Carlos D. Hunter

Malondialdehyde–acetaldehyde adduct is the dominant epitope after MDA modification of proteins in atherosclerosis

Antibodies to malondialdehyde (MDA)-modified macromolecules (adducts) have been detected in the serum of patients with atherosclerosis and correlate with the progression of this disease. However, the epitope and its formation have not been characterized. Studies have shown that excess MDA can be degraded to acetaldehyde, which combines with proteins to from a stable dihydropyridine adduct. To investigate, mice were immunized with MDA adducts in the absence of adjuvant and showed an increase in antibodies to MDA adducts and the carrier protein as the concentration of MDA was increased. In fact, a number of the commercially available antibodies to MDA-modified proteins were able to be inhibited by a chemical analogue, hexyl-MAA. Also, MDA-MAA adducts were detected in the serum and aortic tissue of JCR diabetic/atherosclerotic rats. These studies determined that commercially available antibodies to MDA predominantly react with the MAA adduct and are present in the JCR model of atherosclerosis in both the serum and the aortic tissue. Therefore, the immune response to MDA-modified proteins is most probably to the dihydropyridine structure (predominant epitope in MAA), which suggests that MAA adducts may play a role in the development and/or progression of atherosclerosis.

Malondialdehyde-Acetaldehyde Adducts and Anti–Malondialdehyde-Acetaldehyde Antibodies in Rheumatoid Arthritis

Malondialdehyde‐acetaldehyde (MAA) adducts are a product of oxidative stress associated with tolerance loss in several disease states. This study was undertaken to investigate the presence of MAA adducts and circulating anti‐MAA antibodies in patients with rheumatoid arthritis (RA).

An in vitro method of alcoholic liver injury using precision-cut liver slices from rats

Alcohol abuse results in liver injury, but investigations into the mechanism(s) for this injury have been hampered by the lack of appropriate in vitro culture models in which to conduct in depth and specific studies. In order to overcome these shortcomings, we have developed the use of precision-cut liver slices (PCLS) as an in vitro culture model in which to investigate how ethanol causes alcohol-induced liver injury. In these studies, it was shown that the PCLS retained excellent viability as determined by lactate dehydrogenase and adenosine triphosphate (ATP) levels over a 96-h period of incubation. More importantly, the major enzymes of ethanol detoxification; alcohol dehydrogenase, aldehyde dehydrogenase, and cytochrome P4502E1, remained active and PCLS readily metabolized ethanol and produced acetaldehyde. Within 24 h and continuing up to 96h the PCLS developed fatty livers and demonstrated an increase in the redox state. These PCLS secreted albumin, and albumin secretion was decreased by ethanol treatment. All of these impairments were reversed following the addition of 4-methylpyrazole, which is an inhibitor of ethanol metabolism. Therefore, this model system appears to mimic the ethanol-induced changes in the liver that have been previously reported in human and animal studies, and may be a useful model for the study of alcoholic liver disease.

Autoimmune Hepatitis Induced by Syngeneic Liver Cytosolic Proteins Biotransformed by Alcohol Metabolites

BACKGROUND AND AIMS Aldehydes that are produced following the breakdown of ethanol (acetaldehyde) and lipid peroxidation of membranes (malondialdehyde) have been shown to bind (adduct) proteins. Additionally, these two aldehydes can combine (MAA) on nonsyngeneic and syngeneic proteins to initiate numerous immune responses to the unmodified part of the protein in the absence of an adjuvant. Therefore, these studies provide a potential mechanism for the development of antigen-specific immune responses resulting in liver damage should syngeneic liver proteins be adducted with MAA. METHODS This study sought to test whether MAA-modified syngeneic liver cytosolic proteins administered daily in the absence of adjuvant into C57BL/6 mice abrogates tolerance to initiate a MAA-induced autoimmune-like hepatitis. RESULTS In mice immunized with MAA-modified cytosols, there was an increase in liver damage as assessed by aspartate aminotransferase/alanine aminotransferase levels that correlated with liver pathology scores and the presence of the pro-fibrotic factors, smooth muscle actin, TGF-β, and collagen. IgG antibodies and T-cell proliferative responses specific for cytosolic proteins were also detected. Pro-inflammatory cytokines were produced in the livers of animals exposed to MAA-modified cytosols. Finally, transfer of immunized T cells to naïve animals caused biochemical and histological evidence of liver damage. CONCLUSIONS These data demonstrate that a disease with an autoimmune-like pathophysiology can be generated in this animal model using soluble MAA-modified syngeneic liver cytosols as the immunogen. These studies provide insight into potential mechanism(s) that the metabolites of alcohol may play in contributing to the onset of an autoimmune-like disease in patients with alcoholic liver disease.

Unique Antibody Responses to Malondialdehyde-Acetaldehyde (MAA)-Protein Adducts Predict Coronary Artery Disease

Malondialdehyde-acetaldehyde adducts (MAA) have been implicated in atherosclerosis. The purpose of this study was to investigate the role of MAA in atherosclerotic disease. Serum samples from controls (n = 82) and patients with; non-obstructive coronary artery disease (CAD), (n = 40), acute myocardial infarction (AMI) (n = 42), or coronary artery bypass graft (CABG) surgery due to obstructive multi-vessel CAD (n = 72), were collected and tested for antibody isotypes to MAA-modifed human serum albumin (MAA-HSA). CAD patients had elevated relative levels of IgG and IgA anti-MAA, compared to control patients (p<0.001). AMI patients had a significantly increased relative levels of circulating IgG anti-MAA-HSA antibodies as compared to stable angina (p<0.03) or CABG patients (p<0.003). CABG patients had significantly increased relative levels of circulating IgA anti-MAA-HSA antibodies as compared to non-obstructive CAD (p<0.001) and AMI patients (p<0.001). Additionally, MAA-modified proteins were detected in the tissue of human AMI lesions. In conclusion, the IgM, IgG and IgA anti-MAA-HSA antibody isotypes are differentially and significantly associated with non-obstructive CAD, AMI, or obstructive multi-vessel CAD and may serve as biomarkers of atherosclerotic disease.

Citrullinated mouse collagen administered to DBA/1J mice in the absence of adjuvant initiates arthritis

INTRODUCTION Citrullinated self-proteins are thought to be involved in the onset/progression of rheumatoid arthritis (RA). Numerous studies have been performed to look for the self-antigen that becomes citrullinated and induces RA. Importantly, these studies have been performed using citrullinated self-antigens injected into an animal model in the presence of a strong adjuvant in order to derive the response. However, to date no studies have been performed to determine if these phenotypes can be induced in the absence of an adjuvant. METHODS To investigate this possibility, mice were immunized with citrullinated or non-citrullinated mouse Type II collagen (Cit-Col or Col) in the presence or absence of Freunds Complete Adjuvant (FCA). RESULTS An autoimmune-like RA response was observed in mice immunized with Cit-Col in the absence of FCA; by the increase in caliper score, visual observation, and micro-CT analysis of bone erosions. Antibody and T-cell responses were increased in the Cit-Col injected mice to Cit-Col as well as antibody to Anti-Citrullinated Peptide Antigens (ACPA) as determined by a commercially available test kit. CONCLUSIONS Therefore, the use of citrullinated mouse collagen induces an autoimmune-like RA in the absence of an adjuvant. These data also suggest that citrullinate self-proteins may be potential molecular adjuvants that assist in driving an inflammatory response, that increases the production of PAD in joint tissue, resulting in the citrullination of other self-proteins to exacerbate the disease.

Direct antioxidant properties of methotrexate: Inhibition of malondialdehyde-acetaldehyde-protein adduct formation and superoxide scavenging

Methotrexate (MTX) is an immunosuppressant commonly used for the treatment of autoimmune diseases. Recent observations have shown that patients treated with MTX also exhibit a reduced risk for the development of cardiovascular disease (CVD). Although MTX reduces systemic inflammation and tissue damage, the mechanisms by which MTX exerts these beneficial effects are not entirely known. We have previously demonstrated that protein adducts formed by the interaction of malondialdehyde (MDA) and acetaldehyde (AA), known as MAA-protein adducts, are present in diseased tissues of individuals with rheumatoid arthritis (RA) or CVD. In previously reported studies, MAA-adducts were shown to be highly immunogenic, supporting the concept that MAA-adducts not only serve as markers of oxidative stress but may have a direct role in the pathogenesis of inflammatory diseases. Because MAA-adducts are commonly detected in diseased tissues and are proposed to mitigate disease progression in both RA and CVD, we tested the hypothesis that MTX inhibits the generation of MAA-protein adducts by scavenging reactive oxygen species. Using a cell free system, we found that MTX reduces MAA-adduct formation by approximately 6-fold, and scavenges free radicals produced during MAA-adduct formation. Further investigation revealed that MTX directly scavenges superoxide, but not hydrogen peroxide. Additionally, using the Nrf2/ARE luciferase reporter cell line, which responds to intracellular redox changes, we observed that MTX inhibits the activation of Nrf2 in cells treated with MDA and AA. These studies define previously unrecognized mechanisms by which MTX can reduce inflammation and subsequent tissue damage, namely, scavenging free radicals, reducing oxidative stress, and inhibiting MAA-adduct formation.

Malondialdehyde–acetaldehyde antibody concentrations in rheumatoid arthritis and other rheumatic conditions

Objective: To compare anti‐malondialdehyde–acetaldehyde (MAA) antibody concentrations between rheumatoid arthritis (RA) patients and healthy and rheumatic disease controls. Methods: Anti‐MAA antibody (IgA, IgM, IgG) was measured using ELISA and banked serum from patients with RA (n = 284), osteoarthritis (OA, n = 330), spondyloarthropathy (SpA, n = 50), and systemic lupus erythematosus (SLE, n = 88) as well as healthy controls (n = 82). Anti‐MAA antibody concentrations and the frequency of positivity were compared across groups. Multivariable linear regression analysis limited to RA and OA patients (due to sample size and data availability) was used to identify factors associated with anti‐MAA antibody concentrations. Results: Although RA patients demonstrated among the highest circulating concentrations across isotypes, only IgA anti‐MAA antibody was significantly higher than all other groups (p ≤ 0.02). Proportions (7% to 74%) of OA and SLE (less so for SpA) samples were positive for anti‐MAA antibody, limiting the discriminatory capacity of anti‐MAA antibody in RA (positive in 18% to 80%). In analyses limited to those with RA or OA, factors associated with higher anti‐MAA antibody concentrations included RA case status, younger age (IgM), male sex (IgG), African American race (IgA, IgG) and current smoking (IgA). C‐reactive protein levels and comorbidities were not associated with anti‐MAA antibody concentrations. Conclusion: With the possible exception of the IgA isotype, serum anti‐MAA antibodies measured with currently available assays do not appear to adequately discriminate RA from other rheumatic conditions. With the identification of specific proteins that are MAA‐modified in diseased tissues and requisite assay refinement, anti‐MAA antibody holds potential promise as a biomarker in RA.

Liver tissue metabolically transformed by alcohol induces immune recognition of liver self-proteins but not in vivo inflammation

Precision-cut liver slices (PCLSs) provide a novel model for studies of alcoholic liver disease (ALD). This is relevant, as in vivo ethanol exposure does not appear to generate significant liver damage in ethanol-fed mice, except in the National Institute on Alcohol Abuse and Alcoholism binge model of ALD. Previous studies have shown that the two metabolites of ethanol consumption, malondialdhyde (MDA) and acetaldehyde (AA), combine to form MDA-AA (MAA) adducts, which have been correlated with the development and progression of ALD. In this study, murine PCLSs were incubated with ethanol and examined for the production of MAA adducts. PCLSs were homogenized, and homogenates were injected into C57BL/6 mice. PCLSs from control-, pair-, and ethanol-fed animals served as targets in in situ cytotoxic assays using primed T cells from mice hyperimmunized with control or ethanol-exposed PCLS homogenates. A CD45.1/CD45.2 passive-transfer model was used to determine whether T cells from the spleens of mice hyperimmunized with PCLS ethanol-exposed homogenates trafficked to the liver. PCLSs incubated with ethanol generated MAA-modified proteins in situ. Cytotoxic (CD8+) T cells from immunized mice killed naïve PCLSs from control- and pair-fed mice in vitro, a response that was blunted in PCLSs from ethanol-fed mice. Furthermore, CD45.1 CD8+ T cells from hyperimmunized mice trafficked to the liver but did not initiate liver damage. This study demonstrates that exposure to liver tissue damaged by ethanol mediates robust immune responses to well-characterized alcohol metabolites and native liver proteins in vitro. Moreover, although these proinflammatory T cells traffic to the liver, these responses appear to be dampened in vivo by locally acting pathways. NEW & NOTEWORTHY This study shows that the metabolites of ethanol and lipid breakdown produce malondialdehyde-acetaldehyde adducts in the precision-cut liver slice model system. Additionally, precision-cut liver slices exposed to ethanol and harboring malondialdehyde-acetaldehyde adducts generate liver-specific antibody and T cell responses in the spleens of naïve mice that could traffic to the liver.

Abstract 1167: Osteolysis, splenic and hepatic extramedullary hematopoiesis, MDSCs, tumor growth, and metastases by orthotopic mammary tumors are increased by alcohol consumption and fatty diets

Fatty diets can induce low-grade inflammation that we report is increased by chronic alcohol consumption (CAC). CAC as 16.6% of total calories when administered in combination with the Lieber-DeCarli high-fat diet increases inflammation included hepatic and splenic extramedullary hematopoiesis (EMH) as assessed by flow cytometry, immunohistochemistry and a colony forming unit-granulocyte macrophage (CFU-GM) assay. Further, an increased number of hepatic myeloid derived suppressor cells (MDSCs) CD11b+Gr1+ cells that were predominantly Ly6cbr are observed. The increase in MDSCs is associated with an increased number of hepatic non-parenchymal cells including adipocytes (Oil Red O). Consistent with the increased number of hepatic MDSCs and EMH is a decrease in bone marrow cellularity and progenitor cells measured by flow cytometry (Lin-CD11b-Gr1-Sca-1+) and CFU-GM/femur. Unexpectedly, we observed demineralization and osteolytic lesions by micro computed tomography (micro CT) in all bones examined including femur, tibia, fibula and vertebral column that was associated with osteoclast activity (Trap+). Osteolysis was most notable in the fibula and vertebral spurs associated with osteoclast channels, and the demineralization appeared to be associated with areas of active myelopoiesis. The low grade, chronic inflammation associated with the Lieber-DeCarli fatty diet and CAC accelerated the induction of orthotopic 4T1 mammary tumors, resulting in extensive bone osteolysis, demineralization and increased metastases at aberrant sites including splenic, cardiac, hepatic, and extensive lymph node foci in addition to peritoneal and pleural effusions. The latter were haemorrhagic with a predominant nucleated cell infiltrate composed of bands, segs and myelocytes, supporting EMH. These results support the suggestion that a high-fat diet and CAC together increase tumor induction, metastasis and pathology in association with MDSC mobilization, expansion and EMH resulting in increased numbers of osteoclasts, associated bone demineralization and suppression of T-cell frequency and function. These results support the development of combination therapy strategies incorporating multiple molecular therapeutics that inhibit OCs, MDSCs and their associated mediators. Citation Format: Anand Dusad, Saraswoti Khadge, Geoffrey M. Thiele, Michael J. Duryee, Holly C. Britton, Lynell W. Klassen, Alicia J. Dafferner, Tracy Farrell, Timothy R. McGuire, Carlos D. Hunter, Karen C. Easterling, Karen J. O9Kane, John Graham Sharp, James E. Talmadge. Osteolysis, splenic and hepatic extramedullary hematopoiesis, MDSCs, tumor growth, and metastases by orthotopic mammary tumors are increased by alcohol consumption and fatty diets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1167. doi:10.1158/1538-7445.AM2014-1167