction of immune tolerance through activation of mucosal immunity to heat shock protein 65/60 and beta2 glycoprotein 1. Recent identification of specific immunoreactive antigenic epitopes in the apolipoprotein B-100 component of low density lipoproetin and early experimental observations have provided proof of concept that active vaccination using specific apolipoprotein B-100-related antigens may emerge as a novel immunomodulating atheroprotective strategy.