Biliary Epithelial Cells Of Patients Research Articles

Increased expression of TNFRSF14 and LIGHT in biliary epithelial cells of patients with primary sclerosing cholangitis

Background and aimsThere is a lack of biliary epithelial molecular markers for primary sclerosing cholangitis (PSC). We analyzed candidates from disease susceptibility genes identified in recent genome-wide association studies (GWAS). MethodsExpression levels of GWAS genes were analyzed in archival liver tissues of patients with PSC and controls. Immunohistochemical analysis was performed to evaluate expression levels in the biliary epithelia of PSC (N = 45) and controls (N = 12). Samples from patients with primary biliary cholangitis (PBC) were used as disease controls (N = 20). ResultsHepatic expression levels of ATXN2, HHEX, PRDX5, MST1, and TNFRSF14 were significantly altered in the PSC group. We focused on the immune-related receptor, TNFRSF14. Immunohistochemistry revealed that high expression of TNFRSF14 in biliary epithelial cells was observed only in the PSC group. In addition, the expression of LIGHT, which encodes a TNFRSF14-activating ligand, was increased in PSC liver. Immunohistochemistry showed that high expression of LIGHT was more common in PSC biliary epithelia (53%) than in the PBC (15%) or control (0%) groups; moreover, it was positively associated with fibrotic progression, although it was not an independent prognostic factor. ConclusionsTNFRSF14 and LIGHT are promising candidate markers for PSC.

CCL5 is induced by TLR 3 signaling in HuCCT1 human biliary epithelial cells: possible involvement in the pathogenesis of biliary atresia.

Biliary atresia (BA) is a disease of the newborn that is characterized by progressive, inflammatory and sclerosing cholangiopathy. Innate immune responses to viral components are thought to be involved in the pathogenesis of BA. It is also reported that some chemokines, such as CCL5, are possibly involved in the pathogenesis of experimental animal model of BA. We treated human biliary epithelial HuCCT1 cells with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA (dsRNA) which mimics viral RNA, and analyzed the CCL5 expression by quantitative reverse transcription-PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). To examine the regulation mechanisms of CCL5, we subjected the cells to RNA interference (siRNA) against Toll-like receptor 3 (TLR3), interferon (IFN)-β, NF-κB p65 and IFN regulatory factor (IRF) 3. Immunohistochemical staining for CCL5 was also performed in tissues from patients with BA. Poly IC induced CCL5 expression in HuCCT1 cells. CCL5 expression induced by poly IC was inhibited by the knockdown of TLR3, p65 or IRF3, but it was not affected by knockdown of IFN-β. Immunohistochemical staining showed that CCL5 was strongly expressed in biliary epithelial cells of patients with BA. The current study suggests that TLR3 signaling induces CCL5 expression via NF-κB and IRF3 in bile duct cells, and this pathway may be involved in the pathogenesis of BA.

146 The Impact of Cirrhosis or Prior Liver Transplant On Obstetrical Outcomes: A National Study

BACKGROUND: TLR signaling in biliary epithelial cells (BECs) is tightly controlled to prevent excessive innate immune responses in the healthy liver. We examined the effect of chronic inflammation on TLR-mediated endotoxin tolerance in human BECs in patients with end-stage chronic inflammatory liver disease (ESLD), which protects the host from permanent BEC activation in the face of ubiquitous intestinal TLR ligands in the bile and portal venous blood. METHODS: We examined patients undergoing liver transplantation for immune-mediated (PBC, PSC), alcoholic and viral (chronic hepatitis B and C) ESLD. Northern blots, real-time RT-PCR, western blots and immunocytochemistry were used for mRNA and protein expression studies of innate immune proteins in whole ESLD tissue and isolated primary BECs. Flow cytometry for incorporated endotoxin, ELISAs for secreted proinflammatory mediators such as IL-6, IL-8, and MCP-1, NF-κB reporter assays and TLR over-expression studies were used for functional studies. RESULTS: Freshly isolated human BECs in ESLD showed increased TLR activation profiles and markedly enhanced ICAM-1 expression, which have recently been linked to decreased hepatic endotoxin tolerance. Whole ESLD tissue exhibited increased expression of IFN-γ and TNF-α. Affected BECs depicted activated IFN-γ-dependent genes such as CXCL9, CXCL10 and CXCL11. We also found enhanced activation of IRF-1 and STAT1 in activated BECs, which have been shown to promote TLR signaling. These findings suggested that chronic inflammation led to enhanced TLR-mediated innate immune responses and decreased endotoxin tolerance in affected BECs in ESLD. In line with this hypothesis, primary human BECs isolated from patients with early disease stages depicted normal TLR and IL-6, IL-8, MCP-1 and ICAM-1 expression. These data suggested that increased PRR signaling and diminished endotoxin tolerance in BECs did not play a role in the primary pathogenesis of these diseases and argued for a secondary phenomenon due to chronic inflammation. Further In Vitro studies confirmed that pro-inflammatory cytokines and repetitive endotoxin challenges disrupted homoand hetero-tolerance to endotoxins in BECs. Enhanced TLR surface expression and subsequently increased endotoxin incorporation in cytokine-primed BECs contributed to increased TLR sensitivity. CONCLUSIONS: Chronic inflammation promotes a state of hyper-responsiveness to TLR ligands in ESLD and breaks the protective endotoxin tolerance in BECs. Loss of TLR tolerance appear to be especially deleterious in the face of increased intestinal endotoxin levels, which have been described in patients with ESLD of different etiologies.

Genomic analysis of differentially expressed genes in liver and biliary epithelial cells of patients with primary biliary cirrhosis: Atsushi Tanaka, Asahi Life Fdn, Tokyo Japan; Patrick S Leung, Thomas P Kenny, University of California, Davis, CA; Janice Au-Young, Incyte Genomics, Inc, Palo Alto, CA; Thomas Prindiville, University of California, Davis, CA; Ross L Coppel, Monash University, Victoria Australia; Aftab A

Genomic analysis of differentially expressed genes in liver and biliary epithelial cells of patients with primary biliary cirrhosis: Atsushi Tanaka, Asahi Life Fdn, Tokyo Japan; Patrick S Leung, Thomas P Kenny, University of California, Davis, CA; Janice Au-Young, Incyte Genomics, Inc, Palo Alto, CA; Thomas Prindiville, University of California, Davis, CA; Ross L Coppel, Monash University, Victoria Australia; Aftab A

Genomic Analysis of Differentially Expressed Genes in Liver and Biliary Epithelial Cells of Patients with Primary Biliary Cirrhosis

The characterization of differentially expressed genes provides a powerful tool for identifying molecules that may be involved in the pathogenesis of disease. We have used two independent techniques to identify overexpressed transcripts in bile duct cells and in liver from patients with primary biliary cirrhosis (PBC). In the first method, we used suppressive subtractive hybridization to compare mRNA from isolated PBC bile duct epithelial cells (BECs) to normal BECs and identified 71 clones as transcribed at higher levels in PBC-BECs. Amongst these clones, 62/71 had matches in a non-redundant nucleotide database and 9/71 had matches in an EST database. Of the 62 clones, 51/62 include a complexity of genes involved in cell proliferation, signal transduction, transcription regulation, RNA processing, carbohydrate metabolism and hypothetical/unknown proteins; 4/62 were identified as interstitial collagenase and collagenase precursors, 4/62 as ribosomal proteins, 3/62 as mitochondrial DNA. The mitochondrial cDNA sequences included cytochrome c oxidase, Wnt-13, and the pHL gene, a c-myc oncogene containing coxIII sequence. In the second method, we constructed cDNA libraries from three different PBC livers and sequenced a total of 12,324 independent clones. These 12,324 clones underwent virtual subtraction with 2,814,148 independent clones from Incyte LifeSeq®libraries. Twenty one sequences were identified as unique to PBC liver. Collectively, these approaches identified a number of genes involved in signalling, RNA processing, mitochondrial function, inflammation, and fibrosis. Interestingly, both Wnt-13 and Notch transcripts are overexpressed in PBC liver. Further studies are needed to focus on the significance of these genes during the natural history of disease.

Monoclonal Antibodies to Mitochondrial E2 Components Define Autoepitopes in Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of antimitochondrial Abs (AMA). The autoantigens recognized by AMA are the E2 components of the pyruvate dehydrogenase complex (PDC-E2), the branched chain 2-oxoacid dehydrogenase complex E (BCOADC-E2), and the 2-oxoglutarate dehydrogenase complex E (OGDC-E2). Previous studies using murine monoclonal and human combinatorial Abs to PDC-E2 have demonstrated an intense linear staining pattern in the apical region of biliary epithelial cells (BEC) in PBC but not control liver. We therefore examined whether mAbs to the other mitochondrial autoantigens BCOADC-E2 and OGDC-E2 demonstrated disease-specific patterns of reactivity. Using an expressed recombinant "trihybrid" protein containing the lipoyl domains of PDC-E2, OGDC-E2, and BCOADC-E2, we immunized BALB/c mice to produce 35 mAbs specific for one or more of the above mitochondrial autoantigens. Seven of these mAbs uniquely stained the apical region of BEC in PBC. Of these seven, one was reactive to PDC-E2, two recognized BCOADC-E2, three were reactive to OGDC-E2, and one recognized all three Ags. Our current data demonstrate that, similar to our previous studies regarding PDC-E2, mAbs to BCOADC-E2 and OGDC-E2, or a molecule that cross-reacts with the inner lipoyl domain of all three enzymes, also show a uniquely intense staining pattern in the apical region of BEC in patients with PBC when compared with diseased controls. The abundance of such disease-specific determinants in the target cells of PBC raises interesting possibilities regarding the role of these autoantigens in the pathogenesis of this disease.

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.

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)

Molecular aspects and the pathological basis of primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) has been considered to be a 'model auto-immune disease' for more than two decades. However, the underlying pathophysiology of PBC and the relationship with the associated serological abnormalities have been hitherto elusive. Beginning in 1987 with the cloning and subsequent identification of the mitochondrial autoantigens of PBC, progress has come rapidly and we can now sketch several potential pathogenic pathways through which disease occurs. More than 90% of patients with PBC produce autoantibodies to mitochondria, and the antoantigens involved have been identified as related components of the 2-oxo-acid dehydrogenase complexes (OADC), including the E2 subunits of the pyruvate dehydrogenase complex (PDC-E2), the branched chain 2-oxo-acid dehdrogenase complex and 2-oxo-glutarate dehydrogenase complex, Protein X and E1 alpha. The cDNAs of each of the E2 subunits of OADC have been cloned and characterized. Moreover, the epitopes of the antimitochondrial antibodies (AMA) have been mapped at the highly conserved lipoyl domain E2 subunits. The use of recombinant peptides produced by these clones has greatly facilitated the detection of AMA. In addition, nucleotide sequence analysis of PDC-E2 specific human monoclonals and combinatorial Fabs strongly suggests that these autoantibodies are derived from clonal selection of a restricted set of somatically mutated immunoglobulin germline genes. Most interestingly, however, the use of monoclonal reagents to PDC-E2 has demonstrated that there is an increased expression of either PDC-E2, or a cross-reactive molecule, on the luminal surface of biliary epithelial cells in patients with PBC. These data provide a scenario to explain the tissue specific pathology associated with PBC and several interesting underlying pathophysiological mechanisms.

Abnormal expression of PDC-E 2 on the apical surface of biliary epithelial cells in patients with antimitochondrial antibody—negative primary biliary cirrhosis

The presence of antimitochondrial antibodies (AMA) is a major criterion for the diagnosis of primary biliary cirrhosis (PBC). Although it is not clear that AMA are involved in the pathogenesis of the disease, the study of these autoantibodies has enabled much information to be accumulated about the specificity of this response. The autoantigens have been identified as components of a functionally related enzyme family, the 2-oxo-acid-dehydrogenase complex. Within this complex, pyruvate dehydrogenase E 2 subunit (PDC-E 2) has been determined to be the immunodominant autoantigen. Using a panel of mouse monoclonal antibodies and human combinatorial autoantibodies, it has been demonstrated that patients with PBC, but not controls, have an abnormal expression of either PDC-E 2 or a cross-reacting molecule in the apical region of biliary epithelium. Others have shown a similar reaction using rabbit sera directed to PDC-E 2. Our previous studies have concentrated on AMA-positive patients. In this study, the presence of PDC-E 2, class II, immunoglobulin (Ig) A, and B7/BB1 in the bile duct epithelial cells of AMA-positive as well as AMA-negative patients is addressed. Most patients with AMA-negative PBC (seven of nine) react in a fashion similar to AMA-positive patients with intense staining of the apical region of the bile duct epithelial cells for “PDC-E 2,” increased IgA expression, and little major histocompatibility complex (MHC) class II staining in the early-stage patients. Interestingly, the two AMA-negative patients that did not express PDC-E 2 on the apical side of their biliary epithelium had anticentromere antibodies and Sjögren's syndrome. Based on these data, it can be concluded that the disease process for both AMA-positive and AMA-negative patients with PBC has a similar pathogenic mechanism, which is likely to involve the abnormal expression of PDC-E 2 or a molecular mimic of PDC-E 2, and does not require MHC class II expression.

Abnormal expression of PDC-E2 on the apical surface of biliary epithelial cells in patients with antimitochondrial antibody-negative primary biliary cirrhosis

The presence of antimitochondrial antibodies (AMA) is a major criterion for the diagnosis of primary biliary cirrhosis (PBC). Although it is not clear that AMA are involved in the pathogenesis of the disease, the study of these autoantibodies has enabled much information to be accumulated about the specificity of this response. The autoantigens have been identified as components of a functionally related enzyme family, the 2-oxo-acid-dehydrogenase complex. Within this complex, pyruvate dehydrogenase E2 subunit (PDC-E2) has been determined to be the immunodominant autoantigen. Using a panel of mouse monoclonal antibodies and human combinatorial autoantibodies, it has been demonstrated that patients with PBC, but not controls, have an abnormal expression of either PDC-E2 or a cross-reacting molecule in the apical region of biliary epithelium. Others have shown a similar reaction using rabbit sera directed to PDC-E2. Our previous studies have concentrated on AMA-positive patients. In this study, the presence of PDC-E2, class II, immunoglobulin (Ig) A, and B7/BB1 in the bile duct epithelial cells of AMA-positive as well as AMA-negative patients is addressed. Most patients with AMA-negative PBC (seven of nine) react in a fashion similar to AMA-positive patients with intense staining of the apical region of the bile duct epithelial cells for “PDC-E2,” increased IgA expression, and little major histocompatibility complex (MHC) class II staining in the early-stage patients. Interestingly, the two AMA-negative patients that did not express PDC-E2 on the apical side of their biliary epithelium had anticentromere antibodies and Sjögren’s syndrome. Based on these data, it can be concluded that the disease process for both AMA-positive and AMA-negative patients with PBC has a similar pathogenic mechanism, which is likely to involve the abnormal expression of PDC-E2 or a molecular mimic of PDC-E2, and does not require MHC class II expression. (Hepatology 1995; 22:1440-1446).

Abnormal expression of the E2 component of the pyruvate dehydrogenase complex on the luminal surface of biliary epithelium occurs before major histocompatibility complex class II and BB1/B7 expression

Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease characterized histologically by nonsuppurative destructive cholangitis. Sera from patients with PBC react with a series of intramitochondrial enzymes with the immunodominant response directed against the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Recently, using tissue sections of late-stage PBC, we showed that there is increased expression in biliary epithelial cells of patients with PDCE2 or a molecule cross-reactive with PDC-E2. Previous work has shown that biliary epithelial cells of patients with PBC express an increased amount of class II. To address the sequence of events in the evolution of PBC, we have focused our attention in this study on early biliary epithelial lesions. In particular, we have studied the liver of 22 female patients with PBC that was diagnosed as either stage I or stage II using both a mouse monoclonal antibody that has reactivity similar to human autoantibodies as well as a human Fab combinatorial prepared from the lymph node of a PBC patient. Tissues were simultaneously stained using antibodies to PDC-E2, class II, and BB1/B7. As a positive control, tissues from late-stage PBC were studied concurrently. By determining the order of expression among the three molecules, PDC-E2, class II, and BB1/B7, we report that the expression of PDC-E2 or a PDC-E2-like molecule on biliary duct epithelium of patients with PBC precedes the expression of BB1/B7 and major histocompatibility complex (MHC) class II molecules. The alteration of an autoantigen in biliary duct epithelial may be the earliest lesion in PBC.

Abnormal expression of the E2 component of the pyruvate dehydrogenase complex on the luminal surface of biliary epithelium occurs before major histocompatibility complex class II and BB1/B7 expression

Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease characterized histologically by non-suppurative destructive cholangitis. Sera from patients with PBC react with a series of intramitochondrial enzymes with the immunodominant response directed against the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Recently, using tissue sections of late-stage PBC, we showed that there is increased expression in biliary epithelial cells of patients with PDC-E2 or a molecule cross-reactive with PDC-E2. Previous work has shown that biliary epithelial cells of patients with PBC express an increased amount of class II. To address the sequence of events in the evolution of PBC, we have focused our attention in this study on early biliary epithelial lesions. In particular, we have studied the liver of 22 female patients with PBC that was diagnosed as either stage I or stage II using both a mouse monoclonal antibody that has reactivity similar to human autoantibodies as well as a human Fab combinatorial prepared from the lymph node of a PBC patient. Tissues were simultaneously stained using antibodies to PDC-E2, class II, and BB1/B7. As a positive control, tissues from late-stage PBC were studied concurrently. By determining the order of expression among the three molecules, PDC-E2, class II, and BB1/B7, we report that the expression of PDC-E2 or a PDC-E2—like molecule on biliary duct epithelium of patients with PBC precedes the expression of BB1/B7 and major histocompatibility complex (MHC) class II molecules. The alteration of an autoantigen in biliary duct epithelium may be the earliest lesion in PBC. (Hepatology 1995; 21:1031-1037.)

Biliary epithelial expression of pyruvate dehydrogenase complex in primary biliary cirrhosis: An immunohistochemical and immunoelectron microscopic study

It has been reported recently that there is a unique distribution of the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2) on biliary epithelial cells in patients with primary biliary cirrhosis (PBC) but not primary sclerosing cholangitis. This distribution has been demonstrated using a mouse monoclonal antibody, coined C355.1. The epitope recognized by C355.1 is near the lipoic acid binding site of PDC-E2. C355.1 inhibits PDC-E2 activity in vitro and, unlike a panel of other monoclonal antibodies against different regions of PDC-E2, appears to bind not only to mitochondria but also to a unique antigen expressed predominantly on the luminal side of biliary epithelial cells in PBC. We have extended these observations by studying the subcellular reactivity of C355.1 using postembedding immunoelectron microscopy on the intrahepatic small bile ducts of PBC livers, extrahepatic biliary obstruction (EBO) livers, and normal livers. We report that the reactivity of C355.1 can be classified into two categories. The first category is characterized by small foci of reaction products that were randomly dispersed in cytoplasm, particularly in supranuclear areas; the ultrastructural characterization of these foci was impossible to define but was similar in PBC and EBO. However, of particular interest was the second category of reactivity, which was characterized by deposition of reaction products around the biliary lumen, including microvilli and adjacent subluminal ectoplasm and secretory substances in the biliary lumen. This staining pattern was frequent in PBC livers, only occasionally evident in EBO livers, and not found in normal livers. These data further define and highlight the unique subcellular distribution of PDC-E2 around the biliary lumen in PBC livers and suggest that this abnormality is related to the pathogenesis of bile duct lesions.

Heterogeneity of combinatorial human autoantibodies against PDC-E2 and biliary epithelial cells in patients with primary biliary cirrhosis

Heterogeneity of combinatorial human autoantibodies against PDC-E2 and biliary epithelial cells in patients with primary biliary cirrhosis

Heterogeneity of Combinatorial Human Autoantibodies Against Pdc–E2 and Biliary Epithelial Cells in Patients With Primary Biliary Cirrhosis

The polyclonal nature of antimitochondrial autoantibodies and the limited success of generating human monoclonal antibodies have made analysis of fine specificity and antibody heterogeneity difficult to define. The major autoantigen of primary biliary cirrhosis is the E2 component of the pyruvate dehydrogenase pathway (PDC-E2). To address the relative importance of the region(s) in the PDC-E2 inner lipoyl domain to antibody binding, we report herein detailed profiles of 12 PDC-E2-specific antigen-binding fragments, SP1 through SP12, derived by screening of a combinatorial immunoglobulin library (derived from a primary biliary cirrhosis patient) with full-length native PDC-E2. All antigen-binding fragments are IgG isotypes and include a similar number of lambda- and kappa-chains. The antigen-binding fragments react specifically to PDC-E2 with high affinity (kappa a = 10(-7) to 10(-10) mol/L-1) and recognize a conformational epitope in the inner lipoyl domain of PDC-E2. Furthermore, the antibodies demonstrate substantial heterogeneity in recognition of different recombinant PDC-E2 fragments and differential recognition patterns against mutant constructs of the human PDC-E2 inner lipoyl domain (amino acid residues 91 to 227). In addition, five of the antigen-binding fragment clones (SP1, 3, 4, 8 and 12) demonstrate different staining patterns on biliary epithelial cells of patients with primary biliary cirrhosis but not control liver disease; some antigen-binding fragments specifically stained the apical region of biliary epithelium, a pattern distinct from that of typical mitochondrial staining. The response to the inner lipoyl domain is not, however, monospecific, and there is much more heterogeneity in fine specificity than could be accounted for by arbitrary reshuffling of variable immunoglobulin heavy and light chains into unnatural combinations.

Would the real mccoy please stand up?

HepatologyVolume 19, Issue 3 p. 793-794 Hepatology ElsewhereFree Access Would the real mccoy please stand up? First published: March 1994 https://doi.org/10.1002/hep.1840190338AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Sera from patients with primary biliary cirrhosis (PBC) react with enzymes of the 2-oxo dehydrogenase pathways, particularly PDC-E2. These enzymes are present in all nucleated cells, yet autoimmune damage is confined to biliary epithelial cells. Using a panel of eight mouse monoclonal antibodies and a human combinatorial antibody specific for PDC-E2, we examined by indirect immunofluorescence and confocal microscopy sections of liver from patients with PBC, progressive sclerosing cholangitis, and hepatocarcinoma. The monoclonal antibodies gave typical mitochondrial immunofluorescence on biliary epithelium and on hepatocytes from patients with either PBC, progressive sclerosing cholangitis, or hepatocarcinoma. However, one of eight mouse monoclonal antibodies (C355.1) and the human combinatorial antibody reacted with great intensity and specificity with the luminal region of biliary epithelial cells from patients with PBC. Simultaneous examination of these sections with an antiisotype reagent for human IgA revealed high IgA staining in the luminal region of biliary epithelial cells in patients with PBC. IgG and IgA antibodies to PDC-E2 were detected in the bile of patients with PBC but not normal controls. We believe that this data may be interpreted as indicating that a molecule cross-reactive with PDC-E2 is expressed at high levels in the luminal region of biliary epithelial cells in PBC. References 1 Walker JG, Doniach D, Roitt IM, et al. Serological tests in diagnosis of primary biliary cirrhosis. Lancet 1965; 1: 827. 2 Gershwin ME, Mackay IR. Primary biliary cirrhosis: paradigm or paradox for autoimmunity. Gastroenterology 1991; 100: 822. 3 Yeaman SJ, Fussey SPM, Danner DJ, et al. Primary biliary cirrhosis: identification of two major M 2 mitochondrial autoantigens. Lancet 1988; 1: 1067– 1069. 4 van de Water J, Turchany J, Leung PSC, Lake J, Munoz S, Surh CD, Coppel R, et al. Molecular mimicry in primary biliary cirrhosis: evidence for biliary epithelial expression of a molecule cross-reactive with pyruvate dehydrogenase complex, E2. J Clin Invest 1993; 91: 2653– 2664. 5 Joplin R, Lindsay JG, Johnson GD, Strain A, Neuberger JM. Membrane dihydrolipoamide acetyltransferase (E2) on human biliary epithelial cells in primary biliary cirrhosis. Lancet 1992; 339: 93– 94. 6 van de Water J, Ansari AA, Surh CD, Coppel R, Roche T, Bonkovsky H, Kaplan M, et al. Evidence for the targeting by 2-oxo-dehydrogenase enzymes in the T cell response on primary biliary cirrhosis. J Immunol 1991; 146: 89– 94. 7 Hopf U, Möller B, Stemerowitz R, et al. Relation between Escherichia coli R (rough) forms in gut, lipid A in liver, and primary biliary cirrhosis. Lancet 1988; 2: 1419. 8 Burroughs AK, Rosenstein IJ, Epstein O, et al. Bacteriuria and primary biliary cirrhosis. Gut 1984; 25: 133. 9 Nickowitz RE, Worman HJ. Autoantibodies against nuclear pore membrane protein GP 210: Evidence for molecular mimicry. Hepatology 1993; 18: 109A. 10 Manns MP, Griffin KJ, Sullvan KF, Johnson EF. LKM autoantibodies recognize a short linear sequence in P 450 IID6, a cytochrome P-4450 monooxygenase. J Clin Invest 1991; 88: 1370– 1378. 11 Manns MP and Krüger M. Immunogenetics in chronic liver diseases. Gastroenterology. In press. 12 Ballardini G, Mirakian R, Bianchi FB. Aberrant expression of HLA-DR antigens on bile duct epithelium in PBC: relevance to pathogenesis. Lancet 1984; 2: 1009. Volume19, Issue3March 1994Pages 793-794 ReferencesRelatedInformation

Molecular mimicry in primary biliary cirrhosis. Evidence for biliary epithelial expression of a molecule cross-reactive with pyruvate dehydrogenase complex-E2.

Sera from patients with primary biliary cirrhosis (PBC) react with enzymes of the 2-oxo dehydrogenase pathways, particularly PDC-E2. These enzymes are present in all nucleated cells, yet autoimmune damage is confined to biliary epithelial cells. Using a panel of eight mouse monoclonal antibodies and a human combinatorial antibody specific for PDC-E2, we examined by indirect immunofluorescence and confocal microscopy sections of liver from patients with PBC, progressive sclerosing cholangitis, and hepatocarcinoma. The monoclonal antibodies gave typical mitochondrial immunofluorescence on biliary epithelium and on hepatocytes from patients with either PBC, progressive sclerosing cholangitis, or hepatocarcinoma. However, one of eight mouse monoclonal antibodies (C355.1) and the human combinatorial antibody reacted with great intensity and specificity with the luminal region of biliary epithelial cells from patients with PBC. Simultaneous examination of these sections with an antiisotype reagent for human IgA revealed high IgA staining in the luminal region of biliary epithelial cells in patients with PBC. IgG and IgA antibodies to PDC-E2 were detected in the bile of patients with PBC but not normal controls. We believe that this data may be interpreted as indicating that a molecule cross-reactive with PDC-E2 is expressed at high levels in the luminal region of biliary epithelial cells in PBC.

Combinatorial autoantibodies to dihydrolipoamide acetyltransferase, the major autoantigen of primary biliary cirrhosis.

mRNA from a regional lymph node of a patient with primary biliary cirrhosis (PBC) was used to construct a combinatorial immunoglobulin library in the lambda phage vector system. Six human monoclonal IgG Fab clones (LC1-LC6) specific for the major autoantigen of PBC--dihydrolipoamide acetyltransferase, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2)--were isolated, appearing at a frequency of 0.01% in the combinatorial immunoglobulin library. These Fab clones recognize human PDC-E2 with high affinity (Ka = 10(-7)-10(-9) M-1). Using both immunoblotting and ELISA, LC1-LC6 showed little cross-reactivity to any of the other autoantigens commonly recognized by PBC sera or to other antigens commonly recognized by PBC sera or to other antigens such as histone, calf thymus DNA, and bovine serum albumin. The Fab monoclonal antibodies show a typical anti-mitochondrial staining pattern in HEp-2 cells but react strongly with the luminal aspect of biliary epithelial cells of patients with PBC. Our results demonstrate that a recombinant combinatorial immunoglobulin library can be used to isolate high-affinity Fabs against a specific autoantigen. Such reagents will facilitate the analysis of immunoglobulin gene structure, idiotype, and antigen-antibody interactions in autoimmune disease.