Human monoclonal Fab and human plasma antibodies to carbamyl-epitopes cross-react with malondialdehyde-adducts.

Oxidized low-density lipoprotein (OxLDL) plays a crucial role in the development of atherosclerosis. Carbamylated LDL has been suggested to promote atherogenesis in patients with chronic kidney disease. Here we observed that plasma IgG and IgM antibodies to carbamylated epitopes were associated with IgG and IgM antibodies to oxidation-specific epitopes (ρ = 0·65-0·86, P < 0·001) in healthy adults, suggesting a cross-reaction between antibodies recognizing carbamyl-epitopes and malondialdehyde (MDA)/malondialdehyde acetaldehyde (MAA) -adducts. We used a phage display technique to clone a human Fab antibody that bound to carbamylated LDL and other carbamylated proteins. Anti-carbamyl-Fab (Fab106) cross-reacted with oxidation-specific epitopes, especially with MDA-LDL and MAA-LDL. We showed that Fab106 bound to apoptotic Jurkat cells known to contain these oxidation-specific epitopes, and the binding was competed with soluble carbamylated and MDA-/MAA-modified LDL and BSA. In addition, Fab106 was able to block the uptake of carbamyl-LDL and MDA-LDL by macrophages and stained mouse atherosclerotic lesions. The observed cross-reaction between carbamylated and MDA-/MAA-modified LDL and its contribution to enhanced atherogenesis in uraemic patients require further investigation.

Natural antibodies of newborns recognize oxidative stress-related malondialdehyde acetaldehyde adducts on apoptotic cells and atherosclerotic plaques.

Malondialdehyde acetaldehyde (MAA) adducts are generated under oxidative stress and shown to be highly immunogenic. Our aim was to investigate the recognition of MAA adducts by human natural antibodies in newborns before or at the time of full-term pregnancy. Plasma samples of pre-term (n = 11) and full-term (n = 36) newborns were enriched in specific IgM binding to MAA adducts compared with the maternal plasma IgM levels. Umbilical cord blood lymphocyte phage display library was generated to clone Fabs that specifically recognized MAA adducts without cross-reactivity to malondialdehyde. Fab clones from the antibody libraries of the pre-term and full-term newborns showed high sequence homology to the germline genes encoding the variable regions of antibodies, confirming that these Fabs represented the natural antibody repertoire of human fetuses. The MAA-specific umbilical cord blood Fabs bound to apoptotic human endothelial cells and the binding was efficiently competed with MAA adducts. The MAA-specific Fabs also recognized epitopes on advanced atherosclerotic lesions, and the uptake of infrared (IR)-labeled MAA-low-density lipoprotein by mouse J774A.1 macrophages was significantly reduced in the presence of these Fabs. In conclusion, MAA adducts were identified as one of the major antigenic targets for human natural antibodies already before the time of birth. MAA-specific natural antibodies are suggested to regulate apoptotic cell clearance starting from fetal development and to participate in the immunomodulation of atherosclerosis development during adulthood.

Carbamyl adducts on low-density lipoprotein induce IgG response in LDLR-/- mice and bind plasma autoantibodies in humans under enhanced carbamylation.

AIMS: Post-translational modification of proteins via carbamylation predicts increased risk for coronary artery disease. Uremia and smoke exposure are known to increase carbamylation. The aim was to investigate the role of carbamylated low-density lipoprotein (LDL) immunization on antibody formation and atherogenesis in LDL receptor-deficient (LDLR-/-) mice, and to study autoantibodies to carbamylated proteins in humans with carbamylative load. RESULTS: LDLR-/- mice immunized with carbamylated mouse LDL (msLDL; n=10) without adjuvant showed specific immunoglobulin G (IgG) antibody levels to carbamyl-LDL and malondialdehyde-modified LDL (MDA-LDL) but not to oxidized LDL or native LDL. Immunization did not influence the atherosclerotic plaque area compared with control LDLR-/- mice immunized with native msLDL (n=10) or phosphate-buffered saline (n=11). Humans with high plasma urea levels, as well as smokers, had increased IgG autoantibody levels to carbamyl-modified proteins compared to the subjects with normal plasma urea levels, or to nonsmokers. INNOVATION: Carbamyl-LDL induced specific IgG antibody response cross-reactive with MDA-LDL in mice. IgG antibodies to carbamyl-LDL were also found in human plasma and related to conditions known to have increased carbamylation, such as uremia and smoking. Plasma antibodies to carbamylated proteins may serve as new indicator of in vivo carbamylation. CONCLUSION: These data give insight into mechanisms of in vivo humoral recognition of post-translationally modified structures. Humoral IgG immune response to carbamylated proteins is suggested to play a role in conditions leading to enhanced carbamylation, such as uremia and smoking.

Recognition of Porphyromonas gingivalis gingipain epitopes by natural IgM binding to malondialdehyde modified low-density lipoprotein.

OBJECTIVE: Increased risk for atherosclerosis is associated with infectious diseases including periodontitis. Natural IgM antibodies recognize pathogen-associated molecular patterns on bacteria, and oxidized lipid and protein epitopes on low-density lipoprotein (LDL) and apoptotic cells. We aimed to identify epitopes on periodontal pathogen Porphyromonas gingivalis recognized by natural IgM binding to malondialdehyde (MDA) modified LDL. METHODS AND RESULTS: Mouse monoclonal IgM (MDmAb) specific for MDA-LDL recognized epitopes on P. gingivalis on flow cytometry and chemiluminescence immunoassays. Immunization of C57BL/6 mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and apoptotic cells. Immunization of LDLR(-/-) mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and diminished aortic lipid deposition. On Western blot MDmAb bound to P. gingivalis fragments identified as arginine-specific gingipain (Rgp) by mass spectrometry. Recombinant domains of Rgp produced in E. coli were devoid of phosphocholine epitopes but contained epitopes recognized by MDmAb and human serum IgM. Serum IgM levels to P. gingivalis were associated with anti-MDA-LDL levels in humans. CONCLUSION: Gingipain of P. gingivalis is recognized by natural IgM and shares molecular identity with epitopes on MDA-LDL. These findings suggest a role for natural antibodies in the pathogenesis of two related inflammatory diseases, atherosclerosis and periodontitis.

Specific recognition of malondialdehyde and malondialdehyde acetaldehyde adducts on oxidized LDL and apoptotic cells by complement anaphylatoxin C3a.

Oxidatively modified low-density lipoproteins (Ox-LDL) and complement anaphylatoxins C3a and C5a are colocalized in atherosclerotic lesions. Anaphylatoxin C3a also binds and breaks bacterial lipid membranes and phosphatidylcholine liposomes. The role of oxidized lipid adducts in C3a binding to Ox-LDL and apoptotic cells was investigated. Recombinant human C3a bound specifically to low-density lipoprotein and bovine serum albumin modified with malondialdehyde (MDA) and malondialdehyde acetaldehyde (MAA) in chemiluminescence immunoassays. No binding was observed to native proteins, LDL oxidized with copper ions (CuOx-LDL), or phosphocholine. C3a binding to MAA-LDL was inhibited by two monoclonal antibodies specific for MAA-LDL. On agarose gel electrophoresis, C3a comigrated with MDA-LDL and MAA-LDL, but not with native LDL or CuOx-LDL. C3a bound to apoptotic cells in flow cytometry. C3a opsonized MAA-LDL and was taken up by J774A.1 macrophages in immunofluorescence analysis. Complement-activated human serum samples (n=30) showed increased C3a binding to MAA-LDL (P<0.001) and MDA-LDL (P<0.001) compared to nonactivated samples. The amount of C3a bound to MAA-LDL was associated with total complement activity, C3a desArg concentration, and IgG antibody levels to MAA-LDL. Proteins containing MDA adducts or MAA adducts may bind C3a in vivo and contribute to inflammatory processes involving activation of the complement system in atherosclerosis.

High plasma immunoglobulin (Ig) A and low IgG antibody titers to oxidized low-density lipoprotein are associated with markers of glucose metabolism.

CONTEXT: Plasma oxidized low-density lipoprotein (OxLDL) is known to be associated with obesity, metabolic syndrome, and diabetes. OBJECTIVE: The objective of the study was to investigate the association between plasma IgA, IgM, and IgG titers to OxLDL, phosphocholine (PC), and streptococcal cell wall polysaccharide (CWPS) and markers of glucose metabolism, type 2 diabetes, and liver adiposity. SETTING AND PARTICIPANTS: A population-based cohort of middle-aged Finns (n = 1039) participated in the study. DESIGN: Plasma IgM, IgG, and IgA to copper oxidized LDL (CuOx-LDL) and malondialdehyde-modified low-density lipoprotein (MDA-LDL), PC, and CWPS were determined with chemiluminescent ELISA and liver adiposity with ultrasonography. RESULTS: IgA autoantibody titers to OxLDL and PC, but not CWPS, were positively associated with fasting blood glucose and plasma insulin levels and inversely with insulin sensitivity index. IgA to OxLDL was significantly higher (P = 0.013 for CuOx-LDL and P = 0.016 for MDA-LDL) and IgG to OxLDL significantly lower (P = 0.036 for CuOx-LDL and P = 0.001 for MDA-LDL) among the subjects with type 2 diabetes compared with subjects with normal or impaired glucose metabolism when adjusted for age, sex, body mass index, smoking, and alcohol consumption. Logistic regression analysis showed that high plasma IgA to OxLDL and low IgG to MDA-LDL were independent risk factors for type 2 diabetes. Plasma IgA titers to OxLDL demonstrated a significant association with liver adiposity (P = 0.012) and gamma-glutamyltransferase levels (P = 0.002). CONCLUSIONS: Plasma IgA titers to OxLDL were positively and IgG titers negatively associated with markers of glucose metabolism. High plasma IgA and low IgG to OxLDL were independent risk factors for type 2 diabetes.