bromochloroacetic-acid and Liver-Diseases

The study of cytokeratin expression has provided a valuable insight into the biliary microanatomy of the liver in health and disease. The canals of Hering are a putative site of origin for progenitor cells, which may repopulate the liver after cellular damage and loss. Normal bile ducts and the bile ductular reaction that occurs in many chronic liver diseases - especially chronic biliary tract disease - express cytokeratin (CK) 7 and CK19. Therefore, both ductopenia and the process of bile ductular reaction can be highlighted with immunohistochemistry for these cytokeratins. Furthermore, CK7 is usually expressed in an increasingly widespread manner by hepatocytes as chronic cholestatic liver disease progresses. For these reasons, CK immunohistochemistry is a very useful adjunct to morphological assessment and histochemical stains for copper retention when a diagnosis of chronic biliary disease is being considered. This review describes the anatomical theory behind the use of CK immunohistochemistry for the assessment of bile duct number and distribution in the liver and provides practical advice for the application of this technique in the diagnostic setting of common medical liver diseases.

Topics: Bile Ducts, Intrahepatic; Biopsy; Gallbladder Diseases; Hepatocytes; Humans; Immunohistochemistry; Keratins; Liver; Liver Diseases

Keratins are cytoskeletal proteins that provide structural support to epithelial cells and tissues. Perturbation causes cell and tissue fragility and accounts for a large number of genetic disorders in humans. In humans, 54 functional keratin genes exist and 21 different keratin genes including hair keratins and hair follicle-specific epithelial keratins have been associated with hereditary disorders. Moreover, keratins have been implicated in more complex traits such as liver disease and inflammatory bowel disease. Understanding the molecular basis of keratin disorders has been the basis for improved diagnosis with prognostic implications, genetic counseling and prenatal testing for severe disorders. Besides their mechanical role, keratins have newly identified functions in apoptosis, cell growth, tissue polarity, wound healing and tissue remodeling. Improved understanding of the regulatory functions of keratins may offer novel approaches to overcome current treatment limitations.

Topics: Corneal Dystrophies, Hereditary; Humans; Inflammatory Bowel Diseases; Keratins; Liver Diseases; Mutation; Skin Diseases, Genetic

Simple epithelial keratins (SEKs) are found primarily in single-layered simple epithelia and include keratin 7 (K7), K8, K18-K20, and K23. Genetically engineered mice that lack SEKs or overexpress mutant SEKs have helped illuminate several keratin functions and served as important disease models. Insight into the contribution of SEKs to human disease has indicated that K8 and K18 are the major constituents of Mallory-Denk bodies, hepatic inclusions associated with several liver diseases, and are essential for inclusion formation. Furthermore, mutations in the genes encoding K8, K18, and K19 predispose individuals to a variety of liver diseases. Hence, as we discuss here, the SEK cytoskeleton is involved in the orchestration of several important cellular functions and contributes to the pathogenesis of human liver disease.

Topics: Animals; Biomarkers, Tumor; Humans; Keratins; Liver Diseases; Mice; Mice, Transgenic; Mutation; Neoplasm Metastasis; Neoplasms, Glandular and Epithelial; Proteins

Inclusion bodies are characteristic morphological features of various neuronal, muscular and other human disorders. They share common molecular constituents such as p62, chaperones and proteasome subunits. The proteins within aggregates are misfolded with increased beta-sheet structure, they are heavily phosphorylated, ubiquitinylated and partially degraded. Furthermore, involvement of proteasomal system represents a common feature of virtually all inclusions. Multiple aggregates contain intermediate filament proteins as their major constituents. Among them, Mallory-Denk bodies (MDBs) are the best studied. MDBs represent hepatic inclusions observed in diverse chronic liver diseases such as alcoholic and non-alcoholic steatohepatitis, chronic cholestasis, metabolic disorders and hepatocellular neoplasms. MDBs are induced in mice fed griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine and resolve after discontinuation of toxin administration. The availability of a drug-induced model makes MDBs a unique tool for studying inclusion formation. Our review summarizes the recent advances gained from this model and shows how they relate to observations in other aggregates. The MDB formation-underlying mechanisms include protein misfolding, chaperone alterations, disproportional protein expression with keratin 8>keratin 18 levels and subsequent keratin 8 crosslinking via transglutaminase. p62 presence is crucial for MDB formation. Proteasome inhibitors precipitate MDB formation, whereas stimulation of autophagy with rapamycin attenuates their formation.

Topics: Animals; Humans; Inclusion Bodies; Keratins; Liver Diseases; Proteasome Endopeptidase Complex

Intermediate filaments (IFs) represent the largest cytoskeletal gene family comprising approximately 70 genes expressed in tissue specific manner. In addition to scaffolding function, they form complex signaling platforms and interact with various kinases, adaptor, and apoptotic proteins. IFs are established cytoprotectants and IF variants are associated with >30 human diseases. Furthermore, IF-containing inclusion bodies are characteristic features of several neurodegenerative, muscular, and other disorders. Acidic (type I) and basic keratins (type II) build obligatory type I and type II heteropolymers and are expressed in epithelial cells. Adult hepatocytes contain K8 and K18 as their only cytoplasmic IF pair, whereas cholangiocytes express K7 and K19 in addition. K8/K18-deficient animals exhibit a marked susceptibility to various toxic agents and Fas-induced apoptosis. In humans, K8/K18 variants predispose to development of end-stage liver disease and acute liver failure (ALF). K8/K18 variants also associate with development of liver fibrosis in patients with chronic hepatitis C. Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated K8/K18, chaperones and sequestosome1/p62 (p62) as their major constituents. MDBs are found in various liver diseases including alcoholic and non-alcoholic steatohepatitis and can be formed in mice by feeding hepatotoxic substances griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDBs also arise in cell culture after transfection with K8/K18, ubiquitin, and p62. Major factors that determine MDB formation in vivo are the type of stress (with oxidative stress as a major player), the extent of stress-induced protein misfolding and resulting chaperone, proteasome and autophagy overload, keratin 8 excess, transglutaminase activation with transamidation of keratin 8 and p62 upregulation.

Topics: Humans; Inclusion Bodies; Intermediate Filaments; Keratins; Liver; Liver Diseases; Organelles; Protein Isoforms

Frank B. Mallory described cytoplasmic hyaline inclusions in hepatocytes of patients with alcoholic hepatitis in 1911. These inclusions became known as Mallory bodies (MBs) and have since been associated with a variety of other liver diseases including non-alcoholic fatty liver disease. Helmut Denk and colleagues described the first animal model of MBs in 1975 that involves feeding mice griseofulvin. Since then, mouse models have been instrumental in helping understand the pathogenesis of MBs. Given the tremendous contributions made by Denk to the field, we propose renaming MBs as Mallory-Denk bodies (MDBs). The major constituents of MDBs include keratins 8 and 18 (K8/18), ubiquitin, and p62. The relevant proteins and cellular processes that contribute to MDB formation and accumulation include the type of chronic stress, the extent of stress-induced protein misfolding and consequent proteasome overload, a K8-greater-than-K18 ratio, transamidation of K8 and other proteins, presence of p62 and autophagy. Although it remains unclear whether MDBs serve a bystander, protective or injury promoting function, they do serve an important role as histological and potential progression markers in several liver diseases.

Topics: Adaptor Proteins, Signal Transducing; Animals; Biomarkers; Hepatocytes; Humans; Inclusion Bodies; Keratins; Liver; Liver Diseases; Proteins; Sequestosome-1 Protein; Ubiquitin

Publications concerning liver histopathology in fatty liver disease and chronic hepatitis C, iron and copper overload, and liver transplantation from the past year have been surveyed to highlight useful concepts and diagnostic information.. Two microscopic forms of pediatric nonalcoholic steatohepatitis have been described: type 1 in which hepatocyte ballooning and/or pericellular fibrosis accompany the steatosis; and type 2 which has portal tract inflammation and/or fibrosis as the salient accompanying feature. In chronic hepatitis C, the ductular reaction appears to be a major factor associated with fibrosis. In patients transplanted for hepatitis C virus-related cirrhosis, immunostaining of post-transplant liver biopsies for alpha-smooth muscle actin (i.e. in activated hepatic stellate cells) may identify those individuals at risk for severe recurrence. Clinicopathological papers on several forms of non-HFE hemochromatosis were published and Wilson's disease was described in individuals of 60 years or more in age. Cholestasis in childhood was expertly reviewed and histopathologic precursor lesions of hepatocellular carcinoma were also examined in a comprehensive article.. Recent publications with impact on liver biopsy interpretation include a morphologic classification of nonalcoholic steatohepatitis in childhood, the differential diagnosis of childhood cholestasis and pathogenetic factors involved in fibrogenesis in chronic hepatitis C.

Topics: Amyloid; Biliary Tract Diseases; Biopsy; Humans; Iron Overload; Keratins; Liver Diseases; Liver Transplantation; Metabolism, Inborn Errors; Mitochondrial Diseases; Mutation; Peroxisomes

Mesenchymal hamartoma is an uncommon cystic mass of the liver which occurs primarily in children. There are a few reports of its occurrence in adulthood. Here, we present two cases in female patients, 54 and 51 years old. Radiological examinations in both patients showed multiple cystic lesions in the liver. Surgically, total cystectomy was performed in the first patient, while an unroofing procedure was done in the second patient (due to misdiagnosis of the lesion as a simple cyst of the liver). On microscopic examinations of the lesion in each patient, a multilocular cyst was observed, lined by flattened epithelium and surrounded by a mesenchymal component composed of mature connective tissue, arterial and venous vascular structures, peripheral nerve bundles, and ductal structures. An immunohistochemical panel consisting of desmin, smooth-muscle actin, S-100, vimentin, CD34, carcinoembryonic antigen, pancytokeratin, cytokeratin 7, cytokeratin 8, cytokeratin 17, cytokeratin 18, cytokeratin 19, and cytokeratin 20 was applied to paraffin sections. Immunoreactivity for cytokeratin 7 and cytokeratin 19 was observed in cystic epithelium and ductal structures. Focal and patchy desmin immunoreactivity was observed in connective tissue. S-100 was positive only in peripheral nerve bundles. In conclusion, mesenchymal hamartoma of the liver in adulthood is a localized tumoral abnormality that precedes birth, and which has delayed clinical presentation. These lesions seems to be related to a maturation process. During this period of maturation, immature edematous stroma rich in mucopolysaccharides may convert to mature paucicellular hyalinized connective tissue. This maturation process may be also related to loss of premalignant potential of these tumors.

Topics: Desmin; Hamartoma; Humans; Immunohistochemistry; Keratins; Liver Diseases; Liver Neoplasms; Middle Aged; S100 Proteins; Tomography, X-Ray Computed

The keratin intermediate filament (IF) cytoskeleton of hepatocytes has continuously gained medical relevance over the last two decades. Originally it was mainly recognized as a differentiation marker for diagnostic purposes in pathology. However, keratin IFs were soon identified as major cellular structures to be affected in a variety of chronic liver diseases, such as alcoholic and non-alcoholic steatohepatitis (ASH, NASH), copper toxicosis, and cholestasis. Based on observations in keratin gene knock-out mice, the insight into the functional role of keratins was extended from a mere structural role providing mechanical stability to hepatocytes, to an additional role as target and modulator of toxic stress and apoptosis. The functional relevance of keratins in human diseases has recently been underlined by the identification of mutations in keratin genes in patients with liver cirrhosis.

Topics: Biliary Tract; Cholestasis; Chronic Disease; Cytoskeleton; Epithelial Cells; Hepatitis; Humans; Keratins; Liver; Liver Diseases; Liver Neoplasms; Mutation

Cytoskeletal research in recent years has revolutionized cell biology and biomedicine. The cytoskeleton spans the cytoplasm and interconnects the cell nucleus with the extracellular matrix, thereby forming a structural link between molecules involved in cell communication on the one hand, and gene expression on the other. Since the cytoskeleton is involved in virtually all cellular processes, abnormalities in this essential cellular component frequently result in disease. In this introduction, the basic structure of the cytoskeleton is briefly outlined. Furthermore, the disease processes in which the cytoskeleton plays a decisive role, and which are reviewed in detail in the papers in this issue, are briefly introduced. The advances in our understanding of the cytoskeleton and its function in disease will lead to new diagnostic and therapeutic applications in the foreseeable future.

Topics: Actins; Cell Nucleus; Cytoskeletal Proteins; Cytoskeleton; Hematologic Diseases; Humans; Intestinal Diseases; Keratins; Liver Diseases; Lymphatic Diseases; Muscular Diseases; Neurodegenerative Diseases; Skin Diseases

Topics: Animals; Cytoskeleton; Genetic Predisposition to Disease; Humans; Intermediate Filament Proteins; Intestinal Diseases; Keratins; Liver; Liver Diseases; Mutation; Tissue Distribution

Liver penetration is a rare but serious complication of peptic ulcer disease. We report a case of a 33-year-old woman who took large doses of nonsteroidal antiinflammatory drugs and developed a giant duodenal ulcer that penetrated into her liver. The diagnosis was based on histologic examination of endoscopic biopsies. She was initially treated with a proton pump inhibitor, but, within 5 weeks, she developed a symptomatic postbulbar stricture that required surgical correction. We also review 11 other reported cases of endoscopically and histologically diagnosed peptic ulcer penetration into the liver.

Topics: Adult; Anti-Inflammatory Agents, Non-Steroidal; Chemical and Drug Induced Liver Injury; Diclofenac; Duodenal Ulcer; Duodenoscopy; Duodenum; Female; Humans; Immunoenzyme Techniques; Intervertebral Disc Displacement; Keratins; Liver; Liver Diseases; Liver Function Tests; Peptic Ulcer Hemorrhage; Peptic Ulcer Perforation

Epithelial cells such as hepatocytes exhibit highly polarized properties as a result of the asymmetric distribution of subsets of receptors at unique portions of the surface membrane. While the proper targeting of these surface receptors and maintenance of the resulting polarity depend on microtubules (MTs), the Golgi sorting compartment, and different actin-filament networks, the contribution of keratin intermediate filaments (IFs) has been unclear. Recent data show that the latter cytoskeletal network plays a predominant role in providing resistance to various forms of stress and to apoptosis targeted to the surface membrane. In this context, we first summarize our knowledge of the domain- or assembly-related features of IF proteins and the dynamic properties of IF networks that may explain how the same keratin pair K8/K18 can exert multiple resistance-related functions in simple epithelial cells. We then examine the contribution of linker protein(s) that integrate interactions of keratin IFs with MTs and the actin-cytoskeleton network, polarity-dependent surface receptors and cytoplasmic organelles. We next address likely molecular mechanisms by which K8/K18 can selectively provide resistance to a mechanical or toxic stress, or to Fas-mediated apoptosis. Finally, these issues on keratin structure-function are examined within a context of pathological anomalies emerging in tissue architecture as a result of natural or targeted mutations, or posttranslational modifications at specific amino acid residues. Clearly. the data accumulated in recent years provide new and significant insights on the role of K8/K18, particularly under conditions where polarized cells resist to stressful or apoptotic insults.

Topics: Animals; Apoptosis; Cell Nucleus; Cytoskeleton; Desmosomes; Epithelial Cells; fas Receptor; Golgi Apparatus; Hepatocytes; Humans; Keratin-8; Keratins; Liver Diseases; Microtubules; Models, Biological; Models, Genetic; Mutation; Neoplasms; Protein Binding; Protein Processing, Post-Translational; Protein Structure, Tertiary; Stress, Mechanical

Topics: Animals; Humans; Immunohistochemistry; Keratins; Liver; Liver Diseases; Liver Neoplasms; Reference Values

Topics: Animals; Humans; Intermediate Filaments; Keratins; Liver; Liver Diseases

Mallory bodies (MBs) are characteristic morphologic features of alcoholic hepatitis but are also associated with non-alcoholic liver diseases including long lasting cholestasis, metabolic and neoplastic disorders. MBs contain in addition to keratins non-keratin components, including microtubule-associated (tau protein) and other not yet characterized proteins in an aggregated form. Aggregation of these components in the cell is promoted by posttranslational modifications, such as partial proteolysis, phosphorylation and cross-linking, and may result in functional and structural disturbances of the cell depending on the physiologic function of the components involved. Several enzymes responsible for these modifications are Ca(++)-dependent. Thus, disturbance of Ca(++)-homeostasis may play an essential role in the pathogenesis of MBs. In some structural aspects MBs closely resemble inclusions associated with degenerative disorders of the central nervous system, including Alzheimer's and Parkinson's disease. Studies on the pathogenesis of MBs, therefore, not only shed light on a peculiar type of liver cell injury but may also assist in the understanding of other chronic degenerative diseases, particularly those of the central nervous system.

Topics: Alzheimer Disease; Animals; Brain Diseases; Calcium; Homeostasis; Humans; Keratins; Liver; Liver Cirrhosis, Alcoholic; Liver Diseases; Liver Diseases, Alcoholic; Parkinson Disease; Protein Processing, Post-Translational; Transcription, Genetic

Proliferation of preexisting bile ducts, ductular metaplasia of hepatocytes and proliferation and differentiation of liver stem cells are discussed in the pathogenesis of neoductular structures in the liver. Under the condition of experimental bile duct obstruction and in extrahepatic bile duct stenosis neoductular structures are first the result of proliferation and sprouting of preexisting ducts and cholangioles. Especially in later stages of cholestasis but also in other chronic progredient liver diseases such as chronic alcoholic liver disease and chronic active hepatitis periportal hepatocytes may show a phenotypic shift towards ductular epithelia. In postnatal liver diseases hepatocytes first express keratin 7 and later keratin 19 during ductular transdifferentiation. This is in contrast to embryonal cholangiogenesis. In alpha-1-antitrypsin-deficiency, hemochromatosis, Wilson's disease, and chronic active hepatitis B cellular deposites typically located in hepatocytes such as alpha-1-AT, siderin, copper, HBs-Ag, and HBc-Ag can also be found in neoductular cells close to hepatocytes. These deposites seem to be retained during the ductular transdifferentiation of hepatocytes. Expression of bile duct-type integrin subtypes and TGF beta 1 in neoductular cells are involved in the changing parenchymal/mesenchymal interplay during neoductogenesis, resulting in periductular basal membrane and periductular fibrosis. In FNH the ductular transdifferentiation of hepatocytes is integrated in the histogenesis of micronodules and portal tract equivalents of these tumor-like lesions. Ductular structures in hepatoblastomas and especially in combined hepatocellular and cholangiocarcinomas (CHCC) may reflect the common embryologic derivation of hepatocytes and biliary epithelia. Non-neoplastic liver tissue in resection specimens of our CHCC showed a lower rate of cirrhosis, and a significantly higher Ki 67-LI of neoductular cells compared to liver tissue in resection specimens of HCC and liver metastases. 3 of 10 CHCC had developed in alpha-1-AT-deficiency, in which this protease-inhibitor was predominantly retained in periportal hepatocytes. These findings in non-neoplastic tumor-bearing liver tissue suggest that CHCC include a special histogenic type of primary liver carcinoma which in analogy to some experimental liver tumors might develop from periportal parenchymal cells.

Topics: alpha 1-Antitrypsin; alpha 1-Antitrypsin Deficiency; Animals; Bile Ducts; Bile Ducts, Extrahepatic; Carcinoma, Hepatocellular; Cell Differentiation; Cell Division; Cholangiocarcinoma; Cholestasis; Copper; Hepatitis B Core Antigens; Hepatitis B Surface Antigens; Hepatoblastoma; Humans; Hyperplasia; Keratins; Liver; Liver Diseases; Liver Neoplasms; Metaplasia

Topics: Autoimmune Diseases; Bile Ducts, Intrahepatic; Cytoskeleton; Humans; Inclusion Bodies; Keratins; Liver; Liver Diseases; Liver Neoplasms

Topics: Autoantibodies; Autoimmune Diseases; Biomarkers; Cytoskeleton; Humans; Infections; Keratins; Liver Diseases; Muscle, Smooth

Topics: Actin Cytoskeleton; Animals; Cytoskeleton; Humans; Intermediate Filaments; Keratins; Liver; Liver Diseases; Liver Neoplasms; Microtubules

Mallory's body filament assembly includes polypeptides of the cytokeratin class of intermediate filaments and also higher molecular weight polypeptides normally found only in the cytokeratins of mature keratinocytes of the epidermis. These additional polypeptides may alter both the morphologic characteristics and increase the resistance to dissolution of the filaments by Ca++-activated protease activity. Thus, it is likely that the kinetics of Mallory's body filament assembly and dissolution favor growth of the filaments. In rodents fed certain carcinogens, Mallory's body formation has been accompanied by the induction of the oncofetoenzyme gamma-glutamyl transpeptidase (GGT), suggesting that Mallory's body formation, like GGT induction, is a phenotypic change related to the process of neoplastic transformation in rodents.

Topics: Animals; Carcinogens; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Cytoskeleton; gamma-Glutamyltransferase; Griseofulvin; Humans; Intermediate Filament Proteins; Keratins; Liver; Liver Diseases; Liver Neoplasms; Liver Neoplasms, Experimental; Lung; Lung Diseases; Phosphorylation; Protein Precursors

Keratins are key structural proteins found in skin and other epithelial tissues. Keratins also protect epithelial cells from damage or stress. Fifty-four human keratins were identified and classified into two families, type I and type II. Accumulating studies showed that keratin expression is highly tissue-specific and used as a diagnostic marker for human diseases. Notably, keratin 79 (KRT79) is type II cytokeratin that was identified as regulator of hair canal morphogenesis and regeneration in skin, but its role in liver remains unclear. KRT79 is undetectable in normal mouse but its expression is significantly increased by the PPARA agonist WY-14643 and fenofibrate, and completely abolished in Ppara-null mice. The Krt79 gene has functional PPARA binding element between exon 1 and exon 2. Hepatic Krt79 is regulated by HNF4A and HER2. Moreover, hepatic KRT79 is also significantly elevated by fasting- and high-fat diet-induced stress, and these increases are completely abolished in Ppara-null mice. These findings suggest that hepatic KRT79 is controlled by PPARA and is highly associated with liver damage. Thus, KRT79 may be considered as a diagnostic marker for human liver diseases.

Topics: Animals; Fasting; Hair; Humans; Keratins; Liver; Liver Diseases; Mice; Mice, Knockout

Keratin 8 (K8) is the cytoskeletal intermediate filament protein of simple-type epithelia. Mutations in K8 predispose the affected individual and transgenic mouse to liver disease. However, the role of K8 in the lung has not been reported in mutant transgenic mouse models. Here, we investigated the susceptibility of two different transgenic mice expressing K8 Gly62-Cys (Gly62 replaced with Cys) or Ser74-Ala (Ser74 replaced with Ala) to lung injury. The mutant transgenic mice were highly susceptible to two independent acute and chronic lung injuries compared with control mice. Both K8 Gly62-Cys mice and K8 Ser74-Ala mice showed markedly increased mouse lethality (∼74% mutant mice versus ∼34% control mice) and more severe lung damage, with increased inflammation and apoptosis, under L-arginine-mediated acute lung injury. Moreover, the K8 Ser74-Ala mice had more severe lung damage, with extensive hemorrhage and prominent fibrosis, under bleomycin-induced chronic lung injury. Our study provides the first direct evidence that K8 mutations predispose to lung injury in transgenic mice.

Topics: Animals; Keratin-18; Keratin-8; Keratins; Liver Diseases; Lung Injury; Mice; Mice, Transgenic; Mutation

Accelerating and regulating collagen formation during wound healing repair is key issues for skin regeneration. Insulin can promote the healing of damaged skin by stimulating cellular migration and angiogenesis. Here, human hair keratin-conjugated insulin was synthesized to enhance full-thickness skin regeneration based on the excellent wound healing and hemostatic effects of keratin and the collagen deposition regulation ability of insulin. The insulin-conjugated keratin (Ins-K) was synthesized through the EDC/NHS reaction, which can supply a sustained release of insulin. The Ins-K hydrogel displayed similar water absorption, porosity and rheology properties to those of the keratin hydrogel. However, the Ins-K hydrogel shows a stronger hemostatic ability than the keratin hydrogel group, with a stronger wound healing effect found for the Ins-K hydrogel in the early regeneration stage (first 2 weeks) than for the keratin hydrogel treatment, resulting in smoother skin tissues at an excision section realized by regulating transforming growth factor β1 (TGF-β1) and hydroxyproline (HYP) expression. The results demonstrate that keratin promotes hemostasis and wound healing after insulin conjugation, which highlights the potential of keratin-based materials in tissue regeneration applications.

Topics: Animals; Hemorrhage; Humans; Hydrogels; Insulin; Keratins; Liver; Liver Diseases; Male; Rats; Rats, Sprague-Dawley; Wound Healing

Keratins (K) constitute the epithelial intermediate filaments. Among them, K7/K19 are widely used markers of the regenerative liver response termed ductular reaction (DR) that consists of activated biliary epithelial cells (BECs) and hepatic progenitor cells (HPCs) and correlates with liver disease severity. In the present study we aimed to characterize K23 in the liver.. We analyzed the expression and localization of K23 in the digestive system under basal conditions as well as in various human and mouse liver diseases/stress models. Cell culture studies were used to study factors regulating K23 expression.. In untreated mice, K23 was restricted to biliary epithelia. It was (together with K7/K19) markedly upregulated in three different DR/cholestatic injury models, i.e., multidrug resistance protein 2 (Mdr2) knockouts, animals treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine or subjected to bile duct ligation. K23 levels correlated with the DR marker Fn14 and immunofluorescence staining showed a distinct co-localization with K7/K19. In chronic human liver disease, K23 expression increased in patients with a more prominent inflammation/fibrosis. A dramatic upregulation (>200times) was observed in patients with acute liver failure (ALF) and end-stage primary biliary cholangitis (PBC). Patients with alcoholic liver cirrhosis displayed increased K23 serum levels. In primary hepatocytes as well as hepatobiliary cell lines, treatment with TNF-related weak inducer of apoptosis (TWEAK), and the type I acute phase inducer interleukin (IL)-1β but not the type II inducer IL-6 elevated K23 expression.. K23 represents a specific, stress-inducible DR marker, whose levels correlate with liver disease severity. K23 may represent a useful non-invasive DR marker.. Ductular reaction represents a basic response to liver injury and correlates with liver disease severity. Our study identifies K23 as a novel ductular reaction marker in mice and humans.

Topics: Animals; Humans; Keratins; Keratins, Type I; Liver; Liver Diseases; Mice; Pyridines

Keratins, among other cytoskeletal intermediate filament proteins, are mutated at a highly conserved arginine with consequent severe disease phenotypes due to disruption of keratin filament organization. We screened a kinase inhibitor library, using A549 cells that are transduced with a lentivirus keratin 18 (K18) construct, to identify compounds that normalize filament disruption due to K18 Arg90Cys mutation at the conserved arginine. High-throughput screening showed that PKC412, a multikinase inhibitor, ameliorated K18 Arg90Cys-mediated keratin filament disruption in cells and in the livers of previously described transgenic mice that overexpress K18 Arg90Cys. Furthermore, PKC412 protected cultured A549 cells that express mutant or wild-type K18 and mouse livers of the K18 Arg90Cys-overexpressing transgenic mice from Fas-induced apoptosis. Proteomic analysis of proteins that associated with keratins after exposure of K18-expressing A549 cells to PKC412 showed that nonmuscle myosin heavy chain-IIA (NMHC-IIA) partitions with the keratin fraction. The nonmuscle myosin-IIA (NM-IIA) association with keratins was confirmed by immune staining and by coimmunoprecipitation. The keratin-myosin association is myosin dephosphorylation-dependent; occurs with K8, the obligate K18 partner; is enhanced by PKC412 in cells and mouse liver; and is blocked by hyperphosphorylation conditions in cultured cells and mouse liver. Furthermore, NMHC-IIA knockdown inhibits PKC412-mediated normalization of K18 R90C filaments.. The inhibitor PKC412 normalizes K18 Arg90Cys mutation-induced filament disruption and disorganization by enhancing keratin association with NM-IIA in a myosin dephosphorylation-regulated manner. Targeting of intermediate filament disorganization by compounds that alter keratin interaction with their associated proteins offers a potential novel therapeutic approach for keratin and possibly other intermediate filament protein-associated diseases.

Topics: Animals; Intermediate Filaments; Keratins; Liver Diseases; Mice; Mice, Transgenic; Mutation; Myosins; Protein Binding; Staurosporine

Topics: Animals; Intermediate Filaments; Keratins; Liver Diseases; Mutation; Myosins; Staurosporine

The aim of this study was to evaluate the possible protective effects of Urtica dioica (UD) against liver damage in the common bile duct-ligated rats. A total of 24 male Sprague Dawley rats were divided into three groups, namely, control, bile duct ligation (BDL) and BDL + received UD groups, containing eight animals in each group. The rats in UD-treated groups were given UD oils (2 ml/kg) once a day intraperitoneally for 2 weeks starting 3 days prior to BDL operation. The change demonstrating the bile duct proliferation and fibrosis in expanded portal tracts includes the extension of proliferated bile ducts into the lobules; inflammatory cell infiltration into the widened portal areas were observed in BDL group. Treatment of BDL with UD attenuated alterations in liver histology. The α-smooth muscle actin, cytokeratin-positive ductular proliferation and the activity of terminal deoxynucleotidyl transferase dUTP nick end labeling in the BDL were observed to be reduced with the UD treatment. The data indicate that UD attenuates BDL-induced cholestatic liver injury, bile duct proliferation and fibrosis.

Topics: Actins; Animals; Antioxidants; Cell Proliferation; Cholestasis; Common Bile Duct; DNA Nucleotidylexotransferase; Fibrosis; Immunohistochemistry; In Situ Nick-End Labeling; Keratins; Liver; Liver Diseases; Male; Rats; Rats, Sprague-Dawley; Urtica dioica

Keratin polypeptide 8 (K8) associates noncovalently with its partners K18 and/or K19 to form the intermediate filament cytoskeleton of hepatocytes and other simple-type epithelial cells. Human K8, K18, and K19 variants predispose to liver disease, whereas site-specific keratin phosphorylation confers hepatoprotection. Because stress-induced protein phosphorylation regulates sumoylation, we hypothesized that keratins are sumoylated in an injury-dependent manner and that keratin sumoylation is an important regulatory modification. We demonstrate that K8/K18/K19, epidermal keratins, and vimentin are sumoylated in vitro. Upon transfection, K8, K18, and K19 are modified by poly-SUMO-2/3 chains on Lys-285/Lys-364 (K8), Lys-207/Lys-372 (K18), and Lys-208 (K19). Sumoylation affects filament organization and stimulus-induced keratin solubility and is partially inhibited upon mutation of one of three known K8 phosphorylation sites. Extensive sumoylation occurs in cells transfected with individual K8, K18, or K19 but is limited upon heterodimerization (K8/K18 or K8/K19) in the absence of stress. In contrast, keratin sumoylation is significantly augmented in cells and tissues during apoptosis, oxidative stress, and phosphatase inhibition. Poly-SUMO-2/3 conjugates are present in chronically injured but not normal, human, and mouse livers along with polyubiquitinated and large insoluble keratin-containing complexes. Notably, common human K8 liver disease-associated variants trigger keratin hypersumoylation with consequent diminished solubility. In contrast, modest sumoylation of wild type K8 promotes solubility. Hence, conformational changes induced by keratin natural mutations and extensive tissue injury result in K8/K18/K19 hypersumoylation, which retains keratins in an insoluble compartment, thereby limiting their cytoprotective function.

Topics: Animals; Biomarkers; Humans; Keratins; Liver; Liver Diseases; Male; Mice; Mice, Transgenic; Mutation; Small Ubiquitin-Related Modifier Proteins; Sumoylation; Ubiquitins

Effective hemostatic dressings that are compatible with tissues are needed. Keratins are a class of biomaterials that can be derived by extraction of proteins from human hair. We have recently discovered that keratin biomaterials have hemostatic characteristics and hypothesize that a keratin hydrogel having the ability to absorb fluid and bind cells may be an effective hemostat. The goal of this study was to test a keratin hydrogel and evaluate it compared to current hemostats. Thirty-two New Zealand white rabbits received a lethal liver injury. Eight animals each were assigned to negative control, QuickClot, HemCon bandage, and keratin treatment groups. Vital stats and other data were recorded during surgery and all surviving animals were sacrificed after 72 h. Histology was conducted on all surviving animals. Twenty-four-hour survival rates were 0%, 62.5%, 62.5%, and 75% for the negative control, QuickClot, HemCon, and keratin groups, respectively. Other outcomes included blood loss, mean arterial pressure, heart rate, shock index, and liver histology. All of the hemostats were statistically better than the negative control group at late operative time points. The keratin group consistently performed as well as, or better than, the commercial hemostats. Histology showed an interesting healing response at the hemostat-liver interface in the keratin group.

Topics: Amino Acids; Animals; Biocompatible Materials; Blood Pressure; Disease Models, Animal; Electrocardiography; Electrophoresis, Polyacrylamide Gel; Hair; Heart Rate; Hemostatics; Humans; Keratins; Liver Diseases; Rabbits

Mallory bodies (MBs) represent keratin-rich inclusion bodies observed in human alcoholic liver disease and in several chronic non-alcoholic liver diseases. The mechanism of their formation and their relationship to other inclusion bodies such as aggresomes is incompletely understood. We could induce keratin aggregates typical of MBs in cultured clone 9 rat hepatocytes by transgenic expression of wild-type and mutant aquaporin2 or alpha1-antitrypsin and under various forms of other cellular stress. By immunocytochemical analysis, p62 and poly-ubiquitin, components of classical MBs, could be demonstrated in the keratin aggregates of clone 9 hepatocytes. In addition, histone deacetylase 6, a microtubule-associated deacetylase, was identified as a novel component of the keratin aggregates. Thus, together with their ultrastructural appearance as randomly oriented, organelle-free aggregates of keratin filaments, the keratin aggregates in clone 9 hepatocytes correspond to MBs. An imbalance in keratin 8 to 18 with very low levels of keratin 18 appears to be the underlying cause for their formation. The formation of MBs was microtubule-dependent although not depending on the activity of histone deacetylase 6. Forskolin-induced MBs in clone 9 hepatocytes were reversible structures which disappeared upon drug withdrawal. The MBs were not related to aggresomes since overexpressed misfolded transgenic proteins were undetectable in the keratin aggregates and no vimentin fiber cage was detectable, both of which represent hallmarks of aggresomes. Thus, cultured clone 9 hepatocytes are a useful system to study further aspects of the pathobiology of MBs.

Topics: Animals; Cells, Cultured; Hepatocytes; Humans; Inclusion Bodies; Keratins; Liver Diseases; Microtubules; Rats

Human livers contain two pluripotent progenitors: hepatic stem cells and hepatoblasts. The hepatic stem cells uniquely express the combination of epithelial cell adhesion molecule (EpCAM), neural cell adhesion molecule (NCAM), cytokeratin (CK) 19, albumin +/-, and are negative for alpha-fetoprotein (AFP). They are precursors to hepatoblasts, which differ from hepatic stem cells in size, morphology, and in expressing the combination of EpCAM, intercellular cell adhesion molecule (ICAM-1), CK19, albumin++, and AFP++. The hepatic stem cells are located in vivo in stem cell niches: the ductal plates in fetal and neonatal livers and canals of Hering in pediatric and adult livers. The hepatoblasts are contiguous to the niches, decline in numbers with age, wax and wane in numbers with injury responses, and are proposed to be the liver's transit-amplifying cells. In adult livers, intermediates between hepatic stem cells and hepatoblasts and between hepatoblasts and adult parenchyma are observed. Amplification of one or both pluripotent cell subpopulations can occur in diseases; for example, hepatic stem cell amplification occurs in mild forms of liver failure, and hepatoblast amplification occurs in forms of cirrhosis. Liver is, therefore, similar to other tissues in that regenerative processes in postnatal tissues parallel those occurring in development and involve populations of stem cells and progenitor cells that can be identified by anatomic, antigenic, and biochemical profiles.

Topics: Adult; Antigens, Neoplasm; Cell Adhesion Molecules; Cell Culture Techniques; Epithelial Cell Adhesion Molecule; Female; Gestational Age; Humans; Intercellular Adhesion Molecule-1; Keratins; Liver; Liver Diseases; Liver Regeneration; Microscopy, Confocal; Neural Cell Adhesion Molecules; Pregnancy; Serum Albumin; Stem Cells

Rabbit antibodies may have favorable properties compared to mouse antibodies, including high affinities and better antigen recognition. We used a biochemical and reverse immunologic approach to generate and characterize rabbit anti-phospho-keratin and anti-keratin monoclonal antibodies (MAb). Human keratins 8 and 18 (K8/K18) were used as immunogens after isolation from cells pretreated with okadaic acid or pervanadate to promote Ser/Thr or Tyr hyperphosphorylation, respectively. Selected rabbit MAb were tested by immunofluorescence staining, immunoprecipitation, and 2-dimensional gels. Keratin phospho and non-phospho-mutants were used for detailed characterization of two unique antibodies. One antibody recognizes a K8 G61-containing epitope, an important epitope given that K8 G61C is a frequent mutation in human liver diseases. This antibody binds K8 that is not phosphorylated on S73, but its binding is ablated by G61 but not S73 mutation. The second antibody is bispecific in that it simultaneously recognizes two epitopes: one phospho (K8 pS431) conformation-independent and one non-phospho conformation-dependent, with both epitopes residing in the K8 tail domain. Therefore, a reverse immunologic and biochemical approach is a viable tool for generating versatile rabbit MAb for a variety of cell biologic applications including the potential identification of physiologic phosphorylation sites.

Topics: Animals; Antibodies, Bispecific; Antibodies, Monoclonal; Brain; Brain Chemistry; Cell Line; Colonic Neoplasms; Cricetinae; Enzyme Inhibitors; Epitopes; HT29 Cells; Humans; Immunoblotting; Immunohistochemistry; Keratin-18; Keratin-8; Keratins; Kidney; Liver; Liver Diseases; Mice; Mutation; Okadaic Acid; Phosphorylation; Rabbits; Serine; Stomach Neoplasms; Transfection; Vaccination; Vanadates

Mallory bodies (MBs) and intracellular hyaline bodies (IHBs) are cytoplasmic hepatocellular inclusions that consist of aggregated proteins. MBs are characteristically associated with alcoholic and non-alcoholic steatohepatitis, but may also be found in chronic cholestatic and metabolic (eg copper intoxication) diseases and hepatocellular neoplasms, particularly hepatocellular carcinomas. IHBs have hitherto only been described in hepatocellular carcinoma cells. In the present study hepatocellular carcinomas (HCCs) and a case of idiopathic copper toxicosis were evaluated with respect to the presence and mutual relationship of MBs and IHBs. IHBs alone were present in 8.6%, MBs alone in 16.1% and both types of inclusion in 7.5% of HCCs. It is shown that IHBs may also occur in non-neoplastic hepatocytes in association with idiopathic copper toxicosis, together with MBs. In HCCs and idiopathic copper toxicosis, MBs and IHBs may be present within the same cell. Moreover, hybrid inclusions holding an intermediate position between MBs and IHBs regarding light microscopy, ultrastructure and composition exist. MBs and IHBs contain p62, a stress-inducible adapter protein, as the major constituent. In MBs p62 is associated with keratins, whereas classical IHBs lack keratins. Light microscopic, electron microscopic and immunohistochemical data suggest a close pathogenetic relationship between MBs and IHBs. Both types of inclusion are the result of over-expression and accumulation of the stress protein p62. If p62 is induced alone, or at least prevails, IHBs may arise by aggregation. However, if abnormal keratins are present in addition to p62, p62 associates and co-aggregates with keratins, finally leading to classical MBs.

Topics: Adaptor Proteins, Signal Transducing; Carcinoma, Hepatocellular; Chemical and Drug Induced Liver Injury; Copper; Hepatocytes; Humans; Hyalin; Inclusion Bodies; Keratins; Liver Diseases; Liver Neoplasms; Microscopy, Electron; Microscopy, Immunoelectron; Neoplasm Proteins; Sequestosome-1 Protein

Keratins 8 and 18 (KRT8 and KRT18 genes; K8 and K18 proteins) variants are risk factors for developing end-stage liver disease and may be associated with inflammatory bowel disease and chronic pancreatitis. The frequency of K8/K18 variants in American, British, German, and Italian populations differs. For example, one study showed no amino acid-altering K8/K18 mutations in 256 German patients with liver disorders, while another found 58 out of 467 American liver disease patients with K8/K18 mutations. Both studies used the WaveSystem, which utilizes DHPLC. We hypothesized that experimental conditions contribute to the discrepancy, and we tested this hypothesis using previously described K8/K18 variants and a novel KRT18 c.1057C>G variant (K18 p.R353G) to optimize the DHPLC conditions in 10 examined exons under a range of denaturing temperatures. Six of 16 tested variants in three of the 10 exons, including the frequent KRT8 c.184G>T (K8 p.G62C), KRT8 c.187A>G (K8 p.I63V), and KRT8 c.1022G>A (K8 p.R341H), could not be reliably detected when using temperatures suggested by the prediction software, but all these variants were readily detectable at 2 degrees C higher denaturing temperatures. Using optimized temperatures, we then tested available genomic DNA from 151 out of the 256 German liver disease patients for the presence of K8 variants in exons 1 and 6, where most of the American cohort K8 variants occur. We identified 12 exonic and two intronic K8 variants: one KRT8 c.184G>T (K8 p.G62C), two KRT8 c.187A>G (K8 p.I63V), seven KRT8 c.1022G>A (K8 p.R341H), one KRT8 c.1128G>A (K8 p.E376E), two intronic KRT8 c.1202+46 A>T, and one hitherto undescribed KRT8 c.1138G>A (K8 p.V380I). Therefore, although DHPLC offers a robust and high throughput means for mutation analysis, assessment of denaturing temperature ranges, and possible inclusion of control mutants should be considered.

Topics: Chromatography, High Pressure Liquid; Cohort Studies; DNA Mutational Analysis; Humans; Keratin-18; Keratin-8; Keratins; Liver Diseases; Mutation; Nucleic Acid Denaturation; Protein Structure, Tertiary; Temperature

Mallory bodies (MBs) are characteristic of several liver disorders, and consist primarily of keratins with transglutaminase-generated keratin crosslinks. We tested the effect of the transglutaminase-2 (TG2) inhibitor KCC009 on MB formation in a mouse model fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). KCC009 decreased DDC-induced liver enlargement without affecting MB formation or extent of liver injury. TG2 protein and activity increased after DDC feeding and localized within and outside hepatocytes. KCC009 inhibited DDC-induced hepatomegaly by affecting hepatocyte cell size rather than proliferation. Hence, TG2 is a potential mediator of injury-induced hepatomegaly via modulation of hepatocyte hypertrophy, and KCC009-mediated TG2 inhibition does not affect mouse MB formation.

Topics: Animals; Cell Size; Chemical and Drug Induced Liver Injury; Dicarbethoxydihydrocollidine; Enzyme Inhibitors; GTP-Binding Proteins; Hepatomegaly; Humans; Inclusion Bodies; Isoxazoles; Keratins; Liver Diseases; Mice; Mice, Inbred C3H; Protein Glutamine gamma Glutamyltransferase 2; Proteins; Transglutaminases

Keratin 8 (K8) variants predispose to human liver injury via poorly understood mechanisms. We generated transgenic mice that overexpress the human disease-associated K8 Gly61-to-Cys (G61C) variant and showed that G61C predisposes to liver injury and apoptosis and dramatically inhibits K8 phosphorylation at serine 73 (S73) via stress-activated kinases. This led us to generate mice that overexpress K8 S73-to-Ala (S73A), which mimicked the susceptibility of K8 G61C mice to injury, thereby providing a molecular link between K8 phosphorylation and disease-associated mutation. Upon apoptotic stimulation, G61C and S73A hepatocytes have persistent and increased nonkeratin proapoptotic substrate phosphorylation by stress-activated kinases, compared with wild-type hepatocytes, in association with an inability to phosphorylate K8 S73. Our findings provide the first direct link between patient-related human keratin variants and liver disease predisposition. The highly abundant cytoskeletal protein K8, and possibly other keratins with the conserved S73-containing phosphoepitope, can protect tissue from injury by serving as a phosphate "sponge" for stress-activated kinases and thereby provide a novel nonmechanical function for intermediate filament proteins.

Topics: Animals; Apoptosis; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Fas Ligand Protein; Genetic Predisposition to Disease; Genetic Variation; Hepatocytes; Humans; Intermediate Filament Proteins; Keratin-8; Keratins; Liver Diseases; Liver Function Tests; Marine Toxins; Membrane Glycoproteins; Mice; Mice, Knockout; Mice, Transgenic; Microcystins; Mitogen-Activated Protein Kinases; Mutation; Peptides, Cyclic; Phosphorylation; Tumor Necrosis Factors

We examined the response of murine oval cells, that is, the putative liver progenitor cells, to acetaminophen. Female C57BL/6J mice were injected intraperitoneally with varying doses of N-acetyl-paraaminophen (APAP) (250, 500, 750, and 1,000 mg/kg of weight) and sacrificed at 3, 6, 9, 24, and 48 hours. In preliminary studies, we showed that anticytokeratin antibodies detected A6-positive cells with a sensitivity and specificity of greater than 99%. The oval cell reaction was quantified, on immunostaining for biliary-type cytokeratins, as both number and density of oval cells per portal tract, analyzed by size of portal tract. Acetaminophen injury was followed by periportal oval cell accumulation displaying a moderate degree of morphological homogeneity. Oval cell response was biphasic, not temporally correlating with the single wave of injury seen histologically. Increases in oval cells were largely confined to the smallest portal tracts, in keeping with their primary derivation from the canals of Hering, and increased in a dose-dependent fashion. The timing of the two peaks of the oval cell reaction also changed with increasing dose, the first becoming earlier and the second later. In conclusion, our studies indicate a marked oval cell activation during the height of hepatic injury. Oval cells appear to be resistant to acetaminophen injury. The close fidelity of mechanism and histology of acetaminophen injury between mouse and human livers makes it a useful model for investigating liver regeneration and the participation of stem/progenitor cells in that process.

Topics: Acetaminophen; Animals; Chemical and Drug Induced Liver Injury; Dose-Response Relationship, Drug; Female; Immunologic Techniques; Keratins; Liver; Liver Diseases; Mice; Mice, Inbred C57BL; Stem Cells; Time Factors

Oval cells, putative hepatic stem cells, could potentially provide a novel solution to the severe shortage of donor livers, because of their ability to proliferate and differentiate into functional hepatocytes. We have previously demonstrated that oval cells can be induced to differentiate into cells with morphologic, phenotypic, and functional characteristics of mature hepatocytes. In this study, we have established a new model combining ethionine treatment with partial hepatectomy to activate oval cells, then developed a procedure utilizing selective enzymatic digestion and density gradient centrifugation to isolate and purify such cells from heterogeneous liver cell population. We identified oval cells by their morphological characteristics and phenotypic properties, thereby providing definitive evidence of the presence of hepatic stem-like cells in adult rat livers. Viewed by transmission electron microscopy, they were small cells with ovoid nuclei, a high nucleus/cytoplasm ratio and few organelles, including mitochondria and endoplasmic reticulum. Flow cytometric assay showed that these cells highly expressed OV-6, cytokeratin-19 (CK-19) and albumin. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis displayed that the freshly isolated cells co-expressed albumin, cytokeratin-7 (CK-7) and CK-19 mRNA, indicating that they were essentially bipotential hepatic stem-like cells. Furthermore, we set up a culture system containing growth factors and a fibroblast feeder layer, to provide nourishment to these cells. Thus, we were able to culture them in vitro for more than 3 months, with the number of cells doubling 100 times. Gene expressions of albumin, CK-7 and CK-19 in the cells derived from the expanding colonies at day 95 were confirmed by RT-PCR analysis. These data suggested that the hepatic oval cells derived from adult rat livers possess a high potential to proliferate in vitro with a large increase in number, while maintaining the bipotential nature of hepatic stem cells.

Topics: Albumins; Animals; Antigens, Differentiation; Cell Culture Techniques; Cell Differentiation; Cell Division; Cell Nucleus; Cell Separation; Cells, Cultured; Ethionine; Hepatocytes; Keratins; Liver; Liver Diseases; Liver Transplantation; Male; Microscopy, Electron; Microscopy, Electron, Scanning; Rats; Rats, Wistar; RNA, Messenger; Stem Cells

Topics: DNA Mutational Analysis; Gene Frequency; Genetic Predisposition to Disease; Humans; Keratin-8; Keratins; Liver Diseases; Point Mutation; Polymorphism, Genetic

Mallory bodies (MBs) are hyaline inclusions found in a variety of liver diseases. Because the major components of MBs are cytokeratins (CKs), CK profiles of MBs were examined immunohistochemically in 37 autopsied liver specimens (including a variety of nontumor pathological livers and hepatocellular carcinomas), using 13 antibodies against specific CKs: 34betaB4 (CK 1), OV-TL12/30 (CK 7), 34betaH11 (CK 8), LHP1 (CK 10), KS-1A3 (CK 13), LL002 (CK 14), LHK15 (CK 15), LL025 (CK 16), E3 (CK 17), DC10 (CK 18), b170 (CK 19), Ks20.8 (CK 20), and 34betaE12 (CKs 1, 5, 10, and 11). Positive staining rates of MBs in 37 cases were as follows: CK 8, 100% (37/37); CK 18, 100% (37/37); CK 19, 57% (21/37). CK 7, 49% (18/37); CK 20, 35% (13/37); CK 1, CK 5, CK 10, CK 11, CK 13, CK 14, CK 15, CK 16, and CK 17 were all negative in MBs. CK expression patterns in MBs found in tumor or non-tumor hepatocytes was basically similar. Thus, all present MBs were composed of similar material with common antigenic determinants, regardless of underlying disease; in particular, they consistently contained CK 8 and CK 18, and frequently contained CK 7, CK 19, and CK 20.

Topics: Carcinoma, Hepatocellular; Hepatocytes; Humans; Immunohistochemistry; Inclusion Bodies; Keratins; Liver Diseases; Liver Neoplasms

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Child; Chronic Disease; Cytosine; Guanine; Hepatitis, Autoimmune; Hepatitis, Viral, Human; Histidine; Humans; Keratin-8; Keratins; Liver Cirrhosis; Liver Diseases; Middle Aged; Mutation, Missense; Tyrosine

Mallory bodies (MBs) are aggresomes, composed of cytokeratin and various other proteins, which form in diseased liver because of disruption in the ubiquitin-proteasome protein degradation pathway. Heat shock proteins (hsp's) are thought to be involved in this process because it was discovered that MB formation is induced by heat shock in drug-primed mice. It has been reported that ubiquitin and a mutant form of ubiquitin (UBB(+1)) are found in aggresomes formed in the neurons in Alzheimer's disease and in the liver MBs in various liver diseases. In addition, hsp 70 has been found in aggresomes in Alzheimer's and in MBs in drug-primed mice. Therefore, we hypothesized that hsp's might be involved in MB formation in human liver diseases. Liver biopsy sections were double-stained using ubiquitin and hsp 70 or 90b antibodies. Both hsps 70 and 90b were found in MBs in all liver diseases investigated including primary billiary cirrhosis, nonalcoholic steatohepatitis, hepatitis B and C, idiopathic cirrhosis, alcoholic hepatitis, and hepatocellular carcinoma. Ubiquitin and the hsp's colocalized in all MBs in the diseased liver sections. These results indicate that hsp involvement in MB formation is similar to that seen in aggresome formation in other conformational diseases.

Topics: Animals; Antibodies; Biopsy; Chlormethiazole; Dihydropyridines; GABA Modulators; Heat-Shock Proteins; Humans; Inclusion Bodies; Keratins; Liver; Liver Diseases; Male; Mice; Mice, Inbred C3H; Models, Biological; Ubiquitin

Hepatocytes express keratins 8 and 18 (K8/18) as their only cytoskeletal intermediate filament (IF) proteins, and K8/18 mutations predispose their carriers to liver cirrhosis. Transgenic mice that overexpress mutant human K18 (Arg89-->Cys [R89C]) develop mild chronic hepatitis, hepatocyte fragility, keratin filament disruption, and increased susceptibility to drug-induced liver injury. K18 is a major caspase substrate during apoptosis, and K8- or K18-null mice are significantly predisposed to Fas- and possibly tumor necrosis factor (TNF)-mediated apoptosis in the liver. Here we tested the potential role of the K18 R89C mutation on Fas- or TNF-mediated apoptotic liver injury by injecting Fas antibody (Ab) or TNF-alpha plus actinomycin D into mice that overexpress wild-type (WT) human K18 (with intact filament network, termed TG2 mice) or into K18 R89C mice (with disrupted filament network). K18 R89C mice are significantly more susceptible to Fas-mediated liver injury compared with nontransgenic and TG2 mice. This included differences in lethality, histology, apoptosis, and serum transaminase levels. In contrast, K18 WT and R89C mice manifest similar sensitivity to TNF-induced injury. Both Fas- and TNF-induced apoptosis in liver tissues are associated with caspase-mediated K18 degradation and increased keratin phosphorylation on several but not all sites. In conclusion, transgenic mouse K18 mutation and its consequent keratin filament disruption predispose hepatocytes to Fas- but not TNF-mediated apoptotic injury. This supports the association of keratin mutations with cirrhosis in patients with liver disease and suggests that keratins modulate apoptosis induced by Fas but not TNF.

Topics: Actin Cytoskeleton; Animals; Antibodies; Antineoplastic Agents; Apoptosis; fas Receptor; Humans; Keratin-8; Keratins; Liver Diseases; Mice; Mice, Inbred Strains; Mice, Transgenic; Phosphorylation; Tumor Necrosis Factor-alpha

Keratin 8 and 18 (K8K18) mutations are found in patients with cryptogenic cirrhosis, but the role of keratin mutations in noncryptogenic cirrhosis and the incidence of keratin mutations in the general population are not known. We screened for K8K18 mutations in genomic DNA isolated from 314 liver explants of patients who primarily had noncryptogenic cirrhosis, and from 349 blood bank volunteers. Seven unique K8K18 mutations were found in 11 independent patients with biliary atresia, hepatitis BC, alcohol, primary biliary cirrhosis, and fulminant hepatitis. Seven of the 11 patients had mutations previously described in patients with cryptogenic cirrhosis: K8 Tyr-53 --> His, K8 Gly-61 --> Cys, and K18 His-127 --> Leu. The four remaining patients had mutations at one K8 and three other K18 new sites. Of the 349 blood bank control samples, only one contained the Tyr-53 --> His and one the Gly-61 --> Cys K8 mutations (P < 0.004 when comparing cirrhosis versus control groups). Two additional mutations were found in both the liver disease and blood bank groups and, hence, likely represent polymorphisms. Livers with keratin mutations had cytoplasmic filamentous deposits that were less frequent in livers without the mutations (P = 0.03). Therefore, K8K18 are likely susceptibility genes for developing cryptogenic and noncryptogenic forms of liver disease.

Topics: Cohort Studies; DNA; Genetic Predisposition to Disease; Humans; Keratin-8; Keratins; Liver; Liver Diseases; Mutation; Risk Factors

Using an electrochemiluminescence immunoassay, CYFRA 21-1 concentrations were measured in sera from 187 patients with primary liver cancer (164 with hepatocellular carcinoma (HCC) and 23 with intrahepatic cholangiocarcinoma (ICC)) and 87 patients with benign liver diseases. Concentrations of CYFRA 21-1 were significantly higher in patients with ICC (5.0; interquartile range 3.1-10.7 ng ml(-1)) than in those with benign liver disease (1.4; 1.0-1.9; Mann-Whitney U-test, P<0.0001) or HCC (1.7; 1.1-2.7; Mann-Whitney U-test, P<0.0001). Using cutoff values selected for 95% specificity in the benign group (3.0 ng ml(-1)), CYFRA 21-1 showed higher sensitivity for ICC (87.0%) than three commonly used markers including alpha-fetoprotein (17.4%), carcinoembryonic antigen (34.8%), and carbohydrate antigen 19-9 (60.9%). Serum CYFRA 21-1 increased in ICC from stages I/II to IV (Kruskal-Wallis test, P=0.0102). CYFRA 21-1 concentration increased with extent of local invasion, but not nodal status. Serum CYFRA 21-1 represents a useful diagnostic test for ICC that offers high sensitivity. CYFRA 21-1 reflected differences in tumour burden, suggesting applicability to staging and follow-up.

Topics: Adult; Aged; alpha-Fetoproteins; Antigens, Neoplasm; Biomarkers, Tumor; CA-19-9 Antigen; Carcinoembryonic Antigen; Carcinoma, Hepatocellular; Cholangiocarcinoma; Humans; Keratin-19; Keratins; Liver Diseases; Liver Neoplasms; Male; Middle Aged; Sensitivity and Specificity

Proliferative bile ductular reactions occur in a variety of liver diseases in humans. It is a matter of debate whether such reactions result from progenitor cell proliferation with biliary and hepatocytic differentiation, versus biliary metaplasia of damaged hepatocytes. We investigated bile ductular reactions in liver diseases, paying particular attention to the presence of cells with intermediate (hepatocytic/biliary) features (oval-like cells).. Five specimens each were selected of submassive hepatic necrosis and cirrhosis due to hepatitis B, hepatitis C, autoimmune hepatitis, alcohol injury, primary biliary cirrhosis and primary sclerosing cholangitis. Immunohistochemical stains were performed for biliary markers (cytokeratins [CKs] 7 and 19), as well as hepatocytic markers (HepParl and alpha-fetoprotein[AFP]) in sequential sections. The degree of staining of each cell type (biliary, hepatocytic, intermediate) was graded semiquantitatively.. Hepatocytes always stained diffusely for HepParl, occasionally for CK7, and rarely for CK19. Biliary cells were always diffusely positive for CK7 and CK19, and rarely for HepParl. Intermediate cells were identified in all cases and showed widespread staining for both HepParl and CK7, and less commonly for CK19. AFP was not expressed in any cell type. The morphologic and immunohistochemical features of bile ductular reactions were similar in the different diseases.. Proliferating hepatic parenchymal cells with intermediate (hepatocytic/biliary) morphologic features and combined immunophenotype can be identified in a variety of acute and chronic liver diseases. The similarity of bile ductular reactions among chronic hepatitic, alcoholic and biliary diseases suggests that they result from proliferation of oval-like progenitor cells.

Topics: alpha-Fetoproteins; Antibodies, Monoclonal; Bile Ducts, Intrahepatic; Biomarkers; Hepatocytes; Humans; Immunoenzyme Techniques; Keratins; Liver Diseases; Stem Cells

Sera from 93 patients with connective tissue disease, 36 rheumatoid arthritis, 41 systemic lupus erythematosus, 12 polymyositis/dermatomyositis and 4 systemic sclerosis and sera from 12 patients with liver disease, along with sera of 10 healthy subjects, were tested for antikeratin antibodies using enzyme-linked immunosorbent assay in which the wells were coated with human epidermal keratin. Values above the mean+SD of the 10 healthy subjects were found in 8.3% of rheumatoid arthritis, 29.3% of systemic lupus erythematosus, 33.3% of polymyositis/dermatomyositis, 50% of systemic sclerosis, 16.7% of liver disease patients, and 20% of healthy subjects. The results indicated a character of naturally occurring antibody of antikeratin antibodies in human sera.

Topics: Antibodies; Connective Tissue Diseases; Enzyme-Linked Immunosorbent Assay; Humans; Keratins; Liver Diseases

Exposure of cells to stress, particularly oxidative stress, leads to misfolding of proteins and, if they are not refolded or degraded, to cytoplasmic protein aggregates. Protein aggregates are characteristic features of a variety of chronic toxic and degenerative diseases, such as Mallory bodies (MBs) in hepatocytes in alcoholic and non-alcoholic steatohepatitis, neurofibrillary tangles in neurons in Alzheimer's, and Lewy bodies in Parkinson's disease. Using 2D gel electrophoresis and mass spectrometry, we identified p62 as a novel MB component. p62 and cytokeratins (CKs) are major MB constituents; HSP 70, HSP 25, and ubiquitinated CKs are also present. These proteins characterize MBs as a prototype of disease-associated cytoplasmic inclusions generated by stress-induced protein misfolding. As revealed by transfection of tissue culture cells overexpressed p62 did not induce aggregation of regular CK filaments but selectively bound to misfolded and ubiquitinated CKs. The general role of p62 in the cellular response to misfolded proteins was substantiated by detection of p62 in other cytoplasmic inclusions, such as neurofibrillary tangles, Lewy bodies, Rosenthal fibers, intracytoplasmic hyaline bodies in hepatocellular carcinoma, and alpha1-antitrypsin aggregates. The presence of p62 along with other stress proteins and ubiquitin in cytoplasmic inclusions indicates deposition as aggregates as a third line of defense against misfolded proteins in addition to refolding and degradation.

Topics: Adaptor Proteins, Signal Transducing; alpha 1-Antitrypsin Deficiency; Animals; Astrocytoma; Central Nervous System Neoplasms; CHO Cells; Cricetinae; Heat-Shock Proteins; Humans; Inclusion Bodies; Keratins; Liver Diseases; Mice; Neurodegenerative Diseases; Protein Binding; Protein Folding; Proteins; Sequestosome-1 Protein; Stress, Physiological; Ubiquitin

Mallory bodies (MBs) consist of abnormal keratins, ubiquitin, heat shock proteins, and the protein p62. p62 is encoded by an immediate-early response gene that rapidly responds to a variety of extracellular signals involved in cell proliferation, differentiation, and particularly oxidative stress. It acts as an adapter in signal transduction and binds noncovalently to ubiquitin, possibly being involved in the regulation of the fate of ubiquitinated proteins by segregation (i.e., sequestosome or aggresome formation). The presence of p62 together with ubiquitinated abnormal keratins in the MB characterizes MBs as a disease-associated type of sequestosome. A detailed study on the expression of p62 and its relationship to MB formation in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-treated mouse liver is reported based on immunohistochemical, immunoblot, and Northern blot analyses. The results indicate that p62 is rapidly induced in hepatocytes of intoxicated animals preceding MB formation. As suggested by experiments with short-term DDC-treated naive mice and mice refed DDC after recovery from long-term DDC treatment (primed mice), p62 does not exert an initiating effect on MB formation but the appearance of MBs requires the presence of abnormal keratins, which associate with p62 after ubiquitination. The rapid induction of p62 and its association with MBs further support the role of oxidative stress in MB formation. In conclusion, the constant presence of p62 in MBs suggests that binding of p62 to abnormal keratins may allow hepatocytes to dispose potentially harmful proteins in a biologically inert manner.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antibodies; Biopsy; Blotting, Northern; Blotting, Western; Carrier Proteins; Chemical and Drug Induced Liver Injury; Dicarbethoxydihydrocollidine; Humans; Immediate-Early Proteins; Inclusion Bodies; Keratins; Liver; Liver Diseases; Male; Mice; Microscopy, Immunoelectron; Proteins; RNA, Messenger; Sequestosome-1 Protein; Transcription Factor TFIIH; Transcription Factors; Ubiquitin

Liver disease causes significant morbidity and mortality from multilobular cirrhosis in patients with cystic fibrosis. Abnormal bile transport and biliary fibrosis implicate abnormal biliary physiology in the pathogenesis of cystic fibrosis-associated liver disease (CFLD), yet the mediators linking biliary events to fibrosis remain unknown. Activated hepatic stellate cells (HSCs) are the pre-eminent mediators of fibrosis in a range of hepatic disorders. The dominant stimulus for matrix production by HSCs is the cytokine transforming growth factor (TGF)-beta(1). In CFLD, the role of HSCs and the source of TGF-beta(1) have not been evaluated. Liver biopsy tissue obtained from 38 children with CFLD was analyzed. Activated HSCs, identified by co-localization of procollagen alpha(1)(I) mRNA and alpha-smooth muscle actin, were demonstrated as the cellular source of excess collagen production in the fibrosis surrounding the bile ducts and the advancing edge of scar tissue. TGF-beta protein and TGF-beta(1) mRNA expression were shown to be predominantly expressed by bile duct epithelial cells. TGF-beta(1) expression was significantly correlated with both hepatic fibrosis and the percentage of portal tracts showing histological abnormalities associated with CFLD. This study demonstrates a definitive role for HSCs in fibrogenesis associated with CFLD and establishes a potential mechanism for the induction of HSC collagen gene expression through the production of TGF-beta(1) by bile duct epithelial cells.

Topics: Actins; Adolescent; Child; Child, Preschool; Collagen; Cystic Fibrosis; Female; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization; Infant; Keratins; Liver; Liver Diseases; Male; Muscle, Smooth; Procollagen; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

Intraductal papillary neoplasia of the liver (IPN-L) was recently proposed as the name for intraductal papillary proliferation of neoplastic biliary epithelium with a fine fibrovascular stalk resembling intraductal papillary mucinous neoplasm of the pancreas. We histochemically and immunohistochemically examined IPN-L alone or associated with hepatolithiasis, with an emphasis on the gastrointestinal metaplasia, nuclear p53 expression, and histologic progression. A total of 66 cases of IPN-L were divided into 4 groups: group 1, IPN-L with low-grade dysplasia (13 cases); group 2, IPN-L with high-grade dysplasia (20 cases); group 3, IPN-L lined with carcinoma in situ and no or microinvasion (19 cases); and group 4, group 3 with distinct invasive carcinoma (14 cases). It is suggested that IPN-L progresses from group 1 to group 4. As controls, 20 cases of nonneoplastic intrahepatic large bile ducts and 17 cases of nonpapillary invasive intrahepatic cholangiocarcinoma (ICC) were used. Biliary epithelial hypersecretion of sialomucin rather than sulfomucin was prevalent in IPN-L, and this was associated with the progression of INP-L. Immunohistochemically, cytokeratin (CK) 20 and MUC2, a gastrointestinal marker, were expressed more frequently in IPN-L than in nonneoplastic bile ducts and nonpapillary ICC (P <0.01), and their incidence were significantly increased in parallel with the progression of IPN-L (P < 0.01). In contrast, expression of CK 7, a biliary marker, was decreased in IPN-L compared with nonpapillary ICC. Nuclear p53 immunostaining was detected in 30% of IPN-L as a whole and increased in tandem with the progression of IPN-L (P < 0.01). It is suggested that IPN-L forms a spectrum of biliary epithelial neoplasia with frequent gastrointestinal metaplasia, different from the usual nonpapillary ICC, and shows stepwise progression from the perspective of mucin profile, gastrointestinal metaplasia, and p53 nuclear expression.

Topics: Adult; Aged; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cell Nucleus; Cholangiocarcinoma; Disease Progression; Female; Humans; Immunoenzyme Techniques; Keratin-7; Keratins; Lithiasis; Liver Diseases; Male; Metaplasia; Middle Aged; Mucins; Phenotype; Precancerous Conditions; Tumor Suppressor Protein p53

Apoptosis has been implicated in the pathogenesis of many diseases including various forms of liver failure. The apoptotic process is essentially regulated by intracellular proteases, called caspases, which cleave several vital proteins. Despite the rapid elucidation of apoptotic signaling cascades, however, almost no information exists about the activation of caspases in situ. In the present study, a monoclonal antibody was employed which selectively recognized cleavage site-specific fragments of the caspase substrate cytokeratin-18. We demonstrate that this antibody labeled apoptotic hepatocytes in culture and, in addition, could be used to monitor caspase activation in formalin-fixed tissue biopsies. In liver sections of different liver diseases an increased number of early apoptotic cells was detected which were not found in normal tissue. Our data reveal that hepatobiliary diseases are characterized by elevated caspase activation and apoptosis, which can be specifically detected in situ by a cleavage site-specific antibody against cytokeratin-18.

Topics: Antibodies, Monoclonal; Apoptosis; Caspases; DNA Fragmentation; Enzyme Activation; Flow Cytometry; Humans; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Keratins; Liver; Liver Diseases; Liver Neoplasms; Time Factors; Tumor Cells, Cultured

In alcoholic hepatitis, a severe form of alcohol-induced toxic liver injury, as well as in experimental intoxication of mice with the porphyrinogenic drugs griseofulvin and 3,5-diethoxycarbonyl-1, 4-dihydrocollidine, hepatocytes form cytoplasmic protein aggregates (Mallory bodies; MBs) containing cytokeratins (CKs) and non-CK components. Here we report that mice lacking the CK8 gene and hence CK intermediate filaments in hepatocytes, but still expressing the type I partner, ie, the CK18 gene, do not form MBs but suffer from extensive porphyria and progressive toxic liver damage, leading to the death of a considerable number of animals (7 of 12 during 12 weeks of intoxication). Our observations show that 1) in the absence of CK8 as well as in the situation of a relative excess of CK18 over CK8 no MBs are formed; 2) the loss of CK8 is not compensated by other type II CKs; and 3) porphyria and toxic liver damage are drastically enhanced in the absence of CK8. Our results point to a protective role of CKs in certain types of toxic liver injury and suggest that MBs by themselves are not harmful to hepatocytes but may be considered as a product of a novel defense mechanism in hepatocytes.

Topics: Animals; Bile Ducts; Chemical and Drug Induced Liver Injury; Cytoplasm; Cytoskeleton; Dicarbethoxydihydrocollidine; Epithelium; Keratin-7; Keratins; Liver; Liver Diseases; Mice; Mice, Inbred Strains; Mice, Knockout

Topics: Animals; Antibodies, Monoclonal; Apoptosis; Biliary Tract Diseases; Caspases; Enzyme Activation; Humans; Immunohistochemistry; Keratins; Liver Diseases; Mice

Lymphocytic infiltration in the portal triads usually conceals the detection--in haematoxylin and eosin (H&E) stained sections--of bile ducts in two liver diseases: chronic hepatitis and primary biliary cirrhosis. The aim was to assess the number and the characteristics of the bile ducts in those diseases with the aid of an antibody to cytokeratin 7 (CK7).. Consecutive sections from 99 liver biopsies were stained with H&E and anti-CK7.. In H&E sections the total number of central bile ducts in the triads was 52 in primary biliary cirrhosis (n = 37), 69 in chronic hepatitis (n = 43), and 30 in miscellaneous cases (n = 19). Using anti-CK7, the number of central bile ducts was 276 in primary biliary cirrhosis, 348 in chronic hepatitis, and 96 in miscellaneous cases. Central bile ducts with lumen were found in 93.0% of chronic hepatitis cases and in 89.5% of the miscellaneous cases, but in only 13.5% of the primary biliary cirrhosis cases. Peripheral bile ducts in groups of > or = 4/triad were found in all cases of chronic hepatitis (100%) and in 75.7% primary biliary cirrhosis cases, but only in 10.5% of the miscellaneous cases. In 21.6% of primary biliary cirrhosis cases, no bile ducts (central and/or peripheral) were present.. Anti-CK7 detects bile ducts in the triads that are concealed by chronic inflammatory cells. Central and peripheral bile ducts in groups of > or = 4 were significantly more common in primary biliary cirrhosis and chronic hepatitis than in other liver diseases. The lack of lumen in central bile ducts, as well as the absence of central and/or peripheral bile ducts in CK7 stained liver sections, seem to be valuable additional parameters in the differential diagnosis between primary biliary cirrhosis and chronic hepatitis.

Topics: Adult; Aged; Antibodies, Monoclonal; Bile Ducts, Intrahepatic; Biopsy; Diagnosis, Differential; Female; Hepatitis, Chronic; Humans; Immunoenzyme Techniques; Keratin-7; Keratins; Liver Cirrhosis, Biliary; Liver Diseases; Male; Middle Aged

Extracellular matrix (ECM) may affect the function and phenotype of hepatocytes. Phenotypic changes of hepatocytes in diseased liver were investigated with reference to ECM composition.. Immunohistochemistry was performed on biopsied liver samples from chronic viral hepatitis (CVH), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC) and normal patients, using monoclonal antibodies for laminin, type IV collagen, cytokeratin 19 (CK19) and epithelial glycoprotein (EGP), a protein homologous to nidogen.. In normal controls, both EGP and CK 19 were expressed exclusively on biliary epithelia. Laminin and type IV collagen were expressed around portal bile ducts and blood vessels. Although type IV collagen was expressed in Disse's space, laminin was scarcely expressed. In all pathological livers, both EGP and CK 19 were expressed in proliferated bile ductules. In CVH with piecemeal necrosis, EGP was expressed on periportal hepatocytes, while CK19 expression was limited to a few hepatocytes. Laminin was expressed in Disse's space of periportal sinusoids, where EGP was expressed on hepatocytes. EGP expression on hepatocytes and laminin deposition in Disse's space were rare in PBC and PSC liver.. These results suggest that hepatocytes transform into a phenotype similar to biliary epithelia and, laminin deposition in Disse's space (capillarization of sinusoids) may play a role in this phenotypic change.

Topics: Cholangitis, Sclerosing; Collagen; Extracellular Matrix; Hepatitis, Chronic; Hepatitis, Viral, Human; Humans; Immunohistochemistry; Keratins; Liver; Liver Cirrhosis, Biliary; Liver Diseases; Membrane Glycoproteins; Phenotype

Mallory bodies (MBs) are eosinophilic cytoplasmic inclusions observed predominantly in alcoholic liver disease. Although linked to disease activity, their pathogenesis is still unclear. Since intermediate filaments (cytokeratins) are major components of MBs, their cytokeratin polypeptide composition was analysed with monospecific antibodies for cytokeratins 7, 8, 14, 18, 19, and 20 by immunohistology. MBs were identified by light microscopy and ubiquitin immunostaining. All MBs were positive for cytokeratins 8 and 18. A significant percentage of the MBs was strongly positive for cytokeratins 19 and/or 20, which are not detectable in hepatocytes of normal liver and, in the case of cytokeratin 20, in hepatocytes of diseases devoid of MBs. MBs were essentially negative for cytokeratins 7 and 14. De novo expression of cytokeratins 19 and 20 was independent of the aetiology, occurring in all MB-associated diseases analysed, and seemed to precede MB formation, since in some hepatocytes a cytoskeletal-type staining pattern for these cytokeratins was present. In hepatocellular carcinomas cytokeratins 19 and 20 were frequently detected, but their cellular distribution was less closely associated with MBs. The ectopic expression of cytokeratins 19 and 20 appears to be related to MB formation and may take part in the derangement of the intermediate filaments during MB formation.

Topics: Antibodies, Monoclonal; Carcinoma, Hepatocellular; Child; Hepatolenticular Degeneration; Humans; Immunohistochemistry; Inclusion Bodies; Keratins; Liver; Liver Cirrhosis; Liver Diseases; Liver Diseases, Alcoholic; Liver Neoplasms

The existence of progenitor (stem) cells in the human liver remains a matter of debate. In rodent models of hepatocarcinogenesis and injury, oval cells proliferate in the periportal regions of the portal tracts and are suggested to derive from a stem cell compartment, because they are capable of differentiating into hepatocytes or biliary epithelial cells. In this study, the rat oval cell marker, OV-6 has been used to investigate the hypothesis that there are stem cells present in fetal and pediatric human liver. The pattern of OV-6 expression was compared with the established adult biliary cell markers human epithelial antigen-125 (HEA-125) and cytokeratin-19 (CK-19). In normal pediatric liver (n = 7), bile ducts and ductules were immunostained with CK-19 and HEA-125, whereas OV-6 staining was consistently negative. In fetal tissue (n = 10), ductal plate cells, primitive bile ducts, and hepatoblasts were stained with CK-19 and HEA-125 although only some of the ductal plate cells and hepatoblasts were OV-6 positive. In biliary atresia (n = 6) and 1, anti-trypsin deficiency (1,AT) (n = 4), CK-19 and HEA-125 immunostained ductular proliferative cells that tended to form finely anastomosing ductules, whereas OV-6 staining was found more on discrete cells confined to portal tract margins. Additionally, in diseased liver, OV-6 was strongly positive in hepatocyte lobules with greatest intensity in the periseptal regions. This widespread hepatocyte OV-6 positivity suggests that the antibody may identify cells of a less differentiated phenotype (transitional hepatocytes) that have replaced the mature cells. Therefore, it is proposed that in human liver, OV-6 is recognizing cells with a progenitor stem cell-like phenotype with the capacity to differentiate into OV-6 positive ductular cells or lobular hepatocytes.

Topics: Adult; alpha 1-Antitrypsin Deficiency; Animals; Antigens, Differentiation; Antigens, Surface; Bile Ducts; Biliary Atresia; Biomarkers; Biomarkers, Tumor; Child; Embryo, Mammalian; Embryonic and Fetal Development; Fetus; Gestational Age; Humans; Keratins; Liver; Liver Diseases; Rats; Stem Cells

Mesenchymal hamartoma is a rare liver lesion. This lesion was found in a 7-month-old girl with high serum alphafaetoprotein serum levels and was composed of loose connective tissue containing a certain number of epithelial cells of biliary or hepatic origin. Immunohistochemical studies showed that cytokeratins 7 and 19 were localized in bile duct epithelium. The ultrastructural study showed that the hamartoma was composed of well differentiated ductal structures surrounded by a myxoid mesenchyma with cysts formed either from degenerative mesenchymal areas or from dilated ducts. Flow cytometric analysis of nuclei from frozen tissue revealed that the lesion was DNA aneuploid, with a DNA index of 1.28.

Topics: alpha-Fetoproteins; Aneuploidy; DNA; Female; Flow Cytometry; Hamartoma; Humans; Immunohistochemistry; Infant; Keratin-7; Keratins; Liver Diseases; Mitotic Index

Cytokeratin (CK) patterns and albumin messenger RNA (mRNA) are investigated in 24 patients with benign hepatic lesions (7 patients with focal nodular hyperplasia [FNH], 10 with hepatocellular adenomas [HA], 1 with biliary hamartoma, 4 with biliary cysts, 2 with cystadenomas) and in 8 patients with cystadenocarcinoma, a rare liver malignancy. The lesions and surrounding tissue of the hepatocytic components expressed CK 8 and 18 at immunohistochemistry, whereas the biliary elements evidenced CK 8 and 18 and CK 7 and 19. The albumin mRNA, as detected by in situ hybridization (ISH), revealed different distributions in the hepatocytes of FNH and HA. In the benign biliary lesions, the normal hepatocytes surrounding the tumors expressed albumin mRNA, whereas the biliary structures did not. Interestingly, in the cystadenocarcinomas, albumin mRNA was observed not only in the hepatocytes of residual parenchyma, but also in neoplastic bile duct cells lining the carcinomatous cysts; no signal was identified in the nonneoplastic biliary elements. This indicates that cystadenocarcinomas have a mixed biological phenotype and suggests they could arise either from pluripotent cells or from neoplastic cells that reacquire epigenetic features. Our results suggest two possible diagnostic applications for albumin ISH: on routine sections, it could represent an important tool for distinguishing between cystadenoma and cystadenocarcinoma; and on fine needle biopsy specimens, it could reduce uncertainty between FNH and HA.

Topics: Adenoma; Adolescent; Adult; Aged; Albumins; Biliary Tract Neoplasms; Biomarkers, Tumor; Cell Differentiation; Cystadenocarcinoma; Cystadenoma; Cysts; Diagnosis, Differential; Female; Gene Expression; Hamartoma; Humans; Hyperplasia; Immunoenzyme Techniques; In Situ Hybridization; Keratins; Liver; Liver Diseases; Liver Neoplasms; Male; Middle Aged; Neoplasm Proteins; Phenotype; Protein Isoforms; RNA Probes; RNA, Complementary; RNA, Messenger; RNA, Neoplasm; Stem Cells

Recent studies, including our own, suggest that intermediate filament proteins, particularly bile duct-specific cytokeratin 19 (CK19) and the hepatocyte-specific HepPar1 antigen define the developmental stages of hepatic progenitor cells during liver morphogenesis. We hypothesized that the HepPar1+ CK19+ progenitor cells are activated during human liver regeneration after massive hepatic necrosis and proliferate with the formation of so-called ductular hepatocytes or neocholangioles. We demonstrated previously that the ductular hepatocytes proliferate and share phenotypic characteristics with hepatocytes and biliary epithelial cells. In this investigation, we compared the expression pattern of intermediate filament proteins and HepPar1 antigen in ductular hepatocytes with that of bipotential hepatic progenitor cells. CK14, CK19, vimentin, and HepPar1 antigen were localized by immunoperoxidase staining in 13 human livers with regeneration after massive hepatic necrosis. Double immunostaining of three cases for CK14/CK19 and HepPar1/CK19 was also performed. CK19 reaction exhibited diffuse staining of the cytoplasm of many ductular hepatocytes and bile ducts in all cases. CK14 was expressed in the cytoplasm of ductular hepatocytes and few bile ducts in 5 of 12 specimens. HepPar1 staining was positive in many ductular hepatocytes in 11 of 13 cases. Vimentin was detected in the perinuclear cytoplasm of ductular hepatocytes and some bile duct epithelial cells in all regenerating livers. Double immunostaining for HepPar1/CK19 demonstrated that the ductular hepatocytes contained either HepPar1 or CK19 and that some ductular hepatocytes coexpressed both antigens. CK14, CK19, vimentin, and HepPar1 expression in ductular hepatocytes in human liver regeneration resembles the pattern seen in the developing human liver from 4 to 16 weeks' gestation. This suggests that the ductular hepatocytes recapitulate the developmental stages of bipotential liver progenitor cells and differentiate in steps marked by the acquisition or loss of specific phenotypic characteristics.

Topics: Adolescent; Adult; Aged; Female; Humans; Keratins; Liver Diseases; Liver Regeneration; Male; Middle Aged; Necrosis; Stem Cells; Vimentin

A case of granulosa cell tumor of the ovary associated with hepatocytic differentiation is reported in a 45-year-old patient with a torsioned ovarian tumor. Serum alpha-fetoprotein (AFP) levels were normal 6 days postoperatively. Histopathologically, the granulosa cell tumor was typically trabecular. Its cells had nuclear grooves and were positive only for vimentin. Scattered diffusely throughout the tumor were small groups of regular polygonal cells, the cytoplasm of which secreted bile and was strongly positive for keratin, carcinoembryonic antigen (CEA), alpha-1-antitrypsin (A1AT), and ferritin and moderately positive for fibrinogen and ceruloplasmin. These results unequivocally identified them as hepatic cells. The AFP negativity of the hepatic cells was interpreted as a sign of terminal hepatocytic differentiation. The scattered arrangement of the hepatocytes simulated stromal luteinization. As neither a primary liver tumor nor any associated germ cell tumor was found, the histogenesis of the hepatic cells was thought to be metaplastic.

Topics: alpha 1-Antitrypsin; alpha-Fetoproteins; Carcinoembryonic Antigen; Cell Differentiation; Cell Nucleus; Female; Ferritins; Granulosa Cell Tumor; Humans; Immunohistochemistry; Keratins; Liver; Liver Diseases; Luteal Cells; Metaplasia; Middle Aged; Ovarian Neoplasms; Ovary; Vimentin

The proliferative activity and ultrastructural characteristics of proliferating biliary epithelial cells were analysed immunohistocytochemically in 39 biopsied liver specimens from patients with acute viral hepatitis, chronic hepatitis and liver cirrhosis using a monoclonal antibody against DNA polymerase alpha (DNA-PA). In acute viral hepatitis with perivenular confluent necrosis, proliferation of typical bile ducts was found frequently in portal areas. In chronic aggressive hepatitis and cirrhosis, ductular proliferation of both typical and atypical forms was found in enlarged portal and periportal areas and in confluent necrotic areas. The number of proliferating biliary epithelial cells that stained positive for DNA-PA was small. There were very few positively stained cells in atypical bile ducts in confluent necrotic areas of cirrhosis. Atypical bile ducts seen in chronic aggressive hepatitis, cirrhosis and acute hepatitis with confluent necrosis were positively stained for both cytokeratins 8 and 19. In cirrhosis, the number of stained biliary epithelial cells in typical bile ducts was larger than the number of such cells in atypical bile ducts (P < 0.01). By electron microscopy, the cells positively stained for DNA-PA were mostly so-called clear cells with irregular nuclei containing coarse nucleoplasm, and a few small cells with scanty cytoplasm and few organelles.

Topics: Adult; Aged; Antibodies, Monoclonal; Bile Ducts; Biopsy; Cell Division; Chronic Disease; Cytoplasm; DNA Polymerase II; Epithelium; Female; Hepatitis; Hepatitis, Viral, Human; Humans; Immunohistochemistry; Keratins; Liver; Liver Cirrhosis; Liver Diseases; Male; Microscopy, Electron; Middle Aged; Organelles

Biliary epithelial cells express characteristically cytokeratin in their cytoplasm in normal and diseased livers. The present study disclosed that vimentin was frequently expressed in the cytoplasm of proliferating and damaged bile ductules and interlobular bile ducts, while their normal counterparts were negative for vimentin. Although this expression itself seemed nonspecific to any of the hepatobiliary diseases examined, bile ductules and interlobular bile ducts were frequently positive in chronic cholestatic and necroinflammatory liver diseases. In biliary epithelial cells, vimentin was localized around the nucleus or in the subnuclear regions, when present. Immunoelectron microscopically, reaction products for vimentin and for cytokeratin were found on bundles of intermediate filaments in the cytoplasm of biliary epithelial cells. The former was found mostly in the paranuclear and subnuclear regions, while the latter detected around the desmosomes, in addition to the paranuclear cytoplasm. Vimentin and cytokeratin were also seen together under immunoelectron microscopy on the same intermediate filaments. It seems likely that aberrant expression of vimentin in bile ductules and interlobular bile ducts and heterogeneous antigenic expression of intermediate filaments in the same biliary epithelial cells may be related to proliferation of, reorganization of, or damage to the ductular and ductal biliary cells in a variety of hepatobiliary diseases.

Topics: Bile Duct Diseases; Bile Ducts, Intrahepatic; Cell Division; Female; Humans; Immunoenzyme Techniques; Keratins; Liver Diseases; Microscopy, Immunoelectron; Middle Aged; Vimentin

Severe ethanol-induced liver damage is characterized by fibrous dissociation of liver cell plates leading to many apparently isolated hepatocytes. Three-dimensional reconstruction, however, revealed hepatocytes that were surrounded by connective tissue as endpoints of "parenchymal pillars" or in association with liver cell plates and bile ductules. Double immunofluorescence studies displayed the expression of cytokeratin (CK) 7 in bile ducts, including bile ductules, but also in some hepatocytes still organized in liver cell plates. The other bile duct, typical CK, namely CK 19, was only detectable in few hepatocytes. However, the expression of CK 7 and/or CK 19 was less frequent in hepatocytes that were closely associated with bile ductules. CK 7 and CK 19 were also found in some, but not all, Mallory bodies. These observations indicate that the expression of these two CKs is neither related to a transformation of hepatocytes to bile duct-like structures ("ductal metaplasia") nor to the formation of Mallory bodies. Furthermore, double immunofluorescence studies revealed small groups of hepatocytes and bile ductules that were encircled by basement membrane material, thus suggesting the formation of "secretory units."

Topics: Aged; Bile Ducts; Cell Communication; Cell Membrane; Endoplasmic Reticulum; Extracellular Matrix; Female; Fibrosis; Fluorescent Antibody Technique; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Keratins; Liver; Liver Cirrhosis, Alcoholic; Liver Diseases; Male; Mesoderm; Middle Aged

Among 1,098 liver biopsy specimens obtained from patients with various liver diseases characterized by liver injury, 58 epithelial cells whose cytoplasms stained positively by the periodic acid-Schiff stain (digested with diastase) were recognized in the interlobular bile ducts of 37 specimens from 36 patients. Light microscopic study revealed that the cytoplasms of these cells were clear or stained weakly eosinophilic on hematoxylin and eosin staining and that the cell limits were distinct. From their reaction with periodic acid-Schiff stain and from electron microscopic observation it was clear that these cells contained an abundance of glycogen and were located among the normal bile duct cells surrounded by basement membrane. On electron microscopy, these cells had microvilli of equal sizes on their luminal surfaces and many irregularly sized microvilluslike cell membrane projections on their basal surfaces. They rested on basement membrane with basal spaces. These cells varied in size from 25.0 to 452.2 microns 2 (mean = 212.2 microns 2). In contrast, the sizes of normal bile duct cells and hepatocytes ranged from 20.0 to 69.3 microns 2 (mean = 34.2 microns 2) and from 113.0 to 860.3 microns 2 (mean = 447.0 microns 2), respectively. Immunohistochemical study with antiserum to cytokeratin 19, albumin and alpha 1-antitrypsin on serially cut frozen sections showed that some of these cells expressed markers of bile duct cells and hepatocytes. Some cells expressed only the markers of hepatocytes. Computer graphic three-dimensional reconstruction clearly demonstrated that these cells were located sparsely (but sometimes in groups) among normal interlobular bile duct cells, without any connection to the surrounding parenchymal hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adolescent; Adult; Aged; Albumins; alpha 1-Antitrypsin; Bile Ducts, Intrahepatic; Cytoplasm; Epithelium; Female; Humans; Immunohistochemistry; Keratins; Liver; Liver Diseases; Male; Microscopy, Electron; Middle Aged; Periodic Acid-Schiff Reaction; Staining and Labeling

Human liver parenchymal cells have a very simple cytokeratin composition and express only one cytokeratin pair: cytokeratin 8 (a type II cytokeratin, molecular weight 52 kD) and cytokeratin 18 (a type I cytokeratin, molecular weight 45 kD). Intrahepatic bile duct cells contain in addition to cytokeratins 8 and 18 also cytokeratins 7 (a type II cytokeratin, molecular weight 54 kD) and cytokeratin 19 (a type I cytokeratin, molecular weight 40 kD). The paper deals with the cytokeratin expression in various types of benign and malignant primary liver tumors as assessed by immunohistochemical methods or by the use of gel electrophoresis and immunoblotting of cytoskeletal extracts.

Topics: Adenoma, Bile Duct; Biomarkers; Carcinoma, Hepatocellular; Histocytochemistry; Humans; Hyperplasia; Keratins; Liver Diseases; Liver Neoplasms

Proliferation of bile ductules or ductular hepatocytes occurs in a variety of liver diseases. The origin of these ductular structures and the mechanism of their proliferation are controversial. Using cytokeratin as marker for ductular structures, liver diseases in which ductular proliferation was a consistent and prominent feature were studied. Paraffin-embedded sections of livers (five cases each) with acute or chronic obstruction of extrahepatic bile ducts, primary biliary cirrhosis (stage II), drug-induced cholestatic liver disease, liver allograft rejection, vicinity of metastatic carcinoma, and massive hepatic necrosis were studied by immunohistochemical methods using three kinds of antiserum against cytokeratin polypeptides of different molecular weights. Bile ductules in diseases involving bile ducts and ductular hepatocytes in massive hepatic necrosis were closely associated with hepatocytes at the limiting plate or with injured hepatocytes. These findings suggest that hepatocytes play an important role in the proliferation of ductular structures or may represent their origin.

Topics: Biliary Atresia; Cell Division; Epithelium; Hepatic Duct, Common; Humans; Immunoenzyme Techniques; Keratins; Liver; Liver Diseases; Liver Neoplasms; Necrosis

The novel extracellular matrix glycoprotein tenascin was studied immunohistochemically in normal and fibrotic human liver. Its localization was compared to that of laminin, fibronectin and collagen type IV. In the normal liver, a weak staining for tenascin was detected along sinusoids, while portal tracts were negative. In both alcoholic and cholestatic liver disease and acute and chronic hepatitis, sinusoidal immunoreactivity for tenascin was variably increased as compared to the normal liver. Most striking, however, was the preferential accumulation of tenascin at connective tissue-parenchymal interfaces between proliferating ductules and in areas of piecemeal necrosis. As compared to laminin, fibronectin and collagen type IV, tenascin has the most restricted distribution. Our findings indicate that tenascin is a component of the extracellular matrix of the human liver. Its preferential expression at connective tissue-parenchymal interfaces in fibrosing areas in contrast to its absence from mature fibrous septa suggest a transient role in early matrix organization.

Topics: Alcoholism; Cell Adhesion Molecules, Neuronal; Cholestasis; Chronic Disease; Collagen; Extracellular Matrix; Fibronectins; Hepatitis; Humans; Immunohistochemistry; Keratins; Laminin; Liver; Liver Cirrhosis; Liver Diseases; Tenascin

Hepatocytes and bile duct epithelium express several types of cytokeratins, the characteristic intermediate-filament proteins of epithelial cells. The cytokeratin antigen expression was studied in normal and diseased livers, intrahepatic cholangiocarcinomas, and hepatocellular carcinomas by immunohistochemical methods using a panel of polyclonal and monoclonal antibodies to cytokeratins. Ten percent formaldehyde solution-fixed, paraffin-embedded sections obtained from ten patients without liver disease, 18 patients without liver disease, 18 patients with different stages of primary biliary cirrhosis, 14 patients with alcoholic hepatitis, ten patients with fatty liver hepatitis secondary to diabetes mellitus or morbid obesity, five patients with hepatocellular carcinomas, and five patients with cholangiocarcinomas were examined. The results suggested that hepatocytes and bile duct epithelium retain their distinct cytokeratin profiles in liver disease, including malignant transformation. Therefore, demonstration of cytokeratins in the liver is useful in establishing the cellular origin of neoplasms and understanding the pathogenesis of liver diseases.

Topics: Adenoma, Bile Duct; Carcinoma, Hepatocellular; Fatty Liver; Hepatitis; Humans; Keratins; Liver; Liver Cirrhosis, Biliary; Liver Diseases; Liver Neoplasms; Reference Values; Tissue Distribution

A cytokeratin immunohistochemical study was performed on 38 liver biopsies from cases of primary biliary cirrhosis, primary sclerosing cholangitis, extrahepatic biliary obstruction or drug-induced liver disease in order to analyse the cytoskeletal changes in detail. On paraffin sections of 27 cases, a variable number of hepatocytes were reactive with a polyclonal anti-cytokeratin antiserum that, in the normal liver, stains bile duct cells only. On cryostat sections of 23 cases, a variable number of hepatocytes were immunoreactive with a monoclonal antibody specifically directed against cytokeratin no. 7 and were most numerous in cases of long-standing cholestasis irrespective of the aetiology. In three cases of primary sclerosing cholangitis and two cases of primary biliary cirrhosis a few hepatocytes were also weakly positive with a monoclonal antibody specific for cytokeratin no. 19. Since cytokeratins no. 7 and no. 19 are, in the normal liver, restricted to bile duct cells, these results further support the concept of 'ductular metaplasia' of hepatocytes, the mechanism of which remains unclear.

Topics: Antibodies, Monoclonal; Autopsy; Biopsy; Chemical and Drug Induced Liver Injury; Cholangitis, Sclerosing; Cholestasis, Intrahepatic; Cytoskeleton; Frozen Sections; Humans; Keratins; Liver; Liver Cirrhosis, Biliary; Liver Diseases; Metaplasia; Paraffin

Antibodies against thymus epithelial cells (anti-TEC) and the basal cell layer (BCLA) of squamous epithelia have been described in association with HDV-related chronic liver disease (CLD). Data are lacking on their presence during nAnB virus infection. Sera from 51 patients with nAnB post-transfusion hepatitis, including acute and chronic cases diagnosed during a prospective study on candidates for cardiac surgery, and 167 with various forms of CLD were tested for the presence of anti-TEC and BCLA using indirect immunofluorescence on human thymus and rat forestomach sections. Both antibodies mainly occurred in nAnB, HDV and cryptogenic CLD (anti-TEC: 51%, 47% and 42%; BCLA: 29%, 38% and 31%, respectively). The prevalence of anti-TEC in nAnB CLD turned out to be higher than that recorded in alcoholic, HBV-related, autoimmune, liver and kidney microsomal antibody positive CLD and primary biliary cirrhosis (p ranging from less than 0.03 to less than 0.0004). Two monoclonal antibodies (Mabs) to cytokeratins gave a pattern superimposable on that of spontaneous anti-TEC (both Mabs) and BCLA (only one). Antibodies against epithelial constituents, presumably targeting cytokeratin-associated antigens, occur not only in HDV CLD, as previously reported, but also in nAnB CLD, where they might represent a diagnostic aid, due to the unavailability of reliable serological markers of nAnB infection. The close similarity of anti-TEC and BCLA status between nAnB and cryptogenic CLD suggests a nAnB etiology of at least a proportion of chronic liver patients at present scored as cryptogenic.

Topics: Acute Disease; Antibodies, Monoclonal; Antibody Specificity; Autoantibodies; Child; Epithelium; Fluorescent Antibody Technique; Hepatitis C; Hepatitis, Chronic; Hepatitis, Viral, Human; Humans; Keratins; Liver Diseases; Prevalence; Prospective Studies; Thymus Gland; Transfusion Reaction

The lack of information on the state of fetal liver in PiZZ alpha-1-antitrypsin (AAT) deficiency and a single case report claiming a hypoplasia of interlobular bile ducts in a 20-week PiZZ fetus, instigated this histologic study of the liver in five PiZZ fetuses, 17-20 weeks of gestation and five age-matched controls. We found no difference between the percentage of portal tracts with identifiable bile ducts in the PiZZ (median 22.2%, range 21%-23%) and in the control (median 21.4%, range 20%-24%) on hematoxylin- and eosin-stained sections. Immunostaining with AE1, a monoclonal antibody to cytokeratins restricted to normal bile ducts, doubled the number of recognizable ducts in both PiZZ and control livers. In four PiZZ livers, but in none of the controls, granular deposits of AAT could be detected by specific immunoperoxidase staining. We conclude that an apparent paucity of interlobular bile ducts is normal in the 20-week fetal liver, and our data may be taken as reference for future study dealing with similar material. Except for the cytoplasmic deposition of granules immunoreactive to AAT antiserum, there was no evidence of any developmental anomaly, in particular of the bile duct system in these five PiZZ fetal livers.

Topics: alpha 1-Antitrypsin; alpha 1-Antitrypsin Deficiency; Antibodies, Monoclonal; Bile Ducts; Female; Fetal Diseases; Gestational Age; Humans; Immunoenzyme Techniques; Keratins; Liver; Liver Diseases; Male; Phenotype; Pregnancy

A cytokeratin-immunohistochemical study was performed on eight specimens of focal nodular hyperplasia of the liver in order to determine whether hepatocytes in this lesion can express "bile duct type" cytokeratins. Serially cut cryostat sections were reacted with a panel of six monoclonal antibodies specifically reactive with cytokeratin polypeptides nr. 7, 8, 18 and 19, using a 3-step immunoperoxidase procedure. Hepatocytes were positive for cytokeratins nr. 8 and nr. 18, whereas ductules contained in addition to cytokeratins nr. 8 and nr. 18 also polypeptides nr. 7 and nr. 19. In all cases, a variable number of hepatocytes close to fibrous septa or inside the nodules expressed cytokeratin nr. 7. In four cases, a small number of hepatocytes were also immunoreactive for cytokeratin nr. 19. Our data demonstrate that hepatocytes in focal nodular hyperplasia can express one or even two "bile duct type" cytokeratins, supporting the concept that ductular metaplasia of hepatocytes contributes to the ductular "proliferation" observed in this tumor-like lesion.

Topics: Adult; Female; Humans; Hyperplasia; Immunoenzyme Techniques; Immunohistochemistry; Keratins; Liver; Liver Diseases; Male

Aggregation and derangement of cytokeratin intermediate filaments are thought to be the key mechanism in the formation of Mallory bodies in alcoholic liver disease (ALD). To study the incidence and patterns of intracellular distribution of aggregated cytokeratin and to determine its utility as a diagnostic marker of ALD, 108 liver biopsy specimens from patients with various liver abnormalities were examined by an avidin--biotin peroxidase complex technique on paraffin section using a monoclonal antibody to cytokeratins (Hybritech). In normal liver (n = 11), only bile duct epithelium was positive. Both bile ducts and hepatocytes were positive in pathologic livers (n = 97). In ALD, 82 per cent of cases (42 of 51) showed cytokeratin positivity versus 15 per cent (seven of 46) in nonalcoholic liver disease (e.g., chronic hepatitis, nonalcoholic cirrhosis, cholestasis, and primary biliary cirrhosis). The highest incidence (100 per cent, 37 of 37) of positivity was obtained in cases with alcoholic hepatitis and cirrhosis compared with only 36 per cent (five of 14) in alcoholic fatty liver. Mallory bodies were found by the immunoperoxidase method in 71 per cent of cases (30 of 42) versus in 40 per cent (17 cases) by hematoxylin--eosin stain. In alcoholic fatty liver and alcoholic hepatitis, centrilobular hepatocytes showed cytokeratin positivity, whereas such reactivity was seen predominantly at the periphery of the regenerative nodules in alcoholic cirrhosis. A rare periportal hepatocyte was positive in the nonalcoholic group. These findings suggest that the differential distribution patterns of aggregated cytokeratin may be helpful in differentiating alcoholic from nonalcoholic liver diseases.

Topics: Antibodies, Monoclonal; Biopsy; Epitopes; Humans; Immunoenzyme Techniques; Intermediate Filaments; Keratins; Liver; Liver Diseases; Liver Diseases, Alcoholic

The distribution of tissue polypeptide antigen (40 kD molecular weight) in normal adult and fetal liver, and in liver disease was investigated and compared with the distribution of low and high molecular weight cytokeratins. In normal liver tissue polypeptide antigen was found only in bile duct epithelium; this distribution is similar to that of high molecular weight cytokeratin, but differs from that of low molecular weight cytokeratins. In liver disease it was found in areas of ductular transformation; in Mallory's bodies; and in alcoholic liver disease and primary biliary cirrhosis in some hepatocytes that did not contain Mallory's bodies.

Topics: Adult; Antigens; Bile Ducts, Intrahepatic; Fetus; Humans; Keratins; Liver; Liver Cirrhosis, Biliary; Liver Diseases; Liver Diseases, Alcoholic; Molecular Weight; Peptides; Tissue Polypeptide Antigen

A monoclonal murine antibody (KM 54-5) was produced against Mallory body (MB) material isolated from liver tissue of griseofulvin treated mice. The antigen was identified by positive immunofluroescence microscopy of MBs and by the immunoblotting technique on polypeptides separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In immunoblotting experiments, antibody KM 54-5 reacted with cytokeratins A (human no. 8) and D (human no. 18) of murine, bovine, and human hepatocytes as well as with cytokeratin A (no. 8) and its degradation products present in isolated murine MB. In immunofluorescence microscopy the antibody did not react with cytokeratin filaments of normal liver but showed a positive reaction with MBs after a certain stage in MB development had been reached. In a dot blot assay, using individual cytokeratin polypeptides isolated from murine liver and purified by ion exchange chromatography in pH 8 buffer containing 8 M urea, the antibody reacted with the individual polypeptides A (no. 8) and D (no. 18) but not with the heterotypic tetramer (A2D2) reconstituted from these polypeptides in 4 M urea. These findings confirm the cytokeratin nature of MB filaments. In addition, they show that the pathologic process of MB formation involves changes in cytokeratin organization and conformation, resulting in the accessibility of a specific antigenic determinant which is inaccessible ("masked") in the heterotypic tetramer subunit and in the cytokeratin filaments of normal cells. Hence this study presents an example of a pathological change of cytokeratin filaments and illustrates the value of monoclonal antibodies in the detection of such changes.

Topics: Animals; Antibodies, Monoclonal; Cattle; Cell Line; Chemical and Drug Induced Liver Injury; Electrophoresis, Polyacrylamide Gel; Fluorescent Antibody Technique; Griseofulvin; Humans; Immunologic Techniques; Inclusion Bodies; Keratins; Liver; Liver Diseases; Macromolecular Substances; Male; Mice; Rats

Proliferation of the two types of bile ductules, typical and atypical, in the portal and periportal areas was examined in various liver diseases other than cirrhosis to determine any difference in their immunohistochemical properties and presumed histogenesis. While the typical ductules with a well-formed lumen were frequently seen in a large spectrum of diseases, atypical ductules with a poorly defined lumen were encountered much more frequently in prolonged biliary diseases, including primary biliary cirrhosis and primary sclerosing cholangitis, than in nonbiliary hepatic diseases. Immunocytochemically, cytoplasmic keratin was intensively positive in typical ductules, and the degree of its intensity and extent was variable in atypical ductules. Simultaneously, some of the periportal hepatocytes revealed weak staining for keratin. Luminal borders of typical ductules usually revealed an expression of both carcinoembryonic antigen and epithelial membrane antigen, while atypical ductules and periportal hepatocytes lacked epithelial membrane antigen. The atypical ductules, together with the adjoining hepatocytes, appeared on occasion to form anastomosing cords in prolonged biliary diseases. Thus, atypical ductules seem likely to originate from ductular transformation of the periportal hepatocytes and the typical ductules might result from the proliferation of preexisting interlobular bile ducts and ductules.

Topics: Bile Duct Diseases; Bile Ducts, Intrahepatic; Carcinoembryonic Antigen; Histocytochemistry; Humans; Immunoenzyme Techniques; Keratins; Liver Diseases; Membrane Proteins; Mucin-1

Antiperinuclear factor (APF) was detected in 7 out of 38 patients with autoimmune liver disease (AILD) and in 7 out of 83 patients with non-autoimmune liver disease (NAILD). Anti-keratin antibodies were found in 2 out of 18 patients with AILD and in 3 out of 32 patients with NAILD. Neither APF nor antikeratin antibody was significantly more frequent in AILD than in NAILD. The incidence of APF was shown to be greater (p less than 0.02) in patients with rheumatoid factor (RF) and/or antinuclear antibody. This supports the hypothesis that APF is connected with RF rather than with rheumatoid arthritis, although the presence of APF is evidence towards the diagnosis of rheumatoid arthritis.

Topics: Adult; Aged; Antibodies, Antinuclear; Autoantibodies; Autoimmune Diseases; Female; Hepatitis; Humans; Keratins; Liver Diseases; Male; Middle Aged