Hepatic Met-enkephalin immunoreactivity is enhanced in primary biliary cirrhosis

BACKGROUND/AIMS: In contrast to the normal adult liver, the fetal human and rat livers, and the liver of rats with cholestasis secondary to bile duct resection (BDR) express the preproenkephalin (ppENK) mRNA, which codes for the endogenous opioid peptide Met-enkephalin. In addition, Met-enkephalin immunoreactivity (MEIR) is detected in hepatocytes and in proliferating bile ductules in the cholestatic rat liver. These data suggest that cholestasis is associated with the resurgence of cells that produce Met-enkephalin. To explore further the status of opioids in cholestasis, we studied the expression of MEIR in liver tissue. METHODS: The MEIR was sought in paraffin-preserved liver tissues from patients with primary biliary cirrhosis (PBC) (n = 10). RESULTS: The MEIR was detected in all the PBC livers. Its intensity varied from weak to strong on hepatocytes and bile ducts and the strongest expression appeared as coarse granules. The MEIR was either absent or only faintly expressed by some hepatocytes from disease and nondisease control biopsies, but absent from bile ducts. CONCLUSION: These results suggest that the human liver in cholestasis may be a source of endogenous opioids.     

Alterations in tight junctions differ between primary biliary cirrhosis and primary sclerosing cholangitis

Tight junctions (TJ) of biliary epithelial cells (BEC) and hepatocytes prevent bile regurgitation from the biliary tract. Alterations in these TJs may participate in chronic cholestatic liver diseases such as primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). We examined the localization of 2 TJ proteins, ZO-1 and 7H6, in these diseases. Frozen sections from livers of PBC, PSC, extrahepatic cholestasis (Ex-C), and hepatitis C-associated cirrhosis (LC-C), as well as histologically normal livers, were processed for double-fluorescence immunohistochemistry. In controls and cirrhosis, 7H6 and ZO-1 colocalized surrounding the luminal space of the bile ducts and outlined the bile canalicular spaces between hepatocytes. In untreated PBC, immunostaining for ZO-1 in BEC of bile ducts 40 to 80 microm in diameter was preserved, but that for 7H6 was diminished to absent. In PBC treated with ursodeoxycholic acid (UDCA), immunostaining for 7H6 was well preserved. In PSC as well as in Ex-C, immunostaining for both 7H6 and ZO-1 was well preserved in bile ducts. In hepatocytes, ZO-1 showed preserved immunoreactivity, but immunostaining for 7H6 frequently disappeared. The percentage of bile ducts with immunostaining for 7H6 in all bile ducts with immunostaining for ZO-1 was significantly reduced in PBC compared with that in control, LC-C, Ex-C, and PSC (all P <.0001). Substantial alteration in the TJ protein occurs predominantly in bile ducts in PBC and in hepatocytes in PSC, suggesting increased paracellular permeability along different paracellular routes for bile regurgitation in these chronic cholestatic liver diseases.     

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.     

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.     

The histoautoradiographic localization of taurocholate in rat liver after bile duct ligation. Evidence for ongoing secretion and reabsorption processes

To determine whether in complete obstructive cholestasis taurocholate is taken up by hepatocytes and if so whether it is secreted into bile, tritium-labelled taurocholate was localized by histoautoradiography on cryoslices from normal rat livers and from those after bile duct ligation. In non-cholestatic livers the hepatocytes of acinar zones 1 as well as the lumina and the epithelia of bile ductules and ducts became intensely labelled directly after injection of [3H]taurocholate into a mesenterial vein. Four hours and 4 days after bile duct ligation, hepatocytes of all three acinar zones became labelled, but in contrast to the normal state, pericanalicular concentration of silver grains was not observed, not even within 5 min. Fifteen days after bile duct obstruction, cryoslices taken 2 min after injection of [3H]taurocholate exhibited an intense silver grain labelling of all acinar zones, with the highest density at bile canalicular areas of the liver cell plates as well as the proliferated bile ductules and bile ducts. The biliary epithelium of small bile ductules and ducts of non-cholestatic and of bile duct-obstructed livers were also covered with silver grains; the epithelium of larger ducts exhibited significant labelling predominantly at the lateral sites of the cells. The biliary epithelium of the common bile duct was not significantly labelled. The results indicate that in complete obstructive cholestasis (a) taurocholate continues to be taken up from blood by hepatocytes and secreted into bile, but in terms of varying duration of obstruction, (b) all acinar zones are involved in bile salt transport, (c) in the initial phase (4 h and 4 days respectively after bile duct obstruction) hepatocytes fail to concentrate taurocholate at the canalicular site, (d) in a consecutive phase, in which bile ductules and ducts proliferate (demonstrated for a 15-day cholestasis), the taurocholate concentration at the canalicular site of hepatocytes is re-established and biliary secretion seems to be enhanced, (e) the biliary epithelium of bile ductules and ducts may play a significant role in the reabsorption and/or regurgitation of bile salts from bile to blood. Reabsorption/regurgitation of biliary constituents may also be operative in the non-cholestatic state but may become significantly enhanced with bile ductular proliferation.     

Telomere shortening in the damaged small bile ducts in primary biliary cirrhosis reflects ongoing cellular senescence

Telomere shortening is a trigger of cellular senescence. Biliary epithelial cells in damaged small bile ducts in primary biliary cirrhosis (PBC) show senescent features such as the expression of senescence-associated beta-galactosidase and the increased expression of p16(INK4a) and p21(WAF1/Cip1). We investigated whether the telomere shortening is involved in the pathogenesis of biliary cellular senescence in PBC. We analyzed the telomere length of biliary epithelial cells using quantitative fluorescence in situ hybridization in livers taken from the patients with PBC (n = 13) and control livers (n = 13). We also assessed immunohistochemically the prevalence of DNA damage and the expression of p16(INK4a) and p21(WAF1/Cip1). The study showed a significant decrease in telomere length in biliary epithelial cells in the damaged small bile ducts and bile ductules in PBC compared with normal-looking bile ducts and bile ductules in PBC, chronic viral hepatitis, and normal livers (P < 0.01). gammaH2AX-DNA-damage-foci were detected in biliary epithelial cells in damaged small bile ducts and bile ductules in PBC but were absent in biliary epithelial cells in chronic viral hepatitis and normal livers. The expression of p16(INK4a) and p21(WAF1/Cip1) was increased corresponding to telomere shortening and gammaH2AX-DNA-damage-foci in the damaged small bile ducts in PBC. CONCLUSION: Telomere shortening and an accumulation of DNA damage coincide with increased expression of p16(INK4a) and p21(WAF1/Cip1) in the damaged bile ducts, characterize biliary cellular senescence, and may play a role in the following progressive bile duct loss in PBC.     

Apoptosis of biliary epithelial cells in primary biliary cirrhosis and primary sclerosing cholangitis

BACKGROUND/AIMS: Primary biliary cirrhosis (PBC) is an autoimmune disease characterized by inflammatory destruction of small bile ducts. Primary sclerosing cholangitis (PSC) is a different, presumed autoimmune cholestatic liver disease where the bile ducts are also destroyed. In this study, apoptosis and portal triad inflammation in liver tissue from patients with PBC is examined and compared to that from patients with PSC and patients with normal liver. METHODS: Explanted liver tissue from patients with PBC and PSC and normal liver from patients with metastases to liver were examined. The liver samples were stained for apoptosis using the terminal deoxynucleotidyl triphosphate (TdT)-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay. The biliary epithelial cells (BEC) were then scored on the basis of their TUNEL stain and the degree of periductal inflammation. RESULTS: In PBC, apoptosis of BEC, as detected by the TUNEL assay, was significantly increased in the presence of inflammation. Regardless of the presence or absence of inflammation, the small bile ducts in PBC liver tissue exhibited greater evidence of apoptosis than did similar ducts from PSC or control livers. CONCLUSION: These findings suggest that in PBC, unlike PSC, the apoptosis of BEC in PBC is secondary to the invasion of inflammatory cells.     

Differential expression of intestinal trefoil factor in biliary epithelial cells of primary biliary cirrhosis

Intestinal trefoil factor (ITF) promotes epithelial cell migration and mucosal restitution during inflammation. We used real-time quantitative PCR, in situ nucleic acid hybridization, and immunohistochemistry to study the expression of the ITF gene and protein expression in the liver of primary biliary cirrhosis (PBC) and controls. There were significantly higher levels of ITF messenger RNA (mRNA) in PBC liver compared with primary sclerosing cholangitis (PSC) (P <.05) or normal controls (P <.001) and also higher in hepatitis C virus (HCV) liver (P <.05) and cryptogenic cirrhosis (P <.01) compared with normal controls. However, only in PBC was there a significant difference between small (interlobular and bile ductules) and large (intrahepatic and septal) bile ducts. Using in situ hybridization, the highest levels of ITF gene expression were localized to the large bile ducts in PBC. This differential expression of ITF was also noted at the protein level. Thus, in PBC, although 92% of large bile ducts expressed the ITF protein, only 2% of small bile ducts (P <.0001) expressed ITF. In contrast, in control livers, 34% of large bile ducts and 13% of small bile ducts expressed ITF. ITF protein is absent in small bile ducts in all stages of PBC. In conclusion, the expression of ITF may play an important role in bile duct damage. In small bile ducts, ITF production in response to damage is absent, making such cells vulnerable to damage and providing a thesis for the selective loss of small, but not large, bile ducts in PBC.     

In situ nucleic acid hybridization of pyruvate dehydrogenase complex-E2 in primary biliary cirrhosis: Pyruvate dehydrogenase complex-E2 messenger RNA is expressed in hepatocytes but not in biliary epithelium

Pyruvate dehydrogenase-E2, or a cross-reactive molecule, has been shown by a variety of immunohistochemical methods to be present in increased amounts in biliary epithelial cells (BEC) in primary biliary cirrhosis (PBC). In this study, to further understand the nature of the immunoreactive molecule in BEC, we examined the expression of pyruvate dehydrogenase complex-E2 (PDC-E2) messenger RNA (mRNA) and PDC-E2 protein in sections of livers from patients and controls to help identify the molecule found in BEC. We performed in situ hybridization using an antisense probe against the major epitope of PDC-E2. The data were very striking and suggested that there was no increased production of PDC-E2 in BEC. For example, in livers from patients with PBC, PDC-E2 mRNA was found in periportal hepatocytes in 16 of 17 cases (94%). In contrast, interlobular bile ducts and septal bile ducts had detectable levels of PDC-E2 mRNA in only 1 of 17 (6%) and 3 of 8 (38%) cases, respectively. Interestingly, proliferating bile ductules contained detectable levels of mRNA in 12 of 15 cases (80%). In control liver, periportal hepatocytes were positive in 15 of 17 cases (88%). Interlobular bile ducts, septal bile ducts, and proliferating bile ductules expressed mRNA signals in 4 of 17 (24%), 2 of 10 (20%), and 14 of 16 (88%), respectively. When formalin-fixed, paraffin-embedded sections were examined by immunohistochemical staining with anti-PDC-E2 monoclonal antibody (mAb) C355.1, the interlobular bile ducts showed typical aberrant apical staining in all 10 PBC cases, but 0 of 9 liver controls. Periportal hepatocytes, proliferating bile ductules and infiltrating mononuclear cells stained with C355.1 but in a characteristic mitochondrial staining pattern. The presence of a PDC-E2-like molecule recognized by C355.1 is not reflected by the expression levels of PDC-E2 mRNA in the BEC of patients with PBC.(Hepatology 1997 Jan;25(1):27-32)     

Differential expressions of aquaporin proteins in human cholestatic liver diseases.

Aquaporins (AQPs) are the channel forming membranous proteins involved in the biliary physiological homeostasis. Recently, we have reported the heterogeneous expression of AQPs in intrahepatic biliary epithelial cells or cholangiocytes in mice. However, the involvements of AQPs in hepatobiliary disorder are still unclear. Thus, we hypothesized that the AQP protein expressions are altered in human cholestatic disorders. METHODS: The AQP expressions of the immortalized human cholangiocytes cell line (H69) were assessed by immunoblotting. The expression of AQPs in liver biopsy specimens from various human cholestatic diseases as well as viral hepatitis were evaluated immunohistochemically. The degrees of staining were classified into four grades by comparison with staining intensity from controls. RESULTS: AQP1 expression, predominantly membranous, was confirmed by immunoblotting analysis. However, the other subtypes of AQP expression were not detected. In human pathological tissues, AQP1 expression by interlobular bile ducts was similar to normal and viral hepatitis, although this expression was attenuated according to bile duct injuries in PBC. On the contrary, the AQP1 expression by proliferating bile ductile (equivalent for small cholangiocytes) was enhanced. In intrahepatic cholestasis, AQP1 expressions were diminished, which was further associated with the aberrant expressions by periportal hepatocytes. CONCLUSIONS: AQP1 was expressed intensely in smaller proliferating bile ducts in human cholestatic liver disease. Also, the AQP1 expression was decreased in injured duct cells undergoing degeneration in PBC. The AQP1 expression was decreased in intrahepatic cholestasis probably due to negative feed back of the decreased bile flow. The role of AQP expression profiles may help the understanding of the pathogenesis of human cholestatic liver diseases.     

Cellular localization and up-regulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver

The hepatic expression of the ATP-dependent conjugate export pump multidrug resistance-associated protein 2 (Mrp2) is diminished in experimentally induced models of cholestasis. In this study we have examined the localization and expression of Mrp3, another member of the multidrug resistance-associated protein family, in normal liver and after obstructive cholestasis in the rat. Indirect immunofluorescence and confocal microscopy were used to determine the tissue localization and Western blot analysis was performed to quantitate the expression. In normal rat liver Mrp3 was found on the basolateral membrane of cholangiocytes and a single layer of hepatocytes surrounding the central vein. Three and 7 days after bile duct ligation Mrp3 expression was significantly increased, predominantly in hepatocytes in the pericentral region. By 14 days all hepatocytes showed basolateral membrane labeling for Mrp3 at a time when apical Mrp2 staining was significantly diminished. Proliferating bile ducts continued to stain positive, although the intensity of staining did not seem to vary. After 14 days Western blot quantitation showed that Mrp3 had increased approximately 30-fold in total liver membranes. Quantitation of Mrp3 in membranes from isolated hepatocytes of livers of sham and common bile duct-ligated (CBDL) animals showed a significant up-regulation beginning at 1 day and continuing to increase through 14 days postligation. This was in contrast to the progressive decrease in Mrp2 protein. Because Mrp3 is capable of transporting toxic bile acids, up-regulation of Mrp3 may compensate for the down-regulation of Mrp2 in obstructive cholestasis.     

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.     

Decreased anion exchanger 2 immunoreactivity in the liver of patients with primary biliary cirrhosis

Chloride-bicarbonate anion exchanger 2 (AE2) is expressed in a variety of tissues, including the liver and salivary glands, where it may participate in the generation of hydroionic fluxes into secretions. We have previously reported decreased hepatic levels of AE2 messenger RNA in patients with primary biliary cirrhosis (PBC), a cholestatic condition frequently associated with pluriglandular exocrine failure. Here we investigated the expression of AE2 protein in the liver of PBC patients. Using a monoclonal antibody against an AE2 peptide, immunohistochemistry was performed on liver biopsy specimens from subjects with normal liver (n = 7), patients with PBC (n = 13), and patients with cirrhosis or cholestasis other than PBC (n = 17 and 11, respectively). Immunostaining was graded from 0 to 7, according to its intensity and distribution. AE2 immunoreactivity was observed in normal livers, as previously reported, and in many pathological liver biopsy specimens, being mainly restricted to canaliculi and the luminal membrane of terminal and interlobular bile ducts. Canalicular and ductular scores were significantly reduced in the PBC group compared with each control group (normal liver and cirrhosis or cholestasis other than PBC), whereas no differences in immunoreactivity scores were observed among control groups. When four patients with primary sclerosing cholangitis (PSC) were analyzed, they also differed from those with PBC. These results suggest that PBC is characterized by diminished expression of AE2 in the liver. Reduced levels of this transporter protein might be involved in the pathogenesis of cholestasis in PBC.(Hepatology 1997 Jan;25(1):12-7)     

Endogenous opioids modulate hepatocyte apoptosis in a rat model of chronic cholestasis: the role of oxidative stress.

AIMS/BACKGROUND: There are increasing number of evidences indicating the contribution of endogenous opioids in the pathophysiology of cholestatic liver disease. The aim of the present study was to determine the role of the endogenous-opioid system in the modulation of hepatocytes apoptosis and liver oxidant/anti-oxidant balance during chronic cholestasis in rats. METHODS: We induced cholestasis in rats by bile duct ligation (BDL). Naltrexone, an opioid antagonist, was administered at different doses (2.5, 5, 10, 20 and 40 mg/kg/day) to cholestatic animals for 5 weeks. RESULTS: Naltrexone prevented the cholestasis-induced decrease of hepatic glutathione levels at higher doses (20 and 40 mg/kg/day). In the next phase of the study, we evaluated the effects of 20 mg/kg/day naltrexone treatment on hepatic damage indices and liver oxidant/anti-oxidant balance in 5-week BDL rats. There was a marked increase in the number of apoptotic hepatocytes as well as serum liver enzymes and hepatic lipid peroxidation levels in cholestatic rats compared with sham-operated animals 5 weeks after the operation. Liver anti-oxidant enzyme activities were significantly reduced in cholestatic rats compared with controls. Chronic treatment with naltrexone significantly improved all the aforementioned indices in comparison with saline-treated cholestatic rats. CONCLUSION: Our findings demonstrate that the administration of opioid antagonist is protective against hepatic damage in a rat model of chronic cholestasis. We suggest that increased levels of endogenous opioids contribute to hepatocytes apoptosis in cholestasis, possibly through downregulation of liver anti-oxidant defense.     

Significance of CD30-positive lymphocytes in livers in primary biliary cirrhosis

BACKGROUND: Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the destruction of intrahepatic small bile ducts. It is generally believed that cellular immune mechanisms, particularly T cells, cause this bile duct damage. CD30, which is inducible on selected T cells following activation, is regarded as important for B cell hyperactivity in several autoimmune diseases. In this study, we have attempted to examine CD30-expressing lymphocytes in PBC with respect to B cell hyperactivity. METHODS: We surveyed and counted CD30+ lymphocytes in liver sections from 13 patients with PBC and 36 control livers, including chronic viral hepatitis, extrahepatic biliary obstruction and normal liver by immunohistochemical staining. RESULTS: Several CD30+ lymphocytes were localized in inflamed portal tracts and also accentuated around the bile ducts in PBC livers, but they were rarely detected in control liver sections. The numbers of CD30+ lymphocytes in PBC were significantly higher than in control groups (P<0.01). Double immunohistochemical staining revealed that these CD30+ lymphocytes expressed CD3 as well as CD4. The number of CD30+ lymphocytes, moreover, correlated with that of immunoglobulin (Ig)A-containing cells (r=0.72) in PBC, although no such correlation between CD30+ lymphocytes and IgM or IgG-containing cells was obtained. CONCLUSIONS: These findings indicate that intrahepatic CD30+ lymphocytes have a role in IgA type, B cell abnormal hyperactivity with respect to the pathogenesis of portal tract and bile duct lesions in PBC.     

Ursodeoxycholic acid reduces expression of heat shock proteins in primary biliary cirrhosis

BACKGROUND: To identify injured cells in the liver of patients with primary biliary cirrhosis (PBC) and to determine the effects of ursodeoxycholic acid (UDCA) on these cells, we examined the cellular expression of heat shock proteins (HSPs) in PBC both before and after treatment with UDCA. METHODS: Expression of HSP70 and ubiquitin in PBC livers (n=34) was evaluated immunohistochemically as well as by immunoblot analysis, and compared with chronic viral hepatitis type C (n= 9), primary sclerosing cholangitis (n=8), and controls (n=7). RESULTS: Immunoblot analysis demonstrated a marked expression of HSP70 and ubiquitin in PBC. Immunohistochemical staining for both HSP70 and ubiquitin was observed to be strong in biliary epithelial cells (BECs) and moderate in both hepatocytes and arteries in PBC. Cellular labelling rates for HSP70 and ubiquitin of bile ducts in PBC were significantly higher (p<0.01) than those in chronic viral hepatitis type C, primary sclerosing cholangitis, or controls. The labelling rates for HSP70 and ubiquitin in bile ducts and in hepatocytes were significantly decreased (p<0.01) after treatment with UDCA in PBC. CONCLUSIONS: The present data suggest that BECs and hepatocytes significantly express HSPs even in the early stages of PBC, and that UDCA treatment significantly improves their condition. The immunohistochemical evaluation of HSPs is a valid and sensitive means to identify injured cells in PBC.     

Intracellular balance of oxidative stress and cytoprotective molecules in damaged interlobular bile ducts in autoimmune hepatitis and primary biliary cirrhosis: In situ detection of 8-hydroxydeoxyguanosine and glutathione-S-transferase-pi

Aim: Bile duct injury has been thought to be absent in autoimmune hepatitis (AIH), but recent studies have indicated that AIH patients do have bile duct injury. In this study, the intracellular balance of oxidative stress and cytoprotection in biliary epithelial cells was investigated to clarify the pathogenesis of bile duct injury in AIH. Methods: The intracellular status of oxidative DNA damage caused by oxidative stress and glutathione, an endogenous cytoprotective molecule, were examined in patients with AIH, primary biliary cirrhosis (PBC), and normal controls by immunostaining of 8-hydroxydeoxyguanosine (8-OHdG) and glutathione-S-transferase-pi. Results: Immunohistochemically, 8-OHdG expression was detected as abundantly in the damaged bile ducts of AIH patients as in PBC patients. Moreover, in AIH, 8-OHdG expression was detected in damaged bile ducts more than in undamaged bile ducts. Glutathione-S-transferase-pi expression was relatively preserved in the damaged bile ducts of AIH patients compared to PBC patients, reflecting preservation of intracellular glutathione. Conclusions: In AIH, oxidative stress and DNA damage may be involved in the pathogenesis of bile duct injury in a manner similar to that found in PBC. However, relatively preserved intracellular glutathione may play a key role in preventing progressive bile duct loss following bile duct injury in AIH.     

Enhanced hepatic lipid peroxidation in patients with primary biliary cirrhosis

OBJECTIVE: The mechanisms responsible for hepatic injury have not been fully clarified in primary biliary cirrhosis (PBC). It has recently been suggested that hepatic lipid peroxidation may be involved in the pathogenesis of PBC. The aims of the current study were to determine whether patients with PBC have evidence of enhanced hepatic lipid peroxidation and to evaluate its relationship to clinicopathological features. METHODS: Immunohistochemical detection of 4-hydroxynonenal (HNE) protein adducts was performed in the liver biopsies of 20 patients with PBC. Histological stages of PBC were evaluated. Orcein or Victoria blue staining was performed for detection of copper-associated proteins. The size of bile ducts was defined as the smallest diameter between the subepithelial basement membranes. RESULTS: All 20 patients had immunodetectable HNE protein adducts in the cytoplasm of damaged, but also intact, biliary cells. The mean diameter of bile ducts with HNE protein adducts was smaller than those without the adducts (61.0 +/- 1.9 vs 122.5 +/- 24.4 microm, respectively, p < 0.01). Out of 20 patients, 6 (30%) also had immunodetectable HNE protein adducts in hepatocytes preferentially located around the portal tracts. Most of the patients with hepatocytic HNE protein adducts had copper-associated protein granules in hepatocytes around the portal tracts and were classified as histological stage 3, whereas all of the patients without the adducts lacked copper-associated protein granules and were classified as histological stage 1 or 2. The patients with hepatocytic HNE protein adducts had higher levels of serum total bilirubin than did those without the adducts (2.9 +/- 0.9 vs 0.7 +/- 0.1 mg/dl, respectively, p < 0.01). CONCLUSIONS: Hepatic lipid peroxidation can occur in PBC and may be an early event in bile duct destruction. At advanced stages of PBC, hepatocellular lipid peroxidation may play a role in hepatocyte injury during cholestasis.     

Aberrant expression of cytokeratin 7 as a histological marker of progression in primary biliary cirrhosis

AIM: We evaluated the aberrant expression of cytokeratin 7 (CK-7) in hepatocytes as a marker of cholestasis and progression in primary biliary cirrhosis (PBC). PATIENTS AND METHODS: The expression of CK-7 was studied by immunohistochemistry in 83 cases of PBC. This expression was compared with biochemical data, the deposition of copper-associated protein, and previous histological classifications. RESULTS: In normal liver, CK-7 was expressed exclusively in bile duct epithelial cells (BDE). In PBC, the expression was also observed in hepatocytes. The expression pattern was classified as follows: Grade 0, BDE as in normal; Grade 1, proliferated bile ductules; Grade 2, periportal hepatocytes in addition to proliferated bile ductules; Grade 3, intralobular hepatocytes; Grade 4, the majority of hepatocytes. The grades correlated with serum bilirubin levels but not with serum levels of biliary enzymes. A discrepancy between the CK-7 grading and Ludwig's classification was noted in cases with Stage 1 of the CK-7 grading who were considered Stage 2 or 3 in Ludwig's classification, suggesting that cholestasis and inflammatory activity might be independent events. CONCLUSIONS: These results suggest that the aberrant expression of CK-7 in hepatocytes may be a marker of chronic cholestasis and progression in PBC.     

Loss of cooperative function of transforming growth factor-beta signaling proteins, smad3 with embryonic liver fodrin, a beta-spectrin, in primary biliary cirrhosis.

Modulation of fibrogenesis, epithelial, and mesenchymal cell fates are prominent effects of transforming growth factor-beta (TGF-beta) signaling by Smad proteins. We have previously shown that Smad2 and Smad3 insufficiency leads to a loss of bile ducts. In addition, Smad3/4 activity is mediated by embryonic liver fodrin (ELF), a beta-Spectrin. In mouse elf(-/-) mutants and in liver explant cultures, loss of ELF function results in T lymphocytic proliferation and absent intrahepatic bile ducts. A similar phenotype is seen in a number of cholestatic diseases with progressive loss of intrahepatic bile ducts and fibrosis. However, the expression patterns of Smads or role of ELF in cholestatic and fibrotic liver diseases are not yet known. METHODS/RESULTS: We investigated the role of ELF in primary biliary cirrhosis (PBC), autoimmune hepatitis C, chronic viral hepatitis and in livers from mice deficient in Smad2/Smad3. We generated elf(+/-) mutant mice and analyzed for chronic liver disease and hepatocellular cancer (HCC) from 6 to 12 months. Perturbations in ELF expression were consistently seen only in PBC tissues. ELF expression was similarly aberrant in tissues from Smad2(+/-)/Smad3(+/-) mutant mice. Further studies indicated that ELF mislocalization is correlated with aberrant localization of Smad3 in some PBC tissues. Thirteen of 17 elf(+/-) mutant mice developed steatosis, fibrosis, hepatic dysplasia, with HCC in two mice. CONCLUSIONS: These results suggest that a compromised cytoarchitecture and polarized trafficking of TGF-beta signaling molecules, ELF and Smad3 are involved in the pathogenesis of PBC as well as HCC.