ApoA1 Neutralizes Proinflammatory Effects of Dengue Virus NS1 Protein and Modulates Viral Immune Evasion.

Dengue is a mosquito-borne infectious disease that is highly endemic in tropical and subtropical countries. Symptomatic patients can rapidly progress to severe conditions of hemorrhage, plasma extravasation, and hypovolemic shock, which leads to death. The blood tests of patients with severe dengue typically reveal low levels of high-density lipoprotein (HDL), which is responsible for reverse cholesterol transport (RCT) and regulation of the lipid composition in peripheral tissues. It is well known that dengue virus (DENV) depends on membrane cholesterol rafts to infect and to replicate in mammalian cells. Here, we describe the interaction of DENV nonstructural protein 1 (NS1) with apolipoprotein A1 (ApoA1), which is the major protein component of HDL. NS1 is secreted by infected cells and can be found circulating in the serum of patients with the onset of symptoms. NS1 concentrations in plasma are related to dengue severity, which is attributed to immune evasion and an acute inflammatory response. Our data show that the DENV NS1 protein induces an increase of lipid rafts in noninfected cell membranes and enhances further DENV infection. We also show that ApoA1-mediated lipid raft depletion inhibits DENV attachment to the cell surface. In addition, ApoA1 is able to neutralize NS1-induced cell activation and to prevent NS1-mediated enhancement of DENV infection. Furthermore, we demonstrate that the ApoA1 mimetic peptide 4F is also capable of mediating lipid raft depletion to control DENV infection. Taken together, our results suggest the potential of RCT-based therapies for dengue treatment. These results should motivate studies to assess the importance of RCT in DENV infection in vivo. IMPORTANCE DENV is one of the most relevant mosquito-transmitted viruses worldwide, infecting more than 390 million people every year and leading to more than 20 thousand deaths. Although a DENV vaccine has already been approved, its potential side effects have hampered its use in large-scale immunizations. Therefore, new treatment options are urgently needed to prevent disease worsening or to improve current clinical management of severe cases. In this study, we describe a new interaction of the NS1 protein, one of the major viral components, with a key component of HDL, ApoA1. This interaction seems to alter membrane susceptibility to virus infection and modulates the mechanisms triggered by DENV to evade the immune response. We also propose the use of a mimetic peptide named 4F, which was originally developed for atherosclerosis, as a potential therapy for relieving DENV symptoms.

Anti-SARS-CoV-2 mRNA vaccines as inducers of humoral response against apolipoprotein A-1?

BACKGROUND: COVID-19 and some anti-SARS-CoV-2 vaccines trigger a humoral autoimmune response against a broad range of endogenous components, which may affect recipients' prognosis in predisposed individuals. Autoantibodies directed against apolipoprotein A-1 (AAA1 IgG) the major protein fraction of High Density Lipoprotein have been shown to be raised in COVID-19 and in rheumatoid arthritis (RA) patients and other populations where they have been associated with poorer outcomes. We wanted to assess the impact of anti-SARS-CoV-2 mRNA-based vaccination on AAA1 autoimmune biomarkers in RA patients. METHODS: 20 healthy controls and 77 RA mRNA-based vaccinated patients were collected at baseline, 3 weeks after the first vaccination, 2 and 8 weeks after the second vaccination. AAA1 and SARS-CoV-2 serologies were measured by immunoassays. Systemic and local symptoms occurring during the vaccination protocol were recorded. RESULTS: mRNA-based vaccination induced a significant increase in median AAA1 IgG levels in both healthy controls and RA patients overtime. However, in both populations, these medians trend did not translate into significant increase in AAA1 IgG seropositivity rates despite evolving from 5 to 10% in healthy controls, and from 9 to 12.9% in RA patients. No associations were retrieved between AAA1 IgG and symptoms of any kind during the vaccination protocol. CONCLUSIONS: mRNA-based vaccination seems to induce a light AAA1 IgG response in immunocompetent individuals within 2 months after the last injection. Although we did not observe any warning signs, the formal demonstration of the harmlessness of such biological warrants further studies.

SARS-CoV-2 infection as a trigger of humoral response against apolipoprotein A-1.

BACKGROUND: Unravelling autoimmune targets triggered by SARS-CoV-2 infection may provide crucial insights into the physiopathology of the disease and foster the development of potential therapeutic candidate targets and prognostic tools. We aimed at determining (a) the association between anti-SARS-CoV-2 and anti-apoA-1 humoral response and (b) the degree of linear homology between SARS-CoV-2, apoA-1 and Toll-like receptor 2 (TLR2) epitopes. DESIGN: Bioinformatics modelling coupled with mimic peptides engineering and competition experiments were used to assess epitopes sequence homologies. Anti-SARS-CoV-2 and anti-apoA-1 IgG as well as cytokines were assessed by immunoassays on a case-control (n = 101), an intensive care unit (ICU; n = 126) and a general population cohort (n = 663) with available samples in the pre and post-pandemic period. RESULTS: Using bioinformatics modelling, linear sequence homologies between apoA-1, TLR2 and Spike epitopes were identified but without experimental evidence of cross-reactivity. Overall, anti-apoA-1 IgG levels were higher in COVID-19 patients or anti-SARS-CoV-2 seropositive individuals than in healthy donors or anti-SARS-CoV-2 seronegative individuals (P < .0001). Significant and similar associations were noted between anti-apoA-1, anti-SARS-CoV-2 IgG, cytokines and lipid profile. In ICU patients, anti-SARS-CoV-2 and anti-apoA-1 seroconversion rates displayed similar 7-day kinetics, reaching 82% for anti-apoA-1 seropositivity. In the general population, SARS-CoV-2-exposed individuals displayed higher anti-apoA-1 IgG seropositivity rates than nonexposed ones (34% vs 16.8%; P = .004). CONCLUSION: COVID-19 induces a marked humoral response against the major protein of high-density lipoproteins. As a correlate of poorer prognosis in other clinical settings, such autoimmunity signatures may relate to long-term COVID-19 prognosis assessment and warrant further scrutiny in the current COVID-19 pandemic.

Association Between ApoA-I (Apolipoprotein A-I) Immune Complexes and Adverse Cardiovascular Events-Brief Report.

OBJECTIVE: The immune response is linked to the progression of atherosclerotic cardiovascular disease (CVD). Free autoantibodies targeting ApoA-I (apolipoprotein A-I) have been identified as a component of the inflammatory milieu in patients and have a moderate association with CVD progression. Based on the presence of these antibodies and the high concentration of circulating ApoA-I, we hypothesized that antibodies bound to ApoA-I as an immune complex would be predictive of incident adverse CVD outcomes. Approach and Results: The presence of ApoA-I/IgG immune complexes (ICs) in plasma was confirmed by ELISA in 3 subject cohorts. Characterization of the protein components of ApoAI/IgG ICs indicate that ICs are not correlated with total ApoA-I concentration and are enriched in the anti-inflammatory subclass, IgG4, relative to total plasma IgG (>30% versus 6%). In 359 patients with coronary artery disease (CAD), there were 71 incident adverse CVD events (death, myocardial infarction, and stroke) during a median 4.1-year follow-up. In Cox proportional hazard regression analysis, low levels of ApoA-I/IgG ICs were independent predictors of adverse cardiovascular outcomes after adjustment for age, sex, diabetes mellitus, estimated glomerular filtration rate, presence of obstructive CAD, heart failure, total cholesterol, and HDL (high-density lipoprotein) cholesterol (adjusted hazard ratio of 1.90 [95% CI, 1.03-3.49; P=0.038] between the lowest and the highest tertiles). CONCLUSIONS: Low levels of ApoA-I/IgG ICs are associated with an increased risk of adverse events in patients with CAD, raising their potential to be used as a biomarker to predict CVD progression.

Anti-Apolipoprotein A-1 IgG Influences Neutrophil Extracellular Trap Content at Distinct Regions of Human Carotid Plaques.

BACKGROUND: Neutrophils accumulate in atherosclerotic plaques. Neutrophil extracellular traps (NET) were recently identified in experimental atherosclerosis and in complex human lesions. However, not much is known about the NET marker citrullinated histone-3 (H3Cit) expression and functionality in human carotid plaques. Moreover, the association between the proatherosclerotic autoantibody anti-apolipoprotein A-1 (anti-ApoA-1 IgG) and NET has never been investigated. METHODS: Atherosclerotic plaques have been obtained from 36 patients with severe carotid stenosis that underwent carotid endarterectomy for severe carotid stenosis. Samples were sectioned into upstream and downstream regions from the same artery segment. Plaque composition and expression of NET markers neutrophil elastase (NE) and H3Cit were quantified by immunohistochemistry. H3Cit expression and function was evaluated by immunofluorescence and confocal analysis in a subset of patients. RESULTS: Pathological features of vulnerable phenotypes were exacerbated in plaques developed at downstream regions, including higher accumulation of neutrophils and enhanced expression of NE and H3Cit, as compared to plaques from upstream regions. The H3Cit signal was also more intense in downstream regions, with significant extracellular distribution in spaces outside of neutrophils. The percentage of H3Cit colocalization with CD66b (neutrophils) was markedly lower in downstream portions of carotid plaques, confirming the extrusion of NET in this region. In agreement, the maximum distance of the H3Cit signal from neutrophils, extrapolated from vortex distance calculation in all possible directions, was also higher in downstream plaques. The serum anti-ApoA-1index positively correlated with the expression of H3Cit in downstream segments of plaques. Expression of the H3Cit signal outside of neutrophils and H3Cit maximal distance from CD66b-positive cells increased in plaques from serum positive anti-ApoA-1 patients compared with serum negative patients. CONCLUSION: NET elements are differentially expressed in upstream versus downstream regions of human carotid plaques and may be influenced by circulating levels of anti-ApoA-1 IgG. These findings could warrant the investigation of NET elements as potential markers of vulnerability.

Anti-ApoA-I IgG antibodies are not associated with carotid artery disease progression and first-time cardiovascular events in middle-aged individuals.

OBJECTIVE: IgG antibodies against apolipoprotein A-I (ApoA-I) have been found to be elevated in subjects from the general population with clinically manifest cardiovascular disease and in myocardial infarction patients with an adverse prognosis. Here, we investigated whether these antibodies are prospectively associated with carotid artery disease progression and with the risk for first-time cardiovascular events in individuals with no previous history of cardiovascular disease. APPROACH AND RESULTS: We selected 383 subjects from the cardiovascular cohort of Malmö Diet and Cancer study who suffered a coronary event during a median follow-up period of 15.4 (10.3-16.4) years and 395 age- and sex-matched controls. None of the study participants had a previous history of coronary artery disease or stroke. Anti-ApoA-I IgG were measured by ELISA in serum samples collected at baseline. Intima-media thickness (IMT) was measured in the common carotid artery and in the carotid bifurcation at baseline and after 15.9 (±1.5) years. We found no associations between anti-ApoA-I IgG and carotid artery IMT at baseline or with IMT progression during follow-up. In Cox proportional hazards analyses adjusted for traditional cardiovascular risk factors, the hazard ratio (HR 95%CI) for the primary outcome, incident coronary events, was 0.97 (0.75-1.25), P = 0.782, in subjects with anti-ApoA-I IgG within the highest tertile compared with the lowest tertile. Similarly, we did not find any associations with the secondary outcome, incident first-time stroke. CONCLUSIONS: Serum autoantibodies against ApoA-I do not correlate with disease progression and adverse events in cardiovascular disease-free individuals from the general population.

Impact of CD14 Polymorphisms on Anti-Apolipoprotein A-1 IgG-Related Coronary Artery Disease Prediction in the General Population.

OBJECTIVE: We aimed to determine whether autoantibodies against apoA-1 (apolipoprotein A-1; anti-apoA-1 IgG) predict incident coronary artery disease (CAD), defined as adjudicated incident myocardial infarction, angina, percutaneous coronary revascularization, or bypass grafting, in the general population. We further investigated whether this association is modulated by a functional CD14 receptor single nucleotide polymorphism. APPROACH AND RESULTS: In a prospectively studied, population-based cohort of 5220 subjects (mean age 52.6±10.7 years, 47.4% males), followed over a median period of 5.6 years, subjects positive versus negative for anti-apoA-1 IgG presented a total CAD rate of 3.9% versus 2.8% (P=0.077) and a nonfatal CAD rate of 3.6% versus 2.3% (P=0.018), respectively. After multivariate adjustment for established cardiovascular risk factors, the hazard ratios of anti-apoA-1 IgG for total and nonfatal CAD were: hazard ratio=1.36 (95% confidence interval, 0.94-1.97; P=0.105) and hazard ratio=1.53 (95% confidence interval, 1.03-2.26; P=0.034), respectively. In subjects with available genetic data for the C260T rs2569190 single nucleotide polymorphism in the CD14 receptor gene (n=4247), we observed a significant interaction between anti-apoA-1 IgG and rs2569190 allele status with regards to CAD risk, with anti-apoA-1 IgG conferring the highest risk for total and nonfatal CAD in non-TT carriers, whereas being associated with the lowest risk for total and nonfatal CAD in TT homozygotes (P for interaction =0.011 and P for interaction =0.033, respectively). CONCLUSIONS: Anti-apoA-1 IgG are independent predictors of nonfatal incident CAD in the general population. The strength of this association is dependent on a functional polymorphism of the CD14 receptor gene, a finding suggesting a gene-autoantibody interaction for the development of CAD.

Synthetic Amphipathic Helical Peptides Targeting CD36 Attenuate Lipopolysaccharide-Induced Inflammation and Acute Lung Injury.

Synthetic amphipathic helical peptides (SAHPs) designed as apolipoprotein A-I mimetics are known to bind to class B scavenger receptors (SR-Bs), SR-BI, SR-BII, and CD36, receptors that mediate lipid transport and facilitate pathogen recognition. In this study, we evaluated SAHPs, selected for targeting human CD36, by their ability to attenuate LPS-induced inflammation, endothelial barrier dysfunction, and acute lung injury (ALI). L37pA, which targets CD36 and SR-BI equally, inhibited LPS-induced IL-8 secretion and barrier dysfunction in cultured endothelial cells while reducing lung neutrophil infiltration by 40% in a mouse model of LPS-induced ALI. A panel of 20 SAHPs was tested in HEK293 cell lines stably transfected with various SR-Bs to identify SAHPs with preferential selectivity toward CD36. Among several SAHPs targeting both SR-BI/BII and CD36 receptors, ELK-B acted predominantly through CD36. Compared with L37pA, 5A, and ELK SAHPs, ELK-B was most effective in reducing the pulmonary barrier dysfunction, neutrophil migration into the lung, and lung inflammation induced by LPS. We conclude that SAHPs with relative selectivity toward CD36 are more potent at inhibiting acute pulmonary inflammation and dysfunction. These data indicate that therapeutic strategies using SAHPs targeting CD36, but not necessarily mimicking all apolipoprotein A-I functions, may be considered a possible new treatment approach for inflammation-induced ALI and pulmonary edema.

Predictive Models for the Risk of Dyslipidemia in Adolescents with Essential Arterial Hypertension.

Predictive models of comorbidity, dyslipidemic disorders and essential arterial hypertension, in Russian adolescents aged 12 to 18 years (mean 15.48±1.53) were formulated with consideration for biochemical (lipid profiles) and genetic parameters (carrier state of gene polymorphic variants of apolipoprotein genes ApoA1 (-75G/A and +83C/T), ApoB (Ins/Del), ApoC3 (S1/S2), and ApoE (ε2/ε3/ε4). Significant prognostic risk factors for the mentioned comorbid pathologies were lipid metabolism parameters HDL-Ch, LDL-Ch, VLDL-Ch and carrier state of the +83T allele of the ApoA1 gene and Del allele of the ApoB gene. The obtained mathematical model is characterized by high predictive accuracy: the percentage of correct classification or the rate of correct assignment of each participant to the proper group was 96.33%.

Extended-release niacin increases anti-apolipoprotein A-I antibodies that block the antioxidant effect of high-density lipoprotein-cholesterol: the EXPLORE clinical trial.

AIMS: Extended-release niacin (ERN) is the most effective agent for increasing high-density lipoprotein-cholesterol (HDL-C). Having previously identified anti-HDL antibodies, we investigated whether ERN affected the antioxidant capacity of HDL and whether ERN was associated with the production of antibodies against HDL (aHDL) and apolipoprotein A-I (aApoA-I). METHODS: Twenty-one patients older than 18 years, with HDL-C ≤40 mg dl-1 (men) or ≤50 mg dl-1 (women) were randomly assigned to receive daily ERN (n = 10) or placebo (n = 11) for two sequential 12-week periods, with 4 weeks of wash-out before cross-over. Primary outcome was change of paraoxonase-1 (PON1) activity and secondary outcomes were changes in aHDL and aApoA-I antibodies. Clinical Trial Unique Identifier: EudraCT 2006-006889-42. RESULTS: The effect of ERN on PON1 activity was nonsignificant (coefficient estimate 20.83 U l-1 , 95% confidence interval [CI] -9.88 to 51.53; P = 0.184). ERN was associated with an increase in HDL-C levels (coefficient estimate 5.21 mg dl-1 , 95% CI 1.16 to 9.25; P = 0.012) and its subclasses HDL2 (coefficient estimate 2.46 mg dl-1 , 95% CI 0.57 to 4.34; P = 0.011) and HDL3 (coefficient estimate 2.73 mg dl-1 , 95% CI 0.47 to 4.98; P = 0.018). ERN was significantly associated with the production of aApoA-I antibodies (coefficient estimate 0.25 µg ml-1 , 95% CI 0.09-0.40; P = 0.001). aApoA-I titres at baseline were correlated with decreased PON activity. CONCLUSIONS: The rise in HDL-C achieved with ERN was not matched by improved antioxidant capacity, eventually hampered by the emergence of aApoA-I antibodies. These results may explain why Niacin and other lipid lowering agents fail to reduce cardiovascular risk.

Cholesterol-Independent Suppression of Lymphocyte Activation, Autoimmunity, and Glomerulonephritis by Apolipoprotein A-I in Normocholesterolemic Lupus-Prone Mice.

Apolipoprotein (Apo)A-I, the major lipid-binding protein of high-density lipoprotein, can prevent autoimmunity and suppress inflammation in hypercholesterolemic mice by attenuating lymphocyte cholesterol accumulation and removing tissue-oxidized lipids. However, whether ApoA-I mediates immune-suppressive or anti-inflammatory effects under normocholesterolemic conditions and the mechanisms involved remain unresolved. We transferred bone marrow from systemic lupus erythematosus (SLE)-prone Sle123 mice into normal, ApoA-I-knockout (ApoA-I(-/-)) and ApoA-I-transgenic (ApoA-I(tg)) mice. Increased ApoA-I in ApoA-I(tg) mice suppressed CD4(+) T and B cell activation without changing lymphocyte cholesterol levels or reducing major ApoA-I-binding oxidized fatty acids. Unexpectedly, oxidized fatty acid peroxisome proliferator-activated receptor γ ligands 13- and 9-hydroxyoctadecadienoic acid were increased in lymphocytes of autoimmune ApoA-I(tg) mice. ApoA-I reduced Th1 cells independently of changes in CD4(+)Foxp3(+) regulatory T cells or CD11c(+) dendritic cell activation and migration. Follicular helper T cells, germinal center B cells, and autoantibodies were also lower in ApoA-I(tg) mice. Transgenic ApoA-I also improved SLE-mediated glomerulonephritis. However, ApoA-I deficiency did not have the opposite effects on autoimmunity or glomerulonephritis, possibly as the result of compensatory increases in ApoE on high-density lipoprotein. We conclude that, although compensatory mechanisms prevent the proinflammatory effects of ApoA-I deficiency in normocholesterolemic mice, increasing ApoA-I can attenuate lymphocyte activation and autoimmunity in SLE independently of cholesterol transport, possibly through oxidized fatty acid peroxisome proliferator-activated receptor γ ligands, and it can reduce renal inflammation in glomerulonephritis.

ApoA1 and ApoA1-specific self-antibodies in cardiovascular disease.

Apolipoprotein A1 (ApoA1) is a main protein moiety in high-density lipoprotein (HDL) particles. Generally, ApoA1 and HDL are considered as atheroprotective. In prooxidant and inflammatory microenvironment in the vicinity to the atherosclerotic lesion, ApoA1/HDL are subjected to modification. The chemical modifications such as oxidation, nitration, etc result in altering native architecture of ApoA1 toward dysfunctionality and abnormality. Neutrophil myeloperoxidase has a prominent role in this mechanism. Neo-epitopes could be formed and then exposed that makes them immunogenic. Indeed, these epitopes may be recognized by immune cells and induce production of proatherogenic ApoA1-specific IgG antibodies. These antibodies are biologically relevant because they are able to react with Toll-like receptor (TLR)-2 and TLR4 in target cells and induce a variety of pro-inflammatory responses. Epidemiological and functional studies underline a prognostic value of ApoA1 self-antibodies for several cardiovascular diseases, including myocardial infarction, acute coronary syndrome, and severe carotid stenosis.

Anti-ApoA-1 IgG serum levels predict worse poststroke outcomes.

BACKGROUND: Autoantibodies to apolipoprotein A-1 (anti-ApoA-1 IgG) were shown to predict major adverse cardiovascular events and promote atherogenesis. However, their potential relationship with clinical disability and ischaemic lesion volume after acute ischaemic stroke (AIS) remains unexplored. MATERIALS AND METHODS: We included n = 76 patients admitted for AIS and we investigated whether baseline serum anti-ApoA-1 IgG levels could predict (i) AIS-induced clinical disability [assessed by the modified Rankin Scale (mRS)], and (ii) AIS-related ischaemic lesion volume [assessed by Computed Tomography (CT)]. We also evaluated the possible pro-apoptotic and pro-necrotic effects of anti-ApoA-1 IgG on human astrocytoma cell line (U251) using flow cytometry. RESULTS: High levels of anti-ApoA-1 IgG were retrieved in 15·8% (12/76) of patients. Increased baseline levels of anti-ApoA-1 IgG were independently correlated with worse mRS [ß = 0·364; P = 0·002; adjusted odds ratio (OR): 1·05 (95% CI 1·01-1·09); P = 0·017] and CT-assessed ischaemic lesion volume [ß = 0·333; P < 0·001; adjusted OR: 1·06 (95% CI 1·01-1·12); P = 0·048] at 3 months. No difference in baseline clinical, biochemical and radiological characteristics was observed between patients with high vs. low levels of anti-ApoA-1 IgG. Incubating human astrocytoma cells with anti-ApoA-1 IgG dose dependently induced necrosis and apoptosis of U251 cells in vitro. CONCLUSION: Anti-ApoA-1 IgG serum levels at AIS onset are associated with poorer clinical recovery and worse brain lesion volume 3 months after AIS. These observations could be partly explained by the deleterious effect of anti-ApoA-1 IgG on human brain cell survival in vitro and may have clinical implication in the prediction of poor outcome in AIS.

Quantification of serum apolipoproteins A-I and B-100 in clinical samples using an automated SISCAPA-MALDI-TOF-MS workflow.

A fully automated workflow was developed and validated for simultaneous quantification of the cardiovascular disease risk markers apolipoproteins A-I (apoA-I) and B-100 (apoB-100) in clinical sera. By coupling of stable-isotope standards and capture by anti-peptide antibodies (SISCAPA) for enrichment of proteotypic peptides from serum digests to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS detection, the standardized platform enabled rapid, liquid chromatography-free quantification at a relatively high throughput of 96 samples in 12h. The average imprecision in normo- and triglyceridemic serum pools was 3.8% for apoA-I and 4.2% for apoB-100 (4 replicates over 5 days). If stored properly, the MALDI target containing enriched apoA-1 and apoB-100 peptides could be re-analyzed without any effect on bias or imprecision for at least 7 days after initial analysis. Validation of the workflow revealed excellent linearity for daily calibration with external, serum-based calibrators (R(2) of 0.984 for apoA-I and 0.976 for apoB-100 as average over five days), and absence of matrix effects or interference from triglycerides, protein content, hemolysates, or bilirubins. Quantification of apoA-I in 93 normo- and hypertriglyceridemic clinical sera showed good agreement with immunoturbidimetric analysis (slope = 1.01, R(2) = 0.95, mean bias = 4.0%). Measurement of apoB-100 in the same clinical sera using both methods, however, revealed several outliers in SISCAPA-MALDI-TOF-MS measurements, possibly as a result of the lower MALDI-TOF-MS signal intensity (slope = 1.09, R(2) = 0.91, mean bias = 2.0%). The combination of analytical performance, rapid cycle time and automation potential validate the SISCAPA-MALDI-TOF-MS platform as a valuable approach for standardized and high-throughput quantification of apoA-I and apoB-100 in large sample cohorts.

Definition of human apolipoprotein A-I epitopes recognized by autoantibodies present in patients with cardiovascular diseases.

Autoantibodies to apolipoprotein A-I (anti-apoA-I IgG) have been shown to be both markers and mediators of cardiovascular disease, promoting atherogenesis and unstable atherosclerotic plaque. Previous studies have shown that high levels of anti-apoA-I IgGs are independently associated with major adverse cardiovascular events in patients with myocardial infarction. Autoantibody responses to apoA-I can be polyclonal and it is likely that more than one epitope may exist. To identify the specific immunoreactive peptides in apoA-I, we have developed a set of methodologies and procedures to isolate, purify, and identify novel apoA-I endogenous epitopes. First, we generated high purity apoA-I from human plasma, using thiophilic interaction chromatography followed by enzymatic digestion specifically at lysine or arginine residues. Immunoreactivity to the different peptides generated was tested by ELISA using serum obtained from patients with acute myocardial infarction and high titers of autoantibodies to native apoA-I. The immunoreactive peptides were further sequenced by mass spectrometry. Our approach successfully identified two novel immunoreactive peptides, recognized by autoantibodies from patients suffering from myocardial infarction, who contain a high titer of anti-apoA-I IgG. The discovery of these epitopes may open innovative prognostic and therapeutic opportunities potentially suitable to improve current cardiovascular risk stratification.

Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas.

A fusion protein comprising an α-CD20 single chain variable fragment (scFv) antibody, a spacer peptide, and human apolipoprotein (apo) A-I was constructed and expressed in Escherichia coli. The lipid interaction properties intrinsic to apoA-I as well as the antigen recognition properties of the scFv were retained by the chimera. scFv•apoA-I was formulated into nanoscale reconstituted high-density lipoprotein particles (termed nanodisks; ND) and incubated with cultured cells. α-CD20 scFv•apoA-I ND bound to CD20-positive non-Hodgkins lymphoma (NHL) cells (Ramos and Granta) but not to CD20-negative T lymphocytes (i.e., Jurkat). Binding to NHL cells was partially inhibited by pre-incubation with rituximab, a monoclonal antibody directed against CD20. Confocal fluorescence microscopy analysis of Granta cells following incubation with α-CD20 scFv•apoA-I ND formulated with the intrinsically fluorescent hydrophobic polyphenol, curcumin, revealed α-CD20 scFv•apoA-I localizes to the cell surface, while curcumin off-loads and gains entry to the cell. Compared to control incubations, viability of cultured NHL cells was decreased upon incubation with α-CD20 scFv•apoA-I ND harboring curcumin. Thus, formulation of curcumin ND with α-CD20 scFv•apoA-I as the scaffold component confers cell targeting and enhanced bioactive agent delivery, providing a strategy to minimize toxicity associated with chemotherapeutic agents.

Nanobody-Based Electrochemical Immunoassay for Ultrasensitive Determination of Apolipoprotein-A1 Using Silver Nanoparticles Loaded Nanohydroxyapatite as Label.

Nanobodies (Nbs), derived from camelid heavy-chain antibodies, have distinct advantages over conventional antibodies in immunoassay. In this work, Nbs (Nb11 and Nb19) that can bind to different epitopes on apolipoprotein-A1 (Apo-A1) were screened out from an immunized Bactrian camel for the first time. Nb11 was used as capture antibody and fixed on gold nanoparticles (Au NPs) modified screen-printed carbon electrode (SPCE). The silver nanoparticles loaded nanohydroxyapatite (Ag-nHAP) was used as signal tag to label secondary antibody Nb19. A sandwich-type immunological reaction occurred between Apo-A1 and the two Nbs, which brought the Ag-nHAP to the SPCE surface. After the Ag-nHAP were acidically dissolved in the microelectrolytic cell of the SPCE, stripping voltammetric measurement for the released silver ions was performed to obtain an amplified signal. The peak current values increased by the logarithmic values of Apo-A1 concentrations from 10(-4) to 50 ng mL(-1) under optimal conditions. The detection limit was calculated to be 0.02 pg mL(-1). This method was used for the serum samples analysis and achieved satisfactory results. The low cost and high sensitivity make the electrochemical immunosensor suitable for the Apo-A1 detection, which may find promising application in other fields.

Overexpression of apolipoprotein A-I fused to an anti-transforming growth factor beta peptide modulates the tumorigenicity and immunogenicity of mouse colon cancer cells.

Transforming growth factor beta (TGF-ß) promotes tumor growth, invasion and metastasis in established tumors. In this study, we analyzed the effect of overexpressing an anti-TGF-ß peptide fused to apolipoprotein A-I (ApoA-I) as a scaffold molecule. We generated and characterized stable MC38 colon carcinoma clones expressing ApoA-I fused to the anti-TGF-ß peptide P144 and ApoA-I as control cells. We evaluated in vitro the gene expression profile, cell cycle and anchorage-independent growth. The in vivo tumorigenic potential and immunogenicity were analyzed inoculating the MC38 clones into C57BL/6 mice, recombination-activating gene 1 knockout mice or mice deficient in NK cells either subcutaneously or intrasplenically to generate hepatic metastases. While overexpression of ApoA-I had no effect on the parameters analyzed, ApoA-I fused to P144 markedly diminished the tumorigenic capacity and metastatic potential of MC38 in vitro and in vivo, thus generating a highly immunogenic cell line. MC38 cells transfected with ApoA-I fused to P144 triggered memory T cell responses able to eliminate the parental cell line upon re-challenge. In summary, expression of ApoA-I fused to P144 is a novel strategy to modulate TGF-ß in tumor cells. These results highlight the potential of TGF-ß as a target in the development of new antitumor treatments.

Diagnostic and prognostic value of autoantibodies anti-apolipoprotein A-1 and anti-phosphorylcholine in acute non-ST elevation myocardial infarction.

BACKGROUND: Autoantibodies have been shown to play a critical role in predicting major adverse cardiovascular events in atherosclerotic patients. We aimed to assess the diagnostic accuracy of autoantibodies to apolipoprotein A-1 (anti-apoA-1 IgG) and to phosphorylcholine (anti-PC IgM) for non-ST segment elevation acute myocardial infarction (NSTEMI) and to explore their potential prognostic value. METHODS: This prospective multicentre study included 1072 patients presenting to the emergency department for suspected NSTEMI. The final diagnosis was adjudicated by two independent cardiologists. For both antibodies alone or expressed as a ratio (anti-apoA-1 IgG/anti-PC IgM), we determined their (i) diagnostic accuracy for NSTEMI and (ii) prognostic accuracy for major adverse cardiovascular events (MACE) during 1-year follow-up. RESULTS: A total of 154 patients (14%) had a final diagnosis of NSTEMI. Diagnostic accuracy for the diagnosis of NSTEMI as quantified by the area under the receiver operating characteristics curve (AUC) was very low for both autoantibodies separately as well as combined as a ratio: AUC anti-apoA-1 IgG 0.50 (95%CI, 0.47-0.53, P = 0.99), AUC anti-PC IgM 0.53 (95%CI, 0.50-0.56, P = 0.30) and AUC of the ratio 0.52 (95%CI, 0.49-0.55, P = 0.47). Adding the anti-apoA-1 IgG/Anti-PC IgM ratio to hs-cTnT did not provide incremental diagnostic value over hs-cTnT alone. MACE occurred in 221 patients (21%) during follow-up. The autoantibodies, separately or expressed as ratio, also had very low accuracy to predict MACE (p=ns). CONCLUSIONS: Anti-apoA-1 IgG and anti-PC IgM autoantibodies did not have diagnostic or prognostic value in patients with NSTEMI.

Evidence for the presence of lipid-free monomolecular apolipoprotein A-1 in plasma.

The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. However the structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. We previously obtained a monoclonal antibody (MAb) that specifically recognizes apoA-1 in preß1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preß1-HDL ELISA. Here, we purified preß1-HDL from fresh normal plasma using said antibody, and analyzed the composition and structure. ApoA-1 was detected, but neither phospholipid nor cholesterol were detected in the purified preß1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preß1-HDL and original plasma preß1-HDL, or between the preß1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preß1-HDL MAbs, reactivity with intact plasma preß1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. These results suggest that plasma preß1-HDL is lipid-free monomolecular apoA-1.