Induction of innate immune response and absence of subsequent tolerance to dsRNA in biliary epithelial cells relate to the pathogenesis of biliary atresia

Background/Aims: Biliary epithelial cells (BECs) possess negative regulatory mechanisms of Toll‐like receptor (TLR)‐based tolerance to bacteria (e.g. endotoxin tolerance). Viral infections of the Reoviridae genus with a dsRNA genome are suspected to be part of the aetiology of biliary atresia (BA), but the negative biliary mechanisms remain unexplored. Methods: Cultured human intrahepatic BECs (HIBECs) pretreated with polyinosinic–polycytidylic acid [poly(I:C)] (a synthetic analogue of viral dsRNA) for 24 h were exposed to poly(I:C) in fresh medium. The activation of nuclear factor‐κB (NF‐κB) and the expression of myxovirus resistance protein A (MxA) and tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) mRNAs were evaluated. Moreover, after the pretreatment, the transition of these molecules was examined in poly(I:C)‐free conditions. Results: Treatment with poly(I:C) significantly upregulated NF‐κB activity in fresh HIBECs, and pretreatment failed to show tolerance to poly(I:C). The production of MxA and TRAIL was also preserved. Moreover, upregulation in the pretreated HIBECs was well preserved in poly(I:C)‐free medium for at least 72 h. Conclusions: BECs fail to show tolerance to poly(I:C), and once innate immunity is activated it is sustained in poly(I:C)‐free conditions, suggesting that the initiation of the immune response to dsRNA in HIBECs and its presence after the clearance of virus are closely associated with the progression of BA.     

Interaction between Toll-like receptors and natural killer cells in the destruction of bile ducts in primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is characterized by chronic nonsuppurative destructive cholangitis (CNSDC) associated with destruction of small bile ducts. Although there have been significant advances in the dissection of the adaptive immune response against the mitochondrial autoantigens, there are increasing data that suggest a contribution of innate immune mechanisms in inducing chronic biliary pathology. We have taken advantage of our ability to isolate subpopulations of liver mononuclear cells (LMC) and examined herein the role of Toll-like receptors (TLRs), their ligands, and natural killer (NK) cells in modulating cytotoxic activity against biliary epithelial cells (BECs). In particular, we demonstrate that Toll-like receptor 4 ligand (TLR4-L)-stimulated NK cells destroy autologous BECs in the presence of interferon alpha (IFN-α) synthesized by TLR 3 ligand (TLR3-L)-stimulated monocytes (Mo). Indeed, IFN-α production by hepatic Mo is significantly increased in patients with PBC compared to disease controls. There were also marked increases in the cytotoxic activity of hepatic NK cells from PBC patients compared to NK cells from controls but only when the NK cells were prepared following ligation of both TLR3-L- and TLR4-L-stimulated LMC. These functional data are supported by the immunohistochemical observation of an increased presence of CD56-positive NK cells scattered around destroyed small bile ducts more frequently in liver tissues from PBC patients than controls. CONCLUSION: These data highlight critical differences in the varied roles of Mo and NK cells following TLR3-L and TLR4-L stimulation.     

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.     

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

Abstract: 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.     

Temporal-spatial activation of apoptosis and epithelial injury in murine experimental biliary atresia

Biliary atresia is a fibro-inflammatory cholangiopathy that obstructs the extrahepatic bile ducts in young infants. Although the pathogenesis of the disease is undefined, studies in livers from affected children and neonatal mice with experimental biliary atresia have shown increased expression of proapoptosis molecules. Therefore, we hypothesized that apoptosis is a significant mechanism of injury to duct epithelium. To test this hypothesis, we quantified apoptosis using terminal transferase dUTP nick end labeling and active caspase-3 staining in livers and extrahepatic bile ducts from Balb/c mice infected with Rhesus rotavirus (RRV) within 24 hours of birth. RRV induced a significant increase in labeled cells in the portal tracts and in epithelial and subepithelial compartments of extrahepatic bile ducts, with onset within 3 days and peaks at 5-10 days. Exploring mechanisms of injury, we found increased biliary expression of caspases 1 and 4 and of interferon-gamma (IFNgamma)-related and tumor necrosis factor-alpha (TNFalpha)-related genes. Using a cholangiocyte cell line, we found that neither IFNgamma nor TNFalpha alone affected cell viability; however, simultaneous exposure to IFNgamma and TNFalpha activated caspase-3 and decreased cell viability. Inhibition of caspase activity blocked apoptosis and restored viability to cultured cholangiocytes. In vivo, administration of the caspase inhibitor IDN-8050 decreased apoptosis in the duct epithelium and the extent of epithelial injury after RRV challenge. Conclusion: The biliary epithelium undergoes early activation of apoptosis in a mouse model of biliary atresia. The synergistic role of IFNgamma and TNFalpha in activating caspase-3 in cholangiocytes and the decreased apoptosis following pharmacologic inhibition of caspases support a prominent role for apoptosis in the pathogenesis of experimental biliary atresia.     

Fractalkine and CX3CR1 are involved in the recruitment of intraepithelial lymphocytes of intrahepatic bile ducts

Fractalkine is a chemokine with both chemoattractant and cell-adhesive functions, and in the intestine it is involved with its receptor CX3CR1 in the chemoattraction and recruitment of intraepithelial lymphocytes. We examined the pathophysiological roles of fractalkine and CX3CR1 in normal and diseased bile ducts. Expression of fractalkine and CX3CR1 were examined in liver tissues from patients with primary biliary cirrhosis (17 cases) and controls (9 cases of primary sclerosing cholangitis, 10 cases of extrahepatic biliary obstruction, 20 cases of chronic viral hepatitis C, and 18 cases of histologically normal livers). Expression of fractalkine in biliary epithelial cells (BECs) in response to cytokine treatments was examined using a human cholangiocarcinoma cell line (HuCC-T1) and human intrahepatic BEC line. The chemotaxis of CX3CR1-expressing monocytes (THP-1) toward fractalkine was assayed using chemotaxis chambers. Fractalkine messenger RNA/protein were expressed on BECs of normal and diseased bile ducts, and their expression was upregulated in injured bile ducts of primary biliary cirrhosis. CX3CR1 was expressed on infiltrating mononuclear cells in portal tracts and on CD3(+), CD4(+), and CD8(+) intraepithelial lymphocytes of injured bile ducts in primary biliary cirrhosis. Fractalkine messenger RNA expression was upregulated in two cultured BECs on treatment with lipopolysaccharide and Th1-cytokines (interleukin 1beta, interferon gamma, and tumor necrosis factor alpha). THP-1 cells showed chemotaxis toward fractalkine secreted by cultured cells. In conclusion, Th1-cytokine predominance and lipopolysaccharide in the microenvironment of injured bile ducts resulting from primary biliary cirrhosis induce the upregulation of fractalkine expression in BECs, followed by the chemoattraction of CX3CR1-expressing mononuclear cells, including CD4(+) and CD8(+) T cells, and their adhesion to BECs and the accumulation of biliary intraepithelial lymphocytes.     

An involvement of Hippo-yes-associated protein pathway in biliary epithelial senescence in primary biliary cholangitis

Background & AimsAccumulating evidence suggest that Hippo-yes-associated protein (YAP) pathway plays important roles in development and repair after injuries in biliary system. We disclosed that senescent biliary epithelial cells (BECs) participate in the pathogenesis of primary biliary cholangitis (PBC). We hypothesized that dysregulation of Hippo-YAP pathway may be associated with biliary epithelial senescence in pathogenesis of PBC.Approach & ResultsCellular senescence was induced in cultured BECs by treatment with serum depletion or glycochenodeoxycholic acid. The expression and activity of YAP1 were significantly decreased in senescent BECs (p<0.01). Cellular senescence and apoptosis were significantly increased (p<0.01) and a proliferation activity and a 3D-cyst formation activity were significantly decreased (p<0.01) by a knockdown of YAP1 in BECs. The expression of YAP1 were immunohistochemically determined in livers taken from the patients with PBC (n = 79) and 79 control diseased and normal livers and its association with senescent markers p16^INK4a and p21^WAF1/Cip1 was analyzed. The nuclear expression of YAP1, which indicates activation of YAP1, was significantly decreased in BECs in small bile ducts involved in cholangitis and ductular reactions in PBC, compared to control livers (p<0.01). The decreased expression of YAP1 was seen in senescent BECs showing expression of p16^INK4a and p21^WAF1/Cip1 in bile duct lesions.ConclusionDysregulation of Hippo-YAP1 pathway may be involved in the pathogenesis of PBC in association with biliary epithelial senescence.     

Biliary epithelial cells and primary biliary cirrhosis: the role of liver-infiltrating mononuclear cells

Primary biliary cirrhosis (PBC) is characterized by the highly selective autoimmune injury of small intrahepatic bile ducts, despite widespread distribution of mitochondrial autoantigens. On this basis, it has been suggested that the targeted biliary epithelial cells (BECs) play an active role in the perpetuation of autoimmunity by attracting immune cells via chemokine secretion. To address this issue, we challenged BECs from patients with PBC and controls using multiple Toll-like receptor (TLR) ligands as well as autologous liver-infiltrating mononuclear cells (LMNCs) with subsequent measurement of BEC phenotype and chemokine production and LMNC chemotaxis by quantifying specific chemokines. Our data reflect that BECs from PBC patients and controls express similar levels of TLR subtypes, CD40, and human leukocyte antigen DRalpha (HLA-DRalpha) and produce equivalent amounts of chemokines in our experimental conditions. Interestingly, however, BEC-expressed chemokines elicit enhanced transmigration of PBC LMNCs compared with controls. Furthermore, the addition of autologous LMNCs to PBC BECs led to the production of higher levels of chemokines and enhanced the expression of CD40 and HLA-DRalpha. CONCLUSION: We submit that the proinflammatory activity of BECs in PBC is secondary to the intervention of LMNCs and is not determined per se. These data support the hypothesis that BECs are in fact "innocent victims" of autoimmune injury and that the adaptive immune response is critical in PBC.     

Apoptosis of murine cultured biliary epithelial cells induced by glycochenodeoxycholic acid involves Fas receptor and its ligand

Fas-mediated apoptosis of biliary epithelial cells (BECs) is suggested as a main effector process in immune-mediated biliary diseases. Glycochenodeoxycholic acid (GCDCA), a conjugated hydrophobic bile acid, is known to cause apoptosis of hepatocytes via the direct activation of Fas receptor (FasR). In this study, we investigated the apoptotic effect of GCDCA on cultured murine intrahepatic BECs. It was found that GCDCA induced apoptosis of BECs derived from BALB/c mice in a dose- and incubation time-dependent manner. The morphology and TUNEL positivity for the apoptotic process induced by GCDCA were similar to those induced by beauvericin (BV) which is known to cause apoptosis by a direct activation of caspase-3, a member of the caspase family, at its downstream. The GCDCA-induced apoptosis of BECs was accompanied by an up-regulation of FasR, Fas ligand (FasL), and also caspase-3 expression. GCDCA did not induce the apoptosis of BECs derived from C3H · MRL-Fas^lpr mice possessing a non-functioning FasR, supporting that the GCDCA-induced apoptosis of BECs in BALB/c involves the Fas system. Furthermore, GCDCA induced an increase in interleukin-18 (IL-18) mRNA in BECs and secretion of activated IL-18 from BECs of BALB/c, which might have led to an up-regulation of FasL mRNA and protein expression in BECs followed by FasR/FasL interaction. These results suggest that GCDCA induces apoptosis of BECs through FasR/FasL interaction via an autocrine/paracrine effect, IL-18 is responsible for the expression of FasL in BEC, and the up-regulation of caspase-3 expression is involved in this model. This model could be useful for molecular and genetic studies of Fas-mediated apoptosis of BECs.     

Electron microscopic observation of destruction of biliary epithelium in primary biliary cirrhosis

ABSTRACT— Electron microscopic studies of the intrahepatic biliary tree in 16 patients with primary biliary cirrhosis (PBC) disclosed four types of biliary epithelial injury suggesting cell death in the ducts: 1) coagulative and 2) lytic necrosis without detachment of affected cells from the biliary epithelial layer, and 3) apoptosis and 4) detachment of several adjoining biliary cells from the basement membrane and neighboring biliary cells. Lesions 1), 2) and 3) were also found in livers with extrahepatic cholestasis without bile duct loss, and 1) and 2) were found in PBC livers irrespective of the degree of bile duct loss. 3) was rare and mostly confined to bile ductules, when present. By contrast, 4) was only observed in PBC, especially in livers with a moderate degree of bile duct loss in which extensive bile duct destruction appeared to be progressing. Detached biliary cells in lesion 4) were occasionally in contact with and/or surrounded by migrating lymphocytes with pseudopod formation, suggesting lymphocyte-target cell interactions. It therefore seems possible that epithelial detachment is an important ultrastructural lesion associated with extensive bile duct destruction in PBC livers.     

Myeloperoxidase-positive inflammatory cells participate in bile duct damage in primary biliary cirrhosis through nitric oxide-mediated reactions

Previous studies have suggested that increased nitric oxide (NO)-mediated products are found in the livers of subjects with primary biliary cirrhosis (PBC), but the mechanisms involved remain enigmatic. We took advantage of immunohistochemistry and several unique monoclonal antibodies to study inflammatory cells responsible for the generation of NO, the enzymes responsible for NO production, the expression of 3-nitrotyrosine, and the presence of CD68(+) and/or myeloperoxidase (MPO)(+) cells. We examined a total of 113 liver specimens, including 64 with PBC, 19 with primary sclerosing cholangitis (PSC), 6 with non-A, non-B hepatitis, 6 with alcoholic liver disease, 4 with cryptogenic cirrhosis, 4 with biliary atresia, and 10 normal subjects. Twenty-two percent of PBC had elevated expression of 3-nitrotyrosine in their bile duct epithelial cells (BECs) (P =.0316). Furthermore, the BECs in PBC also demonstrated apoptotic changes. MPO-positive inflammatory cells were also noted adjacent to the basement membrane. In contrast, the liver of normal subjects showed few apoptotic changes in the bile ducts, with no evidence of MPO staining in the portal area. Furthermore, sections from livers of subjects with stage I or stage II PBC demonstrated significantly increased inflammatory cell infiltration (P =.0064) and elevated 3-nitrotyrosine expression in BECs (P =.0246) compared with stage III and IV. The presence of 3-nitrotyrosine was closely associated with infiltrating CD68- and/or MPO-positive cells. There was also a stage-associated difference in the presence of bile duct infiltrating cells and 3-nitrotyrosine in PBC with an increase dominant in early stage disease. In conclusion, NO and reactive oxygen species, collectively determined as 3-nitrotyrosine, are associated with bile duct destruction in PBC and are particularly prevalent in early stage disease.     

Loss of interleukin-12 modifies the pro-inflammatory response but does not prevent duct obstruction in experimental biliary atresia

Background  

Livers of infants with biliary atresia and of neonatal mice infected with rotavirus (RRV) have increased expression of interferon-gamma (IFNγ) and interleukin (IL)-12. While the expression of IFNγ regulates the obstruction of extrahepatic bile ducts by lymphocytes, the role of IL-12 in the pathogenesis of biliary obstruction is unknown. Based on the role of IL-12 as a key proinflammatory cytokine, we hypothesized that loss of IL-12 prevents the obstruction of extrahepatic bile ducts.     

Immune pathogenesis of sclerosing cholangitis in biliary atresia

Biliary atresia (BA) is the most frequent single life-threatening hepatic disorder in early childhood in Japan, Europe and North America. It affects one in 9000 live-born infants in our country. BA is characterized by complete inability to excrete bile associated with obstruction, destruction or absence of the extrahepatic bile ducts. Pathologic study of tissues removed from the porta hepatis and of the proximal extrahepatic bile ducts suggests that BA in the vast majority of infants results from a sclerosing, inflammatory lesion initiated in ductular tissues. Kasai procedure (KP) is in the first line of treatment for BA infants in the developed countries all over the world. In KP, bile duct remnants or fibrous tissues in front of the hepatic artery are dissected and a Roux-en -Y anastomosis is fashioned between the area of the porta hepatic and the jejunum. KP has provided opportunities to survive years for some infants with BA with reasonable quality of life. However in BA, bile duct injury and sclerosing inflammation is ongoing even after apparently successful KP. A 10-year survival rates with native liver is still 53% in 1999 according to multi-centre analysis in Japan. BA is still the leading cause of paediatric liver transplantation. Poor prognosis of BA is, of course, due to unknown aetiology. Potential aetiologies for more common perinatal form of BA include viral infections, immune-mediated injuries and autoimmune disease involving biliary systems. Details of each theory including our updated findings will be reviewed and discussed.     

The immunobiology of bile and biliary epithelium.

Long thought to be just a simple pipe involved in the delivery of bile from hepatocytes to the gallbladder and intestine, bile ducts are now regarded as highly dynamic structures consisting of cell populations involved in formation, transport and modification of bile by both secretory and absorptive processes. In fact, both bile and biliary epithelium appear to have active immunologic roles in both innate and adaptive immune responses. These roles are becoming increasingly clear as techniques have been developed allowing for the study of bile and biliary epithelial cells (BECs) in mucosal immunity. Bile is actively involved in the transport of immunoglobulin to the intestine, while BECs secrete chemokines and cytokines and serve to localize the immune response by expressing critical cell adhesion molecules. Evidence suggests that BECs may also function as professional antigen-presenting cells (APC) and, in the process, contribute to the modulation of inflammatory reactions. Bile ducts and, in particular, BECs, are the primary site of damage in several immunologically mediated liver diseases. Progress in these important areas has been rapid and forms the basis of this review.     

Bile duct cell apoptosis is a rare event in primary biliary cirrhosis

BACKGROUND: The frequency of apoptosis in bile duct cells of primary biliary cirrhosis is still unclear spanning from rare to 50% in the various reports. AIM: To study bile duct cell apoptosis in stage I primary biliary cirrhosis lesions. PATIENTS: Nine stage I-II biopsies with a total number of 26 bile ducts of different sizes, selected from a larger series on the basis of the expression on serial frozen sections of HLA-DR and Fas antigens. METHODS: Apoptosis was evaluated by a DNA fragmentation assay on frozen sections, according to the manufacturer's protocol and by expression of apoptosis related cytokeratin neoepitopes. Bile duct cell proliferation was assessed by MIB1 (Ki-67) expression. RESULTS: Apoptosis was frequently found in inflammatory cells of portal tracts and sinusoids. Apoptosis of hepatocytes was also systematically observed. Only 4 positive bile duct cells were found in 3 bile ducts from 3 biopsies. Quantitative evaluation was not attempted. Cholangiocyte proliferation was observed in the same ducts and occasionally in other biopsies. CONCLUSIONS: These data suggest that cholangiocyte death by apoptosis at the level of typical primary biliary cirrhosis lesions is a rare event, at least in early stages of the disease. The observed rate of proliferation was consistent with the rate of apoptosis.     

Generation of monoclonal antibodies to murine bile duct epithelial cells: identification of annexin V as a new marker of small intrahepatic bile ducts

Biliary epithelial cells (BECs) are distributed along the length of both the extrahepatic and intrahepatic biliary tree, but have distinctly different phenotypes and functions according to their anatomical location. It has been reasoned that the distinct appearance of pathogenic lesions in different biliary diseases may be associated with the expression of distinct proteins. These data prompted us to immunize rats with cultured murine BECs with the objective of determining if there are unique antigens on BECs. Of the 45 monoclonal antibodies (mAbs) produced, 12 mAbs (MBEC 1-12) were selected for detailed study based on their classification into three major groups. These groups included four antibodies (MBEC 1-4) that reacted in a staining pattern typical of mucin. A second group of mAbs, MBECs 5 to 8, reacted strongly along the biliary tract and by immunoblot analysis, reacted with several bands ranging from 44 kd to 64 kd. These antibodies were considered as markers of pan BECs and their staining pattern proved similar to that of a control polyclonal pan-cytokeratin. The final group of mAbs, MBECs 9 to 12, recognized a 36-kd antigen using lysates of murine BECs. These antibodies also predominantly stained small peripheral bile ducts. The reactive antigen was purified by immunoprecipitation and microsequenced; the peptides sequenced showed 100% homology with murine annexin V. The identification of annexin V with predominantly intrahepatic bile ducts, is of significant interest because of the multiple roles of annexin V, including that of membrane cytoskeletal interactions during transport and apoptosis.     

Endoscopic retrograde cholangiography in the diagnosis of biliary malformations in infants

The differentiation of infantile biliary malformations from primary parenchymal diseases is difficult. The recent development of a pediatric side-viewing endoscope (PJF Endoscope; Olympus Corporation of America) provided an opportunity to investigate the usefulness of endoscopic retrograde cholangiography (ERC) for precise visualization of the extrahepatic biliary passages in infants with persistent cryptogenic cholestasis. ERC was performed in 12 patients, with visualization of the existing extrahepatic bile ducts in 4. The entire biliary system was visualized in one, excluding extrahepatic biliary atresia and choledochal cyst. The reduced caliber of the intrahepatic bile ducts and histological observations in a percutaneous liver biopsy supported the diagnosis of intrahepatic biliary hypoplasia in this case. An intact hepatic portochole cystostomy was documented in one, although the intrahepatic biliary system was not delineated. Atresia of the hepatic bile ducts proximal to the gallbladder was documented in two. Of the eight patients in whom extrahepatic bile ducts were not visualized by ERC, six had extrahepatic biliary atresia confirmed at exploratory laparotomy. The papilla of Vater could not be located in four of these six infants. The remaining two had neonatal hepatitis. ERC may offer a useful alternative to operative cholangiography in selected infants with persistent cholestasis and acholic stools.     

Destruction of bile ducts in primary biliary cirrhosis.

Primary biliary cirrhosis is characterized by the immune-mediated, progressive destruction of interlobular bile ducts. Lymphoid cells migrate into the biliary epithelial layer through integrin alpha(4)/fibronectin interaction and are responsible for chronic destructive cholangitis. The bile ducts express intercellular adhesion molecule-1 (ICAM-1) and vascular cellular adhesion molecule-1 (VCAM-1), and infiltrating lymphocytes express LFA1 and VLA4, facilitating their interaction. Epithelioid granulomas contain foamy cells ingesting biliary lipids, and CD1d was detectable in epithelioid granulomas, suggesting that the biliary substance(s) which are leaked is a trigger for chronic destructive cholangitis. Apoptotic biliary destruction is brought about by antigen-specific and non-specific reactions. Shrunken biliary epithelial cells with pyknotic nuclei positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) may reflect apoptotic processes. Increased expression of caspase-3 and -8 with DNA fragmentation factor on the bile ducts may reflect molecular events during apoptosis, and down-regulation of Bcl-2 of biliary epithelial cells seems to facilitate apoptosis. Multiple factors, particularly the Fas system, are stimuli of apoptosis. Anoikis with decreased biliary expression of integrin 6, a ligand for laminin, may also be involved in biliary epithelial apoptosis.