Sildenafil protects against bile duct ligation induced hepatic fibrosis in rats: Potential role for silent information regulator 1 (SIRT1).

Hepatic fibrosis is a potential health problem that may end with life-threatening cirrhosis and primary liver cancer. Recent studies point out to the protective effects of silent information regulator1 (SIRT1), against different models of organs fibrosis. This work aimed to investigate the possible protective effect of sildenafil (SIRT1 activator) against hepatic fibrosis induced by bile duct ligation (BDL). Firstly, three different doses of sildenafil (5, 10, 20mg/kg/day) were investigated; to detect the most protective one against BDL induced liver dysfunction and hepatic fibrosis. The most protective dose is then used; to study its effect on BDL induced SIRT1 downregulation, imbalance of oxidant/antioxidant status, increased inflammatory cytokines and fibrosis. Sildenafil (20mg/kg/day) was the most protective one, it caused upregulation of SIRT1, reduction of hepatic malondialdehyde (MDA) content, increase in expression of nuclear factor erythroid 2-related factor 2 (Nrf2), hemeoxygenease (HO)-1, reduced glutathione (GSH) content and superoxide dismutase (SOD) activity. Hepatic content of tumor necrosis factor-α (TNF-α) and nuclear factor κB (NFκB) expression & content displayed significant reductions with sildenafil treatment, Furthermore, sildenafil caused marked reductions of transforming growth factor (TGF)-ß content, expression of plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), α-smooth muscle actin (α-SMA), fibronectin, collagen I (α1) and hydroxyproline content. However, sildenafil protective effects were significantly reduced by co-administration of EX527 (SIRT1 inhibitor). Our work showed, for the first time that, sildenafil has promising protective effects against BDL induced liver dysfunction and hepatic fibrosis. These effects may be, in part, mediated by up regulation of SIRT1.

Liver alkaline phosphatase: a missing link between choleresis and biliary inflammation.

Several lines of evidence show that serum alkaline phosphatase (AP) is not only a signpost of cholestasis but also a surrogate marker of the severity of primary biliary cirrhosis and primary sclerosing cholangitis. In the present opinion article, we review and discuss the putative role of liver AP in health and in cholestatic diseases. In inflammatory cholestatic conditions, loss of activity of liver AP (resulting from its relocation from canaliculi and the acidic milieu) might promote hyper-adenosine triphosphate-bilia, lipopolysaccharide overload, and subsequent exacerbation and perpetuation of inflammation. Drugs that can restore the polarity of hepatocytes and canalicular export of bile acids or act as bile alkalinity modifiers are predicted to exert anti-inflammatory effects and to benefit both primary biliary cirrhosis and primary sclerosing cholangitis. Oral administration of intestinal AP could be a valid therapeutic intervention that deserves further study under experimental conditions as well as in human diseases. Overall, the key role of the liver microenvironment that might shape the different facets of the inflammatory processes in fibrosing cholangiopathies is highlighted.

Bezafibrate normalizes alkaline phosphatase in primary biliary cirrhosis patients with incomplete response to ursodeoxycholic acid.

BACKGROUND & AIMS: Ursodeoxycholic acid (UDCA) is the standard treatment for primary biliary cirrhosis (PBC) but excellent response is not observed in all cases. Since potential favourable effects of fibrates have been reported in short series with inconclusive results, we have carried out a pilot study to analyse the effects of bezafibrate in patients with suboptimal response to UDCA. METHODS: Thirty women (age 52.3 ± 2.3 years) treated with UDCA and abnormal alkaline phosphatase (AP) levels received bezafibrate (400 mg/d) for 1 year. Changes were measured every 3 months during the study period of 12 months, 3 months after discontinuation and 3 months after resuming bezafibrate. RESULTS: Two patients discontinued the treatment after few days, three at 6 and one at 9 months. Bezafibrate treatment resulted in a significant decrease in AP as early as 3 months. Normalization or decrease of AP below 1.5 times normal levels was observed in 13 and 4 patients respectively. There was also a significant decrease in γ-glutamyl transferase and alanine aminotransferase, cholesterol and triglyceride levels. Bezafibrate treatment resulted in significant improvement of pruritus. A rebound in liver biochemistries and pruritus occurred upon drug discontinuation, changes which improved again after resuming bezafibrate. Response to bezafibrate was associated with lower liver stiffness and severity of cholestasis. No severe adverse effects were observed. CONCLUSIONS: Combination treatment of bezafibrate and UDCA is associated with marked decrease or normalization of alkaline phosphatase as early as 3 months in patients with PBC. Better biochemical response was observed in patients with early disease and lower cholestasis.

Causes and evaluation of mildly elevated liver transaminase levels.

Mild elevations in levels of the liver enzymes alanine transaminase and aspartate transaminase are commonly discovered in asymptomatic patients in primary care. Evidence to guide the diagnostic workup is limited. If the history and physical examination do not suggest a cause, a stepwise evaluation should be initiated based on the prevalence of diseases that cause mild elevations in transaminase levels. The most common cause is nonalcoholic fatty liver disease, which can affect up to 30 percent of the population. Other common causes include alcoholic liver disease, medication-associated liver injury, viral hepatitis (hepatitis B and C), and hemochromatosis. Less common causes include α(1)-antitrypsin deficiency, autoimmune hepatitis, and Wilson disease. Extrahepatic conditions (e.g., thyroid disorders, celiac disease, hemolysis, muscle disorders) can also cause elevated liver transaminase levels. Initial testing should include a fasting lipid profile; measurement of glucose, serum iron, and ferritin; total iron-binding capacity; and hepatitis B surface antigen and hepatitis C virus antibody testing. If test results are normal, a trial of lifestyle modification with observation or further testing for less common causes is appropriate. Additional testing may include ultrasonography; measurement of α(1)-antitrypsin and ceruloplasmin; serum protein electrophoresis; and antinuclear antibody, smooth muscle antibody, and liver/kidney microsomal antibody type 1 testing. Referral for further evaluation and possible liver biopsy is recommended if transaminase levels remain elevated for six months or more.

A Multicenter, Open-Label, Single-Arm Study to Evaluate the Efficacy and Safety of Saroglitazar in Patients With Primary Biliary Cholangitis.

INTRODUCTION: Patients with primary biliary cholangitis (PBC) without biochemical response to ursodeoxycholic acid (UDCA) are at increased risk of liver-related mortality. Saroglitazar is a novel peroxisome proliferator-activated receptor (PPAR) agonist with dual PPAR agonistic properties (α/γ). There is a strong mechanistic rationale for studying saroglitazar in PBC because PPARα is a molecular target of fibrates that showed improvements in liver tests in patients with PBC. METHODS: In this 16-week, open-label, phase 3 study, 37 patients were screened across 3 clinical centers to enroll 7 patients. All patients received daily dose of saroglitazar 4 mg for 16 weeks in addition to their ongoing treatment with UDCA. The primary efficacy endpoint was the reduction in alkaline phosphatase (ALP) level at week 16 as compared to baseline. RESULTS: Mean age of the study population was 51.1 ± 10.0 years, all patients were female of Mexican descent, and mean body mass index was 25.5± = 4.8 kg/m2. Six (85.7%) patients reported taking ursodiol at baseline and continued throughout the study with a mean daily dosage of 417 mg. Among these, the daily dosage of UDCA 500 mg in 4 and 250 mg in 2 subjects, respectively. The mean baseline ALP level was 230 ± 103 U/L. The primary efficacy endpoint, mean change (reduction) from baseline in ALP concentration at week 16 based on the modified intent-to-treat population was -94 ± 53 U/L (P = 0.003), corresponding to a reduction of 48 ± 23%. Treatment with saroglitazar 4 mg resulted in a rapid and sustained decrease of ALP levels at week 4 (-84 ± 47 U/L, P = 0.003). Six patients who completed the study achieved mean ALP reduction of at least 40% at week 4 and all subsequent visits. DISCUSSION: Although the study was terminated because of lack of enrollment, saroglitazar daily for 16 weeks resulted in rapid and sustained improvements in ALP with an acceptable safety profile in patients with PBC.

Heterogeneity of autoreactive T cell clones specific for the E2 component of the pyruvate dehydrogenase complex in primary biliary cirrhosis.

The extraordinary specificity of bile duct destruction in primary biliary cirrhosis (PBC) and the presence of T cell infiltrates in the portal tracts have suggested that biliary epithelial cells are the targets of an autoimmune response. The immunodominant antimitochondrial response in patients with PBC is directed against the E2 component of pyruvate dehydrogenase (PDC-E2). Hitherto, there have only been limited reports on the characterization and V beta usage of PDC-E2-specific cloned T cell lines. In this study, we examined peripheral blood mononuclear cells (PBMC) for their reactivity to the entire PDC complex as well as to the E1- and E2-specific components. We also examined the phenotype, lymphokine profile, and V beta usage of PDC-specific T cell clones isolated from cellular infiltrates from the livers of PBC patients. We report that PBMC from 16/19 patients with PBC, but not 12 control patients, respond to the PDC-E2 subunit. Interestingly, this response was directed to the inner and/or the outer lipoyl domains, despite the serologic observation that the autoantibody response is directed predominantly to the inner lipoyl domain. Additionally, lymphokine analysis of interleukin (IL) 2/IL-4/interferon gamma production from individual liver-derived autoantigen-specific T cell clones suggests that both T helper cell Th1- and Th2-like clones are present in the liver. Moreover, there was considerable heterogeneity in the T cell receptor for antigen (TCR) V beta usage of these antigen-specific autoreactive T cell clones. This is in contrast to murine studies in which animals are induced to develop autoimmunity by specific immunization and have an extremely limited T cell V beta repertoire. Thus, our data suggest that in human organ-specific autoimmune diseases, such as PBC, the TCR V beta repertoire is heterogenous.

Portal hypertension and primary biliary cirrhosis: effect of long-term ursodeoxycholic acid treatment.

BACKGROUND & AIMS: Portal hypertension can complicate primary biliary cirrhosis, but studies evaluating the direct measurement of the portohepatic gradient (PHG) are rare. The aim of the study was to determine the prevalence and prognostic value of portal hypertension in patients treated with ursodeoxycholic acid. METHODS: A total of 132 patients from a local "PBC clinic" were enrolled in this cohort study. The PHG and biochemical values were measured at inclusion and every 2 years. Factors associated with survival were analyzed. RESULTS: Mean PHG at inclusion was 7.2 +/- 5.8 mm Hg. It was higher than normal (6 mm Hg) in 46 patients (34.9%) and higher than 12 mm Hg (variceal bleeding risk limit) in 26 patients (19.7%). There was a difference between the 3 subgroups in the probability of survival free of liver transplantation (P < .0003). After 2 years of treatment, a decreased or stable PHG (hazard ratio, 4.64; 95% confidence interval, 2.01-10.72) and normalization of aspartate aminotransferase (AST) level (hazard ratio, 2.89; 95% confidence interval, 1.03-8.05) were predictive of better survival on multivariate analysis. "Responders" (stable or improved PHG and normalized AST level at 2 years) have a 15-year survival similar to that of a control Quebec female population. CONCLUSIONS: Significant portal hypertension is a common complication of primary biliary cirrhosis. Changes in the PHG and normalized AST level after 2 years of ursodeoxycholic acid treatment can be used to identify a subgroup of responders with survival comparable to that of a control population.

Defective endothelial nitric oxide synthase signaling is mediated by rho-kinase activation in rats with secondary biliary cirrhosis.

In liver cirrhosis, down-regulation of endothelial nitric oxide synthase (eNOS) has been implicated as a cause of increased intrahepatic resistance. We investigated whether Rho-kinase activation is one of the molecular mechanisms involved in defective eNOS signaling in secondary biliary cirrhosis. Liver cirrhosis was induced by bile duct ligation (BDL). We measured mean arterial pressure (MAP), portal venous pressure (PVP), and hepatic tissue blood flow (HTBF) during intravenous infusion of saline (control), 0.3, 1, or 2 mg/kg/hour fasudil for 60 minutes. In BDL rats, 1 and 2 mg/kg/hour fasudil significantly reduced PVP by 20% compared with controls but had no effect on HTBF. MAP was significantly reduced in response to 2 mg/kg/hour fasudil. In the livers of BDL rats, 1 and 2 mg/kg/hour fasudil significantly suppressed Rho-kinase activity and significantly increased eNOS phosphorylation, compared with controls. Fasudil significantly reduced the binding of serine/threonine Akt/PKB (Akt) to Rho-kinase and increased the binding of Akt to eNOS. These results show in secondary biliary cirrhosis that (1) Rho-kinase activation with resultant eNOS down-regulation is substantially involved in the pathogenesis of portal hypertension and (2) Rho-kinase might interact with Akt and subsequently inhibit the binding of Akt to eNOS.

Ceramidase deficiency in Farber's disease (lipogranulomatosis).

Ceramidase activity could not be demonstrated in the kidney and cerebellum from a deceased patient with Farber's disease, whereas the activities of six control acid hydrolase enzymes appeared normal. This enzyme defect presumably accounts for the accumulation that has been described in two patients and may represent the biochemical basis of this disorder.

Predictive value of aspartate aminotransferase to alanine aminotransferase ratio for hepatic fibrosis and clinical adverse outcomes in patients with primary biliary cirrhosis.

BACKGROUND: The aspartate aminotransferase to alanine aminotransferase ratio (AAR) was seldom applied for fibrosis assessment in primary biliary cirrhosis (PBC) patients. GOALS: To validate the AAR for evaluating hepatic fibrosis, disease severity, and prognosis in patients with PBC. STUDY: Ninety-two consecutive PBC patients were retrospectively evaluated to validate the AAR for assessing the severity of liver function, the degrees of hepatic fibrosis, and predicting outcomes. RESULTS: AAR showed modest correlations to Mayo score, model for end-stage liver disease score, and Child-Pugh score (r2=0.156,P<0.001; r2=0.084, P=0.005; r2=0.142, P<0.001, respectively)in evaluating the severity of liver function. For 46 patients who underwent liver biopsy, 35 were in early stage fibrosis and the other 11 were in advanced fibrosis. AAR was significantly higher in patients with advanced fibrosis than those with early fibrosis (mean+/-standard deviation; 1.40+/-0.44 vs. 0.98+/-0.65,P=0.001). The AAR yielded the highest area under the receiver operating curve of 0.847 than Mayo score, model for end-stage liver disease score, and Child-Pugh score in predicting advanced fibrosis. During a median follow-up of 44.5 months, 24 patients expired and 68 patients were alive. Patients with an AAR of 1 or less had significantly better prognosis than their counterparts(P=0.043). CONCLUSIONS: AAR is a simple and reliable marker to assess liver function and hepatic fibrosis as well as to predict outcomes in PBC patients.

Increased expression and altered localization of cathepsin Z are associated with progression to jaundice stage in primary biliary cholangitis.

Our recent genome-wide association study found that the NELFCD/CTSZ locus was significantly associated with progression of primary biliary cholangitis (PBC) to jaundice stage in the Japanese population. In this study, we investigated the role of cathepsin Z in the etiology and pathology of PBC. Serum cathepsin Z levels were measured using enzyme-linked immunosorbent assay. The expression and localization of cathepsin Z in liver specimens were analyzed by western blotting and immunohistochemistry. In PBC patients, serum cathepsin Z levels were significantly increased with disease progression. In addition, its levels were positively correlated with alanine transaminase, aspartate transaminase and total bilirubin, and were negatively correlated with platelet count and albumin. Cathepsin Z expression was markedly increased in hepatocytes at later stages of PBC, and its localization was altered from the peri-bile canaliculus to the cytoplasm, where a fraction was no longer colocalized with endosomal/lysosomal vesicles. Similar altered expression of cathepsin Z was observed in end-stage of other cholestatic liver diseases including sepsis, obstructive jaundice, and Alagille syndrome. Our results indicate that altered expression and localization of cathepsin Z in hepatocytes are characteristic features of PBC and other cholestatic liver diseases, and are implicated in the progression of PBC.

Characterization of overlap syndrome between primary biliary cirrhosis and autoimmune hepatitis according to antimitochondrial antibodies status.

AIMS: Codification of variant forms between Primary Biliary Cirrhosis (PBC) and Autoimmune Hepatitis (AIH) has not been definitively standardized. The aim of this study was to compare among 102 consecutive patients, 2 subsets of overlap syndrome (OS, N=21) with and without antimitochondrial antibody (AMA) to two groups of patients with typical PBC (N=43) or AIH (N=38). METHODS: OS was defined by the presence in the same patient of at least 2 of 3 accepted criteria of PBC and AIH. Twelve patients with OS were AMA negative and 9 were AMA positive. RESULTS: A lower level of alanine transaminase (139+/-48 vs 269+/-154 IU/L, P<0.05) and a trend towards a higher level of alkaline phosphatase or gamma-glutamyl transpeptidase was observed in OS without AMA than in OS with AMA (693+/-200 vs 544+/-124 IU/L; 370+/-66 vs 241+/-77 IU/L, respectively). All AMA-negative patients with OS had antinuclear and/or anti-smooth muscle antibodies. OS without AMA differed from those with AMA in that they had more severe bile duct damage including destructive cholangitis (P<0.05), ductopenia (P<0.05), ductular hyperplasia (P<0.05) and a higher METAVIR fibrosis score (2.5+/-0.3 vs 1.3+/-0.3, P<0.05). The response to therapy was not different between PBC, AIH and OS. CONCLUSIONS: According to the presence of AMA, 2 homogeneous subgroups of patients with overlap syndrome between PBC and AIH may be identified. AMA status affects clinical presentation and liver disease severity of OS.

IgA class antibodies to 2-oxo-acid dehydrogenase complex are not predictive markers of histopathological progression in primary biliary cirrhosis.

Although antimitochondrial antibody (AMA) is the characteristic serological feature of primary biliary cirrhosis (PBC), its pathogenic role remains unclear. In our previous study, we reported a positive correlation between immunoglobulin (Ig) A class anti-2-oxo-acid dehydrogenase complex (2-OADC) and histopathological stage. To determine whether the appearance of IgA class anti-2-OADC by immunoblotting represents an early marker of more aggressive disease or whether it is late finding during the disease course of PBC, we tested not only the entire IgA class but also IgA1, IgA2 and secretory IgA class anti-2-OADC in serial serum samples from 15 patients with PBC. During the median observation period of 51 months, four cases showed histopathological progression (from stage 1 to 2, stage 1 to 3, stage 1 to 4 and stage 2 to 4). There was no statistically significant correlation between the above IgA class anti-2-OADCs and histopathological progression. There was no significant correlation between histopathological stages and IgA2 class anti-2-OADC or secretory IgA class anti-2-OADC by immunoblotting. IgA class anti-2-OADC was more frequent in stages 3-4 than in stages 1-2 (p = 0.0049), but IgA1 class anti-2-OADC was more frequent in stages 1-2 than in stages 3-4 (p = 0.0232). Our present study demonstrated that serum IgA class 2-OADC was not a predictive marker of histopathological progression but was associated with the histopathological stage of PBC. Although the IgA class AMA may have a specific pathogenic role for PBC, the discrepant results between IgA and IgA1 class anti-2-OADC should be further assessed to investigate different functional activities depending on their molecular form.

Endothelial nitric oxide synthase regulation is altered in pancreas from cirrhotic rats.

AIM: To determine whether biliary cirrhosis could induce pancreatic dysfunction such as modifications in endothelial nitric oxide synthase(eNOS) expression and whether the regulation of eNOS could be altered by the regulatory proteins caveolin and heat shock protein 90 (Hsp90), as well as by the modifications of calmodulin binding to eNOS. METHODS: Immunoprecipitations and Western blotting analysis were performed in pancreas isolated from sham and cirrhotic rats. RESULTS: Pancreatic injury was minor in cirrhotic rats but eNOS expression importantly decreased with the length (and the severity) of the disease. Because co-immunoprecipitation of eNOS with both Hsp90 and caveolin similarly decreased in cirrhotic rats, eNOS activity was not modified by this mechanism. In contrast, cirrhosis decreased the calmodulin binding to eNOS with a concomitant decrease in eNOS activity. CONCLUSION: In biliary cirrhosis, pancreatic injury is minor but the pancreatic nitric oxide (NO) production is significantly decreased by two mechanisms: a decreased expression of the enzyme and a decreased binding of calmodulin to eNOS.

The human pyruvate dehydrogenase complex: a polymorphic region of the lipoate acetyl transferase (E2) subunit gene.

A major issue in the study of the pathogenesis of primary biliary cirrhosis is whether the E2 subunit of the pyruvate dehydrogenase complex (PDH-E2), the major autoantigen in the disease, exists as a tissue-specific isoform. cDNA clones spanning a segment of the 3'-catalytic region of PDH-E2 (nt 1158-1361) have been isolated from human kidney, placenta and bile epithelium cells. Nucleotide sequence analysis of the clones showed differences consistent with the presence of normal variants of PDH-E2 in the human population. However, the existence of tissue-specific isoforms of PDH-E2 cannot yet be discounted.

Hexokinase isoenzymes in normal and cirrhotic human liver: suppression of glucokinase in cirrhosis.

The activities of hexokinase isoenzymes I-IV (EC 2.7.1.1) and of N-acetylglucosamine kinase (EC 2.7.1.59) were determined in normal human liver and in alcoholic liver disease and primary biliary cirrhosis after FPLC fractionation of high-speed supernatants on Mono-Q with a linear NaCl gradient. In control human liver the hexokinase activities were: I, 3.6; II, 0.7; III, 3.5, IV, 4.8 (mUnits/mg supernatant protein). The activity of N-acetylglucosamine kinase was 8 mU/mg of protein. In alcoholic liver disease and primary biliary cirrhosis, the activity of hexokinase IV (glucokinase) was suppressed to less than 10% of control activity and the activity of hexokinase I was increased 3-fold. The activity of hexokinase II was increased approximately 7-fold in alcoholic liver disease. The activities of hexokinase III and N-acetylglucosamine kinase were unchanged in cirrhosis. Hexokinase III showed 50% substrate inhibition at 100 mM glucose as compared with 0.5mM glucose. The high activity of hexokinase III in human liver (approximately 50% of the low-Km activity and 70% of glucokinase activity) results in a significant underestimation of glucokinase activity as determined by the conventional spectrometric assay while the activity of N-acetylglucosamine kinase may contribute to an overestimation of glucokinase activity in the radiochemical assay. Furthermore glucokinase is dramatically suppressed in liver disease, which although partly compensated for by the increase in hexokinase I (and II), accounts in part for the well-known glucose intolerance of liver cirrhosis.

Enzyme inhibition assay for pyruvate dehydrogenase complex: clinical utility for the diagnosis of primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is usually diagnosed by the presence of characteristic histopathological features of the liver and/or antimitochondrial antibodies (AMA) in the serum traditionally detected by immunofluorescence. Recently, new and more accurate serological assays for the detection of AMA, such as enzyme-linked immunosorbent assay (ELISA), immunoblotting, and enzyme inhibition assay, have been developed. Of these, the enzyme inhibition assay for the detection of anti- pyruvate dehydrogenase complex (PDC) antibodies offers certain advantages such as objectivity, rapidity, simplicity, and low cost. Since this assay has almost 100% specificity, it may have particular applicability in screening the at-risk segment of the population in developing countries. Moreover, this assay could be also used for monitoring the disease course in PBC. Almost all sera of PBC-suspected patients can be confirmed for PBC or non-PBC by the combination results of immunoblotting and enzyme inhibition assay without histopathological examination. For the development of a "complete" or "gold standard" diagnostic assay for PBC, similar assays of the enzyme inhibition for anti-2-oxoglutarate dehydrogenase complex (OGDC) and anti-branched chain oxo-acid dehydrogenase complex (BCOADC) antibodies will be needed in future.

Serum bile acids in primary biliary cirrhosis.

Fasting serum bile acid levels were measured by gas-liquid chromatography in 56 patients with primary biliary cirrhosis. Of these, 52 (93%) had increased levels (greater than 2mug/ml), including 14 of the 18 with normal serum bilirubin concentrations. The four patients with normal bile acid levels had early lesions as judged by histological and clinical criteria. With progression of the disease, as indicated by the histological features of the lesions, total bile acid levels increased, and the ratio of serum cholic-to-chenodeoxycholic acid decreased. Ratios of serum cholic-to-chenodeoxycholic acid below 1 occurred predominantly in patients with advanced or terminal disease. These studies suggest that serial measurement of serum bile acids may aid in the evaluation of primary biliary cirrhosis.

Role of Lipoylation of the Immunodominant Epitope of Pyruvate Dehydrogenase Complex: Toward a Peptide-Based Diagnostic Assay for Primary Biliary Cirrhosis.

Primary biliary cirrhosis is an immune-mediated chronic liver disease whose diagnosis relies on the detection of serum antimitochondrial antibodies directed against a complex set of proteins, among which pyruvate dehydrogenase complex is considered the main autoantigen. We studied the immunological role of the lipoyl domain of this protein using synthetic lipoylated peptides, showing that the lipoyl chain chirality does not affect autoantibody recognition and, most importantly, confirming that both lipoylated and unlipoylated peptides are able to recognize specific autoantibodies in patients sera. In fact, 74% of patients sera recognize at least one of the tested peptides but very few positive sera recognized exclusively the lipoylated peptide, suggesting that the lipoamide moiety plays a marginal role within the autoreactive epitope. These results are supported by a conformational analysis showing that the lipoyl moiety of pyruvate dehydrogenase complex appears to be involved in hydrophobic interactions, which may limit its exposition and thus its contribution to the complex antigenic epitope. A preliminary analysis of the specificity of the two most active peptides indicates that they could be part of a panel of synthetic antigens collectively able to mimic in a simple immunoenzymatic assay the complex positivity pattern detected in immunofluorescence.

Xanthine oxidoreductase is present in bile ducts of normal and cirrhotic liver.

Xanthine oxidoreductase (XOR) is a widely distributed enzyme, involved in the metabolism of purines, which generates superoxide and is thought to be involved in free radical-generated tissue injury. It is present at high concentrations in the liver, from where it may be released during liver injury into the circulation, binding to vascular endothelium and causing vascular dysfunction. The cellular localization of the enzyme, essential to understanding its function, is, however, still debated. The present study has used a highly specific mouse monoclonal antibody to define the cellular distribution of XOR in normal and cirrhotic human liver. As shown previously, XOR is present in hepatocytes. However, the novel finding of this study is that XOR is present in bile duct epithelial cells, where it is concentrated toward the luminal surface. Moreover, in liver disease, proliferating bile ducts are also strongly positive for XOR. These findings suggest that the enzyme is secreted into bile, and this was confirmed by analysis of human and rat bile. Xanthine oxidase activity was 10 to 20-fold higher in liver tissue obtained from patients with liver disease, than in healthy liver. We conclude that XOR is expressed primarily in hepatocytes, but is also present in bile duct epithelial cells and is secreted into bile. Its role in bile is unknown but it may be involved in innate immunity of the bowel muscosa.