Multifocal granular cell tumour of the biliary tree.

Fewer than 90 cases of granular cell tumour (GCT) of the biliary tract have been reported, including only five cases of multiple GCTs. We present the unusual case of a 40-year-old woman with multifocal GCTs affecting the intrahepatic biliary tree, which were initially suspected to be hepatic multiple metastases from a malignancy of unknown origin. The surgical specimen consisted of a hepatic segment in which five whitish nodular lesions were observed. On microscopic examination, nodular lesions were found in the portal tracts; these were composed of large polygonal cells with abundant highly granular cytoplasm. The nuclei were small and centrally located. The tumour cells tested diffusely positive for CD68-PGM1, S100 protein and α-inhibin, so a diagnosis of multifocal GCT of the biliary tree was made. Three years later, the patient is still alive and a MRI has shown no changes.

A new clarification method to visualize biliary degeneration during liver metamorphosis in Sea Lamprey (Petromyzon marinus).

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey (Petromyzon marinus) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called "CLARITY" was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.

Pediatric biliary interventions.

The most common indication for percutaneous biliary evaluation and intervention in children is for the diagnosis and treatment of liver transplant complications, including strictures and bile leaks. Because liver transplants in children are commonly performed using a Roux-en-Y biliary-enteric anastomosis, endoscopic retrograde cholangiopancreatography is not technically possible; therefore, the first-line procedure for evaluation and treatment of biliary obstruction in this population is percutaneous transhepatic cholangiography (PTC). Percutaneous biliary intervention can be challenging in these patients, because ductal dilation may be minimal or altogether absent in pediatric transplant livers even in the setting of severe obstruction. However, with proper technique, including the use of ultrasound guidance, technical success rates for PTC and biliary drainage can be similar to those in adults. Biliary drainage and biliary stenosis management is a long-term commitment that usually takes several months to more than a year and may require multiple repeat cholangioplasties and biliary drainage catheter exchanges. Due to its minimally invasive nature and relatively low morbidity and mortality compared with open surgical alternatives, percutaneous biliary intervention should be considered the first-line treatment option in children with biliary stenosis who have had previous liver transplant, and for those nontransplant patients who cannot be treated endoscopically.

Crystalline inclusions in hepatocytes and associated interhepatocytic macrophages from female Ohrid trout (Salmo letnica Kar.).

When characterizing the liver ultrastructure in Ohrid trout, we noticed that cells often displayed unusual cytoplasmic crystalline inclusions. Their morphology varied much, concerning the size, shape and electron density, showing aspects never reported in fish liver. In hepatocytes, the inclusions existed in close topographical relationship with dense bodies (often within them). Such "crystals" inclusions also existed in macrophages and in biliary passages lumina. Data revealed seasonal/breeding-related changes of the dense bodies and crystalline inclusions within hepatocytes; decreasing from the earliest to the more advanced ovary maturation stages. Additionally, based on the negative correlations between the amounts of dense bodies and of "crystals" and the ovary somatic ratio, we suggested there might be connections between the sex steroids status and the inclusions formation. A positive correlation between the quantities of "crystals" and of dense bodies further suggested that the inclusions might derive from normal processes, e.g., the females' cyclic liver changes, that involves dense bodies' turnover. However, a toxicological aetiology cannot be excluded. Additionally, multiple mechanisms can contribute to the "crystals" genesis. Facing literature reports and because the inclusions content seemed washed out during tissue processing, they are likely lipid in nature, but their exact composition and genesis require further analyses.

An in vivo look at vertebrate liver architecture: three-dimensional reconstructions from medaka (Oryzias latipes).

Understanding three-dimensional (3D) hepatobiliary architecture is fundamental to elucidating structure/function relationships relevant to hepatobiliary metabolism, transport, and toxicity. To date, factual information on vertebrate liver architecture in 3 dimensions has remained limited. Applying noninvasive in vivo imaging to a living small fish animal model we elucidated, and present here, the 3D architecture of this lower vertebrate liver. Our investigations show that hepatobiliary architecture in medaka is based on a polyhedral (hexagonal) structural motif, that the intrahepatic biliary system is an interconnected network of canaliculi and bile-preductules, and that parenchymal architecture in this lower vertebrate is more related to that of the mammalian liver than previously believed. The in vivo findings presented advance our comparative 3D understanding of vertebrate liver structure/function, help clarify previous discrepancies among vertebrate liver conceptual models, and pose interesting questions regarding the "functional unit" of the vertebrate liver.

Morphological and histological study of the ostrich (Struthio Camelus L.) liver and biliary system.

The peculiarity of the digestive system of the ostrich (Struthio Camelus L.), which is characterized by the continuous production of bile, led us to undertake macroscopical and histological studies of the liver and its biliary system, since very little bibliographic data exist on the subject. For this purpose we observed the organs of male and female ostriches 16-18 months of age, in situ, in order to describe their location, relationships and morphology. Samples of the liver were processed for observation by light microscopy; samples of the hepatoenteric duct were processed for observation by light and electron microscopy. Our findings regarding the liver revealed the presence of two lobes: a left lobe, subdivided into three lobes, and a right undivided lobe. There was no gall-bladder. The histological picture showed unlimited hepatic lobules, with hepatocytes arranged in cord-like fashion two cells thick. A large hepatoenteric duct arose from the porta hepatis, and opened into a papilla in the descending limb of the duodenum. The mucosa of the duct was lined by simple columnar epithelium consisting of cells having the same morphological cytoplasmatic features but distinguished by either a light or a dark nucleus.

Hepatic differentiation of mouse ES cells into BE cells in vitro.

To date, the hepatic differentiation of embryonic stem (ES) cells into biliary epithelial (BE) cells has only been identified in hepatocytes. In this study, an attempt was made to induce the differentiation of ES cells to BE cells. In order to induce hepatic, and then BE cell differentiation, growth factors such as TGF, FGF, HGF and EGF were added to the culture medium supplied to embryonic bodies (EBs) that were derived from ES cells. The marker genes and corresponding proteins of hepatocytes and BE cells such as AFP, ALB, CK8, CK18, CK7, CK19 and GGT, etc. were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), immunocytochemistry (ICC) and enzymatic histochemistry. Lastly, the ratio of BE-like cells to all EBs cells was analyzed and determined by flow cytometry (FCM). Hepatocyte and BE cell marker genes and proteins were found to be expressed in the cytoplasm of differentiated cells. On day 10 of differentiation, many round structures appeared in the EBs culture system and the marker proteins of BE cells were found to be expressed in these structures. The BE cell differentiation ratio continually increased from its initial value of 1.7% on day 13 to 7.4% on day 21. ES cells were found to be able to differentiate into BE cells when cultured using medium with appropriate cell growth factors. These BE cells may be a novel source of differentiated cell types for liver engineering.

Morphological features and modulation of the intrahepatic biliary epithelium.

Much has been learned in the past few years concerning the morphology and function of the intrahepatic biliary epithelium. Immunohistochemistry, together with ultrastructural studies has allowed a better identification or the smallest branches of the biliary tree and of subcellular components (cytoskeleton, specializations of the cell membrane, specific receptors). Modulation of the biliary epithelium in response to physiological or pathological stimuli has renewed the interest concerning the existence of facultative stem cells in the liver. More information is needed however concerning the mechanisms of cell loss in vanishing bile duct syndromes.

Biliary tract of the rat as observed by scanning electron microscopy of cast samples.

The three-dimensional distribution of the biliary tract in the rat was studied by scanning electron microscopy of biliary casts. The casts were prepared by a retrograde infusion of a low viscosity or monomeric methacrylate resin mixture into the common bile duct. No resin flow from the bile canaliculi to sinusoidal capillaries was ever noted. Bile canaliculi formed intricate meshworks and drained via the Hering's canals into the bile ductules. The bile canalicular meshworks of adjacent lobules intercommunicated with each other. The bile ductules formed a marked periportal plexus around the portal vein branch, and drained into the intrahepatic bile duct running along the portal vein branch. The junctional zone of the Hering's canal and bile ductule usually showed an ampullary dilation. When the Hering's canal directly drained into a thick bile ductule or into a periportal plexus of bile ductules, such an ampullary dilation at the origin of the bile ductule was never replicated. The extrahepatic bile duct protruded many crypt-like projections which presumably corresponded to parietal glands. It is suggested that the periportal plexus of bile ductules may store the bile as a substitute for the gallbladder.

La gammagrafía hepatobiliar en el diagnóstico diferencial de la atresia de vías biliares / Hepatobiliary scinatigraphy in the differential diagnosis of biliary atresia and neonatal hepatitis

La gammagrafía hepatobiliar es capaz de diferenciar entre atresia de vías biliares y hepatitis neonatal si el procedimiento se realiza cuando aún el daño hepatocelular del paciente no es grave. La gammagrafía, el ultrasonido y la biopsia hepática son todos métodos complementarios con indicaciones y limitaciones, por lo que deben ser empleados en conjunto y en el orden adecuado para establecer un diagnóstico temprano que repercuta en el tratamiento oportuno de estos pacientes.

Wholesale hepatocytic differentiation in the rat from ductular oval cells, the progeny of biliary stem cells.

BACKGROUND/AIMS: Biliary epithelial cells (ductular oval cells) migrate into the periportal and midzonal parenchyma when hepatocyte regeneration after injury is significantly impeded. The potential of oval cells to differentiate into hepatocytes has been questioned. We have sought to resolve this issue using the modified Solt-Farber procedure in which 2-acetylaminofluorene is used to block hepatocyte regeneration in partially hepatectomized rats. METHODS: Rats received 2-acetylaminofluorene by oral gavage for 6 days before and up to 7 days after a two-thirds hepatectomy. The cellular reaction was visualized by the immunohistochemical localization of intermediate filaments cytokeratins 8 and 19 and vimentin, cytochrome P450 enzymatic proteins and alpha-foetoprotein. Expression of albumin and alpha-foetoprotein mRNA transcripts were observed in situ using antisense riboprobes. RESULTS: During the first 9 days after partial hepatectomy long strings of ductular cells spread outwards from the portal areas. These cells exhibited strong diffuse cytoplasmic staining with the anticytokeratin 8 and 19 antibodies, like authentic bile ducts, but in addition also expressed vimentin and alpha-foetoprotein (protein and mRNA)-collectively termed the "oval cell phenotype". Thereafter, these ducts rapidly vanished to be replaced by basophilic hepatocytes which lacked the oval cell phenotype, but which acquired strong expression of albumin mRNA. At 14 days after partial hepatectomy the oval cell phenotype was restricted to the peripheral margins of the newborn periportal hepatocytes, the distal tips of the oval cell ducts, and these too had disappeared within another 7 days. CONCLUSIONS: Ductular oval cells will differentiate into hepatocytes under appropriate experimental conditions.

New insights into the pathogenesis of copper toxicosis in Wilson's disease: evidence for copper incorporation and defective canalicular transport of caeruloplasmin.

Previous studies have suggested that copper is incompletely incorporated into caeruloplasmin, the major plasma form of copper-transporting protein, in the genetic copper toxic condition, Wilson's disease. In this paper we have investigated the role of copper and caeruloplasmin in the abnormal biliary copper transport and characterizes Wilson's disease. Using SDS/PAGE and Western blotting, we have demonstrated the presence of holocaeruloplasmin in liver samples from patients with Wilson's disease (abnormal biliary copper excretion) and in control patients (normal biliary copper excretion). The presence of holocaeruloplasmin was also confirmed by measurement of caeruloplasmin oxidase activity using staining with o'Dianisidine. In contrast with the findings in liver tissue, holocaeruloplasmin was absent from bile from patients with Wilson's disease, but as expected it was present in the bile from control subjects. We have also identified and partially characterized a 189-200 kDa protein from purified human biliary canalicular membranes which binds copper and possesses caeruloplasmin-like activity when probed with a specific human anti-caeruloplasmin antibody. In conclusion, we have demonstrated that copper incorporation in caeruloplasmin is normal in patients with Wilson's disease contrary to previous reports. Secondly, we have shown that the defect in Wilson's disease appears to lie in the biliary canalicular excretion of holocaeruloplasmin resulting in its retention within the hepatocyte causing copper toxicosis. Finally we have identified and partially characterized a caeruloplasmin-binding protein from biliary canalicular membranes which may provide a link to the gene defect in Wilson's disease.

The liver of the brown trout, Salmo trutta fario: a light and electron microscope study.

A qualitative study by light and electron microscopy was undertaken on the liver of the brown trout, Salmo trutta fario. Vessels and bile ducts were observed to be scattered without any apparent order within the parenchyma. Venous profiles appeared either isolated or included in 'venous-arteriolar tracts' (VAT) and 'venous-biliary-arteriolar tracts' (VBAT). Bile ducts also appeared either isolated or in groups which often included an arteriole. The parenchyma was organised in tubules of hepatocytes encircling biliary passages radially. Those cells were uninucleate and contained large cytoplasmic areas of rough endoplasmic reticulum; lipid droplets and dense bodies sometimes also occupied a considerable portion of the cytoplasm. Microvilli extended from hepatocytes into biliary passages and towards the space of Disse. Other cell types encountered comprised biliary epithelial cells, macrophages (including melanin laden cells), fat-storing cells and endothelial cells. The biliary tree was formed sequentially by intra- and intercellular canaliculi, preductules, ductules and ducts. Canaliculi without microvilli are described for the first time in fishes. Structural differences between the brown trout and other fishes were noted. In contradistinction to other fishes, in brown trout the triads (i.e. the VBAT) are not just occasional structures; also, they probably transmit portal veins. Our observations support the concept of a tubular arrangement of hepatocytes in fish. The possibility that the axis of the tubule may be a sinusoid instead of a biliary passage is questioned. Homology between, on one hand, the segment formed by preductules and ductules and, on the other, the canal of Hering of mammals is defended. It is concluded that among salmonids notable interspecific differences do not seem to exist.

Renal and biliary abnormalities in a new murine model of autosomal recessive polycystic kidney disease.

Current models of autosomal recessive polycystic kidney disease (ARPKD) fail to demonstrate biliary abnormalities in association with renal cysts. We therefore studied a new murine model of ARPKD in which dual renal tubular and biliary epithelial abnormalities are present. Affected homozygous animals typically die 1 month postnatally in renal failure with progressively enlarged kidneys. Renal cysts shift in site from inner cortical proximal tubules at birth to collecting tubules 20 days later, as determined by segment-specific lectin binding. Increased numbers of mitosis were demonstrated in proximal and collecting tubular cysts. In addition, epithelial hyperplasia was demonstrated morphometrically in the intra- and extrahepatic biliary tract of affected animals. The number of intrahepatic biliary epithelial cells was increased by 50% on postnatal day 5 and by 100% on postnatal day 25 (P < 0.01). Despite an increased frequency of "chaotic" portal areas in mice with renal cysts, no intrahepatic cysts or shape abnormalities of the biliary lumen were detected using biliary casts and morphometry. Additionally there was nonobstructive hyperplastic dilatation of the extrahepatic biliary tract which was linked in all animals to the presence of renal cysts. The hyperplastic abnormalities in both renal and biliary epithelium make this new mouse strain a good model for the study of the dual organ cellular pathophysiology of ARPKD.

Three-dimensional fine structure of the biliary tract: scanning electron microscopy of biliary casts.

The three-dimensional structure of the biliary tract was studied by scanning electron microscopy (SEM) of biliary casts. The replica of the biliary tract was successfully prepared by retrograde injection of low viscosity resin into the common bile duct. Bile canaliculi are intricate networks in which hexagonal and pentagonal meshworks are interconnected. Each hexagonal or pentagonal meshwork is on a plane, but adjoining meshworks are on different planes. Bile canalicular networks connect with bile ductules at the periphery of the portal tract. The intrahepatic bile duct showed considerable interspecies variation. The human bile duct has plexiform side branches and periductal sacculi, which are most numerous near the liver hilum and fewest in the smaller portal tracts. The hilar plexus and sacculi are present on opposite sides of the bile duct. The plexus formed at the bifurcation of the bile ducts exhibits a plane. Periductal sacculi were also observed in the monkey and pig bile ducts, particularly the latter, while rat bile ducts possess a peculiar portal bile ductular plexus situated between the portal tract and the surrounding liver parenchyma. No such structures were observed in either the dog or rabbit bile ducts. SEM of the biliary casts showed that the biliary tract was not a simple draining tube but had additional structures, such as periductal sacculi and plexiform side branches. These structures, together with the peribiliary vascular plexus, may be implicated in the modification of bile.

The ultrastructure and histology of cholangiocellular carcinomas in English sole (Parophrys vetulus) from Puget Sound, Washington.

The ultrastructure and histology of cholangiocellular carcinomas from feral English sole (Parophyrs vetulus) living in polluted waterways of Puget Sound, WA. are described. Electron microscopy confirmed that biliary epithelial cells were the main proliferative cell type composing this variety of neoplasm. The arrangement of these cells varied from well-organized multiple bile duct-like structures to disorganized multilayered sheets of poorly differentiated biliary epithelial cells. A fibrous stroma consisting of multiple layers of collagen fibers and fibroblasts, with macrophages and various blood cell types scattered among these layers occurred between bile duct-like structures or aggregates of biliary epithelial cells. Hepatocytes were not apparent in these neoplasms except within small necrotic regions surrounded by neoplastic biliary epithelial cells. No virus-like particles were observed among the cases examined in this study.

Immunohistochemical localization of proteoglycans in interstitial elements of human pancreas and biliary system.

The immunohistochemical localization of large proteoglycan and small proteoglycan was observed, using antibodies 2B1 and 6B6 (Sobue et al., 1988, 1989a), in fetal and adult pancreas and biliary system as well as in tumour tissues, obtained from 11 autopsies and 74 biopsies. The distribution of chondroitin 4- and 6-sulphate side chains, type I and IV collagen and elastin were also studied. In adult pancreas and all the biliary tracts examined, periductal fibrous tissues consisted mainly of dermatan sulphate small proteoglycan with networks of fibrous elements, which were composed of large proteoglycan, elastin, type I collagen and type IV collagen. In the interstitial components of cystadenoma of pancreas and biliary duct carcinoma, similar small proteoglycan-rich components were relatively abundant, although large proteoglycan was present in much larger amounts than that in non-neoplastic adult tissues. In some cholangiomas, the extra- and intracellular hyaline globules formed by the carcinoma cells were found to contain chondroitin sulphate large proteoglycan, laminin and fibronectin. The distribution of proteoglycans was observed to be different in the arterial walls of the interlobular tissues of the adult and the fetal pancreas. The biological significance of large and small proteoglycans in the interstitial connective tissues was discussed.

Three-dimensional arrangement of ductular structures formed by oval cells during hepatocarcinogenesis.

The three-dimensional arrangement of ductular structures formed by oval cells in rats fed 2-acetylaminofluorene (2-AAF) was studied by scanning electron microscopy (SEM) of biliary tract casts and light microscopy of sections of liver injected with india ink via the biliary tract. Both resin and india ink were well injected up to bile ductules, and the findings of each method correlated with each other. By the second week after 2-AAF administration, a few oval cells appeared in the periportal areas forming ductular structures which connected with the portal bile ducts. At the 4th week, increased ductular structures occupied two thirds of the lobule and formed networks communicating with each other, and with the portal bile ducts. At the 8th week, such ductular structures were compressed around hyperplastic nodules and appeared like a basket in biliary casts examined by SEM. Although a histochemical study of gamma-glutamyl transpeptidase revealed activity both on the luminal side of the ductular structures and hepatocytes in hyperplastic nodules, no transition was observed between these two cell populations. These results suggest that oval cells have characteristics more similar to those of biliary epithelia than of hepatocytes, and have no relation to the development of hyperplastic nodules.

Early ultrastructural changes in the biliary epithelial cells of BALB/c and DDY mice immunized with swine serum.

Electron microscopic observations were carried out on the biliary epithelial cells of BALB/c and DDY mice which had received an intraperitoneal injection of 0.2 ml of swine serum twice a week for 2 or 4 weeks. The most characteristic feature of the biliary epithelial cells of BALB/c mice was a marked increase in the number of vesicles having a close spatial relationship with the well-developed Golgi apparatus or rough endoplasmic reticulum (rER). In contrast, marked dilation of rER filled with moderately electron-dense material was conspicuous in the biliary epithelial cells of DDY mice. A prominent increase in the number of blebs and lateral and basal cytoplasmic protrusions in the dilated intercellular space of the biliary epithelium, and submucosal eosinophil infiltration, collagen fiber proliferation and gland hyperplasia with increased mucin secretion were common to both strains. This experimental model of bile duct disease also seems to be useful for investigating alteration of protein synthesis and secretion in epithelial cells.