The monoclonal antibody Ca37, developed against Candida albicans alcohol dehydrogenase, inhibits the yeast in vitro and in vivo.

Candida albicans is a commensal yeast able to cause life threatening invasive infections particularly in immunocompromised patients. Despite the availability of antifungal treatments, mortality rates are still unacceptably high and drug resistance is increasing. We, therefore, generated the Ca37 monoclonal antibody against the C. albicans alcohol dehydrogenase (Adh) 1. Our data showed that Ca37 was able to detect C. albicans cells, and it bound to Adh1 in yeast and Adh2 in hyphae among the cell wall-associated proteins. Moreover, Ca37 was able to inhibit candidal growth following 18 h incubation time and reduced the minimal inhibitory concentration of amphotericin B or fluconazole when used in combination with those antifungals. In addition, the antibody prolonged the survival of C. albicans infected-Galleria mellonella larvae, when C. albicans was exposed to antibody prior to inoculating G. mellonella or by direct application as a therapeutic agent on infected larvae. In conclusion, the Ca37 monoclonal antibody proved to be effective against C. albicans, both in vitro and in vivo, and to act together with antifungal drugs, suggesting Adh proteins could be interesting therapeutic targets against this pathogen.

Active immunization against serum alcohol dehydrogenase normalizes brain dopamine metabolism disturbed during chronic alcohol consumption.

Chronic ethanol consumption in high doses is associated with constitutively elevated activity of the serum alcohol dehydrogenase I (ADH I) isoform, which demonstrates a high affinity not only for ethanol but also for a number of bioamine metabolites. Such excessive ADH activity is probably associated with disruptions in the metabolism of neurotransmitters (dopamine, serotonin, and norepinephrine) and subsequent long-term changes in the activity of their receptors. Ultimately, a stable depressive-like condition contributes to the development of patients' craving for ethanol intake, frequent disruptions during therapy, and low efficacy of treatment. We applied active immunization against ADH to investigate its efficacy in the reduction of excessive serum ADH activity and regulation of ethanol consumption by chronically ethanol-fed Wistar rats (15% ethanol, 4 months, free-choice method), and we analyzed its ability to influence the levels of bioamines in the brain. Immunization (2 injections, 2-week intervals) was performed using a combination of recombinant horse ADH isozyme as an antigen and 2% aluminum hydroxide-based adjuvant. The efficacy of immunization was demonstrated by the production of high titers of ADH-specific antibodies, which was consistent with the significantly reduced ADH activity in the serum of chronically ethanol-fed rats. On the 26th day after the first vaccine injection, we registered significantly lower levels of alcohol consumption compared to ethanol-fed control animals, and the difference reached 16% on the 49th day of the experiment. These observations were accompanied by data that showed reduced levels of ethanol preference in immunized rats. Chronic alcohol drinking led to a decrease in dopamine and DOPAL (a direct dopamine metabolite and a high-affinity ADH substrate) levels in the striatum,while immunization neutralized this effect. Additionally, we observed that inhibition of serum ADH activity caused a decrease in peak dopamine levels during acute alcohol intake in chronically ethanol-fed rats during ethanol withdrawal that was associated with reduced tyrosine hydroxylase activity in the striatum. The obtained data suggest a significant contribution of ADH to the changes in neurotransmitter systems during chronic alcohol consumption and make available new prospects for developing innovative strategies for treatment of excessive alcohol intake.

Identification of Glyceraldehyde-3-Phosphate and Alcohol Dehydrogenases as Autoantigens in Doberman Hepatitis.

An autoimmune background is suspected for Doberman hepatitis (DH). It is based on the finding of mononuclear cell infiltrates in the liver, strong female bias, association to the homozygous risk factor dog leucocyte antigen (DLA) allele DRB1*00601 and aberrant major histocompatibility complex (MHC) class II expression on hepatocytes that correlates with the degree of inflammation in the liver. The aim of this study was to search for autoantibodies against liver-related antigens associated with DH. Twenty-five Dobermans with subclinical DH (SDH), 13 that clinically manifest DH (CDH) and 17 healthy controls were studied. Immunoblotting analysis detected specific antibodies in the DH sera. By mass spectrometry the targets were identified as liver-related enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and alcohol dehydrogenase (ADH). Using ELISA, anti-GAPDH IgG was detected in 36% (9/25) of SDH dogs and 69.2% (9/13) of the CDH dogs compared to healthy controls (0/17) (P < 0.0005). Anti-ADH IgG was detected in 72% (18/25) of SDH dogs and 76.9% (10/13) of CDH dogs and only in one (1/17) control (P < 0.0005). The finding of novel autoantigens, GAPDH and ADH strengthen the hypothesis that DH is an autoimmune disease of the liver. These findings suggest that DH could be diagnosed by screening for autoantibodies against the defined antigens.

Individual roles of brain and serum alcohol dehydrogenase isoforms in regulation of alcohol consumption in SPF Wistar rats.

Alcohol dehydrogenases (ADH) are key enzymes of ethanol metabolism that mediate its oxidation to acetaldehyde. ADHs are also able to oxidize some types of neurotransmitters such as dopamine, serotonin and norepinephrine. Increased level of ADHs activity, induced by chronic alcohol consumption, is presumably associated with disturbed neurotransmitters metabolism that leads to stable alcohol craving. As earlier reported, intraperitoneal administration of 4-methilpirasole (non-specific inhibitor of ADHs) has shown to provide a short-term anti-alcoholic effect, but individual roles of ADH isoforms in this process were still unclear. The aim of this work was to study the roles of brain and serum ADH isoforms in alcohol consumption and neurotransmitter metabolism in the rats. In the study we used specific-pathogen-free (SPF) Wistar rats chronically alcoholized with 15% ethanol. 4-methilpirasole intranasal administration in small doses led to local inhibition of ADH III activity in the brain estimated by spectrophotometric assay. It correlated with dose-dependent reduction of dopamine concentration and increased level of its metabolic products in the brain but did not influence alcohol consumption. These data allowed us to propose an important role of brain ADHs (predominantly ADH III) in metabolism of dopamine in chronically alcoholized rats but not in regulation of alcohol consumption. To evaluate the role of serum ADH isoforms we immunized the rats with recombinant horse ADH that led to production of high levels of cross-reactive anti-ADH antibodies verified by ELISA assay. Immunization led to 30% decrease in alcohol consumption and recovery of general behavioral parameters such as motor activity, anxiety and depression level. At the same time active immunization did not cause any impairments in animal blood composition. We can conclude that immunization against ADHs appeared to be a safe way to decrease alcohol consumption that could be possibly associated with neurotransmitters metabolism correction.

Proteomic surveillance of putative new autoantigens in thyroid orbitopathy.

AIMS: Thyroid orbitopathy (TO) is an autoimmune inflammatory disorder characterised by several ocular manifestations. Several autoantigens have been proposed to be involved in the pathogenesis of TO, but the autoantigen system and the mechanism of TO would be rather complex. In this study, an immunoproteomic method was used to survey novel autoantigens expressed in the orbital fat tissue of patients with TO. METHODS: We used immunoproteomic, ELISA and immunohistochemical staining methods to survey novel autoantigens expressed in the orbital fat tissue of patients with TO. RESULTS: Six protein spots showing high reactivity with the serum from the patients with TO were detected as candidate orbital autoantigens, and two of them (carbonic anhydrase 1 (CA1) and alcohol dehydrogenase 1B (ADH1B)) were further verified by ELISA and immunohistochemical staining. We found that CA1 and ADH1B could attribute target autoantigens in this autoimmune disease. We discovered anti-CA1 and anti-ADH1B antibody prevalence to be higher in patients with TO (68.57%/51.43%) or Graves' disease (GD) (72%/48%) than in healthy controls respectively. Immunohistochemical staining study revealed the significantly enhanced expressions of CA1 and ADH1B in orbital fat of TO compared with that in healthy controls. CONCLUSIONS: We found that CA1 and ADH1B could attribute target autoantigens in this autoimmune disease. The high prevalence of these autoantibodies against CA1 and ADH1B in patients with TO and GD clarifies the potential clinical role for anti-CA1 and anti-ADH1B antibodies as biomarkers for GD and TO.

Oxidation by neutrophils-derived HOCl increases immunogenicity of proteins by converting them into ligands of several endocytic receptors involved in antigen uptake by dendritic cells and macrophages.

The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect infections caused by pathogens not recognized by pattern recognition receptors.

Alcohol dehydrogenase-specific T-cell responses are associated with alcohol consumption in patients with alcohol-related cirrhosis.

UNLABELLED: Patients with alcohol-related liver disease (ALD) have antibodies directed to alcohol dehydrogenase (ADH), anti-ADH titers being associated with disease severity and active alcohol consumption. ADH-specific T-cell responses have not been characterized. We aimed to define anti-ADH cellular immune responses and their association with active alcohol consumption and disease severity. Using cultures of peripheral blood mononuclear cells (PBMCs) from 25 patients with alcohol-related cirrhosis (ARC; 12 were actively drinking or abstinent for <6 months, and 13 were abstinent for >6 months) and hepatic mononuclear cells (HMCs) from 14 patients with ARC who were undergoing transplantation, we investigated T-cell reactivity to 25 overlapping peptides representing the full human ADH protein (beta 1 subunit). ADH-specific peripheral T-cell responses were assessed by the quantification of T-cell proliferation and cytokine production and were correlated with the clinical course. In active alcohol consumers, proliferative T-cell responses targeted ADH31-95 and other discontinuous sequences in the ADH peptide, whereas only one sequence was targeted in abstinents. ADH peptides induced the production of interferon-γ (IFN-γ), interleukin-4 (IL-4), and IL-17. IL-4 production was lower in active drinkers versus abstinents, and IL-17 production was higher. Peptides inducing IFN-γ production outnumbered those inducing T-cell proliferation. The intensity of the predominantly T helper 1 (Th 1) responses directly correlated with disease severity. Similar to PBMCs in abstinents, ADH peptides induced weak T-cell proliferation and a similar level of IL-4 production in HMCs but less vigorous Th 1 and T helper 17 responses. CONCLUSION: This suggests that Th 1 responses to ADH in ARC are induced by alcohol consumption. A Th 1/T helper 2 imbalance characterizes T-cell responses in active drinkers with ARC, whereas IL-4 production prevails in abstinents. This identifies new targets for immunoregulatory therapies in ALD patients for halting detrimental effector T-cell responses, which may encourage liver fibrogenesis and progression to end-stage liver disease.

Identification of B cell epitopes of alcohol dehydrogenase allergen of Curvularia lunata.

BACKGROUND/OBJECTIVE: Epitope identification assists in developing molecules for clinical applications and is useful in defining molecular features of allergens for understanding structure/function relationship. The present study was aimed to identify the B cell epitopes of alcohol dehydrogenase (ADH) allergen from Curvularia lunata using in-silico methods and immunoassay. METHOD: B cell epitopes of ADH were predicted by sequence and structure based methods and protein-protein interaction tools while T cell epitopes by inhibitory concentration and binding score methods. The epitopes were superimposed on a three dimensional model of ADH generated by homology modeling and analyzed for antigenic characteristics. Peptides corresponding to predicted epitopes were synthesized and immunoreactivity assessed by ELISA using individual and pooled patients' sera. RESULT: The homology model showed GroES like catalytic domain joined to Rossmann superfamily domain by an alpha helix. Stereochemical quality was confirmed by Procheck which showed 90% residues in most favorable region of Ramachandran plot while Errat gave a quality score of 92.733%. Six B cell (P1-P6) and four T cell (P7-P10) epitopes were predicted by a combination of methods. Peptide P2 (epitope P2) showed E(X)(2)GGP(X)(3)KKI conserved pattern among allergens of pathogenesis related family. It was predicted as high affinity binder based on electronegativity and low hydrophobicity. The computational methods employed were validated using Bet v 1 and Der p 2 allergens where 67% and 60% of the epitope residues were predicted correctly. Among B cell epitopes, Peptide P2 showed maximum IgE binding with individual and pooled patients' sera (mean OD 0.604±0.059 and 0.506±0.0035, respectively) followed by P1, P4 and P3 epitopes. All T cell epitopes showed lower IgE binding. CONCLUSION: Four B cell epitopes of C. lunata ADH were identified. Peptide P2 can serve as a potential candidate for diagnosis of allergic diseases.

[Identification of protein antigen for antibody DBD02 by biopanning of T7 phage display library].

AIM: To identify the specificity of mAb DBD02, we developed and optimized a new method by biopanning of T7 Select human liver cDNA phage display library. METHODS: After two rounds of biopanning, eluted phages were subjected to Dot blot using mAb DBD02 as primary antibody. The positive PFUs (plaque forming unit) which recognized by DBD02 were further confirmed by Western blot, PCR and sequencing. RESULTS: The antigen recognized by DBD02 was identified as alcohol dehydrogenase. And the epitope for mAb DBD02 was further mapped within a peptide of 22 amino acids (QDYKKPIQEVLKEMTDG-GVDFS). CONCLUSION: Our data indicate that biopanning of T7 phage display library by mAbs is time, cost, and labor-saving and specific tool for protein antigen identification.

Immunochemical features in the classification of human alcohol dehydrogenase family.

Human alcohol dehydrogenase (ADH) constitutes a complex family with diversified functions. Rabbit antihuman class I, II, III, and IV ADH antisera were prepared and used as probes to compare cross-reactivity with the isozymes across classes by semiquantitative Western blotting and quantitative enzyme-linked immunosorbent assay (ELISA). The interclass cross-reactivities with the noncognate isozymes by ELISA, generally approximately 0-35%, appeared considerably lower than those of the intraclass cross-reactivities except with the class IV isozyme. The anti-ADH1B1, ADH1C1, and ADH3 antisera, but not the anti-ADH2, exhibited approximately 80% cross-reactivity with ADH4. The intraclass cross-reactivities among class I isozymes ADH1A, ADH1B1, and ADH1C1 with anti-ADH1B1 or anti-ADH1C1 antisera were approximately 90%. Immunohistochemistry detecting with class-specific antibodies for ADH1-4 isolated from the corresponding antisera demonstrated that ADH4 was the predominant isoform expressed in the basal and suprabasal layer of human esophagus mucosa, whereas it was virtually devoid in the adjacent squamous cell carcinoma. Thus, the setup is more valuable for scanning ADH expression at protein level in different tissues and under different conditions, and maybe not as a tool for classification.

Alcohol dehydrogenase: an autoantibody target in patients with alcoholic liver disease.

The link between alcohol consumption and liver disease is not direct and several factors including autoimmunity to hepatocyte components have been implicated. We have previously identified alcohol dehydrogenase (ADH) as an autoantigen in autoimmune liver disease and in a proportion of patients with alcoholic liver disease. The aim of the present study is to investigate the association between the presence of anti-ADH antibodies, alcohol consumption and severity of liver damage in alcoholic patients. The presence of antibodies to human ADH beta2 and horse ADH was investigated in 108 patients with documented history of alcohol consumption and alcohol related liver disease, 86 being active alcohol abusers and 22 on sustained alcohol withdrawal, 39 with non-alcohol related disease and 22 normal subjects. Antibodies to either ADH form were more frequently detected in active alcohol abusers (55/86, 64%) than in patients on sustained alcohol withdrawal longer than 6 months (1/8, 13 %, P < 0.005), HBV infection (2/8, 25 %, P=0.03), non-alcohol related disease (9/29, 23 %, P < 0.0001) and in normal controls (3/22, 14 %, P < 0.0001); were more frequent in patients with cirrhosis than in those with steatosis (26/34, 76 % vs 34/64, 53 %, P=0.02); and were associated with elevated levels of ALT (anti-ADH beta2, P < 0.05), immunoglobulin A (P < 0.05) and gamma-glutamyl transpeptidase (P=0.01). Anti-ADH antibody positive serum samples were able to inhibit the enzymatic activity of ADH. These findings suggest that anti-ADH antibodies may be triggered by alcohol consumption and act as a disease activity marker in alcoholic liver disease.

[Administration of the omega-3 polyunsaturated fatty acid drug eiferol decreases alcohol motivation in albino rats by elevating the level of antibodies to alcohol dehydrogenase]. / Vvedenie preparata polinenasyshchennykh zhirnykh kislot semeistva omega-3 (éiferola) snizhaet alkogol'nuiu motivatsiiu u belykh krys, povyshaia uroven' antitel k alkogol'degidrogenaze.

The study experimentally assessed the approach proposed by the authors to lower alcohol motivation, which involves enhancement of a specific immunity at the stage of alcoholization when acetaldehydemodified ethanol exchange enzymes [alcohol dehydrogenase (ADH)] and acetaldehyde dehydrogenase may be expected to occur. Omega-3 polyunsaturated fatty acid (PUFA) drugs enhance the formation of autoantibodies to modified ADH and decrease the activity of ADH in the stomach and liver. At the same time, PUFA drugs can, under certain conditions, produce an anti-alcoholic activity and a positive effect on the psychoemotional status of animals after the ethanol deprivation period.

Antibodies to alpha5beta1 and alpha(v)beta3 integrins react with Candida albicans alcohol dehydrogenase.

It has been hypothesized that Candida albicans possesses integrin-like receptors on its cell surface. This is because C. albicans binds numerous fluid-phase extracellular matrix (ECM) proteins on its cell surface and adheres to the same ECM proteins when immobilized. In addition, numerous antibodies to human integrins (receptors for ECM proteins) bind to the fungal cell surface and in so doing inhibit the binding of the respective proteins. To demonstrate the presence of such a cell surface integrin, a cDNA library of C. albicans yeast cells was screened with polyclonal antiserum to the human fibronectin receptor (alpha5beta1 integrin). Clones isolated by this screening technique also reacted specifically to antiserum against the human vitronectin receptor (alpha(v)beta3 integrin). DNA sequence analysis of the cloned insert predicted a 350 aa protein (37 kDa). This predicted protein showed 75% homology at the nucleotide sequence level to alcohol dehydrogenase (ADH) of Saccharomyces cerevisiae. In vitro transcription/translation of the cloned inserts yielded a 37 kDa protein that was immunoprecipitated with antibodies to the alpha5beta1 and alpha(v)beta3 integrins and an antibody to a C. albicans fibronectin receptor. These antibodies and an mAb to the human vitronectin receptor demonstrated an antigen of -37 kDa present in the cell-wall preparations of C. albicans and in spent growth medium. All four antibodies reacted with authentic ADH. The possible significance of these results in relation to C. albicans adherence is discussed.

[Mechanisms of suppression of alcohol motivation after immunization of albino rats against alcohol dehydrogenase I: The role of ADH epitopes and ADH activity in the adrenal glands]. / O mekhanizmakh podavleniia alkogol'noi motivatsii pri immunizatsii belykh krys k alkogol'degidrogenaze I: rol' épitopov ADG i aktivnosti ADG v nadpochechnikakh.

Experimental results have demonstrated a significant decrease in the level of alcohol consumption by albino rats immunized with heterologous horse alcohol dehydrogenase. The role of ADH epitopes 9-14, 93-115, and 265-276 in this phenomenon was examined, and it was established that the latter sequence (265-276) plays the biggest role. The inhibition of ADH activity in the adrenals of immunized rats was much higher compared to the liver. We propose a hypothesis that the effect of alcohol dehydrogenase on alcohol consumption is connected with its role in catecholamine metabolism.

Identification of two liver proteins recognized by autoantibodies elicited in mice infected with mouse hepatitis virus A59.

Western blot experiments showed that sera from mice infected with the mouse hepatitis virus strain A59 (MHV-A59) contained autoantibodies (autoAb) that bound to a 40-kDa protein present in liver and kidney extracts. No reaction was observed with extracts of the heart, muscles, spleen, brain and lung. The Ab cross-reacted with a 40-kDa protein from human, rat and sheep liver, but not with liver extracts from the silver side fish (Odontesthes bonariensis). No correlation was found between the development of the hypergammaglobulinemia that followed the viral infection and the occurrence of the autoAb. Reactive immunoglobulins pertained to the IgG1, IgG2a and IgG2b subclasses, recognized cryptic epitopes and were detected from 10 days up to 8 weeks after MHV-infection. The 40-kDa protein was purified from mouse liver extracts by ion-exchange chromatography, gel filtration and SDS-PAGE. Because the N-terminal was blocked, we digested the protein in-gel with trypsin and sequenced various peptides. Results indicated a 100% homology of sequence between the protein recognized by the autoAb and liver fumarylacetoacetate hydrolase (FAH), the enzyme that mediates the last step of tyrosine catabolism. Additionally, a second protein recognized by the autoAb was detected during FAH purification steps and was identified as liver alcohol dehydrogenase.

Structure-function relationships in Drosophila melanogaster alcohol dehydrogenase allozymes ADH(S), ADH(F) and ADH(UF), and distantly related forms.

Drosophila melanogaster alcohol dehydrogenase (ADH), a paradigm for gene-enzyme molecular evolution and natural selection studies, presents three main alleloforms (ADHS, ADHF and ADHUF) differing by one or two substitutions that render different biochemical properties to the allelozymes. A three-dimensional molecular model of the three allozymes was built by homology modeling using as a template the available crystal structure of the orthologous D. lebanonensis ADH, which shares a sequence identity of 82.2%. Comparison between D. lebanonensis and D. melanogaster structures showed that there is almost no amino-acid change near the substrate or coenzyme binding sites and that the hydrophobic active site cavity is strictly conserved. Nevertheless, substitutions are not distributed at random in nonconstricted positions, or located in external loops, but they appear clustered mainly in secondary structure elements. From comparisons between D. melanogaster allozymes and with D. simulans, a very closely related species, a model based on changes in the electrostatic potential distribution is presented to explain their differential behavior. The depth of knowledge on Drosophila ADH genetics and kinetics, together with the recently obtained structural information, could provide a better understanding of the mechanisms underlying molecular evolution and population genetics.

Structural and functional divergence of class II alcohol dehydrogenase--cloning and characterisation of rabbit liver isoforms of the enzyme.

cDNAs coding for class II alcohol dehydrogenase were isolated from a rabbit-liver cDNA library. Deduced amino acid sequences show that isozymic forms of rabbit class II alcohol dehydrogenase exist, with a positional identity of 88.4%. A high variability in structure of class II alcohol dehydrogenase between the species is also reflected in function. The rabbit II-1 isozyme shows common characteristics with the human enzyme, but has a lower Km value for ethanol, 4.2 mM. The II-2 isozyme shows restriction for aliphatic alcohols longer than pentanol. For shorter alcohols the II-2 form has similar Km values as the II-1 isozyme, 5.5 mM for ethanol, but is a low activity variant with a 10-fold decrease in k(cat) values compared with II-1. Nevertheless, II-2 has a higher specificity for benzoquinone than II-1 due to a lower Km value, 80 microM compared with 1 mM, and is in this sense more like the human class II enzyme. In addition a rabbit class III alcohol dehydrogenase cDNA was isolated that encodes a typical class III enzyme/glutathione-dependent formaldehyde dehydrogenase. The finding of isozymic forms of class II alcohol dehydrogenase is in line with the evolution of the system of medium-chain alcohol dehydrogenases with different enzymes, different classes and different isozymes and further underline the complexity of the entire mammalian alcohol dehydrogenase system.

Alcohol dehydrogenase: a target of humoral autoimmune response in liver disease.

BACKGROUND & AIMS: Liver-specific membrane lipoprotein (LSP) is a heterogeneous liver preparation that has been widely used to study autoreactivity in liver disease. The aim of this study was to identify autoantigens in LSP. METHODS: Guinea pig anti-LSP serum was used to screen a human liver complementary DNA (cDNA) library. Humoral immune responses to isolated potential autoantigens were investigated by immunoblotting in 91 pediatric patients with various liver diseases, 20 adult patients with alcoholic liver disease and 20 with autoimmune thyroid disease, 37 healthy children, and 20 healthy adults. RESULTS: A 1.6-kilobase cDNA insert isolated from the cDNA library was found to encode amino acids 61-374 of the human alcohol dehydrogenase (ADH)-gamma 1 subunit. Antibodies to this or other ADH subunits were found significantly more frequently in autoimmune liver diseases (19 of 39 patients; 49%), Wilson's disease (5 of 13 patients; 38%), and alcoholic liver disease (10 of 20 patients; 50%) than in normal controls (P < 0.0001, P < 0.005, and P < 0.05, respectively) and correlated with disease activity in autoimmune liver disease. CONCLUSIONS: ADH has been identified as a new antigenic component of the LSP using a xenogeneic antiserum to immunoprobe a human cDNA liver library and seems to be a target autoantigen in liver disease. This approach may be useful in identifying other potential autoantigens.

Structure and regulation of the Candida albicans ADH1 gene encoding an immunogenic alcohol dehydrogenase.

The Candida albicans ADH1 gene encodes an alcohol dehydrogenase which is immunogenic during infections in humans. The ADH1 gene was isolated and sequenced, and the 5'- and 3'-ends of its mRNA were mapped. The gene encodes a 350 amino acid polypeptide with strong homology (70.5-85.2% identity) to alcohol dehydrogenases from Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe. The cloned C. albicans ADH1 gene was shown to be functional through complementation of adh mutations and efficient production of active alcohol dehydrogenase in S. cerevisiae. Northern analysis of C. albicans RNA revealed that ADH1 mRNA levels were regulated in response to carbon source and during batch growth. During growth on glucose, ADH1 mRNA levels rose to maximum levels during late exponential growth phase and declined to low levels in stationary phase. The ADH1 mRNA was relatively abundant during growth on galactose, glycerol, pyruvate, lactate or succinate, and less abundant during growth on glucose or ethanol. Alcohol dehydrogenase levels did not correlate closely with ADH1 mRNA levels under the growth conditions studied, suggesting either that this locus is controlled at both transcriptional and post-transcriptional levels, or that other differentially regulated ADH loci exist in C. albicans.