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Y Nakanuma

Publications and source records attributed to Y Nakanuma.

At least 55 records · Page 3Linked to original sources
PubMed2000

Expression of Bcl-2 familial proteins is reduced in small bile duct lesions of primary biliary cirrhosis.

In primary biliary cirrhosis, biliary epithelial cell death by apoptosis results in progressive bile duct loss. We examined immunohistochemically 4 apoptosis-regulating bcl-2 familial proteins (bcl-2, mcl-1, bcl-X, and bax) in the biliary epithelium in 19 cases of primary biliary cirrhosis. Ten cases of chronic hepatitis C, 9 cases of extrahepatic biliary obstruction, and 10 cases of normal liver were used as a control. Bcl-2 and mcl-1 are inhibitors of apoptosis, bcl-X, probably bcl-XL in biliary epithelial cells, an inhibitor, and bax, a promoter of apoptosis. First, we clarified the distribution of bcl-2 familial proteins on the intrahepatic biliary tree in normal livers. Bcl-2 was detected in the interlobular bile ducts and bile ductules, but not in the large and septal bile ducts in all cases examined. Mcl-1, bcl-X, and bax were diffusely detectable at the any level of the intrahepatic biliary tree, with a staining pattern that was diffuse and cytoplasmic. This distribution pattern was preserved in extrahepatic biliary obstruction. Bcl-2 expression was lost or markedly reduced in the damaged interlobular bile ducts in primary biliary cirrhosis, whereas the reduction was only focal or mild in the bile ducts with hepatitis-associated damage in chronic hepatitis C. Expression levels of mcl-1, bcl-X, and bax were similarly reduced to that of bcl-2 in these 2 diseases. These findings suggest that bax is not important as a proapoptotic factor in the damaged bile ducts and that downregulation of bcl-2 and mcl-1, and probably that of bcl-XL, leads to a decrease in the threshold of apoptosis and increase in the vulnerability to apoptotic stimuli in these bile ducts, followed by the progressive apoptotic loss of interlobular bile ducts, in primary biliary cirrhosis.

Apoptosis↗
PubMed2000

Amplification and sequence analysis of partial bacterial 16S ribosomal RNA gene in gallbladder bile from patients with primary biliary cirrhosis.

BACKGROUND/AIMS: The etiopathogenesis of bile duct lesion in primary biliary cirrhosis is unknown, though the participation of bacteria and/or their components and products is suspected. In this study, we tried to detect and identify bacteria in the bile of patients with primary biliary cirrhosis by polymerase chain reaction using universal bacterial primers of the 16S ribosomal RNA gene. METHODS: Gallbladder bile samples from 15 patients with primary biliary cirrhosis, 5 with primary sclerosing cholangitis, 5 with hepatitis C virus-related liver cirrhosis, 11 with cholecystolithiasis, and from 12 normal adult gallbladders were used. In addition to the culture study, partial bacterial 16S ribosomal RNA gene was amplified by polymerase chain reaction (PCR) taking advantage of universal primers that can amplify the gene of almost all bacterial species, and the amplicons were cloned and sequenced. Sequence homology with specific bacterial species was analyzed by database research. Bacterial contamination at every step of the bile sampling, DNA extraction and PCR study was avoided. Furthermore, to confirm whether bacterial DNA is detectable in liver explants, the same analysis was performed using 10 liver explants of patients with primary biliary cirrhosis. RESULTS: In primary biliary cirrhosis, 75% (p<0.0001) of 100 clones were identified as so-called gram-positive cocci while these cocci were positive in only 5% in cholecystolithiasis (p<0.0001). In cholecystolithiasis gram-negative rods were predominant instead. One bacterial species detected in a normal adult was not related to those detected in primary biliary cirrhosis and cholecystolithiasis patients. No bacterial DNA was detected by PCR amplification in 10 liver explants of patients with primary biliary cirrhosis. CONCLUSIONS: The present results raise several possible roles of gram-positive bacteria in bile in the etiopathogenesis of primary biliary cirrhosis. However, these results could also reflect an epiphenomenon due to decreased bile flow in the patients with primary biliary cirrhosis at an advanced stage.

Adult↗
PubMed2000

Immunological similarities between primary sclerosing cholangitis and chronic sclerosing sialadenitis: report of the overlapping of these two autoimmune diseases.

Primary sclerosing cholangitis (PSC) is characterized by destructive inflammation and fibrosis affecting the bile ducts. The etiology of PSC is still unknown, although lymphocytic infiltration in the portal areas suggests an immune-mediated destruction of the bile ducts. Patients with one autoimmune disease often suffer from one or more other autoimmune diseases. It is well known that there is a close relationship between PSC and inflammatory bowel disease, particularly ulcerative colitis(UC). However, the pathological findings in UC and other overlap diseases do not resemble those of PSC. In the present study, we report a patient with chronic sclerosing sialadenitis (Kuttner's tumor) and PSC. It is compared the sclerosing changes in both salivary glands and bile ducts histologically. In addition, the expression pattern of mast cell tryptase, b-FGF, and HLA-DR were examined in both tissues immunohistochemically. Histological features of sclerosing change in both salivary and bile ducts were quite similar. Marked mast cell infiltration and b-FGF expression were seen in the sclerosing areas in both tissues. In active inflammatory areas of the salivary glands, HLA-DR expression was also seen. We hypothesized that similar immune reactions occur in both the salivary gland and bile ducts and are responsible for the fibrosis that follows.

Antibodies, Monoclonal↗
PubMed2000

Primary biliary cirrhosis: an orchestrated immune response against epithelial cells.

Primary biliary cirrhosis (PBC) is an organ-specific autoimmune disease that predominantly affects women and is characterized by chronic progressive destruction of small intrahepatic bile ducts with portal inflammation and ultimately fibrosis. The serologic hallmark of PBC is the presence of antibodies to mitochondria, especially to the E2 component of the pyruvate dehydrogenase complex. The mechanisms by which (and if) such antibodies produce liver tissue injury are unknown. However, the presence of these antibodies has allowed detailed immunological definition of the antigenic epitopes, the nature of reactive autoantibodies and the characterization of T-cell responses. Several mechanisms may now be proposed regarding the immune-mediated bile duct damage in PBC, including the possible role of T-cell-mediated cytotoxicity and intracellular interaction between the IgA class of antimitochondrial antibodies and mitochondrial autoantigens. There are major questions which remain unanswered, including, of course, etiology, but also the reasons for female predominance, the absence of PBC in children, the relative ineffectiveness of immunosuppressive drugs, and the specific role of mitochondrial antigens. The data so far provide suggestive evidence that PBC is a mucosal disease; this thesis provides a basis for discussion of etiology via the enterohepatic circulation of toxins and/or infection.

Acyltransferases↗
PubMed2000

Fas ligand expressing mononuclear cells around intrahepatic bile ducts co-express CD68 in primary biliary cirrhosis.

AIMS/BACKGROUND: Apoptosis, including the Fas system, has been implicated in progressive bile duct loss in primary biliary cirrhosis (PBC). In this study, we attempted to analyze Fas ligand (FasL) expressing mononuclear cells infiltrating in the portal tracts of PBC. METHODS: We immunohistochemically assessed co-expression of leukocyte markers and FasL on infiltrating mononuclear cells in 18 patients with PBC. Twenty-five patients with chronic hepatitis C (CH-C) were used as controls. RESULTS: In PBC, FasL expressing cells were scattered in the portal tracts, and some were accentuated around the damaged bile ducts. In addition, these cells co-expressed CD68 (71%), a marker of monocytes, but not UCHL-1, CD3 and CD57, markers of activated T cells and natural killer cells. By contrast, in CH-C, the biliocentric pattern of FasL expression was not evident, and about half of FasL expressing cells (42-56%) co-expressed UCHL-1, CD3 or CD57. CD14, a receptor for bacterial products such as lipopolysaccharides, was also detected on a proportion of FasL expressing mononuclear cells around the damaged bile ducts in PBC. CONCLUSION: The results suggest that in PBC, FasL expressing CD68+ monocytes are at least partly involved in apoptotic bile duct loss mediated by the Fas system, and a surface molecule, CD14, participates in this process.

Antigens, CD↗
PubMed2000

Evidence of the participation of peribiliary mast cells in regulation of the peribiliary vascular plexus along the intrahepatic biliary tree.

Our pilot study disclosed that tryptase-positive mast cells (MC) were densely distributed around the intrahepatic bile ducts (peribiliary MC). In this study, the pathophysiologic roles of these MC were examined with respect to the microcirculation around the bile duct in 71 cases of histologically normal liver, 24 cases of chronic hepatitis, and 45 cases of liver cirrhosis. The tryptase-positive MC were very close to the microvessels of the peribiliary vascular plexus (PVP), which supply the intrahepatic biliary tree. The tryptase-positive MC were frequently found adjacent to vascular smooth muscle cells, including pericytes. The location of the tryptase-positive MC was confirmed by ultrastructural analysis. In cirrhosis, the numbers of both microvessels of PVP and peribiliary MC increased in parallel. Peribiliary MC were immunoreactive for endothelin 1 (ET-1), and were variably immunoreactive for histamine, chymase, inducible nitric oxide synthase (iNOS), and endothelin A and B (ET(A) and ET(B)) receptors, particularly in cirrhotic livers. On vascular endothelial cells of PVP, endothelial nitric oxide synthase (eNOS) and ET-1 were consistently detectable, and ET(A) receptors, ET(B) receptors, and iNOS were variably detectable. Pericytes of PVP expressed ET(A) and ET(B) receptors in addition to ET-1 and iNOS. Biliary epithelial cells also focally expressed iNOS, ET-1, and ET(A) and ET(B) receptors. These vasoactive substances were strongly expressed on the cellular components in cirrhotic liver. By in situ hybridization, iNOS mRNA signals were observed on iNOS-immunoreactive cell components, including peribiliary MC. These morphologic and immunohistochemical findings suggest that the cellular components displaying vasoactive substances in the milieu of the intrahepatic biliary tree are very dynamic in the vasoregulation of PVP in normal livers, even more so in cirrhosis, and that peribiliary MC exert local effects on the microcirculation of PVP, directly and indirectly.

Bile Ducts, Intrahepatic↗
PubMed2000

Absence of Epstein-Barr virus (EBV) in intrahepatic cholangiocarcinoma confirmed by lack of EBV-coded nuclear RNA and latent membrane protein-1.

AIMS: Studies are disclosing that Epstein-Barr virus (EBV) is involved in the aetiology of various neoplasms including undifferentiated carcinomas of the aerodigestive tract. The aetiology of intrahepatic cholangiocarcinoma (ICC), a malignant neoplasm arising from intrahepatic biliary epithelia, has yet to be fully evaluated. To date, two cases of EBV-related ICC have been reported, and they presented foci of lymphoepitheliomatous undifferentiated carcinoma components. METHODS AND RESULTS: To determine whether EBV is commonly involved in the developments of ICC, we performed in-situ hybridization and immunohistochemistry for EBV in 215 cases of ICC in Japan, using a probe against EBV-coded nuclear RNA (EBER) and a specific antibody against latent membrane protein-1 (LMP-1), respectively. We did not detect EBV-infected carcinoma cells in any of the ICC cases examined. No lymphoepitheliomatous undifferentiated carcinoma components were found either. CONCLUSION: The results suggest that EBV infection is unlikely to be involved in the pathogenesis of ICC.

Adult↗
PubMed2000

Cytokeratin profile relates to histological subtypes and intrahepatic location of intrahepatic cholangiocarcinoma and primary sites of metastatic adenocarcinoma of liver.

AIMS: We evaluated the cytokeratin profile of intrahepatic cholangiocarcinoma with respect to its histological classification and intrahepatic location (peripheral vs. hilar), and compared its profile with that of a variety of metastatic adenocarcinomas in liver. METHODS AND RESULTS: Expression of cytokeratins 7, 8, 18, 19 and 20 was immunohistochemically examined in intrahepatic cholangiocarcinoma (n = 77) and metastatic adenocarcinoma in liver (21 colorectal, 14 gastric, three gallbladder and three pancreatic cancers). Materials were autopsy or surgical specimens. Cytokeratins 7, 8, 18 and 19 were expressed in 75 (97%), 75 (97%), 59 (77%) and 71 (92%) cases of intrahepatic cholangiocarcinoma, respectively. Moderate and extensive expression of cytokeratin 18 was more frequent in the peripheral than in the hilar type. Moderate and extensive expression of cytokeratin 19 was seen in almost all cases of well-differentiated intrahepatic cholangiocarcinomas, while expression was decreased relatively in the moderately and decreased more in the poorly differentiated cases. While cytokeratin 20 was not found in non-neoplastic biliary epithelia or in well-differentiated intrahepatic cholangiocarcinomas, this cytokeratin was occasionally detectable in moderately and poorly differentiated intrahepatic cholangiocarcinomas and its expression was more frequent in the hilar type. Cytokeratin 20 expression was observed in 17 (81%) of metastatic adenocarcinomas in liver from colorectal regions, to a lesser degree in those from gastric regions, and was rare in those from gallbladder and pancreatic regions; cytokeratin 7 showed a reverse expression pattern in these metastatic adenocarcinomas in liver. The profile of cytokeratins 7 and 20 of metastatic colorectal and gastric carcinomas differed from that for intrahepatic cholangiocarcinomas, while that of metastatic gallbladder and pancreatic carcinoma was similar to that for intrahepatic cholangiocarcinomas. Moreover, cytokeratin 18 and 19 expression was significantly infrequent in metastatic gastric carcinomas than in intrahepatic cholangiocarcinomas and metastatic colorectal carcinomas. CONCLUSION: The combined immunostaining of cytokeratins 7, 18, 19 and 20 is useful for the characterization of intrahepatic cholangiocarcinomas with respect to histological subtypes and intrahepatic location. It helps to differentiate intrahepatic cholangiocarcinoma from metastatic adenocarcinomas in liver and from colorectal and gastric regions; it also indicates the primary focus metastatic adenocarcinomas in livers.

Adenocarcinoma↗
PubMed2000

Distribution of apoptotic cells and expression of apoptosis-related proteins along the intrahepatic biliary tree in normal and non-biliary diseased liver.

AIMS: The cell kinetics and homeostasis of biliary epithelial cells may be maintained differently along the biliary tree. In this study, the role of apoptosis in the maintenance and homeostasis of the intrahepatic biliary tree was evaluated. METHODS AND RESULTS: By counting apoptotic biliary cells and by immunostaining apoptosis-related proteins in normal liver, fatty liver, and those with acute viral hepatitis, chronic viral hepatitis, and hepatitis virus-related cirrhosis, it was found that the larger the intrahepatic bile ducts became, the more biliary epithelial cells underwent apoptosis. bcl-2, an inhibitor of apoptosis, was diffusely expressed in the interlobular bile ducts, but rarely detectable in the large and septal bile ducts. bcl-XL and mcl-1, inhibitors of apoptosis, and bax, a promoter of apoptosis, were diffusely expressed along the intrahepatic biliary tree. CD95, a direct inducer of apoptosis, was present in the large and septal bile ducts, but rarely in the interlobular bile ducts. CONCLUSION: The ratio of bax to bcl-2, as well as the expression of CD95 which differed at the interlobular versus large and septal bile ducts, may be responsible for the unique distribution of apoptotic biliary cells and involved in the homeostasis of the intrahepatic biliary tree.

Aged↗
PubMed2000

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.

Apoptosis↗
PubMed2000

Up-regulation of fas ligand at early stages and down-regulation of Fas at progressed stages of intrahepatic cholangiocarcinoma reflect evasion from immune surveillance.

We examined immunohistochemically the possible participation of the Fas/Fas ligand (FasL) system in intrahepatic cholangiocarcinoma (ICC) during the escape from immune surveillance, using 68 cases of ICC, 29 cases of normal intrahepatic large bile ducts, and 18 cases of biliary dysplasia. Apoptosis of tumor-infiltrating lymphocytes (TIL) was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Fas was weakly expressed in normal intrahepatic bile ducts. Almost all biliary dysplasia and well-differentiated ICCs showed moderate to marked expression of Fas, while Fas expression was variable in moderately and poorly differentiated ICCs. Down-regulation of Fas expression was significantly correlated with histologic de-differentiation, vascular invasion, the size of ICCs, and short survival of ICC patients. By in situ hybridization, FasL mRNA were frequently and strongly expressed in biliary dysplasia compared with non-neoplastic intrahepatic bile duct. In well-differentiated ICCs, FasL mRNA expression was frequent and intense. But, the expression gradually decreased in moderately and poorly differentiated ICCs. Down-regulation of FasL mRNA expression in ICCs was correlated with perineural invasion and tumor size (over 4 cm) (P <.05). Apoptotic TIL were more frequent in ICC foci than in non-neoplastic foci remote from ICC foci. These findings suggest that a tumor evasion mechanism involving Fas/FasL exists in ICC; frequent and intense expression of FasL mRNA in well-differentiated ICCs enable them to escape immune surveillance by counterattacking Fas-bearing TIL. This counterattack becomes insensitive in poorly differentiated ICCs, in which the down-regulation of Fas gives them a resistance against the FasL-expressing TIL. These mechanisms may be involved in the tumor progression.

Adult↗
PubMed2000

The immunopathology of primary biliary cirrhosis: thoughts for the millennium.

Primary biliary cirrhosis is an organ specific autoimmune disease that produces progressive cholestatic liver failure. It is predominantly a disease of women characterized by chronic progressive destruction of small intrahepatic bile ducts with portal inflammation and ultimately fibrosis. The serologic hallmark of primary biliary cirrhosis (PBC) is the presence of antibodies to mitochondria. The mechanisms by which and if which such antibodies produce liver tissue injury is unknown. However, the presence of these antibodies have allowed detailed immunological definition of the antigenic epitopes, the nature of reacting autoantibodies and the characterization of T cell responses. Several mechanisms may now be proposed regarding the immune mediated bile duct damage in PBC, including the possible role of T cell-mediated cytotoxicity and intracellular interaction between the IgA class of antimitochondrial antibodies (AMA) and mitochondrial autoantigens. The advent of molecular biology, the ability to clone and define epitopes, and the use of in situ nucleic acid hybridization, have all led to advances in understanding the natural history of immunopathology in PBC. There are major questions which remain unanswered, including, of course, etiology, but also including the questions of why there is female predominance, the absence of PBC in children, the relative ineffectiveness of immunosuppressive drugs, and the specific role of mitochondrial antigens. In this review, we focus on these issues and particularly on the immunobiology of patients with this disease.

Apoptosis↗
PubMed1999

Alleviation of lipopolysaccharide-induced acute liver injury in Propionibacterium acnes-primed IFN-gamma-deficient mice by a concomitant reduction of TNF-alpha, IL-12, and IL-18 production.

The present study was designed to investigate the role of IFN-gamma in LPS-induced liver injury following priming with Propionibacterium acnes. At 1 week after priming BALB/c mice with P. acnes, a large number of macrophages (Mphi) and lymphocytes predominantly infiltrated the portal area, resulting in the intrahepatic formation of granulomas consisting of epithelioid and lymphoid cells. In comparison, in IFN-gamma gene-disrupted BALB/c mice (IFN-gamma knockout mice), the number of infiltrated Mphi was decreased, with a significant reduction in the number and size of granulomas. Subsequent elicitation with a low dose of LPS induced massive hepatic necrosis in wild-type BALB/c mice, with a marked increase in the serum levels of TNF-alpha, IL-12, and IL-18 and subsequently of alanine transferase. In contrast, IFN-gamma knockout mice developed scattered focal necrosis of the liver with significantly lower levels of serum alanine transferase as well as drastic decreases in TNF-alpha, IL-12, and IL-18 production. The administration of an anti-IFN-gamma neutralizing mAb at the eliciting phase significantly alleviated liver injury and reduced serum IL-12 and IL-18 levels. Thus, endogenously produced IFN-gamma is involved in the pathogenesis of this liver injury model by regulating Mphi infiltration and granuloma formation in the priming phase as well as cytokine production in the eliciting phase.

Acute Disease↗
PubMed1999

Expression of MUC1 and MUC2 mucin core proteins and their messenger RNA in gall bladder carcinoma: an immunohistochemical and in situ hybridization study.

Expression of mucin core protein MUC1 and MUC2 was examined at the protein and mRNA level in 55 cases of carcinoma and 20 of dysplasia, and in 15 non-dysplastic epithelia of the gall bladder. In non-dysplastic epithelium, MUC1 protein was not expressed, while in dysplasia, MUC1 was focally expressed in ten cases, particularly in those associated with carcinoma. In carcinoma, MUC1 was expressed heterogeneously, and the frequency and extent of MUC1 expression increased with histological dedifferentiation. MUC1 was found on the apical cell surface and also in the cytoplasm in well- and moderately-differentiated carcinoma, and on the cell border in poorly-differentiated cases. In infiltrative regions, MUC1 expression was more predominant and MUC1 frequently leaked outside the foci of carcinoma. By contrast, MUC2 was focally expressed in non-dysplastic as well as in dysplastic epithelia and more frequently in well-differentiated adenocarcinoma. MUC2-positive cells resembled goblet cells, whether in non-dysplastic epithelium, dysplasia or carcinoma. Cell proliferative activity was higher in MUC1-positive than in MUC1-negative carcinoma cells. Distributions of MUC1 and MUC2 mRNA signals and of MUC1 and MUC2 proteins were similar in carcinoma and dysplasia. These results suggest that MUC1 expression by gall bladder carcinoma may reflect histological dedifferentiation, increased proliferative activity, and invasiveness, while MUC2 expression is related to lower proliferative activity and reflects some differentiation towards goblet cells; and that MUC1 expression in gall bladder dysplasia reflects malignant transformation.

Adenocarcinoma↗
PubMed1999

Aberrant expression of stem cell factor on biliary epithelial cells and peribiliary infiltration of c-kit-expressing mast cells in hepatolithiasis and primary sclerosing cholangitis: a possible contribution to bile duct fibrosis.

Hepatolithiasis and primary sclerosing cholangitis (PSC) are intractable chronic biliary diseases. In hepatolithiasis, bilirubin-calcium stones are packed in multiple irregularly dilated intrahepatic bile ducts. In PSC, small bilirubin-calcium stones develop terminally. The progressive periductal fibrosis with dilated and stenotic bile ducts in these two diseases may play a role in their incurability. This immunohistochemical study has investigated the expression of some factors that might be involved in fibrogenesis in hepatolithiasis and PSC. Many mast cells positive for c-kit were found in the periductal and ductal fibrosis around the intrahepatic large bile ducts and also around the proliferative peribiliary glands. These mast cells also expressed basic fibroblast growth factor and/or tumour necrosis factor-alpha, which are known as fibrogenetic factors. It was of interest that the aberrant expression of stem cell factor (SCF), a ligand of c-kit, was demonstrated on biliary epithelia of the dilated and stenotic bile ducts showing periductal fibrosis and inflammation and also of the proliferated peribiliary glands in hepatolithiasis and PSC, while no such expression was seen in non-affected bile ducts in hepatolithiasis or in the bile ducts in normal livers. Some of the infiltrating mononuclear cells around the SCF-expressing bile ducts were also positive for SCF. It seems likely that aberrantly expressed SCF on biliary epithelial cells accumulates and stimulates mast cells via the c-kit receptor and that these up-regulated mast cells induce progressive periductal and portal fibrosis by displaying fibrogenetic factors in hepatolithiasis and PSC.

Bile Ducts, Intrahepatic↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

In situ nucleic acid detection of PDC-E2, BCOADC-E2, OGDC-E2, PDC-E1alpha, BCOADC-E1alpha, OGDC-E1, and the E3 binding protein (protein X) in primary biliary cirrhosis.

The characteristic serological feature of primary biliary cirrhosis (PBC) is the presence of antimitochondrial antibodies (AMAs), and the major proteins recognized by AMAs are subunits of the 2-oxo acid dehydrogenase complexes (2-OADC), including the E2 components of the pyruvate dehydrogenase complex (PDC), the 2-oxo-glutarate dehydrogenase complex (OGDC), the branched-chain 2-oxoacid dehydrogenase complex (BCOADC), the E3 binding protein (E3BP or protein X) and the E1a component of mammalian PDC. Previous work has postulated that either E3BP, or a molecule cross-reactive with the PDC-E2 molecule, is uniquely expressed on the surface of biliary epithelial cells in PBC. To address this issue, we performed in situ hybridization for all of the major 2-OADC components at the mRNA level, including PDC-E2, BCOADC-E2, OGDC-E2, PDC-E1a, BCOADC-E1a, OGDC-E1, and E3BP using 13 PBC and 9 control livers using 7 mitochondrial antisense probes. In both PBC and controls, the expression of all 2-OADC component mRNA studied herein were found in hepatocytes and infiltrating mononuclear cells, without significant differences. Interestingly, however, despite published data on immunohistochemical staining, interlobular bile ducts including the injured bile ducts in PBC were generally negative or only faintly positive, with the exception of 1 bile duct in 1 of 13 cases of PBC and 1 of 9 control liver specimens. Moreover, confocal microscopic examination and image analysis revealed that the mRNA signal intensity of each of the 2-OADC components in the bile ducts of PBC was relatively lower in comparison with control liver diseases. These data suggest that continuous enhanced synthesis of the 2-OADC components is not likely to be occurring in the biliary epithelial cells in PBC, and that an increase of PDC-E2 or E3BP immunoreactivity in PBC is caused by exogenous imported or cross-reactive molecules.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗