Embryology of extra- and intrahepatic bile ducts, the ductal plate.

In the human embryo, the first anlage of the bile ducts and the liver is the hepatic diverticulum or liver bud. For up to 8 weeks of gestation, the extrahepatic biliary tree develops through lengthening of the caudal part of the hepatic diverticulum. This structure is patent from the beginning and remains patent and in continuity with the developing liver at all stages. The hepatic duct (ductus hepaticus) develops from the cranial part (pars hepatica) of the hepatic diverticulum. The distal portions of the right and left hepatic ducts develop from the extrahepatic ducts and are clearly defined tubular structures by 12 weeks of gestation. The proximal portions of the main hilar ducts derive from the first intrahepatic ductal plates. The extrahepatic bile ducts and the developing intrahepatic biliary tree maintain luminal continuity from the very start of organogenesis throughout further development, contradicting a previous study in the mouse suggesting that the extrahepatic bile duct system develops independently from the intrahepatic biliary tree and that the systems are initially discontinuous but join up later. The normal development of intrahepatic bile ducts requires finely timed and precisely tuned epithelial-mesenchymal interactions, which proceed from the hilum of the liver toward its periphery along the branches of the developing portal vein. Lack of remodeling of the ductal plate results in the persistence of an excess of embryonic bile duct structures remaining in their primitive ductal plate configuration. This abnormality has been termed the ductal plate malformation.

The clinicopathological and prognostic relevance of cytokeratin 7 and 19 expression in hepatocellular carcinoma. A possible progenitor cell origin.

AIMS: Cytokeratin (CK) 7 and CK19 expression, present in hepatic progenitor cells (HPCs) and in cholangiocytes but not in normal hepatocytes, has been reported in some hepatocellular carcinomas (HCCs); however, the incidence and relevance of this expression in HCC in Caucasians is not known. Therefore, our aim was to study the occurrence and clinicopathological characteristics of HCC expressing CK7 and/or CK19 in 109 Caucasian patients. METHODS AND RESULTS: The expression of hepatocellular differentiation markers (Hepar, canalicular polyclonal carcinoembryonic antigen), biliary/progenitor cell markers (CK7, CK19), alpha-fetoprotein (AFP), p53 and beta-catenin in HCC was semiquantitatively assessed by immunohistochemistry. Of 109 HCCs, 78 were CK7-/CK19- (72%), 13 CK7+/CK19- (12%), seven CK7-/CK19+ (6%), 11 CK7+/CK19+ (10%). CK19 expression was significantly associated with elevated serum AFP (400 ng/ml) (P = 0.023), tumour AFP expression (P < 0.0001), presence in serum of anti-hepatitis B core (P = 0.016), less fibrosis in non-neoplastic parenchyma (P = 0.009) and less nuclear beta-catenin expression (P = 0.021). CK7 expression was significantly associated with elevated serum bilirubin (> 2 mg/dl) (P = 0.0005) and less nuclear beta-catenin expression (P = 0.003). HCC expressing CK19 had a higher rate of recurrence (P = 0.009, hazard ratio 12.5, n = 31) after liver transplantation compared with CK19- tumours. CONCLUSIONS: In our series, 28% of HCCs contained cells expressing CK7 and/or CK19. They potentially derive from HPCs. The higher recurrence rate of CK19+ HCC after transplantation suggests a worse prognosis for these HCCs compared with CK19- HCC.

In vivo gene transfer of endothelial nitric oxide synthase decreases portal pressure in anaesthetised carbon tetrachloride cirrhotic rats.

BACKGROUND: Portal hypertension in cirrhosis results from enhanced intrahepatic resistance to an augmented inflow. The former is partly due to an imbalance between intrahepatic vasoconstriction and vasodilatation. Enhanced endothelin-1 and decreased activity of hepatic constitutive endothelial nitric oxide synthase (NOS 3) was reported in carbon tetrachloride (CCl(4)) cirrhotic rat liver. AIMS: To study whether an increase in hepatic NOS 3 could be obtained in the CCl(4) cirrhotic rat liver by in vivo cDNA transfer and to investigate a possible effect on portal pressure. METHODS: Hepatic NOS 3 immunohistochemistry and western blotting were used to measure the amount of NOS 3 protein. Recombinant adenovirus, carrying cDNA encoding human NOS 3, was injected into the portal vein of CCl(4) cirrhotic rats. Cirrhotic controls received carrier buffer, naked adenovirus, or adenovirus carrying the lac Z gene. RESULTS: NOS 3 immunoreactivity and amount of protein (western blotting) were significantly decreased in CCl(4) cirrhotic livers. Following cDNA transfer, NOS 3 expression and the amount of protein were partially restored. Portal pressure was 11.4 (1.6) mm Hg in untreated cirrhotic (n=9) and 11.8 (0.6) in lac Z transfected (n=4) cirrhotic rats but was reduced to 7.8 (1.0) mm Hg (n=9) five days after NOS 3 cDNA transfer. No changes were observed in systemic haemodynamics, in liver tests or urinary nitrates, or in NOS 3 expression in lung or kidney, indicating a highly selective transfer. CONCLUSIONS: NOS 3 cDNA transfer to cirrhotic rat liver is feasible and the increase in hepatic NOS 3 leads to a marked decrease in portal hypertension without systemic effects. These data indicate a major haemodynamic role of intrahepatic NOS 3 in the pathogenesis of portal hypertension in CCl(4) cirrhosis.

Hepatic progenitor cells in hepatocellular adenomas.

Hepatocellular adenoma is a benign tumor of the liver that has a small but not negligible risk of malignant transformation into hepatocellular carcinoma. In analogy with the established role of oval cells in hepatocarcinogenesis in rodent models, human hepatic progenitor cells may have a function in the development of liver tumors. To investigate this issue, we performed immunohistochemistry on biopsies of 10 consecutively resected hepatocellular adenomas using markers for hepatic progenitor cells. Sections of paraffin-embedded and frozen biopsies were stained using antibodies against cytokeratins 7, 8, 18, and 19, chromogranin-A, OV-6, and neural cell adhesion molecule. Hepatic progenitor cells were observed in five of 10 hepatocellular adenomas. These five tumors also contained cells with an immunohistochemical phenotype intermediate between hepatic progenitor cells and hepatocytes. Hepatic progenitor cells and intermediate hepatocyte-like cells were scattered throughout the tumors with a density that varied from area to area. Ultrastructural examination confirmed the presence of hepatic progenitor cells. Our study shows that hepatic progenitor cells are present in a considerable proportion of hepatocellular adenomas, supporting the hypothesis that human hepatic progenitor cells can play a role in the development of hepatocellular tumors.

Expression of neural cell adhesion molecule in human liver development and in congenital and acquired liver diseases.

In the liver, neural cell adhesion molecule (NCAM) is a marker of immature cells committed to the biliary lineage and is expressed by reactive bile ductules in human liver diseases. We investigated the possible role of NCAM in the development of intrahepatic bile ducts and aimed at determining whether immature biliary cells can contribute to the repair of damaged bile ducts in chronic liver diseases. Therefore, we performed immunohistochemistry for NCAM and bile duct cell markers cytokeratin 7 and cytokeratin 19 on frozen sections of 85 liver specimens taken from 14 fetuses, 10 donor livers, 18 patients with congenital liver diseases characterized by ductal plate malformations (DPMs), and 43 cirrhotic explant livers. Duplicated ductal plates and incorporating bile ducts during development showed a patchy immunoreactivity for NCAM, while DPMs were continuously positive for NCAM. Bile ducts showing complete or patchy immunoreactivity for NCAM were found in cirrhotic livers, with higher frequency in biliary than in posthepatitic cirrhosis. Our results suggest that NCAM may have a function in the development of the intrahepatic bile ducts and that NCAM-positive immature biliary cells can contribute to the repair of damaged bile ducts in chronic liver diseases.

Predictive value of liver cell dysplasia for development of hepatocellular carcinoma in patients with non-cirrhotic and cirrhotic chronic viral hepatitis.

AIMS: Hepatocellular carcinoma (HCC) frequently develops in patients with chronic viral hepatitis, especially in the cirrhotic stage. We retrospectively studied whether the presence of the putative preneoplastic lesions large liver cell dysplasia (LLCD) and/or small liver cell dysplasia (SLCD) in a needle liver biopsy of these patients are a risk factor for the development of HCC. METHODS AND RESULTS: The presence of LLCD and SLCD in the needle liver biopsy taken at the initial work-up of 115 patients with chronic hepatitis B or C was assessed retrospectively. LLCD and SLCD were present in the initial biopsy of, respectively, 35 (30%) and 25 patients (22%). During a mean follow-up of 107 months, 16 patients (14%) developed HCC and this occurred significantly more frequently in patients with cirrhosis, age > or = 55 years, LLCD or SLCD. Cirrhosis and LLCD were independent risk factors for HCC development. CONCLUSIONS: Our findings indicate that the presence of LLCD in a needle liver biopsy of patients with viral-induced chronic liver disease is an independent risk factor for the development of HCC. If these results are confirmed, the presence of LLCD can be used to identify a subgroup of patients at high risk for HCC requiring more intensive screening.

Human and rat hepatic stellate cells express neurotrophins and neurotrophin receptors.

The expression of neurotrophins and neurotrophin receptors in non-neural tissue is related to tissue remodeling, differentiation, proliferation and migration of target cells. The literature yields contradictory results on neurotrophin and neurotrophin receptor expression in the liver. We show immunoreactivity to antibodies to nerve growth factor (NGF), brain-derived neurotrophin (BDNF), neurotrophin 3 (NT-3), neurotrophin 4/5 (NT-4/5), the low-affinity nerve growth factor receptor p75 and the high-affinity tyrosine kinase receptors (Trk) B and C in hepatic stellate cells and weak reactivity for BDNF, NT-3, and NT-4/5 in hepatocytes, in cryosections of human and rat liver, in normal and varying pathologic conditions. Immunoreactivity is unequivocally localized to hepatic stellate cells by double staining with alpha-smooth muscle actin (alpha-SMA) and desmin, studied by confocal laser scanning microscopy. Finally, the presence of mRNA transcripts for the different neurotrophins and neurotrophin receptors, with the exception of Trk-B, is shown by reverse transcription polymerase chain reaction (RT-PCR) on RNA extracted from freshly isolated rat hepatic stellate cells, compared with hepatocyte RNA. Hepatocyte RNA was found to contain BDNF, NT-3, NT-4/5 mRNA (which is compatible with the immunohistochemical findings) and Trk-A mRNA. In conclusion, hepatic stellate cells are a source of several neurotrophins in the liver and they express neurotrophin receptors. These findings correspond with the known involvement of hepatic stellate cells in tissue remodeling, their production of extracellular matrix components and their proliferation in acute necrotizing liver pathology. In analogy with findings in other organs and systems, neurotrophins are hypothesized to play a role in the pathophysiology of liver disease.

Deep intralobular extension of human hepatic 'progenitor cells' correlates with parenchymal inflammation in chronic viral hepatitis: can 'progenitor cells' migrate?

Ductular reaction and putative progenitor cells (or 'progenitor cells'), which are presumed to be the human counterpart of the oval cells in rat liver, have been discerned in various human liver diseases, including chronic viral hepatitis. Since in experimental models of chronic hepatitis the activation of oval cells is correlated with the inflammatory infiltrate, this study investigated whether there is a correlation in chronic viral hepatitis between the number of 'progenitor cells' extending into the lobule and the severity of parenchymal inflammation, on the one hand, and the extent of ductular reaction and the severity of interface hepatitis, on the other hand. Liver biopsies of 55 patients with chronic hepatitis B and/or C were used. The severity of parenchymal inflammation and of interface hepatitis was semiquantitatively graded on a haematoxylin and eosin-stained paraffin section, while the number of 'progenitor cells' and the extent of the ductular reaction were assessed on a serial section stained for cytokeratin (CK) 7. In addition, more extensive phenotyping of 'progenitor cells' was performed on sections from frozen material from five patients, using antibodies against CK7, CK8, CK18, CK19, chromogranin-A, and the rat oval cell marker OV-6. The number of more centrally located 'progenitor cells' correlated significantly with the severity of the parenchymal inflammation, while the extent of the ductular reaction correlated significantly with the severity of interface hepatitis. These findings suggest that in chronic viral hepatitis, inflammation plays a role in 'progenitor cell' activation and its topography. In cases with moderate and severe lobular inflammation, 'progenitor cells' were strikingly scattered throughout the parenchyma and surrounded by intermediate hepatocyte-like cells, suggesting their migration into the parenchyma and their differentiation towards the hepatocytic lineage.

The immunohistochemical phenotype of dysplastic foci in human liver: correlation with putative progenitor cells.

BACKGROUND/AIMS: In previous studies we found strong evidence for the existence and activation in human liver of putative progenitor cells resembling oval cells in rat liver. In view of the known role of rat oval cells in regeneration and hepatocarcinogenesis, we investigated a possible correlation between human putative progenitor cells and different types of dysplastic foci. METHODS: We determined the immunohistochemical phenotype of dysplastic foci found in 20 cirrhotic liver explants of various etiology, using specific antibodies against hepatocyte-type cytokeratin (CK) 8 and CK18, bile duct-type CK7 and CK19, chromogranin-A (chrom-A) and rat oval cell marker OV-6. RESULTS: All 12 foci of large cell dysplasia had a phenotype similar to that of surrounding parenchyma. Oncocytic foci showed a strong cytoplasmic staining for CK7. Three out of six of these foci contained "progenitor cells", which are small cells immunoreactive for CK18, CK7, CK19, OV-6, chrom-A and stained more intensely for CK8 than surrounding hepatocytes. Four out of eight glycogen-storing foci contained CK7-positive intermediate hepatocyte-like cells and "progenitor cells". Sixteen out of 29 small cell dysplastic foci consisted of "progenitor cells" and intermediate hepatocyte-like cells which were immunoreactive for CK7, CK18, OV-6, chrom-A and showed a stronger cytoplasmic positivity for CK8 than surrounding hepatocytes. CONCLUSIONS: Foci of large cell dysplasia show no correlation with putative progenitor cells. Half of the oncocytic and glycogen-storing foci contain "progenitor cells", while more than half of the foci of small cell dysplasia consist of small cells with the same immunohistochemical phenotype as putative progenitor cells and intermediate hepatocyte-like cells, suggesting that differentiating putative progenitor cells can give rise to foci of small cell dysplasia.

Fas and Fas ligand: strong co-expression in human hepatocytes surrounding hepatocellular carcinoma; can cancer induce suicide in peritumoural cells?

Fas (Apo-1, CD95), a member of the nerve growth factor/tumour necrosis factor receptor superfamily, mediates apoptosis in response to agonistic antibodies or Fas ligand (Fas-L) binding. Fas has been shown to be present on hepatocyte membranes in normal liver and in chronic hepatitis C. At the present time, very limited data are available on the expression of Fas-L. This paper describes a study of 20 cases of active chronic hepatitis of different aetiologies, 20 hepatocellular carcinomas (HCCs) and the adjacent non-tumoural liver parenchyma, and five normal livers. The immunohistochemical expression of Fas and Fas-L was determined using specific monoclonal antibodies. In normal liver, Fas was faintly expressed on membranes of hepatocytes and bile duct cells, while Fas-L was negative. In active chronic hepatitis, Fas expression in hepatocytes was enhanced, resulting in a diffuse honeycomb pattern. Fas-L showed cytoplasmic positivity in hepatocytes in areas of interface hepatitis. Strong expression of Fas as well as Fas-L in the hepatocytes immediately adjacent to HCC was a constant finding. Within the HCCs, Fas-L expression was variable, but present only in a minority of cells. Fas varied from a diffuse honeycomb pattern to focal positivity in occasional cells. There was no correlation between Fas and Fas-L expression in the tumours. In conclusion, hepatocytes can co-express Fas and Fas-L in areas of interface hepatitis and adjacent to HCC, suggesting that they have the ability to induce apoptosis in an autocrine or paracrine way. Within the tumour, the Fas-Fas-L apoptosis pathway seems to be little involved.

Malignant angiomyolipoma of the liver: a hitherto unreported variant.

AIMS: After their original recognition in the kidney, angiomyolipomas (AMLs) have been reported in the liver for more than 20 years. In the kidney, five cases of malignant AML have been reported. We report the first case of malignant hepatic AML. METHODS AND RESULTS: A 70-year-old female patient presented with abdominal discomfort. Clinical examination revealed a palpable liver. CT scan showed a polymorphous hypervascular lesion in the right liver lobe. A biopsy was taken and resulted initially in a differential diagnosis between a hepatocellular carcinoma, a metastatic tumour (possibly of renal origin) and angiomyolipoma (AML). After immunohistochemistry, a hepatic AML was suggested, given the immunoreactivity for HMB45/NKIC-3. The mass was resected 5 years later because of relapsing abscess formation. Gross examination of the resection specimen showed a focally encapsulated brown mass with focal necrosis. Microscopic examination showed a tumour growing in sheets, separated by sinusoidal-like vessels. Most of the tumour cells had a large, polygonal, clear cytoplasm, often with eosinophilic condensation around the nucleus. There was prominent vascular invasion. Immunohistochemistry (reactivity for HMB-45, NKIC-3, S100 and alpha smooth muscle actin, negativity for cytokeratin and vimentin) and electron microscopy confirmed the diagnosis of monomorphic epithelioid AML with prominent vascular invasion. Seven months after tumour resection, the patient died of recurrent disease. CONCLUSIONS: This case highlights the importance of immunohistochemistry and electron microscopy in diagnosing this type of tumour. Possibly, in the past, malignant AML of the liver has been misdiagnosed as HCC.

Pericardial disease is often not recognised as a cause of chronic severe ascites.

BACKGROUND/AIMS: Severe chronic ascites remains a difficult diagnostic and therapeutic problem. Even in the current era, constrictive pericarditis is an underestimated and sometimes unrecognised cause. Moreover, missing the diagnosis deprives patients of remedial therapy. METHODS: Two cases of calcified constrictive pericarditis, complicated with cirrhosis and diagnosed in a late stage, are described. Due to insufficient clinical appreciation and lack of trust in echocardiography results, performed by cardiologists who were insufficiently familiar with the echocardiographic features of constrictive pericarditis, diagnosis was delayed in the two patients RESULTS: The diagnosis of constrictive pericarditis as a cause of ascites is based upon the clinical signs of right heart failure in a patient with normal systolic left and right ventricular function and a high, serumascitic albumin-content difference. Complementary workup with complete Doppler echocardiography study, right and left heart catheterisation and MRI or cine CT of the heart is necessary to confirm the diagnosis. CONCLUSION: Careful history taking and clinical examination remain the cornerstone of any diagnostic work-up, even in this era of technological refinement.

Synaptophysin: A novel marker for human and rat hepatic stellate cells.

Synaptophysin is a protein involved in neurotransmitter exocytosis and is a neuroendocrine marker. We studied synaptophysin immunohistochemical expression in 35 human liver specimens (normal and different pathological conditions), in rat models of galactosamine hepatitis and carbon tetrachloride-induced cirrhosis, and in freshly isolated rat stellate cells. Synaptophysin reactivity was present in perisinusoidal stellate cells in both human and rat normal liver biopsies. The number of synaptophysin-reactive perisinusoidal cells increased in pathological conditions. Double staining for alpha-smooth muscle actin and synaptophysin, detected by confocal laser scanning microscopy, unequivocally demonstrated colocalization of both markers in lobular stellate cells. In addition, freshly isolated rat stellate cells expressed synaptophysin mRNA (detected by polymerase chain reaction) and protein. Finally, electron microscopy showed the presence of small electron translucent vesicles, comparable to the synaptophysin-reactive synaptic vesicles in neurons, in stellate cell projections. We conclude that synaptophysin is a novel marker for quiescent as well as activated hepatic stellate cells. Together with the stellate cell's expression of neural cell adhesion molecule, glial fibrillary acidic protein, and nestin, this finding raises questions about its embryonic origin and its differentiation. In addition, the presence of synaptic vesicles in stellate cell processes suggests a hitherto unknown mechanism of interaction with neighboring cells.