Peter Schirmacher

Bile duct epithelia as target cells in primary biliary cirrhosis and primary sclerosing cholangitis

Abstract Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are chronic autoimmune-mediated diseases of the biliary tree, resulting in a loss of bile ducts. There are morphological features that clearly distinguish them from each other: in PBC, there is overt destruction of the bile ducts with disruption of the basement membrane; in PSC there is abundant periductular fibrosis with shrinkage and subsequent loss of the bile ducts. In order to see if the disparate histopathology is paralleled by different immunohistology we looked at a panel of epitopes on bile duct epithelia especially to see if biliary epithelial cells may present as targets for cell mediated immune respone. In PBC bile duct epithelial cells mostly expressed CD58 (lymphocyte function-associated antigen 3), CD80 (B7 BB1), and CD95 (Fas). In PSC, however, these epitopes were only expressed in a few examples to a lower degree. The respective effector T lymphocytes were positive for CD2 and CD28. Subtyping of the lymphocytes in the liver tissue further showed a predominance of CD4 positive T cells over CD8 cells up to 2-to-1 in both diseases. Determination of lymphocytes by cytokines to Th1 or Th2 subtype showed a majority of Th1 lymphocytes in PBC and PSC. We conclude that in PBC bile duct epithelial cells may display features of target cells of a T cell-mediated immune reaction with the Th1 cells predominating. In PSC other mechanisms of bile duct loss may play a role, since in this disease the majority of cells lack essential epitopes that constitute targets of cell mediated immunity.

Hepatocellular Hyperplasia, Plasmacytoma Formation, and Extramedullary Hematopoiesis in Interleukin (IL)-6/Soluble IL-6 Receptor Double-Transgenic Mice

Cytokines interact not only with membrane anchored receptors, but also with specific soluble receptors which circulate in the bloodstream. In general, soluble cytokine receptors such as soluble tumor necrosis factor receptor, soluble interleukin 1 receptor, and soluble interleukin 4 receptor compete with their membrane-bound counterparts for the ligands and therefore act as antagonists. In contrast, soluble receptors for cytokines of the interleukin-6 (IL-6) family complex with their ligands act agonistically. Interestingly, the complex of IL-6 and the soluble interleukin 6 receptor (sIL-6R) activates target cells that do not express the membrane-bound IL-6R and therefore cannot respond to IL-6. To identify cellular responses that are due to IL-6/sIL-6R but not to IL-6 alone, IL-6/sIL-6R double-transgenic mice were generated and compared with IL-6 single-transgenic mice. IL-6/sIL-6R transgenic mice develop a severe phenotype showing 1) marked hepatocellular hyperplasia frequently surrounded by peliosis and necrosis, 2) significant acceleration and aggravation of plasmacytoma formation, and 3) excessive activation of extramedullary hematopoiesis in spleen and liver followed by a subsequent increase of all cellular components in the peripheral blood. These in vivo data suggest that the sIL-6R recruits primarily unresponsive cell populations such as hematopoietic progenitor cells and hepatocytes to IL-6-induced proliferation, but also enhances the known mitogenic effect of IL-6 on plasma cells and thereby contributes to plasmacytoma formation.

Combined Therapy with Azathioprine, Prednisolone, and Ursodiol in Patients with Primary Sclerosing Cholangitis: A Case Series

Primary sclerosing cholangitis is a progressive cholestatic liver disease (1) that is widely regarded as an autoimmune disease because of its association with HLA-B8, HLA-DR3, and HLA-DR52a (2) and with other immune-mediated diseases, including inflammatory bowel disease (1). Various immunologic abnormalities have been described elsewhere (1, 3). No established medical therapy has been shown to halt the progressive course of this disease (4). Several immunosuppressive or immunomodulating agents, including penicillamine (5), colchicine (6), methotrexate (7), cyclosporine (8), tacrolimus (9), and ursodeoxycholic acid (UDCA) (10) have been tested, with varying results, in the treatment of patients with primary sclerosing cholangitis. Azathioprine treatment has been reported in three patients. Two of these patients improved (11, 12), and one died with a liver abscess found during autopsy (13). Results on the use of steroids have been equally conflicting (4, 14, 15). Several trials of UDCA showed some promise (16-18), but a recent large controlled trial did not show a clinical benefit of treatment with UDCA alone (10). Because of the pathophysiologic consideration that primary sclerosing cholangitis is an immune-mediated disease, further study of immunosuppressive therapy is warranted (19). The combination of azathioprine and prednisolone has not yet been tested in the treatment of primary sclerosing cholangitis. We present a pilot study of immunosuppression with azathioprine and prednisolone combined with UDCA for patients with primary sclerosing cholangitis. Methods Fifteen patients (10 male, 5 female; mean age SD, 39 4 years) were included in this pilot study (Table). They had had well-defined primary sclerosing cholangitis for a mean of 46 months before treatment was started. Before study entry, 14 patients underwent endoscopic retrograde cholangiography that showed the typical primary sclerosing cholangitis pattern. The 15th patient had magnetic resonance imaging because an endoscopy was not successful. Liver tissue specimens were obtained directly before study entry in 13 patients either by laparoscopy or by percutaneous needle biopsy using the Menghini technique. Informed consent was obtained from every patient before study entry, and the local ethics committee approved the study. Table. Variables at Study Entry and during Treatment Treatment consisted of azathioprine, 1 to 1.5 mg/kg of body weight per day (Glaxo Wellcome GmbH, Hamburg, Germany); prednisolone, 1 mg/kg per day initially, tapering to 5 to 10 mg per day (Merck KGaA, Darmstadt, Germany); and UDCA, 500 to 750 mg per day (mean, 650 mg per day) (Dr. Falk GmbH, Freiburg, Germany). Before study entry and immunosuppressive treatment, seven patients were treated with UDCA, 500 to 750 mg per day (mean, 714 mg per day) for a median of 33 months (range, 3 to 60 months). Patients underwent a complete history, physical examination, and abdominal ultrasonography. They were followed after 6 and 12 weeks and every 3 to 6 months thereafter. Laboratory tests were done during these examinations. Follow-up endoscopic retrograde cholangiography was not considered mandatory within the study period for asymptomatic patients without clinical or biochemical signs of progression. Therefore, 10 patients were followed endoscopically; a change in stenosis of greater than 30% was considered relevant. Liver biopsy specimens were scored according to the criteria of Ludwig and colleagues (20). Specificity for antineutrophil cytoplasmic antibody was tested by enzyme-linked immunosorbent assay. Data were collected for 3 to 81 months of treatment (median, 41 months). Statistical significance was calculated by using the nonparametric Wilcoxon signed-rank test. Values are given as the mean SE. Results Fifteen patients were treated for 3 to 81 months (median, 41) months. Patient characteristics are summarized in the Table. Nine patients were symptomatic at study entry; the most common symptoms were pruritus and fatigue (4 patients each). Eight patients had associated inflammatory bowel disease: Seven had ulcerative colitis and 1 had Crohn disease. Other autoimmune phenomena seen were pyoderma gangrenosum, the sicca syndrome, and arthralgia, each seen in 1 patient. One patient had a cholangiogram typical for primary sclerosing cholangitis 2 years after the diagnosis of autoimmune hepatitis. Of the 15 patients included in the study, 14 underwent endoscopic retrograde cholangiography and 1 had magnetic resonance cholangiography. Thirteen patients had liver biopsies, and 2 patients declined this procedure; however, the 2 patients who did not have biopsy were included in the study because of the typical cholangiographic and laboratory findings. The decision of radiographic or histologic follow-up was left to the treating physician; therefore, only 10 patients had repeated cholangiography and liver biopsies. Two patients had to discontinue therapy with the study medications after 3 and 13 months because of adverse reactions. One patient with ulcerative colitis had to be switched to a different immunosuppressive regimen after 3 years because his inflammatory bowel disease was not sufficiently controlled. Median follow-up was 41 months (range, 3 to 81 months). No patients were lost to follow-up. We observed a rapid and significant decline in liver enzyme levels that persisted throughout the observation period (Figure, left): Alkaline phosphatase and aspartate aminotransferase levels decreased by 56%, alanine aminotransferase levels by 65%, and total bilirubin levels by 27%. In 5 patients, alkaline phosphatase and aspartate aminotransferase levels completely returned to normal. Cholangiographic findings remained stable in 9 of 10 patients. One patient required endoscopic dilatation of a common bile duct stenosis 1 year after treatment began and has since remained stable. Histologic follow-up of 10 patients showed stable disease (Table). Of these 10 patients, 6 showed histologic improvement, 2 remained stable, and 2 worsened. At last follow-up, only 2 of 15 patients remained symptomatic. Figure. Biochemical changes under immunosuppressive therapy. Left. Right. Seven patients had been treated with UDCA for a median of 33 months (range, 3 to 60 months) before immunosuppressive therapy was added at study entry. Before study entry, total bilirubin levels had increased in four of the seven patients; in one patient, the bilirubin level remained stable. In these five patients, total bilirubin levels declined after immunosuppressive therapy began. All seven patients from this subgroup showed a decline in levels of alkaline phosphatase, total bilirubin, and alanine aminotransferase; this result was statistically significant after 3 months of immunosuppressive therapy (Figure, right). Two patients showed radiographic progression during UDCA therapy, but no further progression could be seen after 1 year of combined immunosuppressive therapy. Treatment was well tolerated in 13 of 15 patients. One patient had to discontinue azathioprine therapy after 3 months of therapy because of fever. This patient, whose disease has since progressed with two episodes of bleeding from esophageal varices, is the only study group patient awaiting liver transplantation. Another patient showed signs of hepatic toxicity; therefore, immunosuppression was stopped after 13 months of therapy. Myelotoxicity was monitored by platelet and leukocyte counts, and no adverse effects were noted (Table). An episode of bacterial cholangitis in 1 patient was managed by temporarily decreasing immunosuppression and by adding standard antibiotic treatment. He has remained free of subsequent infections for more than 30 months with continued immunosuppressive therapy. Discussion Primary sclerosing cholangitis is thought to be an immune-mediated disease (2). It is likely that immunosuppression influences the course of this disease, but controlled trials have not yet found effective therapy, perhaps because of the small number of patients studied. Furthermore, earlier reports have used various monotherapies rather than combined immunosuppression (7-9, 11-15). Our pilot study combined corticosteroids with azathioprine. This combination is standard treatment for autoimmune hepatitis and severe inflammatory bowel diseases, two conditions related to primary sclerosing cholangitis. The combination treatment was effective in halting disease progression in almost all patients treated over the follow-up period (median, 41 months). Only 1 patient exhibited progression of a single extrahepatic bile duct stenosis. Among the 10 patients who had follow-up biopsies, no progression was seen, and 6 of 10 patients had improved liver histologic findings. Seven patients from this group, with histologic stage III or IV disease, had stable or improved findings on follow-up biopsies. This suggests that immunosuppressive therapy might be helpful even in advanced stages of disease, although sampling error is possible because primary sclerosing cholangitis tends to have a focal appearance (1). Laboratory values improved markedly, and this improvement remained stable throughout the study. Treatment was well tolerated in 13 of 15 patients. Use of azathioprine had to be stopped for 1 patient because of fever and for another patient because of suspected hepatic toxicity. Allergic or toxic reactions to azathioprine are common and necessitate drug withdrawal in 5% to 10% of patients. Subsequent monotherapy with steroids did not result in improvement in 1 patient whose disease has since progressed and who is now awaiting liver transplantation. Other side effects, such as weight gain, were related solely to corticosteroids and were minor and mostly transient during the initial high-dose therapy. We used UDCA in our treatment protocol because it is standard therapy in Germany despite disappointing trial data. Ursodeoxycholic acid can improve laboratory values in most patients bu

Hepatitis C virus associated primary hepatocellular carcinoma in a noncirrhotic liver

SummaryThe case of a 71-year-old man with a primary hepatocellular carcinoma in a non-cirrhotic liver is reported. There were no risk factors of hepatocellular carcinoma (HCC)-like liver cirrhosis, alcohol drinking, tobacco smoking, exposure to vinyl chloride, thorotrast, aflatoxin or α1-antitrypsin deficiency. Serologically, the patient was positive for antibodies to the hepatitis B virus (anti-HBc, anti-HBs) and for anti-hepatitis C virus (HCV) antibodies. Virologically, positive and negative strands of HCV RNA could be detected in the patients serum and tumorous liver tissue by reverse transcription polymerase chain reaction as a sign of persistent HCV replication. Histologically, the HCC was completely surrounded by liver tissue which showed the signs of nodular regenerative hyperplasia. Indeed, the mechanism of hepatocarcinogenesis remains to be clarified. However, this case supports the observation that HCC may also develop in patients with HCV infection without preexisting liver cirrhosis.

De novo expression of nonhepatocellular cytokeratins in Mallory body formation

Abstract Mallory bodies (MBs) are eosinophilic cytoplasmic inclusions observed predominantly in alcoholic liver disease. Although linked to disease activity, their pathogenesis is still unclear. Since intermediate filaments (cytokeratins) are major components of MBs, their cytokeratin polypeptide composition was analysed with monospecific antibodies for cytokeratins 7, 8, 14, 18, 19, and 20 by immunohistology. MBs were identified by light microscopy and ubiquitin immunostaining. All MBs were positive for cytokeratins 8 and 18. A significant percentage of the MBs was strongly positive for cytokeratins 19 and/or 20, which are not detectable in hepatocytes of normal liver and, in the case of cytokeratin 20, in hepatocytes of diseases devoid of MBs. MBs were essentially negative for cytokeratins 7 and 14. De novo expression of cytokeratins 19 and 20 was independent of the aetiology, occurring in all MB-associated diseases analysed, and seemed to precede MB formation, since in some hepatocytes a cytoskeletal-type staining pattern for these cytokeratins was present. In hepatocellular carcinomas cytokeratins 19 and 20 were frequently detected, but their cellular distribution was less closely associated with MBs. The ectopic expression of cytokeratins 19 and 20 appears to be related to MB formation and may take part in the derangement of the intermediate filaments during MB formation.

HEPATOCELLULAR CARCINOMA IN A PATIENT WITH HEREDITARY HEMOCHROMATOSIS AND NONCIRRHOTIC LIVER. A CASE REPORT

A case of a 62-year-old patient with hereditary hemochromatosis is reported, who developed hepatocellular carcinoma (HCC) in the absence of cirrhosis and other potential risk factors for HCC. Occurrence of HCC in patients with genetic hemochromatosis and noncirrhotic liver is a rare event which has previously been described only six times and appears to be limited to male patients.

Progressive Focal Nodular Hyperplasia of the Liver in a Patient with Genetic Hemochromatosis-Growth Promotion by Iron Overload?

A 27-year-old driving instructor was referred to our outpatient department because of an unclear mass in the right lobe of the liver. The lesion had first been diagnosed eight years before referral by an ultrasound scan and had increased in size from 3 cm diameter to 9 cm. The mass was of enhanced echogenicity and had well-defined borders. A recent blood test had shown an elevated ferritin (545 mg/l normal 34–310 mg/l). Further evaluation of the patient showed a transferrin saturation of 57% and elevated g-GT of 44 units/l (normal 6–28 u/l). Serology for hepatitis A, B, and C virus infection was negative. All other blood tests were normal. The patient was operated and the tumor excised. Histopathological and Experimental Findings. Histological examination of the resected tumor specimen confirmed the picture of a focal nodular hyperplasia (FNH, solid type) (Figure 1). The peritumerous liver tissue displayed marked hepatocellular iron deposition (grade IV) in contrast to cells of the FNH, which showed no iron deposition at all (Figure 2). The portal tracts and liver parenchyma showed no significant fibrosis. Since hemochromatosis was likely, peritumerous liver tissue was analyzed for absolute iron concentration. The hepatic iron content was 13.82 mg Fe/g liver dry weight, resulting in an hepatic iron index of 10.3 (normal ,1.9), which is diagnostic for genetic hemochromatosis. In addition, PCR analysis of DNA from peripheral blood cells was performed and showed homozygosity for the Cys282Tyr mutation of the HLA-HFE gene. From the resected tumor specimen, we could establish an epithelial cell line that was phenotypically stable for up to 10 passages. These FNH cells were also homozygous for the Cys282Tyr mutation. Since the tumor had shown a rapid increase in size, we performed immunohistology for the Ki-67 antigen. In keeping with that, the number of Ki-67 positive cells among 500 randomly selected hepatocytes was elevated with 4%, although the distribution of positive cells was focally accentuated (Figure 3).

Intracellular accumulation of incompletely processed transforming growth factor-alpha polypeptides in ground glass hepatocytes of chronic hepatitis B virus infection

BACKGROUND Transforming growth factor-alpha is an intracellularly processed and secreted polypeptide that induces a proliferative response in epithelial target cells and represents a potential regulatory factor in embryonic development, liver regeneration, and also hepatocarcinogenesis. We have observed focal transforming growth factor-alpha expression in liver tissues with chronic hepatitis B virus infection. METHODS To further elucidate the nature of this focal transforming growth factor-alpha accumulation we have analyzed overall 23 different liver tissues with chronic hepatitis B virus and hepatitis C virus infection as well as normal liver tissues by immunohistology, ELISA, and Western immunoblot with and without immunoprecipitation. RESULTS By immunohistology transforming growth factor-alpha polypeptides showed focal subcellular accumulation in ground glass hepatocytes, the histological hallmark of chronic hepatitis B virus infection, in co-localization with HBV-preS1 antigen. By ELISA and Western immunoblot increased tissue concentrations of transforming growth factor-alpha were demonstrated in chronically hepatitis B virus-infected liver tissues with ground glass hepatocytes, especially a 15-kD polypeptide, most likely representing an incompletely processed transforming growth factor-alpha polypeptide. Transforming growth factor-alpha retention in ground glass hepatocytes is not a general unspecific effect, since it was not observed for several other secretory liver proteins. Accumulated transforming growth factor-alpha in ground glass hepatocytes does not co-localize with Epidermal Growth Factor Receptor expression. CONCLUSION Thus evidence is presented that a principally secreted (viral) polypeptide (HBV-preS1) can interfere with the secretion and processing of a second (cellular) protein (transforming growth factor-alpha). Accumulation of transforming growth factor-alpha may result from alteration of the endoplasmic reticulum due to storage of hepatitis B virus surface antigen particles. No evidence was found for transforming growth factor-alpha in ground glass hepatocytes to intracellularly interact with the Epidermal Growth Factor Receptor.

Persistent expression of hepatitis C virus genome in primary tumor and adrenal metastasis of a hepatocellular carcinoma developed in a non-cirrhotic liver

To the Editor: There is increasing evidence that chronic infection with hepatitis C virus (HCV) is a risk factor for hepatocellular carcinoma (HCC) in patients seronegative for hepatitis B virus (HBV) surface antigen (HBsAg). In western countries, HCCs occur in anti-HCV positive patients mostly in association with cirrhosis, which can be considered as a precancerous condition (1). However, there are rare cases of HCC that were found in anti-HCV positive patients without pre-existing liver cirrhosis (2). We report here the detection of HCV RNA in a primary HCC derived from an HCV-infected patient with a non-cirrhotic liver and its persistent expression in an adrenal metastasis that developed 3 years later. In January, 1992, the 7 l-year-old symptomless patient was admitted to our hospital because of a solitary inhomogeneous tumor in the right lobe of the liver with a size of 5×4x3 cm. The serum aminotransferase levels were slightly increased not exceeding 3 times normal. All other liver function parameters were within normal range. The c~-fetoprotein (c~-FP) level was elevated to 86 ng/ml. The patients serum was positive for antiHCV antibodies (Ab) as measured by second-generation immunoassay. He had no history of non-viral risk factors of liver disease. The tumor staging was free from metastases in other organs, so a right-sided hemihepatectomy was performed. The histological examination of tumor tissue showed a moderately differentiated, trabecular HCC. The surrounding nontumorous liver tissue showed no cirrhosis, but nodular regenerative hyperplasia. The postoperative ot-FP level returned to normal range. In March, 1995, an increase in ~-FP level to 50 ng/ml was observed. The tumor staging revealed a singular right adrenal mass that was completely resected. The histological examination of the tumor showed metastatic tissue of the previously described HCC. A liver biopsy was still free from cirrhosis and tumor reactivation. In 1992 and 1995, we found HCV RNA using reverse transcription polymerase chain reaction (RT-PCR) (3) in the patients serum, lymphocytes, nontumorous liver tissue, primary HCC or adrenal metastasis, respectively. In contrast, the patients serum and tumor were negative for HBV DNA using PCR assay performed as recently published by our group (4). Our case shows in a prospective manner that HCV RNA detected in a primary HCC without cirrhosis can be found again in an adrenal metastasis occurring 3 years later. This observation is in line with results of a retrospective study about HCV RNA expression in HCV-associated HCCs without cirrhosis previously reported by Brechots group (5). In conclusion, these data support the view that HCV may exert a direct oncogenic effect during long-lasting infection, and, therefore, itself play an important role in the pathogenesis of HCCs arising in non-cirrhotic livers.

Sustained elimination of hepatitis B virus from serum induced in a patient with chronic hepatitis B and advanced human immunodeficiency virus infection

A 48-year-old male patient was admitted with acquired immunodeficiency syndrome (stage III, Centers for Disease Control 1993) and viremic hepatitis B. Blood CD4 count was 15/μl. Discontinuation of prednisolone, previously prescribed by the patients family practitioner because of elevated liver enzymes, resulted in severe hepatitis (alanine aminotransferase > 300U/1). Administration of interferon-α, (9 × 106U s.c. 3 × weekly) was initiated. Serum markers of viral replication disappeared, and aminotransferase levels returned to normal within a few weeks. The patients serum was found negative for HBsAg after 3 months. Immunohistochemical analysis of liver biopsies before and during interferon therapy showed disappearance of all hepatitis B virus antigens and a marked reduction in inflammatory activity. Hepatitis B virus seroconversion remained stable until the patient died from the syndrome 2 years later. This case shows that in spite of severe HIV-associated immune deficiency with CD4 counts constantly below 100/μl, interferon-α can lead to sustained serological and histological improvement of viremic hepatitis B. Previous administration and discontinuation of cortisone may have helped to reach this effect.