Primary sclerosing cholangitis: new approaches to diagnosis, surveillance and treatment.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory bile duct disease of unknown etiology, frequently associated with inflammatory bowel disease and leading to end-stage liver disease requiring liver transplantation. Moreover, PSC is a premalignant condition associated with an increased risk for hepatobiliary and colorectal malignancy. Since effective medical therapy for PSC is still lacking, this disorder represents a potentially fatal disease with poor prognosis. This article is a summary of an overview given at the 5th Falk Gastro Conference in Munich 2012 and reviews the challenges associated with diagnosis, surveillance and therapy of PSC.

Characterization of the antibodies to p62 nucleoporin in primary biliary cirrhosis using human recombinant antigen.

Reactivity of sera from patients with primary biliary cirrhosis (PBC) with a 60 kDa component of nuclear pore complexes (NPCs), purified by affinity chromatography on wheat-germ agglutinin (WGA)-Sepharose, was previously detected. Recently, clinical significance of the anti-NPC antibodies in PBC became evident. In the light of recent reports, indicating the correlation of the anti-NPC antibodies with severity and progression of the disease, the characterization of the reactive antigens is becoming essential in the clinical management of patients with PBC. Since accurate autoantibody detection represents one of the fundamental requirements for a reliable testing, we have generated a human recombinant p62 protein and validated an immunoprecipitation assay for the detection of anti-p62. We also demonstrated that the generated human recombinant p62 nucleoporin was modified by N-acetylglucosamine residues. More than 50% of tested PBC sera precipitated (35)S-radioactively labeled p62 recombinant nucleoporin and 40% recognized this recombinant antigen by immunoblotting. We compared the reactivity of PBC sera with rat and human nucleoporin. The incidence of anti-p62 nucleoporin positive PBC sera increased by 15% when human recombinant antigen was used. The titer of autoantibodies in p62-positive PBC samples strongly varied. Preadsorption of the PBC sera with p62 recombinant protein completely abolished their reactivity with the antigen. In conclusion, this study unequivocally proves that autoantibodies reacting with the 60 kDa component of NPCs target p62 nucleoporin and, more importantly, provide a better antigen source for future evaluations of the clinical role of anti-p62 in PBC.

Characterization of autoantibodies against components of the nuclear pore complexes: high frequency of anti-p62 nucleoporin antibodies.

Antibodies against nuclear components (ANAs) occur in sera of approximately 50% of patients with primary biliary cirrhosis (PBC). By indirect immunofluorescence (IIF) ANA-positive PBC sera generate most frequently, homogeneous, speckled, centromere, and rim-like staining patterns. A perinuclear staining pattern is indicative for the reactivity of the sera with the components of the nuclear envelope. A substantial subset of PBC patients develops antibodies against constituents of the nuclear pore complexes (NPCs). These autoantibodies target two major autoantigens: gp210 glycoprotein and p62 kDa nucleoporin. Originally, a strong reaction of PBC with a 60 kDa protein of NPCs that was affinity purified on wheat-germ agglutinin (WGA)-Sepharose was described. Recently, using human recombinant p62 nucleoporin the identity of the reactivity was confirmed. In this work we compared by immunoprecipitation the reactivity of 20 PBC sera with the two recombinant autoantigens of the NPCs. Two out of 20 (10%) PBC sera precipitated recombinant gp210 glycoprotein and 11 out of 20 (55%) PBC sera reacted with p62 nucleoporin. These results evidence that anti-p62 antibodies occur more frequently than the autoantibodies against gp210 glycoprotein. Considering the recently reported clinical significance of ANAs in PBC, the prognostic value of the anti-NPC antibodies and their correlation with severity and progression of the disease is under evaluation.

Correlation of initial autoantibody profile and clinical outcome in primary biliary cirrhosis.

Although there have been significant advances in understanding the clinical and biochemical features of primary biliary cirrhosis (PBC), there is still a paucity of data on the usefulness of biomarkers as prognostic indicators. This is particularly important at the time of initial diagnosis. Indeed, the widespread use of antimitochondrial antibody testing has led to an earlier diagnosis of asymptomatic PBC and it is difficult to predict which patients will experience a benign versus a rapidly progressive course. To address this issue, we examined a unique population of 127 newly diagnosed patients with PBC during a 15-year period of observation that began in January 1990. Sera from these patients were analyzed for antimitochondrial, antinuclear, and anti-smooth muscle antibodies, and immunoblotting was performed for nuclear pore complex (NPC). The patients were then followed up longitudinally using biochemical liver function tests. No patient was under any medical therapy for PBC at the time of the initial sera collection. Data were analyzed based not only on the clinical features, but also the Mayo score and specific outcome measures, including time to death, need for liver transplantation, and complication free survival. Among patients with early disease, bilirubin increased to >2 mg/dL in the anti-NPC(+) patients (26% vs. 5%, P = .019). Anti-NPC antibodies remained stable or slightly increased over the period of observation. In condusion, anti-NPC identifies patients likely to experience an unfavorable clinical course and more rapid disease progression.

TGF-beta1 codon 25 gene polymorphism is associated with cirrhosis in patients with hereditary hemochromatosis.

Hereditary hemochromatosis (HHC) is an autosomal recessive disorder of iron metabolism with variable penetrance. Only a minority of C282Y homozygotes develop clinical overt disease and cirrhosis. The phenotypic heterogeneity of HHC may be due to host genetic factors influencing fibrogenesis such as cytokine gene polymorphisms. In this respect, we investigated the impact of functional genetic polymorphisms of TGF-beta1 (codon 10 Leu/Pro, codon 25 Arg/Pro), TNF-alpha (-308 G/A, -238 G/A) and angiotensinogen (-6 G/A) on the development of cirrhosis in HHC. One hundred and forty-nine (111 male, mean age: 51.0+/-12.9) C282Y homozygotes who underwent liver biopsy were studied. Genotyping was performed by RFLP analysis. TGF-beta1 codon 25 genotypes Arg/Pro and Pro/Pro were more common in patients with cirrhosis than in those without (23.6% vs. 7.4%, p = 0.005). In contrast, the distribution of TGF-beta1 codon 10, TNF-alpha and angiotensinogen genotypes was not different. Logistic regression analysis identified male sex, age, serum ferritin and TGF-beta1 codon 25 Arg/Pro and Pro/Pro as independent predictors for the presence of cirrhosis. The adjusted odds ratio for TGF-beta1 codon 25 Arg/Pro and Pro/Pro was 2.8 (95% CI 1.4-5.7, p = 0.004). In conclusion, C282Y homozygotes carrying TGF-beta1 genotypes Arg/Pro and Pro/Pro are more likely to develop cirrhosis than those with genotype Arg/Arg.

Association of myeloperoxidase promotor polymorphism with cirrhosis in patients with hereditary hemochromatosis.

BACKGROUND/AIMS: Hereditary hemochromatosis (HHC) is a disorder of iron metabolism with variable penetrance. Oxidative stress plays a central role in the progression to cirrhosis. Several enzymes involved in the production or degradation of reactive oxidants, like myeloperoxidase (MPO) and heme oxygenase (HO)-1 are influenced by promotor polymorphisms. This study assessed the impact of polymorphisms of the MPO (-463G/A) and the HO-1 promotors of Vienna (GT)n on the evolution of cirrhosis in patients with HHC. METHODS: One-hundred and fifty-eight C282Y homozygotes without cofactors for fibrosis progression (119 males; mean age: 51.0+/-13.3) were studied. All patients underwent liver biopsy. Hepatic iron content was measured by atom absorption spectrophotometry. MPO polymorphism was assessed by RFLP analysis; HO-1 microsatellite polymorphism by a laser-based semi-automated DNA sequencer. RESULTS: The MPO genotypes GG, GA, and AA were found in 102 (64.6%), 45 and 11 patients, respectively. The GG-genotype was more common in patients with cirrhosis than in those without (78.7 vs. 55.7%, P=0.003). The distribution of HO-1 genotypes was not different. Logistic regression analysis revealed MPO genotype-GG, serum ferritin, age and male sex as independent predictors for cirrhosis. CONCLUSIONS: MPO genotype GG is associated with cirrhosis in patients with hereditary hemochromatosis.

Anti-SLA seropositive autoimmune hepatitis sera recognize distinct subunits of glutathione S-transferase: high prevalence of the Ya autoantigen.

We have recently reported that anti-SLA seropositive autoimmune hepatitis (AIH) patients develop autoantibodies against glutathione S-transferase (GST). GSTs are multifunctional enzymes mediating hepatic detoxification of cytotoxic and genotoxic compounds and are also involved in biliary secretion. We have observed varying reactivity of individual AIH sera towards several GST isoenzymes. Since the GST subunits have very similar molar masses and therefore are not satisfactorily resolved by one-dimensional gel electrophoresis, we have performed their fractionation by reverse-phase high performance liquid chromatography (HPLC) to better separate the individual GST isoenzymes. 4 individual GST subunits were isolated as judged by electrophoretic analysis of the 4 distinct peaks. The identity of isolated proteins was unequivocally determined by protein sequencing. Isolated subtypes were loaded on 15% SDS gels and blotted. Immunoblotting was performed with eleven anti-SLA positive sera that displayed differential reactivity with total GSTs. Fractionation of the GSTs by HPLC did not impair their ability to react with specific autoantibodies. Interestingly, the majority of GST-positive AIH sera reacted with one or two GST subtypes, only two sera recognized 3 subunits. Ya was most prevalent autoantigen. Autoantibodies against Yb2 were detected solely in one serum. This pattern of reactivity indicates that individual patients' sera discriminate between GST subunits despite their sequence homology. It is well known that the GST variants differ within their amino-terminal part while the residual moiety is highly conserved. It would suggest that autoantibodies recognize distinct epitopes located within amino-terminus of individual GST variants.

Liver pathology in compound heterozygous patients for hemochromatosis mutations.

BACKGROUND: While hepatic pathology of homozygous carriers of the C282Y mutation of the HFE haemochromatosis gene is well defined, the impact of the C282Y/H63D compound heterozygous carrier state is unknown. AIMS: To evaluate the range of hepatic pathology in C282Y/H63D compound heterozygous patients. PATIENTS: 25 C282Y/H63D compound heterozygous patients with and without known underlying liver disease underwent liver biopsies for evaluation or abnormal liver tests. Eleven cadaveric liver donors with HFE wild type served as controls. METHODS: Mutations in the HFE gene were detected by polyacrylamide gel electrophoresis (PAGE) separation of digested polymerase chain reaction (PCR)-amplificates. The extent of light microscopic changes of liver architecture were studied on haematoxylin, eosin (H. E.) stains. In addition, the extent and the distribution of iron deposition was graded on Prussian blue-stained sections and hepatic iron was quantified by atom absorption spectroscopy. Serum ferritin concentration and the transferrin saturation index were measured using routine laboratory methods. RESULTS: Patients without underlying liver disease (n = 15): Hepatic inflammation was seen in only 8% but fibrosis was found in 36% of compound heterozygous patients. Eighty six percent of those patients had stainable iron predominantly found in Rappaport's zone 1 and 2, but all had a liver iron-index < 1.9. Transferrin saturation was found elevated in 36% of compound heterozygous patients. Patients with liver fibrosis showed significantly higher ferritin levels than patients without liver fibrosis (1110 ng/mL versus 307 ng/mL, p < 0.05). Patients with underlying disease (n = 10): In compound heterozygous patients, 77% had hepatic inflammation and 88% fibrosis. Stainable iron (44%) was less frequently found than in patients without underlying liver disease. Hepatic iron-index in patients with underlying liver disease was always below 1.17; transferrin saturation was elevated in only 22% of the compound heterozygous patients. Histologic hepatic iron-index was significantly lower in patients with underlying disease (median 0.047) as compared to patients without underlying liver disease (median 0.274, P < 0.05). CONCLUSIONS: The underlying liver disease determines the extent of hepatic pathology seen in livers of compound heterozygous patients. However, considerable histologic fibrosis can also be found in compound heterozygous patients without underlying liver disease.

Subcellular localization of the ABCG2 transporter in normal and malignant human gallbladder epithelium.

Epithelium of the gallbladder and biliary tract is exposed to high concentrations of potentially harmful exogenous and endogenous compounds excreted into primary bile. As the ATP-dependent efflux pump ABCG2 can prevent cellular accumulation of anticancer drugs, estrogen sulfate, xenobiotics, porphyrins, and sterols, its expression in the biliary tract might mediate protection by hindering their penetration. We therefore investigated the expression and subcellular distribution of ABCG2 in normal and malignant human gallbladder. After demonstrating ABCG2 expression in gallbladder epithelium by RT-PCR and Western blotting, we analyzed the subcellular localization of ABCG2 by indirect immunofluorescence in gallbladder adenocarcinoma specimens, and compared it to that in cholelithiasis, and normal gallbladder samples (n = 54). In control, cholelithiasis, and well-differentiated tumor samples (grade 1, T1-3), ABCG2 is present at the luminal membrane of epithelial cells, which was proven by colocalization of apical-bound TRITC-labeled lectin (wheat germ agglutinin). In poorly differentiated gallbladder adenocarcinomas, intracellular ABCG2, in addition to luminal ABCG2 immunoreactivity, was found in 13/21 carcinoma samples (grade 2 and 3, T2-4, P < 0.01). In 3/11 of grade 3 tumors, ABCG2 was present in the cytoplasmatic compartment only (P < 0.01). In proliferating bile ducts of cholangiocarcinomas, ABCG2 showed an analogous staining pattern with presence in cytosolic compartments. However, the apical marker enzyme neutral endopeptidase remained on the membrane in all samples. To study whether phosphatidylinositol 3-kinase (PI3K) signaling might be necessary for ABCG2 membrane insertion, we treated freshly isolated human gallbladder epithelial cells with the PI3K inhibitor wortmannin. As assessed by indirect immunofluorescence, this maneuver redistributes ABCG2 to intracellular compartments. In conclusion, our data suggest a protective role for ABCG2 in well-differentiated gallbladder epithelial cells. Cytoplasmatic accumulation of ABCG2 in poorly differentiated carcinomas might coincide with malfunctioning of PI3K-signaling pathways during tumor progression.

Lack of evidence for involvement of fetal microchimerism in pathogenesis of primary biliary cirrhosis.

Microchimerism may be involved in the etiopathogenesis of autoimmune diseases such as scleroderma. Primary biliary cirrhosis (PBC) shares some features with scleroderma, including a female predominance and a histologic picture reminiscent of chronic graft-versus-host disease. Our aim was to detect Y-chromosome-specific sequences as a marker for microchimerism in liver tissue of female patients with PBC. Liver biopsies of 105 female patients were investigated (28 patients with primary biliary cirrhosis, 25 patients with chronic hepatitis C, 6 patients with chronic hepatitis B, 9 with autoimmune hepatitis, and 37 patients with other liver diseases) by a sensitive Y-chromosome-specific polymerase chain reaction and/or fluorescence in situ hybridization (FISH) technique for the detection of the Y chromosome on a single cell level. In the liver of 9 (8.6%) female patients Y-chromosome-specific sequences were detected by PCR. Five of the patients had PBC as underlying disease, 2 had chronic hepatitis C, and 2 other liver diseases. No significant difference in the positivity rate for Y-specific sequences in females with PBC and patients with other liver diseases was found (P > 0.05). By FISH, single cells with one Y chromosome were detected in liver specimens from 3 of 21 patients suffering from PBC and from 1 of 13 patients with other liver diseases. In summary, microchimerism can be detected in livers of patients with hepatic diseases. However, in our study we found no evidence for an increased prevalence of microchimerism in the livers of patients with primary biliary cirrhosis. Our data suggest that microchimerism does not play a significant role in the development of PBC.

Spontaneous viral clearance in patients with acute hepatitis C can be predicted by repeated measurements of serum viral load.

Early interferon (IFN) therapy prevents viral persistence in acute hepatitis C, but in view of the resulting costs and morbidity patients who really need therapy have to be identified. Twelve consecutive patients with acute hepatitis C (9 women, 3 men, mean age: 39.5 +/- 18.8 y, genotype 1: 7, genotype 3a: 3, 2 could not be genotyped) were studied. The sources of infection were medical procedures in 6, sexual transmission in 3, and intravenous drug abuse in 3 patients. Viral load was measured by Cobas Amplicor HCV Monitor v2.0 (Roche Diagnostic Systems, Branchburg, NY). The time from infection to clinical symptoms was 43.3 +/- 8.6 (mean +/- SD) days. Eight patients cleared hepatitis C virus (HCV) spontaneously and remained HCV-RNA negative with a follow-up of 9.0 +/- 3.9 months. In these patients viral load declined fast and continuously. The time from exposure to HCV-RNA negativity was 77.4 +/- 25.3 and from the first symptoms was 34.7 +/- 22.1 days. In 4 patients HCV-RNA levels remained high or even increased. Two of them became sustained responders to treatment initiated after a 6-week observation period. The 2 remaining patients were not treated (one because of contraindications for IFN, the other declined therapy) and are still HCV-RNA positive. In conclusion, patients with acute icteric hepatitis C have a high rate of spontaneous viral clearance within the first month after the onset of symptoms. IFN therapy appears only needed in patients who fail to clear the virus within 35 days after onset of symptoms. By this approach, IFN therapy was not necessary in two thirds of patients with acute hepatitis C.

The melatonin receptor subtype MT1 is expressed in human gallbladder epithelia.

Based on the fact that human bile and, particularly gallbladder bile, contains high physiological levels of the antioxidant melatonin, the aim of this study was to investigate whether the melatonin receptor MT1 is present in human gallbladder. Expression and localization of MT1 was assessed by RT-PCR, Western blotting and immunofluorescence analysis in gallbladder samples from patients with cholelithiasis and with advanced gallbladder carcinoma. Additionally, we monitored mRNA expression of the two key enzymes of melatonin synthesis, i.e. arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT). MT1 mRNA and protein were present in all cholelithiasis (n = 10) and gallbladder carcinoma (n = 5) samples. As indicated from RT-PCR and Western blot studies, MT1 is located in gallbladder epithelia. Epithelial expression was further proven by immunofluorescence staining of MT1 in paraffin-embedded cholelithiasis and gallbladder carcinoma sections. Analysis of AANAT and HIOMT mRNA expression showed that HIOMT mRNA is present in gallbladder. Surprisingly, AANAT was not detectable under conditions where it was found in a human colon specimen. The absence of AANAT suggests that in human gallbladder, HIOMT might be involved in the formation of 5-hydroxytryptamine products other than melatonin. In summary, our results provide the first evidence for the presence of MT1 in human gallbladder epithelia. Therefore, in addition to its profound antioxidative effects in the biliary system, melatonin might also act through MT1-mediated signal transduction pathways. Thereby, it might be involved in the regulation of gallbladder function.