Natural IgM Antibodies Research Articles

Characterization of a natural mouse monoclonal antibody recognizing epitopes shared by oxidized low-density lipoprotein and chaperonin 60 of Aggregatibacter actinomycetemcomitans.

Natural antibodies are predominantly antibodies of the IgM isotype present in the circulation of all vertebrates that have not been previously exposed to exogenous antigens. They are often directed against highly conserved epitopes and bind to ligands of varying chemical composition with low affinity. In this study we cloned and characterized a natural mouse monoclonal IgM antibody selected by binding to malondialdehyde acetaldehyde epitopes on low-density lipoprotein (LDL). Interestingly, the IgM antibody cross-reacted with Aggregatibacter actinomycetemcomitans (Aa) bacteria, a key pathogenic microbe in periodontitis reported to be associated with risk factor for atherosclerosis, thus being named as Aa_Mab. It is more intriguing that the binding molecule of Aa to Aa_Mab IgM was found to be Aa chaperonin 60 or HSP60, a member of heat-shock protein family, behaving not only as a chaperone for correct protein folding but also as a powerful virulence factor of the bacteria for inducing bone resorption and as a putative pathogenic factor in atherosclerosis. The findings will highlight the question of whether molecular mimicry between pathogen components and oxidized LDL could lead to atheroprotective immune activity, and also would be of great importance in potential application of immune response-based preventive and therapeutic strategies against atherosclerosis and periodontal disease.

IL-17A Promotes Pulmonary B-1a Cell Differentiation via Induction of Blimp-1 Expression during Influenza Virus Infection

B-1 cells play a critical role in early protection during influenza infections by producing natural IgM antibodies. However, the underlying mechanisms involved in regulating this process are largely unknown. Here we found that during influenza infection pleural cavity B-1a cells rapidly infiltrated lungs, where they underwent plasmacytic differentiation with enhanced IgM production. This process was promoted by IL-17A signaling via induction of Blimp-1 expression and NF-κB activation in B-1a cells. Deficiency of IL-17A led to severely impaired B-1a-derived antibody production in the respiratory tract, resulting in a deficiency in viral clearance. Transfer of B-1a-derived natural antibodies rescued Il17a -/- mice from otherwise lethal infections. Together, we identify a critical function of IL-17A in promoting the plasmacytic differentiation of B-1a cells. Our findings provide new insights into the mechanisms underlying the regulation of pulmonary B-1a cell response against influenza infection.

Oxidation-specific epitopes are major targets of innate immunity in atherothrombosis.

Atherosclerosis is a chronic inflammatory disease of the vascular wall that results from disturbed lipoprotein metabolism and increased oxidative stress. A major consequence of this is lipid peroxidation, which generates a number of breakdown products of membrane lipids that form so called oxidation-specific epitopes (OSE). OSE have been documented in oxidized lipoproteins and on the surface of dying cells and circulating microparticles, and their ability to trigger robust pro-inflammatory and pro-thrombotic responses has been demonstrated extensively. Recent studies have identified specific OSE as major targets of both cellular and soluble pattern recognition receptors of the innate immune system, including innate natural IgM antibodies. This allows the immune system to identify metabolic waste and mediate important physiological house keeping functions, e.g. by promoting the removal of cellular debris and by neutralizing oxidized molecules. Indeed, innate B1 cells and B1 cell derived natural IgM with specificity for OSE have been shown to protect mice from the development of atherosclerotic lesions. Moreover, OSE-specific natural IgM antibodies bind and neutralize the pro-inflammatory and pro-thrombotic effects of OSE, and low levels of OSE-specific IgM are associated with an increased risk for myocardial infarction. Understanding the molecular components and mechanisms involved in this process, will help identify individuals with increased risk for atherothrombosis and indicate novel points for therapeutic intervention.

Variation and Repeatability of Natural Antibodies Against Keyhole Limpet Hemocyanin of Indigenous Chicken of Kenya

The immune system is designed to provide protection to the body by combating pathogens. Identifying animals with superior immune responses reduces disease occurrences, increases farm profit and improves product quality and safety. Consequently, there is need to breed disease resistant animals that will eliminate the danger of currently used disease management strategies; drug prophylaxis and vaccination, which are unsafe and ineffective respectively. studies aimed at investigating the mechanisms involved in genetic resistance have been done, however a standardized biological parameter indicative of disease resistance or susceptibility remains elusive. The objectives of the study were to determine presence and variation of IgA, IgG and IgM among indigenous chicken. Estimate repeatability within the indigenous chicken over time of IgA, IgM and IgG natural antibody isotypes against Keyhole limpet hemocyanin. Blood samples from 24 indigenous chickens of the same age and sex were collected four times within three weeks. IgA, IgM and IgG titer values were measured by indirect ELISA from the sera. A mixed model with repeated measures was performed to determine variation and repeatability. All the immunoglubulin isotypes binding KLH in chicken serum were recorded. There was significant difference between isotypes concentrations with IgM and IgA being the lowest and highest titre values, respectively. Repeatability was 0.68, 0.99 and 0.99 for IgM, IgG and IgA, respectively. The isotypes were detectable and variable in serum of indigenous chicken and consistently and repeatedly measurable in blood serum. This finding may lay the basis for genetic improvement of immune response in the indigenous chicken.

Concomitant Use of Neuroprotective Drugs in Neuro Rehabilitation of Multiple Sclerosis.

We provide an overview of rehabilitation in neurological diseases. A large amount of literature available on neurorehabilitation is based from the rehabilitative work on stroke and spinal cord injuries. After a brief description of rehabilitation, the potential application of neurorehabilitation in neurodegenerative diseases specifically multiple sclerosis (MS) is summarized. Since MS causes a wide variety of symptoms, the rehabilitation in MS patients may benefit from an interdisciplinary approach that encloses physiotherapy, cognitive rehabilitation, psychological therapy, occupational therapy, and other methods to improve fatigue. Neurorehabilitation helps patients to reach and maintain their optimal physical, psychological and intellectual, levels but it does not reverse long-term disabilities that arise from neurological disorders. This calls for the need of better neuroregenerative and neuroprotective treatment strategies in addition to neurorehabilitation. We discuss neuroprotective drugs aimed at preventing axonal, neuronal, myelin and oligodendrocyte damage and cell death that are approved and others that are currently in clinical trials, with an emphasis on human derived natural antibodies with remyleination potential. Our investigative group developed recombinant natural human IgM antibodies against oligodendrocytes and neurons with a potential for CNS repair and remyleination. One such recombinant antibody, rHIgM22 completed a phase 1 clinical trial with no toxicity and with an objective of promoting remyleination in multiple sclerosis. Inclusion of these drugs as a multifaceted approach may further enhance the efficacy of neurorehabilitation in neuroinflammatory and neurodegenerative disorders.

Malondialdehyde Epitopes as Targets of Immunity and the Implications for Atherosclerosis.

Accumulating evidence suggests that oxidation-specific epitopes (OSEs) constitute a novel class of damage-associated molecular patterns (DAMPs) generated during high oxidative stress but also in the physiological process of apoptosis. To deal with the potentially harmful consequences of such epitopes, the immune system has developed several mechanisms to protect from OSEs and to orchestrate their clearance, including IgM natural antibodies and both cellular- and membrane-bound receptors. Here, we focus on malondialdehyde (MDA) epitopes as prominent examples of OSEs that trigger both innate and adaptive immune responses. First, we review the mechanisms of MDA generation, the different types of adducts on various biomolecules and provide relevant examples for physiological carriers of MDA such as apoptotic cells, microvesicles, or oxidized low-density lipoproteins. Based on recent insights, we argue that MDA epitopes contribute to the maintenance of homeostatic functions by acting as markers of elevated oxidative stress and tissue damage. We discuss multiple lines of evidence that MDA epitopes are proinflammatory and thus important targets of innate and adaptive immune responses. Finally, we illustrate the relevance of MDA epitopes in human pathologies by describing their capacity to drive inflammatory processes in atherosclerosis and highlighting protective mechanisms of immunity that could be exploited for therapeutic purposes.

FRI-343 - Low Levels of Natural IGM Antibodies Towards Oxidation-Specific Epitopes are Associated with Human Non-Alcoholic Steatohepatitis

FRI-343 - Low Levels of Natural IGM Antibodies Towards Oxidation-Specific Epitopes are Associated with Human Non-Alcoholic Steatohepatitis

Aberrant levels of natural IgM antibodies in osteoarthritis and rheumatoid arthritis patients in comparison to healthy controls

Natural IgM antibodies (nIgM) are polyreactive autoantibodies that have diverse roles in regulating autoimmunity, systemic inflammation and removal of oxidized low-density lipoproteins (oxLDL). We hypothesized that aberrant states of nIgM may exist in persons with osteoarthritis (OA) and rheumatoid arthritis (RA). Herein, we characterized and compared the levels of nIgM specific for phosphorylcholine (anti-PC), double-stranded DNA (anti-dsDNA), and galactosyl (anti-Gal) in persons with OA, RA and healthy controls (HC). Levels of anti-PC nIgM in OA patients were significantly lower than both HC and RA patients in an age-adjusted analysis (P<0.05). In contrast, anti-Gal nIgM levels were significantly higher in RA patients than OA patients (P<0.05) and markedly increased in comparison to HC. Anti-PC nIgM significantly correlated with anti-dsDNA and anti-Gal nIgM levels in HC and RA (P<0.05) but not in OA patients. Elevated CRP levels were associated with RA conditions and old ages in general. There was no significant correlation between anti-PC nIgM and CRP or oxLDL levels. Our study highlights for the first time the evidence of aberrant state of nIgM in human OA compared to healthy individuals that implicates a deficiency in immune responses to oxLDL which may contribute to the metabolic syndromes in the development of OA.

Atherosclerosis Susceptibility in Mice Is Independent of the V1 Immunoglobulin Heavy Chain Gene.

The V1 (VHS107.1.42) immunoglobulin heavy chain gene is thought to be critical in producing IgM natural antibodies of the T15-idiotype that protect against both atherosclerosis and infection from Streptococcus pneumoniae. Our aim was to determine whether genetic loss of the V1 gene increased atherosclerotic plaque burden in vivo because of a reduction in the T15-idiotype or other atheroprotective antibodies. We crossed VHS107.1.42-deficient mice with the atherosclerosis-prone Apoe(-/-) and Ldlr(-/-) strains. Although these double knockout strains manifested no defects in B-cell development, we did observe a substantial reduction in early immune responses against phosphocholine after immunization. However, the titers of plasma antibodies reacting against defined atherosclerotic antigens such as oxidized low-density lipoprotein, as well as the T15-idiotype, were unaffected by loss of the VHS107.1.42 gene in hypercholesterolemic mice. Furthermore, we observed no increase in atherosclerotic lesion formation, either within the aortic arch or aortic root. Robust deposition of IgM within atherosclerotic plaques could also be readily observed in both control and experimental mice. Our data indicate that IgM-dependent protection against atherosclerosis is unlikely to be dependent on antibodies that use the VHS107.1.42 gene, in contrast to the acute immune response conferred by this heavy chain in the response to phosphocholine and in providing resistance against lethal S pneumoniae infection.

Identification of Cell Surface Straight Chain Poly-N-Acetyl-Lactosamine Bearing Protein Ligands for VH4-34-Encoded Natural IgM Antibodies.

B cell binding and cytotoxicity by human VH4-34-encoded Abs of the IgM isotype has been well documented. A VH4-34-IgM has recently shown a favorable early response in a phase 1 trial for treatment of B cell acute lymphoblastic leukemia. Although its B cell ligand has been identified as straight chain poly-N-acetyl-lactosamine (SC-PNAL), the carrier of the sugar moiety has not been identified. Using nanoelectrospray ionization mass spectrometry, we identify the metabolic activation related protein complex of CD147-CD98 as a major carrier of poly-N-acetyl-lactosamine (SC-PNAL) on human pre-B cell line Nalm-6. Previous studies have suggested CD45 as the SC-PNAL carrier for VH4-34-encoded IgG Abs. Because Nalm-6 is CD45 negative, human peripheral blood B lymphocytes and human B cell line, Reh, with high CD45 expression, were examined for SC-PNAL carrier proteins. Western blot analysis shows that the CD147-98 complex is indeed immunoprecipitated by VH4-34-encoded IgMs from human peripheral blood B lymphocytes and human B cell lines, Reh, OCI-Ly8, and Nalm-6. However, CD45 is immunoprecipitated only from peripheral B lymphocytes, but not from Reh despite the high expression of CD45. These results suggest that human B cells retain SC-PNAL on the CD147-98 complex, but modulate the sugar moiety on CD45. Because the carbohydrate moiety may act as a selecting Ag for VH4-34 autoantibody repertoire, its differential expression on proteins may provide a clue to the intricate atypical regulation of the VH4-34 gene.

Naturally Occurring Monoclonal Antibodies and Their Therapeutic Potential for Neurologic Diseases

Modulating the immune system does not reverse long-term disability in neurologic disorders. Better neuroregenerative and neuroprotective treatment strategies are needed for neuroinflammatory and neurodegenerative diseases. To review the role of monoclonal, naturally occurring antibodies (NAbs) as novel therapeutic molecules for treatment of neurologic disorders. Peer-reviewed articles, including case reports, case series, retrospective reviews, prospective randomized clinical trials, and basic science reports, were identified in a PubMed search for articles about NAbs and neurologic disorders that were published from January 1, 1964, through June 30, 2015. We concentrated our review on multiple sclerosis, Parkinson disease, Alzheimer disease, and amyotrophic lateral sclerosis. Many insults, including trauma, ischemia, infection, inflammation, and neurodegeneration, result in irreversible damage to the central nervous system. Central nervous system injury often results in a pervasive inhibitory microenvironment that hinders regeneration. A common targeted drug development strategy is to identify molecules with high potency in animal models. Many approaches often fail in the clinical setting owing to a lack of efficacy in human diseases (eg, less than the response demonstrated in animal models) or a high incidence of toxic effects. An alternative approach is to identify NAbs in humans because these therapeutic molecules have potential physiologic function without toxic effects. NAbs of the IgG, IgA, or IgM isotype contain germline or close to germline sequences and are reactive to self-components, altered self-components, or foreign antigens. Our investigative group developed recombinant, autoreactive, natural human IgM antibodies directed against oligodendrocytes or neurons with therapeutic potential for central nervous system repair. One such molecule, recombinant HIgM22, directed against myelin and oligodendrocytes completed a successful phase 1 clinical trial without toxic effects with the goal of promoting remyelination in multiple sclerosis. Animal studies demonstrate that certain monoclonal NAbs are beneficial as therapeutic agents for neurologic diseases. This class of antibodies represents a unique source from which to develop a new class of disease-modifying therapies.

Low marginal zone-like B lymphocytes and natural antibodies characterize skewed B-lymphocyte subpopulations in del22q11 DiGeorge patients

PurposePatients with DiGeorge syndrome suffer from T-lymphopenia. T-cells are important for the maturation and regulation of B-cell function. Our aim was to characterize the B-cell compartment in DiGeorge syndrome patients. MethodsB-cell subset phenotypization using flow cytometry. Serum BAFF (B-cell activating factor) and serum anti-alpha-galactosyl IgM measurement using ELISA. Serum IgG measurement using nephelometry. ResultsWe observed a significantly increased number of naïve B-cells and decreased number of switched memory B-cells in DiGeorge patients. Furthermore, we observed increased BAFF levels and a trend toward hypergammaglobulinemia later in life. Surprisingly, we detected a decrease in marginal zone-like (MZ-like) B-cells and natural antibodies in DiGeorge patients. ConclusionThe maturation of B-cells is impaired in DiGeorge patients, with high naïve and low switched memory B-cell numbers being observed. There is a clear trend toward hypergammaglobulinemia later in life, coupled with increased serum BAFF levels. Surprisingly, the T-independent humoral response is also impaired, with low numbers of MZ-like B-cell and low levels of anti-alpha-galactosyl IgM natural antibodies being detected.

Cytoplasmic myosin-exposed apoptotic cells appear with caspase-3 activation and enhance CLL cell viability.

The degree of chronic lymphocytic leukemia (CLL) B-cell antigen receptor (BCR) binding to myosin exposed apoptotic cells (MEACs) correlates with worse patient outcomes, suggesting a link to disease activity. Therefore, we studied MEAC formation and the effects of MEAC binding on CLL cells. In cell line studies, both intrinsic (spontaneous or camptothecin-induced) and extrinsic (FasL- or anti-Fas-induced) apoptosis created a high percent of MEACs over time in a process associated with caspase-3 activation, leading to cytoplasmic myosin cleavage and trafficking to cell membranes. The involvement of common apoptosis pathways suggests that most cells can produce MEACs and indeed CLL cells themselves form MEACs. Consistent with the idea that MEAC formation may be a signal to remove dying cells, we found that natural IgM antibodies bind to MEACs. Functionally, co-culture of MEACs with CLL cells, regardless of immunoglobulin heavy chain variable region gene mutation status, improved leukemic cell viability. Based on inhibitor studies, this improved viability involved BCR signaling molecules. These results support the hypothesis that stimulation of CLL cells with antigen, such as those on MEACs, promotes CLL cell viability, which in turn could lead to progression to worse disease.

Natural and adaptive IgM antibodies in the recognition of tumor-associated antigens of breast cancer (Review).

For early detection of cancer, education and screening are important, but the most critical factor is the development of early diagnostic tools. Methods that recognize the warning signs of cancer and take prompt action lead to an early diagnosis; simple tests can identify individuals in a healthy population who have the disease but have not developed symptoms. Early detection of cancer is significant and is one of the most promising approaches by which to reduce the growing cancer burden and guide curative treatment. The early diagnosis of patients with breast cancer is challenging, since it is the most common cancer in women worldwide. Despite the advent of mammography in screening for breast cancer, low-resource, low-cost alternative tools must be implemented to complement mammography findings. IgM is part of the first line of defense of an organism and is responsible for recognizing and eliminating infectious particles and removing transformed cells. Most studies on breast cancer have focused on the development of IgG-like molecules as biomarkers or as a treatment for the advanced stages of cancer, but autoantibodies (IgM) and tumor-associated antigens (proteins or carbohydrates with aberrant structures) have not been examined as early diagnostic tools for breast cancer. The present review summarizes the function of natural and adaptive IgM in eliminating cancer cells in the early stages of pathology and their value as early diagnostic tools. IgM, as a component of the immune system, is being used to identify tumor-associated antigens and tumor-associated carbohydrate antigens.

Protective natural autoantibodies to apoptotic cells: evidence of convergent selection of recurrent innate-like clones.

During murine immune development, recurrent B cell clones arise in a predictable fashion. Among these B cells, an archetypical clonotypic set that recognizes phosphorylcholine (PC) antigens and produces anti-PC IgM, first implicated for roles in microbial protection, was later found to become expanded in hyperlipidemic mice and in response to an increased in vivo burden of apoptotic cells. These IgM natural antibodies can enhance clearance of damaged cells and induce intracellular blockade of inflammatory signaling cascades. In clinical populations, raised levels of anti-PC IgM correlate with protection from atherosclerosis and may also downmodulate the severity of autoimmune disease. Human anti-PC-producing clones without hypermutation have been isolated that can similarly discriminate apoptotic from healthy cells. An independent report on unrelated adults has described anti-PC-producing B cells with IgM genes that have conserved CDR3 motifs, similar to stereotypic clonal sets of B cell chronic lymphocytic leukemia. Taken together, emerging evidence suggests that, despite the capacity to form an effectively limitless range of Ig receptors, the human immune system may often recurrently generate lymphocytes expressing structurally convergent B cell receptors with protective and homeostatic roles.

Atheroprotective potential of CXCR4 on B1 cells (CAM4P.145)

Abstract Atherosclerosis is a chronic inflammatory condition in which lipid deposition in arteries leads to the recruitment, activation, and accumulation of immune cells in the artery wall. Immune cells can have protective or detrimental effects on disease development. Existing evidence demonstrates that B1 cells hinder atherosclerotic lesion formation by secreting natural IgM antibodies that recognize modified self-epitopes present in diseased arteries. However, the mechanisms underlying B1 cell migration to arteries require further study. Our lab utilizes flow cytometry of circulating human immune cells in patients undergoing intravascular ultrasound of coronary arteries, in order to identify key surface markers that are associated with plaque burden. Expression of the chemokine receptor CXCR4 on human CD20+ CD3- CD27+ CD43+ B1 cells associated with reduced plaque burden in coronary arteries. No correlation was found between plaque volume and CXCR4 expression on naïve or memory B cell subsets. Prior evidence demonstrates that the helix-loop-helix transcription factor Id3 regulates B cell homing to the aorta and B cell-mediated atheroprotection. Loss of Id3 in the Apoe-/- mouse model of atherosclerosis led to systemic increase in surface expression of CXCR4 on B1 cells. Loss of Id3 additionally led to attenuated lesion formation, as measured by immunohistochemical lesion analysis of the aorta. These data suggest a potential atheroprotective function of CXCR4 on B1 cells.

Targeting pathogenic postischemic self-recognition by natural IgM to protect against posttransplantation cardiac reperfusion injury.

Natural IgM antibodies represent a class of innate pattern recognition receptors that recognize danger-associated molecular patterns expressed on stressed or dying cells. They play important roles in tissue homeostasis by disposing of prenecrotic cells and suppressing inflammation. However, ischemic insult leads to a pathogenic level of IgM binding and complement activation, resulting in inflammation and injury. We investigate the role of self-reactive IgM in the unique setting of transplantation where the donor organ undergoes both cold and warm ischemia and global ischemic insult. By transplanting hearts from wild-type donor mice into antibody-deficient mice reconstituted with specific self-reactive IgM monoclonal antibodies, we identified neoepitopes expressed after transplantation and demonstrated a key role for IgM recognition of these epitopes in graft injury. With this information, we developed and characterized a therapeutic strategy that exploited the postischemia recognition system of natural antibodies. On the basis of neoepitope identification, we constructed an anti-annexin IV single-chain antibody (scFv) and an scFv linked to Crry, an inhibitor of C3 activation (scFv-Crry). In an allograft transplantation model in which recipients contain a full natural antibody repertoire, both constructs blocked graft IgM binding and complement activation and significantly reduced graft inflammation and injury. Furthermore, scFv-Crry specifically targeted to the transplanted heart and, unlike complement deficiency, did not affect immunity to infection, an important consideration for immunosuppressed transplant recipients. We identified pathophysiologically important epitopes expressed within the heart after transplantation and described a novel translatable strategy for targeted complement inhibition that has several advantages over currently available approaches.

Anti-Gal IgM, IgA and IgG natural antibodies in childhood

The target for the most abundant xenoreactive natural antibodies in humans is the α-Gal epitope. Anti-Gal could provide natural immune defense against pathogens that express the α-Gal epitope. Anti-Gal natural antibodies are usually studied in adult individuals. Data demonstrating the incidence and concentration of anti-Gal natural antibodies in childhood are in short supply and incomplete. In the present study we prospectively quantified anti-Gal IgM, IgA and IgG levels in different age groups of children from delivery to 24 months of age and compared these levels to the level of these antibodies in their respective mothers. Measurement of anti-Gal antibodies may broaden the spectrum of specific antibodies that are available for determination of specific antibody responses in physiological and pathological conditions in children.Plasma was collected from umbilical cord blood of full term newborn, from blood of infants at age 6, 12 and 24 months and from their respective mothers at time of delivery. Quantitative determination of anti-Gal antibodies IgM, IgA and IgG were made with the enzyme immunoassays Human Anti-Alpha Galactosyl IgM ELISA, IgG ELISA and IgA ELISA. Hemagglutination activity was titrated against rabbit erythrocytes.The kinetic processes for the formation of natural antibodies in the first two years of life, in general, compared with the kinetics for the formation of total immunoglobulins IgM, IgA and IgG. There were no detectable anti-Gal IgM and IgA in the cord blood, whereas anti-Gal IgG were found at similar levels in both neonate cord blood and peripheral blood of their respective mothers. When comparing the percentage of natural antibodies in the plasma of children, the level of natural antibodies in children at the age of two years was approximately 37% for IgM, 25% for IgG and 15% for IgA. The titration of antibodies required for agglutination of rabbit red blood cells over the 24 month period followed the same trend observed for the formation of natural antibodies.Our study demonstrates the kinetics of formation of anti-Gal IgM, IgA and IgG natural antibodies in the first two years of life. The relative lack of these antibodies in this period should be taken into account when assessing for humoral immunodeficiencies, particularly with regards to the potential for children to mount an anti-carbohydrate response.

Peptide mimotopes of malondialdehyde-epitopes for clinical applications in cardiovascular disease

IntroductionAutoantibodies specific for malondialdehyde modified LDL (MDA-LDL) represent potential biomarkers to predict cardiovascular risk. However, the generation of MDA-LDL results in the formation of many different epitopes with high variability. Because it is not known, which MDA epitope is biological important, the aim of this study was to identify and characterize peptide mimotopes of MDA-LDL that could be used as antigens to improve the reproducible detection of MDA-specific autoantibodies. Methods and ResultsPeptide phage display libraries were screened for phages binding to the MDA-LDL specific natural IgM antibody LR04. After biopanning two consensus sequences (P1 & P2) of binding phages were synthesized. P1 and P2 were specifically bound by LR04, as the binding of LR04 to coated peptides was fully competed by MDA-LDL but not native LDL. P1 and P2 were also bound by other MDA specific murine (EN1) and human (IK17) antibodies. Furthermore, the binding of LR04 to late apoptotic cells was completely inhibited by both peptides, identifying them as mimotopes of naturally occurring epitopes on dying cells. Immunization of C57BL/6 mice with P2 conjugated to BSA, but not BSA alone, resulted in the robust induction of IgG1 and IgM antibodies against MDA-LDL. Moreover, serum IgG of immunized mice specifically stained epitopes in atherosclerotic lesion of rabbits and humans. Finally, we measured anti-mimotope antibody titers in serum samples previously collected from healthy subjects (n=17) and from patients (n=140) with stable angina pectoris undergoing percutaneous coronary intervention (Tsimikas, 2004). In patients a significant positive correlation was observed between anti-MDA-LDL and anti-mimotope IgM (P1, r=0.8; P2, r=0.6; p<0.0001) and IgG (P1, r=0.4; P2, r=0.3; p<0.0001) antibodies. A similar correlation was also found in sera of healthy subjects with IgM (P1, r=0.6; P2, r=0.4; p<0.0004) and IgG (P1, r=0.7; P2, r=0.5; p<0.0001) antibodies. ConclusionsThus, we have identified specific mimotopes of MDA-LDL that serve as highly reproducible antigens to assess autoantibody titers in patients with cardiovascular disease. Future studies will reveal their usefulness for therapeutic vaccination approaches against atherosclerosis.

Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies

Oxidation-specific epitopes (OSEs) present on apoptotic cells and oxidized low density lipoprotein (OxLDL) represent danger-associated molecular patterns that are recognized by different arcs of innate immunity, including natural IgM antibodies. Here, we investigated whether circulating microparticles (MPs), which are small membrane vesicles released by apoptotic or activated cells, are physiological carriers of OSEs. OSEs on circulating MPs isolated from healthy donors and patients with ST-segment elevation myocardial infarction (STE-MI) were characterized by flow cytometry using a panel of OSE-specific monoclonal antibodies. We found that a subset of MPs carry OSEs on their surface, predominantly malondialdehyde (MDA) epitopes. Consistent with this, a majority of IgM antibodies bound on the surface of circulating MPs were found to have specificity for MDA-modified LDL. Moreover, we show that MPs can stimulate THP-1 (human acute monocytic leukemia cell line) and human primary monocytes to produce interleukin 8, which can be inhibited by a monoclonal IgM with specificity for MDA epitopes. Finally, we show that MDA+ MPs are elevated at the culprit lesion site of patients with STE-MI. Our results identify a subset of OSE+ MPs that are bound by OxLDL-specific IgM. These findings demonstrate a novel mechanism by which anti-OxLDL IgM antibodies could mediate protective functions in CVD.