Genetic epidemiology of gallbladder disease in Mexican Americans and cholesterol 7$\alpha$-hydroxylase gene sequence variation

Among Mexican Americans, the second largest minority group in the United States, the prevalence of gallbladder disease is markedly elevated. Previous data from both genetic admixture and family studies indicate that there is a genetic component to the occurrence of gallbladder disease in Mexican Americans. However, prior to this thesis no formal genetic analysis of gallbladder disease had been carried out nor had any contributing genes been identified.^ The results of complex segregation analysis in a sample of 232 Mexican American pedigrees documented the existence of a major gene having two alleles with age- and gender-specific effects influencing the occurrence of gallbladder disease. The estimated frequency of the allele increasing susceptibility was 0.39. The lifetime probabilities that an individual will be affected by gallbladder disease were 1.0, 0.54, and 0.00 for females of genotypes "AA", "Aa", and "aa", respectively, and 0.68, 0.30, and 0.00 for males, respectively. This analysis provided the first conclusive evidence for the existence of a common single gene having a large effect on the occurrence of gallbladder disease.^ Human cholesterol 7$\alpha$-hydroxylase is the rate-limiting enzyme in bile acid synthesis. The results of an association study in both a random sample and a matched case/control sample showed that there is a significant association between cholesterol 7$\alpha$-hydroxylase gene variation and the occurrence of gallbladder disease in Mexican Americans males but not in females. These data have implicated a specific gene, 7$\alpha$-hydroxylase, in the etiology of gallbladder disease in this population.^ Finally, I asked whether the inferred major gene from complex segregation analysis is genetically linked to the cholesterol 7$\alpha$-hydroxylase gene. Three pedigrees predicted to be informative for linkage analysis by virtue of supporting the major gene hypothesis and having parents with informative genotypes and multiple offspring were selected for this linkage analysis. In each of these pedigrees, the recombination fractions maximized at 0 with a positive, albeit low, LOD score. The results of this linkage analysis provide preliminary and suggestive evidence that the cholesterol 7$\alpha$-hydroxylase gene and the inferred gallbladder disease susceptibility gene are genetically linked. ^

REGULATION OF TOXIN SYNTHESIS BY CLOSTRIDIUM DIFFICILE

Clostridium difficile is the leading definable cause of nosocomial diarrhea worldwide due to its virulence, multi-drug resistance, spore-forming ability, and environmental persistence. The incidence of C. difficile infection (CDI) has been increasing exponentially in the last decade. Virulent strains of C. difficile produce either toxin A and/or toxin B, which are essential for the pathogenesis of this bacterium. Current methods for diagnosing CDI are mostly qualitative tests that detect the bacterium, the toxins, or the toxin genes. These methods do not differentiate virulent C. difficile strains that produce active toxins from non-virulent strains that do not produce toxins or produce inactive toxins. Based on the knowledge that C. difficile toxins A and B cleave a substrate that is stereochemically similar to the native substrate of the toxins, uridine diphosphoglucose, a quantitative, cost-efficient assay, the Cdifftox activity assay, was developed to measure C. difficile toxin activity. The concept behind the activity assay was modified to develop a novel, rapid, sensitive, and specific assay for C. difficile toxins in the form of a selective and differential agar plate culture medium, the Cdifftox Plate assay (CDPA). This assay combines in a single step the specific identification of C. difficile strains and the detection of active toxin(s). The CDPA was determined to be extremely accurate (99.8% effective) at detecting toxin-producing strains based on the analysis of 528 C. difficile isolates selected from 50 tissue culture cytotoxicity assay-positive clinical stool samples. This new assay advances and improves the culture methodology in that only C. difficile strains will grow on this medium and virulent strains producing active toxins can be differentiated from non-virulent strains. This new method reduces the time and effort required to isolate and confirm toxin-producing C. difficile strains and provides a clinical isolate for antibiotic susceptibility testing and strain typing. The Cdifftox activity assay was used to screen for inhibitors of toxin activity. Physiological levels of the common human conjugated bile salt, taurocholate, was found to inhibit toxin A and B in vitro activities. When co-incubated ex vivo with purified toxin B, taurocholate protected Caco-2 colonic epithelial cells from the damaging effects of the toxin. Furthermore, using a caspase-3 detection assay, taurocholate reduced the extent of toxin B-induced Caco-2 cell apoptosis. These results suggest that bile salts can be effective in protecting the gut epithelium from C. difficile toxin damage, thus, the delivery of physiologic amounts of taurocholate to the colon, where it is normally in low concentration, could be useful in CDI treatment. These findings may help to explain why bile rich small intestine is spared damage in CDI, while the bile salt poor colon is vulnerable in CDI. Toxin synthesis in C. difficile occurs during the stationary phase, but little is known about the regulation of these toxins. It was hypothesized that C. difficile toxin synthesis is regulated by a quorum sensing mechanism. Two lines of evidence supported this hypothesis. First, a small (KDa), diffusible, heat-stable toxin-inducing activity accumulates in the medium of high-density C. difficile cells. This conditioned medium when incubated with low-density log-phase cells causes them to produce toxin early (2-4 hrs instead of 12-16 hrs) and at elevated levels when compared with cells grown in fresh medium. These data suggested that C. difficile cells extracellularly release an inducing molecule during growth that is able to activate toxin synthesis prematurely and demonstrates for the first time that toxin synthesis in C. difficile is regulated by quorum signaling. Second, this toxin-inducing activity was partially purified from high-density stationary-phase culture supernatant fluid by HPLC and confirmed to induce early toxin synthesis, even in C. difficile virulent strains that over-produce the toxins. Mass spectrometry analysis of the purified toxin-inducing fraction from HPLC revealed a cyclic compound with a mass of 655.8 Da. It is anticipated that identification of this toxin-inducing compound will advance our understanding of the mechanism involved in the quorum-dependent regulation of C. difficile toxin synthesis. This finding should lead to the development of even more sensitive tests to diagnose CDI and may lead to the discovery of promising novel therapeutic targets that could be harnessed for the treatment C. difficile infections.

Intercellular adhesion and membrane polarity in rat hepatocytes: Localization of cell -CAM 105 and other domain-specific membrane proteins {\it in situ\/} and {\it in vitro\/} by electron microscopy

Cell-CAM 105 has been identified as a cell adhesion molecule (CAM) based on the ability of monospecific and monovalent anti-cell-CAM 105 antibodies to inhibit the reaggregation of rat hepatocytes. Although one would expect to find CAMs concentrated in the lateral membrane domain where adhesive interactions predominate, immunofluorescence analysis of rat liver frozen sections revealed that cell-CAM 105 was present exclusively in the bile canalicular (BC) domain of the hepatocyte. To more precisely define the in situ localization of cell-CAM 105, immunoperoxidase and electron microscopy were used to analyze intact and mechanically dissociated fixed liver tissue. Results indicate that although cell-CAM 105 is apparently restricted to the BC domain in situ, it can be detected in the pericanalicular region of the lateral membranes when accessibility to lateral membranes is provided by mechanical dissociation. In contrast, when hepatocytes were labeled following incubation in vitro under conditions used during adhesion assays, cell-CAM 105 had redistributed to all areas of the plasma membrane. Immunofluorescence analysis of primary hepatocyte cultures revealed that cell-CAM 105 and two other BC proteins were localized in discrete domains reminscent of BC while cell-CAM 105 was also present in regions of intercellular contact. These results indicate that the distribution of cell-CAM 105 under the experimental conditions used for cell adhesion assays differs from that in situ and raises the possibility that its adhesive function may be modulated by its cell surface distribution. The implications of these and other findings are discussed with regard to a model for BC formation.^ Analysis of molecular events involved in BC formation would be accelerated if an in vitro model system were available. Although BC formation in culture has previously been observed, repolarization of cell-CAM 105 and two other domain-specific membrane proteins was incomplete. Since DMSO had been used by Isom et al. to maintain liver-specific gene expression in vitro, the effect of this differentiation system on the polarity of these membrane proteins was examined. Based on findings presented here, DMSO apparently prolongs the expression and facilitates polarization of hepatocyte membrane proteins in vitro. ^

Altered cholesterol metabolism in a hypercholesterolemia -resistant rabbit colony

A colony of rabbits has been developed at the University of Texas Medical School at Houston that is resistant to dietary-induced hypercholesterolemia. The liver of resistant rabbits had higher levels of ($\sp{125}$I) $\beta$-VLDL binding and 3-hydroxy-3-methylglutaryl (HMGCoA) reductase activity, but lower acyl coenzyme A:cholesterol acyltransferase (ACAT) activity than normal rabbits. Direct quantitation of intracellular cholesterol content of the liver revealed that the resistant rabbits had $<$10% of the intracellular free cholesterol present in normal rabbits. Fibroblasts isolated from normal and resistant rabbits exhibited differences in ($\sp{125}$I) LDL binding, HMGCoA reductase activity and ACAT activity that were similar to those found in the liver. No structural differences were found in the LDL receptor of normal and resistant fibroblasts that would account for the increased binding capacity of the resistant cells. The regulation of LDL receptor levels by exogenous oxygenated sterols was similar in normal and resistant fibroblasts. The regulation of LDL receptor binding capacity by LDL was attenuated in the resistant compared to normal fibroblasts, suggesting that the resistant fibroblasts have an alternate pathway for processing lipoprotein-derived cholesterol. Sterol-balance studies revealed that the cholesterol-fed resistant rabbits increased lithocholic acid excretion compared to the basal state, and had higher levels of deoxycholic acid excretion than cholesterol-fed normal rabbits. In addition, the specific activity and mRNA levels of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H) were higher in resistant rabbits than normal rabbits, suggesting that the increased bile acid excretion was due to an increase in bile acid synthesis. Increased clearance of cholesterol relieves the negative feedback inhibition cholesterol exerts on expression of the LDL receptor. The number of cell surface LDL receptors is then increased in resistant rabbits and allows rapid clearance of lipoproteins from the plasma compartment, thereby reducing plasma cholesterol levels. The low intracellular cholesterol level also relieves the negative feedback inhibition cholesterol exerts on HMGCoA reductase. Increased synthesis of cholesterol from acetate provides cells with cholesterol for bile acid synthesis and/or homeostasis. The activity of ACAT is then decreased due to the flux of cholesterol through the bile acid synthetic pathways. ^

Characterization of the cholesterol 7$\alpha$-hydroxylase promoter in hypercholesterolemia -resistant rabbits

Our laboratory has developed and partially characterized a strain of New Zealand white rabbits that are resistant to the hypercholesterolemia which typically occurs in normal rabbits when fed a cholesterol-enriched diet. This phenotype is most likely attributed to an increase in bile acid excretion by hypercholesterolemia-resistant (CRT) rabbits as a result of elevated enzyme activity of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H), the rate-limiting enzyme in bile acid synthesis. Northern analysis revealed that CRT rabbits, in comparison to normal rabbits, have a 7-fold greater steady-state C7$\alpha$H mRNA levels irrespective of dietary regimen. The C7$\alpha$H gene in both phenotypes was determined to be a single copy gene. The hypothesis was that the elevated C7$\alpha$H mRNA levels in CRT rabbits, in comparison to normal animals, was due to an increase in the transcription rate of the C7$\alpha$H gene as a result of a mutation in a cis-acting element and/or a trans-acting factor within the hepatocyte. To isolate the C7$\alpha$H gene from both normal and CRT rabbits, genomic libraries were prepared from both phenotypes into $\lambda$GEM12 vectors using conventional techniques. Three CRT and one normal phage clones that contained the C7$\alpha$H gene were identified by screening the library with a series of probes located within different exons of the C7$\alpha$H cDNA. Sequencing analysis confirmed that approximately 1100 bp of the C7$\alpha$H 5'-flanking region from both normal and CRT phenotypes was identical. The increase in C7$\alpha$H mRNA levels was not attributed to a cis-acting mutation within this region. Liver nuclear extracts were prepared from normal and CRT rabbits maintained either on a basal or 0.25% cholesterol-enriched diet and incubated with several radiolabeled DNA fragments from the C7$\alpha$H gene. A 37 basepair region, located between nucleotides $-$452 to $-$416 was identified that had altered binding patterns between normal and CRT rabbits as a function of diet. Two additional regions, $-$747 to $-$575 and $-$580 to $-$442, produced banding patterns which were identical, irrespective of phenotype or diet. In conclusion, these studies suggested that the increase in C7$\alpha$H mRNA in CRT rabbits was due to differences in binding of a cholesterol-responsive transcription factor to the C7$\alpha$H promoter. ^

(Table 3) Liver weight, blood liver parameters, and blood plasma PCB concentrations in 14 sledge dog bitches and their 14 pups in Aasiaat, west Greenland

We assessed the relationship between exposure to organohalogen polluted minke whale (Balaenoptera acutorostrata) blubber and liver morphology and function in a generational controlled study of 28 Greenland sledge dogs (Canis familiaris). The prevalence of portal fibrosis, mild bile duct hyperplasia, and vascular leukocyte infiltrations was significantly higher in the exposed group (all Chi-square: p<0.05). In case of granulomas, the frequency was significantly highest in the bitches (P generation) while the prevalence of portal fibrosis was highest in the F generation (pups) (both Chi-square: p<0.05). No significant difference between exposed and controls was found for bile acid, ALAT, and ALKP, while ASAT and LDH were significantly highest in the control group (both ANOVA: p<0.05). We therefore suggest that a daily intake of 50-200 g environmentally organohalogen polluted minke whale blubber can cause liver lesions in Greenland sledge dogs. It is reasonable to infer that other apex predators such as polar bears (Ursus maritimus) and humans may suffer from similar impacts.

In vivo inhibition of rat stellate cell activation by soluble transforming growth factor β type II receptor: A potential new therapy for hepatic fibrosis

Transforming growth factor β (TGF-β) is a well characterized cytokine that appears to play a major role in directing the cellular response to injury, driving fibrogenesis, and, thus, potentially underlying the progression of chronic injury to fibrosis. In this study, we report the use of a novel TGF-β receptor antagonist to block fibrogenesis induced by ligation of the common bile duct in rats. The antagonist consisted of a chimeric IgG containing the extracellular portion of the TGF-β type II receptor. This “soluble receptor” was infused at the time of injury; in some experiments it was given at 4 days after injury, as a test of its ability to reverse fibrogenesis. The latter was assessed by expression of collagen, both as the mRNA in stellate cells isolated from control or injured liver and also by quantitative histochemistry of tissue sections. When the soluble receptor was administered at the time of injury, collagen I mRNA in stellate cells from the injured liver was 26% of that from animals receiving control IgG (P < 0.0002); when soluble receptor was given after injury induction, collagen I expression was 35% of that in control stellate cells (P < 0.0001). By quantitative histochemistry, hepatic fibrosis in treated animals was 55% of that in controls. We conclude that soluble TGF-β receptor is an effective inhibitor of experimental fibrogenesis in vivo and merits clinical evaluation as a novel agent for controlling hepatic fibrosis in chronic liver injury.

Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain

A novel multispecific organic anion transporting polypeptide (oatp2) has been isolated from rat brain. The cloned cDNA contains 3,640 bp. The coding region extends over 1,983 nucleotides, thus encoding a polypeptide of 661 amino acids. Oatp2 is homologous to other members of the oatp gene family of membrane transporters with 12 predicted transmembrane domains, five potential glycosylation, and six potential protein kinase C phosphorylation sites. In functional expression studies in Xenopus laevis oocytes, oatp2 mediated uptake of the bile acids taurocholate (Km ≈ 35 μM) and cholate (Km ≈ 46 μM), the estrogen conjugates 17β-estradiol-glucuronide (Km ≈ 3 μM) and estrone-3-sulfate (Km ≈ 11 μM), and the cardiac gylcosides ouabain (Km ≈ 470 μM) and digoxin (Km ≈ 0.24 μM). Although most of the tested compounds are common substrates of several oatp-related transporters, high-affinity uptake of digoxin is a unique feature of the newly cloned oatp2. On the basis of Northern blot analysis under high-stringency conditions, oatp2 is highly expressed in brain, liver, and kidney but not in heart, spleen, lung, skeletal muscle, and testes. These results provide further support for the overall significance of oatps as a new family of multispecific organic anion transporters. They indicate that oatp2 may play an especially important role in the brain accumulation and toxicity of digoxin and in the hepatobiliary and renal excretion of cardiac glycosides from the body.

Design of compounds that increase the absorption of polar molecules

Hydrophilic drugs are often poorly absorbed when administered orally. There has been considerable interest in the possibility of using absorption enhancers to promote absorption of polar molecules across membrane surfaces. The bile acids are one of the most widely investigated classes of absorption enhancers, but there is disagreement about what features of bile acid enhancers are responsible for their efficacy. We have designed a class of glycosylated bile acid derivatives to evaluate how increasing the hydrophilicity of the steroid nucleus affects the ability to transport polar molecules across membranes. Some of the glycosylated molecules are significantly more effective than taurocholate in promoting the intestinal absorption of a range of drugs, showing that hydrophobicity is not a critical parameter in transport efficacy, as previously suggested. Furthermore, the most effective glycosylated compound is also far less damaging to membranes than the best bile acid absorption promoters, presumably because it is more hydrophilic. The results reported here show that it is possible to decouple absorption-promoting activity from membrane damage, a finding that should spark interest in the design of new compounds to facilitate the delivery of polar drugs.

Disruption of the murine gene encoding phosphatidylethanolamine N-methyltransferase

All nucleated cells make phosphatidylcholine via the CDP-choline pathway. Liver has an alternative pathway in which phosphatidylcholine is made by methylation of phosphatidylethanolamine catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). We investigated the function of PEMT and its role in animal physiology by targeted disruption of its gene, Pempt2. A targeting vector that interrupts exon 2 was constructed and introduced into mice yielding three genotypes: normal (+/+), heterozygotes (+/−), and homozygotes (−/−) for the disrupted PEMT gene. Only a trace of PE methylation activity remained in Pempt2(−/−) mice. Antibody to one form of the enzyme, PEMT2, indicated complete loss of this protein from Pempt2(−/−) mice and a decrease in Pempt2(+/−) mice, compared with Pempt2(+/+) mice. The levels of hepatic phosphatidylethanolamine and phosphatidylcholine were minimally affected. The active form of CTP:phosphocholine cytidylyltransferase, the regulated enzyme in the CDP-choline pathway, was increased 60% in the PEMT-deficient mice. Injection of [l-methyl-3H]methionine demonstrated that the in vivo PEMT activity was eliminated in the Pempt2(−/−) mice and markedly decreased in the Pempt2(+/−) mice. This experiment also demonstrated that the choline moiety derived from PEMT in the liver can be distributed via the plasma throughout the mouse where it is found as phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin. Mice homozygous for the disrupted Pempt2 gene displayed no abnormal phenotype, normal hepatocyte morphology, normal plasma lipid levels and no differences in bile composition. This is the first application of the “knockout mouse” technique to a gene for phospholipid biosynthesis.

Actin Filaments and Microtubules are Involved in Different Membrane Traffic Pathways That Transport Sphingolipids to the Apical Surface of Polarized HepG2 Cells

In polarized HepG2 hepatoma cells, sphingolipids are transported to the apical, bile canalicular membrane by two different transport routes, as revealed with fluorescently tagged sphingolipid analogs. One route involves direct, transcytosis-independent transport of Golgi-derived glucosylceramide and sphingomyelin, whereas the other involves basolateral to apical transcytosis of both sphingolipids. We show that these distinct routes display a different sensitivity toward nocodazole and cytochalasin D, implying a specific transport dependence on either microtubules or actin filaments, respectively. Thus, nocodazole strongly inhibited the direct route, whereas sphingolipid transport by transcytosis was hardly affected. Moreover, nocodazole blocked “hyperpolarization,” i.e., the enlargement of the apical membrane surface, which is induced by treating cells with dibutyryl-cAMP. By contrast, the transcytotic route but not the direct route was inhibited by cytochalasin D. The actin-dependent step during transcytotic lipid transport probably occurs at an early endocytic event at the basolateral plasma membrane, because total lipid uptake and fluid phase endocytosis of horseradish peroxidase from this membrane were inhibited by cytochalasin D as well. In summary, the results show that the two sphingolipid transport pathways to the apical membrane must have a different requirement for cytoskeletal elements.

Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis

Mutations in the sister of P-glycoprotein (Spgp) or bile salt export pump (BSEP) are associated with Progressive Familial Intrahepatic Cholestasis (PFIC2). Spgp is predominantly expressed in the canalicular membranes of liver. Consistent with in vitro evidence demonstrating the involvement of Spgp in bile salt transport, PFIC2 patients secrete less than 1% of biliary bile salts compared with normal infants. The disease rapidly progresses to hepatic failure requiring liver transplantation before adolescence. In this study, we show that the knockout of spgp gene in mice results in intrahepatic cholestasis, but with significantly less severity than PFIC2 in humans. Some unexpected characteristics are observed. Notably, although the secretion of cholic acid in mutant mice is greatly reduced (6% of wild-type), total bile salt output in mutant mice is about 30% of wild-type. Also, secretion of an unexpectedly large amount of tetra-hydroxylated bile acids (not detected in wild-type) is observed. These results suggest that hydroxylation and an alternative canalicular transport mechanism for bile acids compensate for the absence of Spgp function and protect the mutant mice from severe cholestatic damage. In addition, the spgp−/− mice display a significant increase in the secretion of cholesterol and phospholipids into the bile. This latter observation in spgp−/− mice suggests that intrahepatic, rather than intracanalicular, bile salts are the major driving force for the biliary lipid secretion. The spgp−/− mice thus provide a unique model for gaining new insights into therapeutic intervention for intrahepatic cholestasis and understanding mechanisms associated with lipid homeostasis.

The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity

The pregnane X receptor (PXR) is the molecular target for catatoxic steroids such as pregnenolone 16α-carbonitrile (PCN), which induce cytochrome P450 3A (CYP3A) expression and protect the body from harmful chemicals. In this study, we demonstrate that PXR is activated by the toxic bile acid lithocholic acid (LCA) and its 3-keto metabolite. Furthermore, we show that PXR regulates the expression of genes involved in the biosynthesis, transport, and metabolism of bile acids including cholesterol 7α-hydroxylase (Cyp7a1) and the Na+-independent organic anion transporter 2 (Oatp2). Finally, we demonstrate that activation of PXR protects against severe liver damage induced by LCA. Based on these data, we propose that PXR serves as a physiological sensor of LCA, and coordinately regulates gene expression to reduce the concentrations of this toxic bile acid. These findings suggest that PXR agonists may prove useful in the treatment of human cholestatic liver disease.

Outer Dense Fiber 2 Is a Widespread Centrosome Scaffold Component Preferentially Associated with Mother Centrioles: Its Identification from Isolated Centrosomes

Because centrosomes were enriched in the bile canaliculi fraction from the chicken liver through their association with apical membranes, we developed a procedure for isolation of centrosomes from this fraction. With the use of the centrosomes, we generated centrosome-specific monoclonal antibodies. Three of the monoclonal antibodies recognized an antigen of ∼90 kDa. Cloning of its cDNA identified this antigen as a chicken homologue of outer dense fiber 2 protein (Odf2), which was initially identified as a sperm outer dense fiber-specific component. Exogenously expressed and endogenous Odf2 were shown to be concentrated at the centrosomes in a microtubule-independent manner in various types of cells at both light and electron microscopic levels. Odf2 exhibited a cell cycle-dependent pattern of localization and was preferentially associated with the mother centrioles in G0/G1-phase. Toward G1/S-phase before centrosome duplication, it became detectable in both mother and daughter centrioles. In the isolated bile canaliculi and centrosomes, Odf2, in contrast to other centrosomal components, was highly resistant to KI extraction. These findings indicate that Odf2 is a widespread KI-insoluble scaffold component of the centrosome matrix, which may be involved in the maturation event of daughter centrioles.

Random phage mimotopes recognized by monoclonal antibodies against the pyruvate dehydrogenase complex-E2 (PDC-E2).

Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), is the autoantigen most commonly recognized by autoantibodies in primary biliary cirrhosis (PBC). We identified a peptide mimotope(s) of PDC-E2 by screening a phage-epitope library expressing random dodecapeptides in the pIII coat protein of fd phage using C355.1, a murine monoclonal antibody (mAb) that recognizes a conformation-dependent epitope in the inner lipoyl domain of PDC-E2 and uniquely stains the apical region of bile duct epithelium (BDE) only in patients with PBC. Eight different sequences were identified in 36 phage clones. WMSYPDRTLRTS was present in 29 clones; WESYPFRVGTSL, APKTYVSVSGMV, LTYVSLQGRQGH, LDYVPLKHRHRH, AALWGVKVRHVS, KVLNRIMAGVRH and GNVALVSSRVNA were singly represented. Three common amino acid motifs (W-SYP, TYVS, and VRH) were shared among all peptide sequences. Competitive inhibition of the immunohistochemical staining of PBC BDE was performed by incubating the peptides WMSYPDRTLRTS, WESYPDRTLRTS, APKTYVSVSGMV, and AALWGVKVRHVS with either C355.1 or a second PDC-E2-specific mAb, C150.1. Both mAbs were originally generated to PDC-E2 but map to distinct regions of PDC-E2. Two of the peptides, although selected by reaction with C355.1, strongly inhibited the staining of BDE by C150.1, whereas the peptide APKTYVSVSGMV consistently inhibited the staining of C355.1 on biliary duct epithelium more strongly than the typical mitochondrial staining of hepatocytes. Rabbit sera raised against the peptide WMSYPDRTLRTS stained BDE of livers and isolated bile duct epithelial cells of PBC patients more intensively than controls. The rabbit sera stained all size ducts in normals, but only small/medium-sized ductules in PBC livers. These studies provide evidence that the antigen present in BDE is a molecular mimic of PDC-E2, and not PDC-E2 itself.