Expression of apolipoprotein A-1 mRNA in normal intrahepatic biliary tree

Abstract: Our previous study demonstrated that apolipoprotein A-1 (apo A-1) immunoreactive peptides were located diffusely in the cytoplasm, not only of human normal hepatocytes, but also of intrahepatic bile ducts and peribiliary glands. It is important to determine whether the presence of these immunoreactive peptides in intrahepatic biliary tree is caused by pinocytosis from the bile, or by intracellular protein synthesis. Thus, we investigated whether apo A-1 is synthesized by cells that line the biliary tree. Normal human liver samples obtained at surgery were used, and the expression and distribution of apo A-1 mRNA in normal human liver tissues were examined, using in situ hybridization histochemistry with a ³⁵S-labeled oligonucleotide probe specific for apo A-1. On the autoradiogram, many silver grains were found to be distributed uniformly in hepatocytes. In addition, an appreciable apo A-1 mRNA signal was also observed in both the surface epithelial lining of the bile ducts and the epithelial cells of the peribiliary glands. In conclusion, these findings suggest that the apo A-1 found in bile is secreted both by hepatocytes and by intrahepatic bile duct cells and peribiliary glands.     

Secretory component and immunoglobulins in the intrahepatic biliary tree and peribiliary gland in normal livers and hepatolithiasis

Distribution of secretory component (SC), IgA, IgM and joining chain (J chain) in the intrahepatic biliary epithelium and peribiliary glandular epithelium were studied in normal livers and hepatolithiasis. In normal livers, SC was immunohistochemically demonstrated homogeneously in the cytoplasm of the biliary lining and peribiliary glandular epithelium. Immunoelectron-microscopically, SC was localized in the basolateral plasma membranes, nuclear membranes, rough endoplasmic reticulums, Golgi complexes and intracytoplasmic small vesicles, suggesting that the biliary lining and peribiliary glandular epithelium has an ability to synthesize SC. On the other hand, IgA and IgM were found heterogeneously in the biliary lining epithelium, and immunoelectron-microscopically they were only localized in the basolateral plasma membranes and vesicles. J chain was found in similar portions, suggesting that IgA and IgM in biliary lining and glandular epithelium were polymeric. It seems likely that polymeric IgA and IgM combine with SC on the biliary lining and glandular epithelial membranes, and subsequent endocytic transport and secretion of these complexes into the bile occur. In hepatolithiasis in which biliary living epithelia are hyperplastic and peribiliary glands proliferate markedly, such endocytic transport and secretion seemed to be increased. From these findings, it was suggested that the peribiliary glandular as well as biliary lining epithelium contributes to a local immunity in the biliary tree, especially in hepatolithiasis.     

Secretory component and immunoglobulins in the intrahepatic biliary tree and peribiliary gland in normal livers and hepatolithiasis

Distribution of secretory component (SC), IgA, IgM and joining chain (J chain) in the intrahepatic biliary epithelium and peribiliary glandular epithelium were studied in normal livers and hepatolithiasis. In normal livers, SC was immunohistochemically demonstrated homogeneously in the cytoplasm of the biliary lining and peribiliary glandular epithelium. Immunoelectron-microscopically, SC was localized in the basolateral plasma membranes, nuclear membranes, rough endoplasmic reticulums, Golgi complexes and intracytoplasmic small vesicles, suggesting that the biliary lining and peribiliary glandular epithelium has an ability to synthesize SC. On the other hand, IgA and IgM were found heterogeneously in the biliary lining epithelium, and immunoelectron-microscopically they were only localized in the basolateral plasma membranes and vesicles. J chain was found in similar portions, suggesting that IgA and IgM in biliary lining and glandular epithelium were polymeric. It seems likely that polymeric IgA and IgM combine with SC on the biliary lining and glandular epithelial membranes, and subsequent endocytic transport and secretion of these complexes into the bile occur. In hepatolithiasis in which biliary living epithelia are hyperplastic and peribiliary glands proliferate markedly, such endocytic transport and secretion seemed to be increased. From these findings, it was suggested that the peribiliary glandular as well as biliary lining epithelium contributes to a local immunity in the biliary tree, especially in hepatolithiasis.     

Blood group antigens in the intrahepatic biliary tree. I. Distribution in the normal liver

The distribution of six blood group-related antigens, A, B, H, Lea, Leb and sialylated Lea antigens, in the intrahepatic biliary tree was studied. These carbohydrate antigens in fixed normal liver tissues were immunostained with the use of highly specific monoclonal antibodies in an avidin-biotin-peroxidase complex method. Bile ducts expressed both ABH and Lewis blood group antigens. However, only Lewis blood group antigens could be detected in the bile ductules. Canaliculo-ductular junctions could be clearly delineated with the anti-Lewis blood group antibodies, especially with anti-Lea antibody. Small Lewis antigen-positive cells were scattered intralobularly. They were adjacent to parenchymal liver cells, mainly in Rappaport zone 1, and apparently in continuity with the portal biliary tree. Sialylated Lea antigen was found in some septal bile ducts. No blood group antigen could be detected in the bile canaliculi. These results indicate that (1) biliary tract of a given size has its own pattern of blood group antigen expression, and (2) biliary epithelial cells are not identical with regard to the phenotypic expression of their structural carbohydrates. In future, it will be possible to classify biliary epithelial cells by their blood group antigen expression.     

Development of the intrahepatic biliary tree

The liver develops from two anlages: the hepatic diverticulum, which buds off the ventral side of the foregut, and the septum transversum, which is the mesenchymal plate that partially separates the embryonic thoracic and abdominal cavities. The endodermal cells of the hepatic diverticulum invade the septum transversum, forming sheets and cords of hepatoblasts arrayed along the sinusoidal vascular channels derived from the vitelline veins emanating from the yolk sac. The vitelline veins fuse to form the portal vein, which ramifies as tributaries within the liver along mesenchymal channels termed portal tracts. Those hepatoblasts immediately adjacent to the mesenchyme of the portal tracts differentiate into a ductal plate, a single circumferential layer of biliary epithelial cells. Mesenchymal cells interpose between the ductal plate and the remaining parenchymal hepatoblasts, which differentiate into hepatocytes. By week 7 the ductal plate begins to reduplicate, forming a double layer of cells around the portal tract. Lumena form between the two cell layers of the ductal plate, forming peripheral biliary tubular structures. These peripheral tubules remodel and, with continued proliferation of the mesenchyme, by the 11th week begin to become more centrally located within portal tracts as terminal bile ducts with a circular cross-section. The remaining ductal plate resorbs, leaving behind only tethers of bile ductules connecting the terminal bile ducts to the parenchyma. Abutting and within the parenchyma are the canals of Hering, ductular structures half-lined by hepatocytes and half-lined by biliary epithelial cells. Maturation of the intrahepatic biliary tree to the mature tubular treelike architecture occurs from the hilum of the liver outward, beginning around the 11th week of gestation and continuing past birth for several months. The architecture of maturation is the same regardless of gestational age or radial location in the liver. Importantly, the immature intrahepatic biliary system maintains patency and continuity with the extrahepatic biliary tree throughout gestation, with no evidence of a solid phase of development. Thus, from the earliest time of hepatocellular bile formation beginning around the 12th week, there is a patent passage to the alimentary canal.     

Dynamics of the vascular profile of the finer branches of the biliary tree in normal and diseased human livers

BACKGROUND/AIMS: Results of our previous studies supported the concept that in the human liver, the smallest ramification of the biliary tree, the bile ductules, might contain hepatic progenitor cells. An insufficient proliferative response and loss of bile ductules preceded bile duct loss whereas preservation of bile ductules mitigated bile duct loss. METHODS: Presently we investigated the vascular profile of the bile ductules in diseased human livers and livers showing normal histological features as controls, using CD34, CK7 and alphaSMA antibodies in a double immunolabeling technique. VEGF-A expression was also studied. In control livers bile ductules traversed the boundaries of the portal tract into the lobule as ductular-vascular units, in a pattern outlining the classic hexagonal lobule, following the vascular septa. The latter are thought to be extensions of portal veins. In diseased states the two structures reacted in unison. Increased or decreased numbers of ductules were consistently accompanied by similar changes of accompanying microvessels. Increased numbers of ductules and microvessels were paralleled by increased ductular expression of VEGF-A. RESULTS: Our data support the concept that the smallest branches of the biliary tree might have their own vascular supply and that the ductules might in turn maintain their vasculature during regenerative processes.     

Blood group antigens in the intrahepatic biliary tree. II. Type 1 chain N-acetyllactosamine-related carbohydrate antigens in the proliferated bile ductules

The expression of the carbohydrate antigens related to type 1 chain N-acetyllactosamine (1NAcLc) in the proliferated bile ductules was immunohistochemically examined in liver tissues of 10 cases of chronic hepatitis (CH), 9 of liver cirrhosis (LC) and 8 of alcoholic hepatitis. The ductular expression of the examined blood group antigens was essentially the same among these pathological conditions. The backbone structure, i.e., 1NAcLc (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----3R), was not detected in the proliferated ductules as in the normal bile ductules. Its fucosylated structures (Le(a), Le(b) and type 1 chain H) were more strongly expressed in the proliferated ductules than in the normal ductules. Sialyl-Le(a) which was not found in the normal ductules was detected weakly but definitely in the proliferated ductules. Besides proliferated ductules, single or small numbers of epithelial cells expressing the 1NAcLc-related antigens were found intralobularly. This suggests possible migration of biliary epithelial cells into the hepatic lobules. In conclusion, within the proliferated bile ductules (a) synthesis of the 1NAcLc-related antigens is increased compared to the normal ductules, (b) the backbone structure is completely sialylated or fucosylated as in the normal ductules and (c) alpha 1----4fucosylation of sialyl-1NAcLc, i.e., sialyl-Lea, formation occurs despite its absence in the normal ductules.     

Ultrastructural and immunohistochemical studies of the intrahepatic peribiliary capillary plexus in normal livers and extrahepatic biliary obstruction in human beings.

Ultrastructural and immunohistochemical study was conducted on the intrahepatic peribiliary capillary plexus in normal livers and in those with extrahepatic biliary obstruction. In both conditions, capillaries positive for Ulex europaeus agglutinin I and type IV collagen were always present in the vicinity of the bile ducts. Immunoelectron microscopy showed the presence of type IV collagen on the basal lamina of these capillaries; Ulex europaeus agglutinin I was also positive on their cytoplasms. Under electron microscope, a considerable number of these capillaries were seen as being composed of fenestrated endothelium with a diaphragm and with extreme cytoplasmic attenuations that were dense at the sides facing the bile duct in comparison with the opposite sides in normal livers. In extrahepatic biliary obstruction, plasmalemmal pinocytic vesicles, multivesicular bodies and other cellular organellae such as rough endoplasmic reticulum and Weibel-Palade bodies increased in number in these capillaries, relative to normal livers, probably reflecting increased permeability and functional activities. These characteristic ultrastructural features of the peribiliary capillary plexus might be arranged to transport substances effectively by way of intrahepatic biliary epithelial cells in normal livers and also might be altered to meet the increased functional demands of extrahepatic biliary obstruction.     

Protein expression of double-stranded RNA-activated protein kinase (PKR) in intrahepatic bile ducts in normal adult livers, fetal livers, primary biliary cirrhosis, hepatolithiasis and intrahepatic cholangiocarcinoma

BACKGROUND/AIM: The protein expression of double-stranded RNA-activated protein kinase (PKR) in intrahepatic bile ducts has not been investigated. METHODS: Immunohistochemistry and a semiquantitative scoring method in normal liver and biliary diseases were used for the investigation. RESULTS: In "normal" adult livers (n=10), intrahepatic bile ducts were negative for PKR. In normal fetal livers (n=25), primitive biliary epithelia were almost negative for PKR. In primary biliary cirrhosis (PBC) (n=30), damaged bile ducts were frequently positive for PKR, while uninvolved bile ducts were negative. In hepatolithiasis (n=27), proliferated bile ducts were positive for PKR, and the PKR score correlated with the degree of proliferation. In cholangiocarcinoma (CC) (n=44), PKR expression was frequently noted, and the PKR score correlated with good differentiation of CC, being highest in well-differentiated CC and lowest in poorly-differentiated CC. The PKR score decreased in the following order: CC (mean PKR score=3.96), hepatolithiasis (2.56), PBC (1.60), normal fetal liver (0.40), and normal adult livers (0.00). The PKR expression in hepatocytes was "baseline" in normal adult livers, while moderately increased in fetal livers, PBC, hepatolithiasis and CC. CONCLUSIONS: Although the significance of these data is unclear, they suggest (i) that PKR is absent in bile ducts in normal adult and fetal livers, (ii) that PKR in bile duct cells newly emerges or increases in PBC, hepatolithiasis, and CC, (iii) that PKR accumulates in damaged bile ducts in PBC, (iv) that PKR increases in parallel with biliary cell proliferation in hepatolithiasis, and (v) that PKR expression correlates with differentiation in CC. PKR expression in intrahepatic bile ducts seems to be associated with inflammation or cell proliferation of the bile duct cells.     

Blood group antigens in the intrahepatic biliary tree : II. Type 1 chain N-acetyllactosarnine-related carbohydrate antigens in the proliferated bile ductules

The expression of the carbohydrate antigens related to type 1 chain N-acetyllactosamine (1NAcLc) in the proliferated bile ductules was immunohistochemically examined in liver tissues of 10 cases of chronic hepatitis (CH), 9 of liver cirrhosis (LC) and 8 of alcoholic hepatitis. The ductular expression of the examined blood group antigens was essentially the same among these pathological conditions. The backbone structure, i.e., 1NAcLc (Galβ1 → 3GlcNAcβ1 → 3Galβ1 → 3R), was not detected in the proliferated ductules as in the normal bile ductules. Its fucosylated structures (Le^a, Le^b and type 1 chain H) were more strongly expressed in the proliferated ductules than in the normal ductules. Sialyl-Le^a which was not found in the normal ductules was detected weakly but definitely in the proliferated ductules. Besides proliferated ductules, single or small numbers of epithelial cells expressing the 1NAcLc-related antigens were found intralobularly. This suggests possible migration of biliary epithelial cells into the hepatic lobules. In conclusion, within the proliferated bile ductules (a) synthesis of the 1NAcLc-related antigens is increased compared to the normal ductules, (b) the backbone structure is completely sialylated or fucosylated as in the normal ductules and (c) α1 → 4fucosylation of sialyl-1NAcLc, i.e., sialylLe^a, formation occurs despite its absence in the normal ductules.     

Carcinoembryonic antigen and blood group-related carbohydrate antigens in glycoproteins in human bile in hepatolithiasi

We investigated whether carbohydrate antigens on biliary glycoproteins and carcinoembryonic antigen (CEA) are related to hepatolithiasis. CEA, ABO, and Lewis blood group-related antigens, as well as sialyl-Tn antigen in hepatic bile, were analyzed by Western blotting in samples from 12 patients with hepatolithiasis and 37 with other biliary diseases (choledocholithiasis,13; cholecystolithiasis, 5; acute cholecystitis, 2; cholangiocarcinoma, 5; common bile duct carcinoma, 4; pancreatic carcinoma, 6; and metastatic carcinoma of liver, 2). CEA was positive on mucinous glycoprotein in six patients (50%) with hepatolithiasis and one case (17%) with pancreatic carcinoma. CEA was also positive on a glycoprotein of approximately 200 kd in eight patients (67%) with hepatolithiasis and two (33%) with pancreatic carcinoma. Lewis X was detected on the mucinous glycoprotein in almost all samples, as well as on glycoproteins of approximately 180 kd in all hepatolithiasis samples and approximately half of those from patients with other diseases. Sialyl-Tn antigen was detected on mucinous glycoprotein in four (80%) with cholangiocarcinoma, two (50%) with common bile duct carcinoma, and all pancreatic carcinoma samples. Mucinous glycoprotein and glycoproteins containing CEA and Lewis X antigens are enriched in hepatic bile of hepatolithiasis, and they may be closely related to the formation of intrahepatic calculi. Sialyl-Tn antigen in biliary mucinous glycoprotein will be a good marker of biliary and pancreatic carcinoma, and probably for cholangiocarcinoma complicated with hepatolithiasis. (Hepatology 1996 Feb;23(2):258-63)     

Histological and ultrastructural examination of the intrahepatic biliary tree in primary sclerosing cholangitis

Intrahepatic bile ducts were examined histologically and ultrastructurally in wedge-biopsied liver specimens from three patients with primary sclerosing cholangitis. Bile ducts with periductal concentric fibrosis, which is a characteristic finding in primary sclerosing cholangitis, revealed ultrastructurally finger-like projections or fine undulations of the basal free surfaces with markedly duplicated basal lamina. The lamina was collared by a layer of elongated fibroblasts and thickened bundles of collagen fibers outwards. These changes were consistently found in all sizes of ducts examined, and might be related to progressive periductal fibrosis. Serial section observations showed that some severely affected ducts actually disappeared when accompanying severe periductal fibrosis. It would therefore appear that progressive periductal fibrosis may interrupt fluid and nutrient exchange between the bile duct epithelia and peribiliary capillary plexus, followed by obliteration of the biliary lumina. Although the bile ducts showed segmentally periductal lymphocytic infiltration and, ultrastructurally, point contacts between infiltrating lymphocytes and biliary epithelial cells were observed occasionally, the exact role of infiltrating lymphocytes in the pathogenesis of primary sclerosing cholangitis remains unclear.     

Primary biliary cystadenocarcinoma perforating the duodenum and left intrahepatic biliary tree -mimicking a hydatid cyst

Abstract: We report the case of a 76-year-old woman with biliary cystadenocarcinoma perforating the left biliary tree and exhibiting intra-tumoral gas bubbles resulting from invasion of the duodenum. The clinical history included subfebrile temperatures of 3 months duration, and pains associated with an abdominal mass in the right upper quadrant. Blood tests showed leucocytosis, and radiological studies revealed the features of a partially calcified septated tumor with nodular components combined with multiple gas-fluid levels, mimicking an infected hydatid cyst. Intraoperative ultrasonography, cholangiography and frozen section histology were necessary to prove the malignant nature of this cystic tumor. Provided that complete resection with strict adherence to oncological precepts is possible, the prognosis of cystadenocarcinoma is better than in hepatocellular or cholangiocellular carcinoma.     

Expression of anti-OV6 antibody and anti-N-CAM antibody along the biliary line of normal and diseased human livers

Following hepatic injury, proliferation of anastomosing ductules can be observed. The origin of this ductular reaction is not completely clear, although there is considerable evidence for proliferation of a putative hepatic progenitor cell, reported to be located in the canals of Hering (CoH) and showing morphologic similarities with rat oval cells. In this study, we analyzed the immunophenotype of solitary oval cell-like cells (SOC), intralobular groups of cuboidal cells that might represent lining cells of CoH, bile ductular cells (BDC), bile duct epithelial cells (BEC), and hepatocytes. We used the antibodies OV6, CK19, and CD56 (NCAM) in a double-staining method in a series of 111 liver specimens. The series consisted of a variety of liver diseases, primary liver tumors, and normal livers. In normal livers, SOC, CoH, BDC, and BEC were uniformly and predominantly CK19+, OV6+, and CD56-. In diseased livers SOC and BDC were CK19+, OV6+, and also CD56+. Occasionally, BEC was CD56+ in damaged bile ducts in diseased liver, e.g., PSC. CoH lining cells were not present in cirrhotic nodules and were indistinguishable from BDC in the fibrous septa. The consistent and uniform staining patterns of SOC, CoH, and BDC support the concept that these cells share the same biliary lineage and might represent one biliary structure. The expression of CD56 on these cells in diseased livers indicates that CD56 is a useful marker for a reparative or regenerative state of the biliary liver-cell constituents but not to discriminate a putative hepatic stem cell.     

Vascular plexus around intrahepatic bile ducts in normal livers and portal hypertension

Vessels around the intrahepatic large bile ducts (peribiliary vascular plexus) were examined by histologic, immunohistochemical and scanning electron microscopic observations. The vessels within duct walls were mainly capillaries, while those around the duct walls were composed of capillaries and venules. A majority of vessels was positive for factor VIII-related antigen and Ulex europaeus lectin I. Scanning electron microscopy of hepatic arterial and biliary casts revealed that bile ducts were surrounded by the vascular plexus derived from hepatic arterial branches, and serial section observations in addition disclosed the vessels connecting the peribiliary plexus with portal venous branches ('internal roots'). The peribiliary vascular plexus was increased considerably in livers with portal hypertension, especially idiopathic portal hypertension, extrahepatic portal venous obstruction and hepatocellular carcinoma with portal venous tumor thrombi. Internal roots were also frequently found in the livers with portal hypertension. These results suggest that altered intrahepatic hemodynamics in portal hypertensive conditions involves the peribiliary vascular plexus, resulting in an increase of the number and frequent occurrence of 'internal roots', these vessels probably operating as intrahepatic collaterals.     

Innervation of intrahepatic bile ducts and peribiliary glands in normal human livers, extrahepatic biliary obstruction and hepatolithiasis. An immunohistochemical study

Innervation of the intrahepatic biliary tree was examined in human normal livers, extrahepatic biliary obstruction and hepatolithiasis. Nerve fibers were immunohistochemically identified on formalin-fixed and paraffin-embedded sections by antibodies to S-100 protein (S-100) and neuron specific enolase (NSE). S-100 and NSE-immunoreactive nerve fibers were present in the walls of intrahepatic large, medium-sized and septal bile ducts as well as in peribiliary glands. Some nerve fibers were in close contact with epithelia of bile ducts and peribiliary glands. Serial section observations showed that the nerve fibers arising from nerve bundles approached, and came to lie in close contact with epithelia of the bile ducts and peribiliary glands. Nerve fibers were sparse around the interlobular bile ducts and bile ductules. These immunoreactive nerve fibers of the intrahepatic biliary tree were rather sparse in normal livers, intermediate in extrahepatic biliary obstruction and dense in hepatolithiasis. These findings suggest that intrahepatic bile ducts and peribiliary glands are innervated and biliary functions are regulated in part by these nerve fibers. Increased nerve fibers may have altered effects on biliary functions in hepatolithiasis.