Associations between an expanded autoantibody profile and treatment responses to biologic therapies in patients with rheumatoid arthritis.

BACKGROUND: Although biologics represent a major advance in rheumatoid arthritis (RA), many patients fail to achieve adequate responses to these agents. We examined whether combined positivity to three well-characterized autoantibodies predicts treatment response among RA patients initiating biologics. METHODS: The study included biologic-naïve patients initiating anti-TNF treatment, biologic-exposed patients switching to rituximab or tocilizumab, and patients (biologic naïve or exposed) initiating abatacept. Rheumatoid factor (RF), anti-cyclic citrullinated peptide (CCP) antibody, and IgG antibodies to malondialdehyde-acetaldehyde (MAA) were measured using banked enrollment serum. The relationship between the number of autoantibodies positive (0-3) and treatment response (absolute improvement in 28-joint Disease Activity Score [DAS28-CRP] or improvement > 1.2) at 6 months was examined using multivariable linear and logistic regression. RESULTS: Of 1,229 patients initiating biologics, 79% were women; 89% were Caucasian. The number of baseline RA-related autoantibodies positive was associated with improved treatment response in a dose-dependent fashion. Compared to patients seronegative for all autoantibodies, adjusting for covariates, those positive for all three were more than twice (OR 2.35; 95% CI 1.57-3.51) as likely to achieve DAS28 improvement > 1.2 units. Associations of autoantibody positivity with biologic treatment response were strongest for anti-CCP antibody, persisted in analyses limited to biologic naïve patients, and did not appear to differ markedly among different agents examined. CONCLUSION: An expanded autoantibody profile appears to significantly predict RA treatment response to biologic treatment in a dose-dependent fashion. Incorporating these serologic profiles with additional biomarkers or other informative patient characteristics could provide an opportunity to personalize RA management.

Convenient method of producing cyclic single-chain Fv antibodies by split-intein-mediated protein ligation and chaperone co-expression.

Single-chain Fv (scFv) is a recombinant antibody in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. Compared with monoclonal antibodies, scFvs have the advantages of low-cost production using Escherichia coli and easy genetic manipulation. ScFvs are, therefore, regarded as useful modules for producing next-generation medical antibodies. The practical use of scFvs has been limited due to their aggregation propensity mediated by interchain VH-VL interactions. To overcome this problem, we recently reported a cyclic scFv whose N-terminus and C-terminus were connected by sortase A-mediated ligation. Preparation of cyclic scFv is, however, a time-consuming process. To accelerate the application study of cyclic scFv, we developed a method to produce cyclic scFv by the combined use of a protein ligation technique based on protein trans-splicing reaction (PTS) by split intein and a chaperone co-expression system. This method allows for the preparation of active cyclic scFv from the cytoplasm of E. coli. The present method was applied to the production of cyclic 73MuL9-scFv, a GA-pyridine antibody, as a kind of advanced glycation end-product. These findings are expected to evoke further application study of cyclic scFv.

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.

Antibodies against malondialdehyde-acetaldehyde adducts can help identify patients with abdominal aortic aneurysm.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a pathologic dilation of the aorta. Inflammation of the aortic wall has been shown to be involved in AAA formation. Malondialdehyde-acetaldehyde (MAA) adducts are MAA/protein hybrids with immunogenic, proinflammatory, and profibrotic properties. Levels of MAA adducts are elevated in patients with coronary artery disease; however, the role of MAA adducts in AAA is unclear. We hypothesize that levels of circulating antibodies against MAA adducts are increased in patients with AAA. METHODS: Plasma samples were collected from mice and patients with AAA and control patients with atherosclerosis but not AAA. AAA was induced in mice by a standard CaCl2 protocol, with matching sham mice. Plasma levels of anti-MAA antibodies were quantified by enzyme-linked immunosorbent assay. RESULTS: Patients with AAA exhibited higher levels of immunoglobulin G and immunoglobulin A anti-MAA antibody subtypes (P = .049 and .026, respectively) compared with control patients. Conversely, immunoglobulin M anti-MAA antibodies in AAA patients were lower compared with control patients (P = .018). In CaCl2-treated mice, immunoglobulin G anti-MAA antibodies were elevated after AAA formation (P = .006). CONCLUSIONS: The pattern of anti-MAA antibodies is able to distinguish between patients with AAA and patients with atherosclerosis but no AAA. These results demonstrate that MAA adducts are associated with AAA and suggest that they may play a role in either initiating or propagating chronic inflammation in AAA.

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.

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.

Plasma IgA antibody levels to malondialdehyde acetaldehyde-adducts are associated with inflammatory mediators, obesity and type 2 diabetes.

AIM: Obesity and type 2 diabetes (T2D) associate with increased oxidative stress. Malondialdehyde acetaldehyde (MAA) adducts have been suggested to be one of the antigenic epitopes in MDA-LDL responsible for the antibody recognition. Our aim was to investigate the associations between plasma IgA antibodies to MAA-LDL, inflammatory markers, adipokines, obesity, and T2D. METHODS: IgA to MAA-LDL were measured in a subsample (n = 1507) of the Finnish Health 2000 survey. The associations between antibody levels, obesity, TNF-α, IL-6, high-sensitivity (hs) CRP, resistin, adiponectin, fasting plasma (fp) glucose, fp-insulin, glycosylated hemoglobin (Hb-A1C), and T2D were investigated. RESULTS: IgA to MAA-LDL associated positively with fasting plasma insulin. IgA to MAA-LDL were higher among subjects with T2D (P < 0.001) compared to subjects with normal glucose metabolism. IgA to MAA-LDL associated with obesity, but was not independently (P = 0.002, not significant after correction for multiple tests) associated with T2D in logistic regression analysis. IgA to MAA-LDL, obesity, and TNF-α all associated with markers of glucose metabolism. CONCLUSIONS: T2D subjects had increased IgA to MAA-LDL compared to subjects with normal glucose metabolism. The data suggest that the associations between IgA to MAA-LDL and markers of glucose metabolism were independent of TNF-α but dependent on components of the metabolic syndrome.

IGHV1-69-encoded antibodies expressed in chronic lymphocytic leukemia react with malondialdehyde-acetaldehyde adduct, an immunodominant oxidation-specific epitope.

The immunoglobulins expressed by chronic lymphocytic leukemia (CLL) B cells are highly restricted, suggesting they are selected for binding either self or foreign antigen. Of the immunoglobulin heavy-chain variable (IGHV) genes expressed in CLL, IGHV1-69 is the most common, and often is expressed with little or no somatic mutation, and restricted IGHD and IGHJ gene usage. We found that antibodies encoded by one particular IGHV1-69 subset, designated CLL69C, with the HCDR3 encoded by the IGHD3-3 gene in reading frame 2 and IGHJ6, specifically bound to oxidation-specific epitopes (OSE), which are products of enhanced lipid peroxidation and a major target of innate natural antibodies. Specifically, CLL69C bound immunodominant OSE adducts termed MAA (malondialdehyde-acetaldehyde-adducts), which are found on apoptotic cells, inflammatory tissues, and atherosclerotic lesions. It also reacted specifically with MAA-specific peptide mimotopes. Light chain shuffling indicated that non-stochastically paired L chain of IGLV3-9 contributes to the antigen binding of CLL69C. A nearly identical CLL69C Ig heavy chain was identified from an MAA-enriched umbilical cord phage displayed Fab library, and a derived Fab with the same HCDR3 rearrangement displayed identical MAA-binding properties. These data support the concept that OSE (MAA-epitopes), which are ubiquitous products of inflammation, may play a role in clonal selection and expansion of CLL B cells.

Increased immunogenicity to P815 cells modified with malondialdehyde and acetaldehyde.

Aldehyde modified proteins have been associated with the development and/or progression of alcoholic liver disease (ALD). These protein adducts are capable of initiating many immunological responses that are harmful to the normal homeostasis of organism function. Previous studies have shown that malondialdehyde (MDA) and acetaldehyde (AA) synergistically form a unique adduct (MAA) with soluble proteins, which are capable of inducing cytokine release, T-cell proliferation, and antibody production. The purpose of this study was to determine whether MAA adduction can elicit similar responses to cells using a well-defined tumor model. The mouse mastocytoma P815 tumor cell line was modified with MAA (P815-MAA) or left unmodified (P815) and 10(6) irradiated cells were injected into DBA/2 mice once a week for 5 weeks. Serum was collected and tested for antibody responses to P815 cells and the MAA epitope. Immunization of MAA adducted P815 cells into syngeneic DBA/2 mice induced a strong antibody response to the MAA epitope as determined by ELISA on Alb and MAA-Alb (508 microg/ml and 1092 microg/ml, respectively). In addition, antibody to unmodified P815 cells was detected by fluorescent technique. Mice immunized with P815 cells or PBS showed little or no reactivity to the MAA epitope or P815 cells. Studies to assess IL-12 stimulation showed that peritoneal macrophages from P815 and PBS immunized animals produced modest amounts of IL-12 (20 and 35 pg/ml) when stimulated with Alb or MAA-Alb. However, macrophage from P815-MAA immunized mice responded to soluble MAA adduct (142 pg/ml). Finally, in tumor survival studies the mean survival was 14.25 days in PBS treated mice; 15.75 days with P815 immunized mice and 18.25 days with P815-MAA immunized mice. Therefore, these data strongly suggest that antibody responses are induced by P815 cells modified with MAA adducts. This may be a possible tool to begin looking at how alcohol metabolites potentially modify cells and/or cellular components making them recognizable to the immune system as foreign. It is thought that these studies define a model system that will be useful in assessing antibody and potentially T-cell responses to cells that are modified by MAA.

Immune Responses to Ethanol Metabolites and Cytokine Profiles Differentiate Alcoholics with or without Liver Disease.

OBJECTIVES: Excessive alcohol consumption is associated with the generation of antibodies against neoantigens induced by ethanol metabolism. However, the associations between such immune responses, ethanol consumption, and liver injury remain unclear. METHODS: Eight-six male alcoholics with (n=54) or without (n=32) liver disease, and 20 male volunteers (6 abstainers, 14 moderate drinkers) underwent clinical, morphological, and biochemical assessments of liver status and ethanol consumption. RESULTS: Antiacetaldehyde adduct IgAs in both groups of alcoholics were significantly higher than those in the controls. Elevated IgGs occurred in patients with liver disease, whereas IgMs were high in the heavy drinkers without apparent liver disease. Liver disease patients had high levels of both proinflammatory (IL-2, IL-6, IL-8, TNF-alpha) and antiinflammatory (IL-10) cytokines, whereas those without liver disease showed elevated IL-6, IL-8, and IL-10 only. Ethanol consumption correlated significantly with antiadduct IgA and IL-6 levels, which also showed parallel changes upon abstinence. CONCLUSIONS: Alcoholic liver disease is associated with the generation of IgAs and IgGs against acetaldehyde-derived antigens and enhanced levels of both pro- and antiinflammatory cytokines, whereas elevated IgA, IL-6, and IL-10 characterize alcoholics without liver disease. These data suggest that immunological mechanisms may play a role in the sequence of events leading to liver disease in some patients with excessive drinking.

Malondialdehyde-acetaldehyde haptenated protein binds macrophage scavenger receptor(s) and induces lysosomal damage.

There is evidence that the chemical modification of proteins (haptens) with malondialdehyde-acetaldehyde (MAA) and the immune response to these haptenated proteins is associated with the initiation and/or progression of alcohol liver disease. Experimentally, proteins modified with MAA induce antibody and T cell responses, which are mediated by scavenger receptor(s). Moreover, macrophages have been shown to play an important role in processing and presenting MAA-haptenated proteins in vitro. In vitro, MAA-modified proteins have been shown to induce both apoptosis and necrosis in a dose- and cell-type-dependent manner. Natural ligands modified by oxidative stress, such as oxidized LDL, similarly initiate not only antibody responses, but also cause cell death by disrupting lysosomes after binding to scavenger receptors and internalization. We therefore investigated the binding, internalization, and lysosomal integrity in a macrophage cell line to a MAA-haptenated protein. We demonstrate for the first time that MAA-haptenated proteins are preferentially bound by scavenger receptors on macrophages, which internalize the ligands and shuttle them to lysosomes. Moreover, MAA-haptenated proteins are demonstrated to be associated with a rapid dose-dependent disruption in lysosomal integrity, resulting in leakage and caspase activation. Similarly, as hen egg lysozyme (HEL)-MAA concentrations increased (>31.3 microg/ml), increased levels of apoptosis and a G1/S cell cycle checkpoint inhibition were identified. This study identifies mechanisms by which MAA-haptenated proteins are taken up by a representative antigen-presenting cell and may delineate steps by which MAA-haptenated proteins induce cell death and induce their immunogenicity to the carrier protein.

Ethanol promotes intestinal tumorigenesis in the MIN mouse. Multiple intestinal neoplasia.

Epidemiological studies suggest that alcohol consumption increases the risk of developing colorectal cancer; however, these data are confounded by numerous cosegregating variables. Previous experimental reports with the rodent carcinogen model have also yielded discordant results. To clarify the alcohol-colon cancer relationship, we used the MIN (multiple intestinal neoplasia) mouse, a genetic model of intestinal tumorigenesis. Twenty-four MIN mice were randomized to ethanol supplementation in the drinking water (15% alternating with 20% on a daily basis) or control. Mice were sacrificed after 10 weeks, and the intestinal tumors were scored under magnification. Tissue sections were assessed for apoptosis and cell proliferation rates, along with the presence of the malondialdehyde-acetaldehyde (MAA) adduct, a mutagenic adduct associated with ethanol consumption. Ethanol supplementation resulted in a significant increase in tumor number (135 +/- 35%; P = 0.027 versus control). The induction of tumorigenesis by ethanol was most dramatic in the distal small bowel (167 +/- 56%; P = 0.01). In the uninvolved intestinal mucosa, there was no difference in proliferative or apoptotic indices. Cytoplasmic and nuclear MAA adducts were detected in both ethanol-treated and control mice. We demonstrated that ethanol ingestion increased intestinal tumorigenesis in the MIN mouse model. Furthermore, whereas mechanisms remain incompletely elucidated, our data implicate formation of MAA adducts. This report provides further support that ethanol consumption is a risk factor for colorectal cancer.

Autoimmune responses against oxidant stress and acetaldehyde-derived epitopes in human alcohol consumers.

BACKGROUND: Studies in experimental animals have indicated that chronic ethanol ingestion triggers the formation of antibodies directed against proteins modified with reactive metabolites of ethanol and products of lipid peroxidation. However, the nature and prevalence of such antibodies have not been compared previously in alcoholic patients. METHODS: Autoantibodies against adducts with acetaldehyde- (AA), malondialdehyde- (MDA), and oxidized epitopes (Ox) were examined from sera of 54 alcohol consumers with (n = 28) or without (n = 26) liver disease, and from 20 nondrinking controls. RESULTS: Anti-AA-adduct IgA and IgG antibodies were elevated in 64% and 31% of patients with biopsy-proven alcoholic liver disease (ALD, n = 28), respectively. The IgA titers were significantly higher than those from nondrinking controls (p < 0.001), or heavy drinkers without significant liver disease (p < 0.001). Anti-MDA adduct titers (IgG) were elevated in 70% of the ALD patients. These titers were significantly higher (p < 0.001) than those from nondrinking controls, or heavy drinkers without liver disease. Antibodies (IgG) against Ox epitopes occurred in 43% of ALD patients, and the titers also were significantly higher (p < 0.05) than those from nondrinking controls. The anti-AA and anti-MDA adduct titers in ALD patients correlated significantly with the combined clinical and laboratory index (CCLI) of liver disease severity (r(s) = 0.449, p < 0.05; r(s) = 0.566, p < 0.01, respectively), the highest prevalences of anti-AA-adducts (73%) and anti-MDA-adducts (76%) occurring in ALD patients with cirrhosis. CONCLUSIONS: The present results indicated that autoantibodies against several distinct types of protein modifications are generated in ALD patients showing an association with the severity of liver disease.

Soluble proteins modified with acetaldehyde and malondialdehyde are immunogenic in the absence of adjuvant.

Recent studies have shown that the alcohol metabolites malondialdehyde and acetaldehyde can combine to form a stable adduct (MAA) on proteins. This adduct has been detected in the livers of rats chronically consuming ethanol, and serum antibodies to MAA have been observed at significantly higher concentrations in ethanol-fed when compared with pair-fed or chow-fed control rats. More recently, preliminary studies have strongly suggested that the MAA adduct is capable of stimulating antibody responses to soluble proteins in the absence of adjuvants. The antibodies produced recognize either the MAA epitope or the carrier protein itself. Therefore, it was the purpose of this study to examine the potential immunogenicity of MAA-modified exogenous proteins in the absence of adjuvants. Balb/c mice were immunized in the presence or absence of adjuvant with different concentrations of unmodified or MAA-modified proteins. The antibody response to both the MAA epitope and unmodified protein epitopes were determined by ELISA. In the absence of adjuvant, significant antibody responses were induced to both the MAA epitope and nonmodified protein epitopes. Smaller immunizing doses of MAA-protein conjugate favored the production of antibodies to nonmodified proteins, whereas larger doses induced a strong anti-MAA response. In studies to begin determining a mechanism for the specificity of the response in the absence of adjuvants, peritoneal macrophages were found to bind and degrade MAA-adducted proteins through the use of a scavenger receptor. This indicated that MAA-adducted proteins may be specifically taken up and epitopes presented to the humoral immune system in the absence of adjuvants. Importantly, these are the first data showing that an alcohol-related metabolite can induce an antibody response in the absence of adjuvant and suggesting a mechanism by which antibody to the MAA adduct or its carrier (exogenous or endogenous) proteins may be generated in vivo.

An enzyme immune assay for serum anti-acetaldehyde adduct antibody using low-density lipoprotein adduct and its significance in alcoholic liver injury.

An acetaldehyde (AcH) adduct was prepared using rabbit low-density lipoprotein as carrier proteins. An antibody against this adduct was raised in Watanabe heritable hyperlipidemic rabbits and cross-reacted with human low-density lipoprotein and bovine serum albumin adducts. Using this antibody, serum anti-AcH-adduct antibody levels were measured by a direct ELISA method in 56 Japanese adults (healthy adults and patients with nonalcoholic gastrointestinal diseases, alcoholic liver injury, or alcoholic pancreatitis). The antibody level (mean +/- SD) was 22 +/- 10 microg/ml in healthy adults, 22 +/- 11 microg/ml in nonalcoholic gastrointestinal diseases, and 16 +/- 13 microg/ml in alcoholic pancreatitis. These antibody levels tended to increase with the progression of alcoholic liver injury, starting from fatty liver via hepatitis to cirrhosis, 29 +/- 24 microg/ml in fatty liver, 35 +/- 29 microg/ml in alcoholic hepatitis, and 46 +/- 54 microg/ml in alcoholic cirrhosis. The antibody level in patients taking 100 g or more of ethanol per day tended to be higher, compared with those in people taking less ethanol. A follow-up observation revealed that alcohol abstinence after hospitalization raised serum anti-AcH-adduct antibody level in some patients and kept it constantly low in other patients. The immunohistochemical study using the anti-AcH-adduct antibody revealed the presence of adduct-like substance in hepatocytes of liver biopsy specimens obtained from patients with alcoholic liver disease. The results indicate that the anti-AcH-adduct antibody may be associated with the progress of alcoholic liver diseases.

Antibodies made against a formaldehyde-protein adduct cross react with an acetaldehyde-protein adduct. Implications for the origin of antibodies in human serum which recognize acetaldehyde-protein adducts.

Acetaldehyde, the major metabolite of ethanol, reacts with lysine and other free amino groups on proteins to form acetaldehyde-protein adducts. The presence of antibodies which recognize such acetaldehyde-protein adducts in sera from alcoholics has been attributed to an immune response to such adducts. Complicating this conclusion is the finding that sera from non-alcoholic control subjects also contain antibodies which recognize acetaldehyde-protein adducts. In the current research we sought to determine whether antibodies which recognize epitopes formed by the reaction of a protein with acetaldehyde can be formed in response to a protein modified with a structurally related protein adduct. We modified lysine residues on apolipoprotein (apo) B-100 with acetaldehyde and formaldehyde under reducing conditions, to form epsilon-N-methyl- and epsilon-N-ethyl-lysine residues, and with acetic anhydride to form epsilon-N-acetyl-lysine residues, and made antibodies against these modified proteins in guinea-pigs. In ELISA assays antibodies made against methylated apoB-100 (Me-apoB) cross-reacted effectively with ethylated apoB-100 (Et-apoB), while antibodies made against acetic anhydride-modified apoB-100 did not cross-react. We conclude that methyl-lysine shares one or more immunoreactive epitopes with ethyl-lysine, and that antibodies which recognize acetaldehyde-modified proteins can be formed in response to formaldehyde-modified proteins. We demonstrate that sera from both alcoholics and non-drinkers contain antibodies which recognize Me-apoB and Et-apoB and that the titres of these antibodies are comparable. These data raise the possibility that some human serum antibodies which recognize acetaldehyde-modified protein epitopes may have been made against formaldehyde-modified protein epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural analysis of peptide-acetaldehyde adducts by mass spectrometry and production of antibodies directed against nonreduced protein-acetaldehyde adducts.

Acetaldehyde can form protein-acetaldehyde adducts (AAs) in vivo and may play a role in the genesis of alcoholic liver disease. The nature of the chemical modification of proteins by acetaldehyde in vivo has not been elucidated. In vitro, acetaldehyde can form reversible adducts including a Schiff's base with lysine (K) and imidazolidinone with terminal amino groups of proteins such as human hemoglobin (Hb). In this study, we used FAB/MS to analyze the products of peptide-AAs (pep-AAs) formed by incubating acetaldehyde with Hb peptides. We then used an octabranched multiple antigen peptide (MAP) system containing Hb peptide-AAs to raise antibodies. Three Hb peptides [i.e., 8-pep consisting of 8 residues (V1HLTPVEK8) at the N-terminus of beta-chain of human sickle-cell Hb, 11-pep-gly consisting of 11 residues (G56NPKVKAHGKK66) in a segment of beta-chain rich in lysine, and 11-pep-pro that consists of the same sequence as 11-pep-gly, except G56 was replaced by proline (P)] were incubated with 1 mM acetaldehyde at 4 degrees C for 7d without NaCNBH3 (nonreduced conditions). Analysis by FAB/MS showed that 8-pep formed an imidazolidinone at the N-terminal valine, 11-pep-gly formed a Schiff's base and imidazolidinone at the N-terminus, whereas 11-pep-pro that lacks a free alpha-amino group formed only a Schiff's base at K59. By contrast, incubation of these Hb peptides with 250 mM acetaldehyde and NaCNBH3 at 37 degrees C for 1 hr (reduced conditions) produced mono- and diethylated modifications of all available K residues, as well as the N-terminal amino group.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde-modified epitopes in liver biopsy specimens of alcoholic and nonalcoholic patients: localization and association with progression of liver fibrosis.

Acetaldehyde, the first product of ethanol oxidation, has been shown to stimulate collagen gene expression and to form protein-acetaldehyde adducts. Because little is known about these adducts in human liver tissue, we assessed, with an immunohistochemical procedure, the presence and location of acetaldehyde-protein adducts in liver biopsy specimens of alcoholic patients. In addition, we correlated the presence of adducts with the progression or subsequent occurrence of liver fibrosis. The group included 106 patients with high alcohol consumption (> 90 gm ethanol/day for the last 5 yr), 10 nonalcoholic patients with normal livers and 23 patients with other liver diseases. Sixty-four of the 106 alcoholic patients had a second liver biopsy, whose specimen was used to assess the progression of liver fibrosis. Polyclonal antibodies were produced against homologous low-density lipoprotein purified from rabbit serum and modified in vitro in the presence of acetaldehyde. Protein-acetaldehyde adducts could be detected by immunohistochemistry in biopsy specimens of 90 alcoholic patients (85%), in none of the 10 nonalcoholic patients with normal livers and in 65% of the patients with nonalcoholic liver disease. Acetaldehyde-modified epitopes were detected in the intracellular and extracellular compartment. Intracellular protein-acetaldehyde adducts were localized in the cytoplasm of hepatocytes with a more intense staining in zone 3. No correlation existed between the intensity of intracellular staining and the histologically assessed severity of liver disease.(ABSTRACT TRUNCATED AT 250 WORDS)