Alcoholic hepatitis

Clinical and pathological data from four cases of fatal non-cirrhotic alcoholic hepatitis are presented. The patients were relatively young but had a long history of excessive alcohol intake, and the disease had an acute onset which progressed inexorably to death in hepatic and renal failure.At autopsy the livers were enlarged but non-cirrhotic. The important histological criteria for the diagnosis of alcoholic hepatitis are a panlobular polymorphonuclear inflammatory cell infiltrate, the presence of Mallory material (alcoholic hyaline) and fine fibrosis surrounding small groups of liver cells. The latter finding may be more marked in the centrolobular zone and result in the picture of central hyaline sclerosis.     

Intermediate filaments: analysis of filamentous aggregates induced by griseofulvin, an antitubulin agent

Mice fed griseofulvin, an antibiotic with antimicrotubular activity, formed hepatocellular aggregates of intermediate filaments, which resembled those associated with human alcoholic liver disease. These aggregates, termed Mallory bodies, were isolated from both human and mouse liver and the composition of these structures compared. Electrophoretic analysis indicated that the mouse filaments were composed of four major polypeptides (51,000, 47,000, 37,000, and 36,000 daltons). Human Mallory bodies possessed a similar number of components but of different molecular weights (56,000, 51,000, 50,000, and 38,000 daltons). Guinea pig antisera prepared against both whole human Mallory bodies and the major human polypeptide (56,000 daltons) crossreacted with mouse Mallory body material in both immunochemical and immunocytochemical systems. Our findings suggest that the two filament systems possess similar biochemical and immunological properties.     

Constitutive aggregates of intermediate-sized filaments of the vimentin and cytokeratin type in cultured hepatoma cells and their dispersal by butyrate

Cloned hepatoma cells ($\text{72}\text{22}$) derived from the liver of a rat treated with the carcinogen, diethylnitrosamine, exhibit a genetically stable, large, acentric, juxtanuclear, hyaline aggregate of loosely packed intermediate-sized (7–11 nm) filaments, interspersed with variable but minor amounts of microtubules, polyribosomes and membranous structures. Immunofluorescence microscopy shows that the these filaments react specifically with antibodies to bovine prekeratin and to murine vimentin. The aggregates contain aster-like foci common to the arrangement of both tonofilament-like and vimentin-containing intermediate-sized filaments, although both filament systems show different fibrillar patterns in other cytoplasmic regions. While the cytokeratin filament system is not significantly altered during exposure to colcemid, the vimentin in the abnormal aggregate is rearranged during such treatment into extensive and complex perinuclear ‘whorls’ of filaments. Treatment of the cells with butyrate results in a markedly flattened, hepatocyte-like morphology, a reappearance of typical actin-containing ‘cables’, and a progressive disintegration of the filament aggregate concomitant with a normal display of filaments of both the cytokeratin and vimentin type. The results show that (i) some cells contain aggregates consisting of two different types of intermediate-sized filaments oriented onto a common focal center; (ii) such an abnormal filament arrangement is clonally stable; (iii) the vimentin-type filaments contained in such aggregates are still susceptible to the action of antimitotic drugs and can be rearranged into characteristic perinuclear whorls; and (iv) this abnormal aggregate of intermediate filaments can be reverted to normal patterns upon treatment of the cells with butyrate.     

Failure of Electron Paramagnetic Resonance Spectroscopy Studies to Detect Elevated Free Radical Signals in Liver Biopsy Specimens from Patients with Alcoholic Liver Disease

Electron paramagnetic resonance spectroscopy (EPR) was used to study free radicals and transition metal complexes in liver tissue taken from patients with liver disease. Samples were frozen to 77K directly following biopsy to prevent deterioration. Our major aim was to compare signals from patients suffering from alcohol abuse with those from patients having liver damage not induced by alcohol. Samples were obtained from 19 chronic alcohol abusers and 7 non-alcoholic liver disease patients. Of the 19 alcoholic patients, 18 had an increased fat content, 6 had Mallory's hyaline, 12 had an acute inflammatory response, 9 had increased stainable iron and 4 had evidence of fibrosis. A signal derived from free radicals with a spectroscopic splitting factor of g = 2.0045 was found in all samples. This signal in the alcoholic patients had a mean amplitude of 2.96 cm (± 1.42 SD), and in patients with non-alcoholic liver disease 2.12cm (±0.82) (p = 0.10NS), measured under identical instrument settings.

The molar proportion of diene conjugated linoleic acid (DCLA), a free radical marker, in the sera of alcoholic patients was 2.68% (±1.93), but did not correlate with the free radical signals obtained by EPR spectroscopy. Also, there was no correlation between the free radical derived EPR signal and fat content, Mallory's hyaline, inflammatory infiltrate, iron or fibrosis in the liver biopsy specimens. Similarly the concentrations of aspartate transaminase, albumin, and gamma-glutamyl transferase in serum samples showed no correlations with free radical concentrations.

The absence of any significant increase in the stable free radical signal in the presence of alcohol induced liver disease and the lack of correlation between the signal and either histological or serological evidence of liver damage, suggests that alcohol derived free radicals may not be involved in the pathogenesis of alcoholic liver disease.

Unusually large sextet features characteristic of MN(II) complexes were observed for all liver samples. Such signals are very rare in human tissue, showing that there is a strong accumulation of Mn (II) in the liver. However, no systematic trends were observed. In some samples signals characteristic of iron-sulphur cluster units were detected, but again no correlations could be discovered.     

Vertebrate liver cytokeratins: a comparative study

The structure and composition of intermediate filaments isolated from liver of representatives of different vertebrate classes have been studied by electron microscopy and biochemical and immunochemical methods. It has been shown that the methodological approach for isolation of rat liver intermediate filaments can be efficiently applied to all other classes of vertebrates. The intermediate filaments studied have the same electron microscopic morphology and are species undistinguishable. The molecular weight of intermediate filament proteins varies from 40,000 to 60,000 and their isoelectric point varies from 5.0 to 6.45. Immunological investigations show that in all animals studied the intermediate filaments are built up of cytokeratins belonging to both types of keratins: type I and type II. Only one protein of the type II cytokeratins is present in all vertebrate classes, whereas in lower vertebrates two or even three type I cytokeratins contribute to the structure of liver intermediate filaments. The biochemical and immunochemical results are discussed with regard to the evolution of liver cytokeratins.     

Actin participates in the structure of liver intermediate filaments

A dominating protein fraction (p45) having molecular weight of 45000 and pI 5.45 was found in the intermediate filaments pellet obtained from rat liver besides the present cytokeratins. Peptide mapping and radioimmunological assays with antibodies against this protein and muscle actin proved that the p45 protein belongs to the actin group. Immunoelectron microscopy revealed that this protein is located on the liver intermediate filaments. By melting of the cytokeratin complexes in urea it was established that p45 protein is complexed with the low molecular weight cytokeratin.     

Serum alpha-mannosidase in patients with alcoholic liver disease

Sera from 9 persons with either biopsy-proven alcoholic liver disease or a history of chronic, excessive ethanol consumption were analyzed for their content of various hydrolases. Compared to controls, significant elevations in the following enzyme activities were seen in sera from the patient population: acid phosphatase (2.0-fold), beta-glucuronidase (2.1-fold), hexosaminidase (1.4-fold), and alpha-L-fucosidase (2.3-fold). In addition, alpha-mannosidase activity, previously reported to be unchanged in cases of hepatic cirrhosis [Reglero et al., Clinica chim. Acta 130: 155-158], (1980) was found to be significantly increased (p less than 0.001) when assays were performed at acid (pH 4.5) or intermediate (pH 5.5) hydrogen ion concentrations. Fractionation of sera on DEAE-Sephadex columns showed that the increase in alpha-mannosidase activity in the serum of patients with alcoholic liver disease was due to increases in the level of at least one 'acid alpha-mannosidase' and two intermediate pH optimum alpha-mannosidases. The general increase in the activity of a group of glycosidases is consistent with a hypothesis involving decreased clearance of glycoproteins from the blood of persons with hepatic cirrhosis.     

Intermediate filaments: Detection of lectin-like activity in purified alcoholic hyaline

Alcoholic hyaline, an intracellular, filamentous (10 nm) aggregate isolated from human alcoholic livers, bound the glycoprotein enzyme, horseradish peroxidase, in a specific and reproducible manner. Using a solid-phase assay system consisting of adsorbed alcoholic hyaline, we have shown that this binding is thermolabile, relatively insensitive to both pH extremes and high ionic strength, and highly sensitive to the presence of neutral and amino sugars. The results suggest that the binding of horseradish peroxidase is not a passive adsorption but rather an “active” phenomenon involving carbohydrate groups on the enzyme. The presence of an intracellular, filament-associated lectin is strongly indicated.     

Ultrastructure of alcoholic hyalin and fate of the affected hepatocytes.

In liver biopsy specimens, foci of satellitosis, i.e., foci of alcoholic hyalin containing hepatocytes surrounded by accumulated leukocytes, were studied by means of electron-microscopic investigation. Within satellitosis hepatocytes, the same morphologic variants of alcoholic hyalin were observed as formerly described in nonsatellitosis liver cells: (1) clusters of randomly oriented smooth filaments of homogenous electron density, (2) bundles of filaments aligned in parallel arrays and exhibiting irregular densities and indistinct boundaries, and (3) masses of a strongly osmiophilic amorphous material, presumably lipidic in nature. The individual hyalin body was composed of one, two, or all three components in varying proportions; an uptake by cytosegresomes was never observed. The leukocytes of satellitosis foci, mostly polymorphonuclear ones without obvious alterations, had close contact to the central hepatocytes, and sometimes covered large holes in the hepatocellular plasma membrane. In other cases, hepatocytes and neutrophils were both destroyed and the hyalin bodies were found unaltered within the sinusoids. It is suggested that alcoholic hyalin represents an inert proteinaceous storage material, deposited at the site of synthesis which in the course of time becomes superimposed by a lipid component. In cases of alcohol-induced hepatocellular lesions, those hepatocytes which produce hyalin bodies may have a special metabolic sensitivity to alcohol which on the one hand may result in hyalin synthesis and accumulation and on the other hand may, under special conditions, lead to plasma membrane destruction with secondary satellitosis formation.     

Differential location of different types of intermediate-sized filaments in various tissues of the chicken embryo.

The location of constitutive proteins of different types of intermediate-sized (about 10 mm) filaments (cytokeratin, vimentin, desmin, brain filament protein) was examined in various tissues of 11--20 day chick embryos, using specific antibodies against the isolated proteins and immunofluorescence microscopy on frozen sections and on isolated serous membrane. The tissues studied which contained epithelia were small intestine, gizzard, esophagus, crop, liver, kidney, thymus, mesenteries, and epidermis. The results show that the different intermediate filament proteins, as seen in the same organ, are characteristic of specific lines of differentiation: Cytokeratin filaments are restricted to--and specific for--epithelial cells; vimentin filaments are seen--at this stage of embryogenesis--only in mesenchymal cells, including connective tissue, endothelial and blood cells, and chondrocytes; filaments containing protein(s) related to the subunit protein prepared from gizzard 10 nm filaments (i.e., desmin) are significant only in muscle cells; and intermediate filament protein of brain, most probably neurofilament protein, is present only in nerve cells. We conclude that for most tissues the expression of filaments of cytokeratin, vimentin, desmin, and neurofilament protein is mutually exclusive, and that these protein structurees provide useful markers for histochemical and cytochemical differentiation of cells of epithelial, mesenchymal, myogenic, and neurogenic differentiation.     

Down-regulation of cytokeratin 14 mRNA in polyoma virus middle T-transformed rat liver epithelial cells.

We have recently shown that rat liver nonparenchymal epithelial cells, such as T51B cells, selectively express cytokeratin (CK) 14 as a partner of CK8 in their intermediate filaments, and we proposed CK14 as a unique cell lineage marker of the liver epithelial cell population (R. Blouin, M-J. Blouin, I. Royal, A. Grenier, A. Loranger, D. R. Roop, and N. Marceau, Differentiation, submitted for publication, 1992). In the present study, T51B-261A (spontaneously transformed) and T51B-261B (aflatoxin B1-treated) clones and clones derived from T51B cells transfected with SV40 large T (LT) and polyoma virus middle T (MT) were used to investigate CK gene expression in nontransformed and transformed liver epithelial cells. T51B-261A, T51B-261B, MT-T51B, and LT/MT-T51B clones all grew in calcium-deficient medium and formed colonies in soft agar, whereas LT-T51B clones did not grow at all in either one of these assays. T51B-261A and T51B-261B clones formed small, slow growing tumors when injected into newborn syngenic rats, whereas the MT-T51B and LT/MT-T51B clones produced rapidly forming, large tumors. There was no effect of cell transformation on CK expression, except in the clones expressing MT, where the CK intermediate filaments were completely lost. Analyses of [35S]methionine incorporation into the Triton-resistant cytoskeleton and of total proteins confirmed that CKs were absent. In contrast, vimentin intermediate filaments remained unaffected in all of the clones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential Location of Different Types of Intermediate-Sized Filaments in Various Tissues of the Chicken Embryo

The location of constitutive proteins of different types of intermediate-sized (about 10 mm) filaments (cytokeratin, vimentin, desmin, brain filament protein) was examined in various tissues of 11–20 day chick embryos, using specific antibodies against the isolated proteins and immunofluorescence microscopy on frozen sections and on isolated serous membrane. The tissues studied which contained epithelia were small intestine, gizzard, esophagus, crop, liver, kidney, thymus, mesenteries, and epidermis. The results show that the different intermediate filament proteins, as seen in the same organ, are characteristic of specific lines of differentiation: Cytokeratin filaments are restricted to – and specific for – epithelial cells; vimentin filaments are seen – at this stage of embryogenesis – only in mesenchymal cells, including connective tissue, endothelial and blood cells, and chondrocytes; filaments containing protein(s) related to the subunit protein prepared from gizzard 10 nm filaments (i.e., desmin) are significant only in muscle cells; and intermediate filament protein of brain, most probably neurofilament protein, is present only in nerve cells. We conclude that for most tissues the expression of filaments of cytokeratin, vimentin, desmin, and neurofilament protein is mutually exclusive, and that these protein structures provide useful markers for histochemical and cytochemical differentiation of cells of epithelial, mesenchymal, myogenic, and neurogenic differentiation.     

Semiquantitative Postembedding Characterization of Intermediate Filaments in Central Nervous System Lesions Using Immunoelectron Microscopy

Standardized postembedding immunoelectron microscopy was performed to demonstrate glial fibrillary acidic protein (GFAP) and vimentin in individual intermediate filaments to determine the diagnostic value of demonstrating ultrastructural and immunophenotypic characteristics of intermediate filaments in routine brain biopsy specimens. Dual expression of GFAP and vimentin was observed in the astroblastoma and astrocytes of Alexander's disease. The antigen availability for vimentin, however, was too low to allow reliable assessment of the GFAP:vimentin ratio in individual intermediate filaments and/or filament bundles. In meningioma, only vimentin positive intermediate filaments were found. GFAP positive intermediate filaments were present in all other specimens except the oligodendroglial components of the mixed glioma, which were devoid of intermediate filaments. GFAP positivity in the filamentous periphery and electron-dense core of Rosenthal fibers was demonstrated. Technical and tissue processing factors had a significant effect on particle density values obtained for individual specimens. Although the number, distribution, and density of glial intermediate filaments varies in different astroglial entities, correlation of particle density values determined by immunoelectron microscopy with relative GFAP concentrations in different lesions requires utmost caution. Nevertheless, application of the postembedding approach to routinely fixed biopsy specimens indicated an association of different entities with the exclusive presence of GFAP and/or vimentin in individual intermediate filaments, thus emphasizing the diagnostic value of intermediate filament typing for pathological characterization.     

Cytoplasmic filaments in the endothelial cells of the sheathed capillary: an ultrastructural and immunocytochemical study in the pig spleen.

Cytoplasmic filaments of the endothelial cells of sheathed capillaries in the pig spleen were identified and their ultrastructure was studied. Two types of cytoplasmic filaments were found: intermediate filaments (diameter: 10 nm) which filled most of the interior of the cells, and thin filaments (diameter: 5 nm) which were located just beneath the cell membrane and filled the lateral cytoplasmic processes. In immunocytochemical preparations, the intermediate filaments were positive for vimentin and desmin, and were negative for keratin. Staining of the thin filaments with heavy meromyosin resulted in arrowhead formations. These observations suggest that the intermediate filaments maintain the cytoarchitecture, possibly protecting the cell from structural alterations induced by blood pressure changes. Concurrently, thin filaments may facilitate the passage of red blood cells and blood platelets through the interendothelial fenestrae of the sheathed endothelial cell to the reticular meshwork in the capillary sheath.     

Ultrastructural and immunohistochemical analysis of proteoglycans in mouse pubic symphysis

Proteoglycans were accurately localized in mouse pubic symphyseal tissues using the cuprolinic blue method. Specific glycosaminoglycans degradative enzymes, together with chondroitin sulfate and decorin antibodies, allowed the identification of glycosaminoglycans. Chondroitin sulfate proteoglycans were the main proteoglycans observed in hyaline cartilage, fibrocartilage, and dense connective tissue. Ultrastructurally, they were seen as electron-dense granules and filaments. The granules, rich in chondroitin sulfate chains, were exclusively found in hyaline cartilage, whereas filaments were present in cartilage, fibrocartilage, and dense connective tissue. The latter were classified by size and susceptibility to enzyme digestion into F1, F2 and F3 filaments: F1 filaments were small, thin, and collagen fibril-associated; F2 filaments were thick, heavily stained, and localized around individual collagen fibrils and between bundles of collagen fibrils; and F3 filaments were scattered throughout elastic fiber surfaces. Considering their localization, susceptibility to chondroitinase AC and immunohistochemical detection, the symphysial F1 filaments were found to be preferentially decorin substituted with chondroitin sulfate side chains. The F2 filaments were also susceptible to chondroitinase AC treatment, whereas F3 filaments could be digested by heparitinase.The data thus obtained on the localization and identification of pubic symphyseal proteoglycans in virgin mice may be useful in the study of structural modifications that occur throughout pregnancy.     

Heavy meromyosin labeling of intermediate filaments in cultured connective tissue cells

Mild treatment with trypsin causes a radical change in the heavy meromyosin (HMM) binding properties of intermediate filaments in glycerinated, myosin-extracted cultured chick embryo connective tissue cells. In non-trypsin-treated cells, HMM labeling of filaments was often indistinct and variable in its distribution. By contrast, in cells treated with trypsin (under conditions which allowed most intermediate filaments to survive), virtually all filaments, including those of intermediate size, decorated with HMM to give distinct arrowhead patterns. We suggest that most intermediate filaments in such cells contain a core of F-actin masked by trypsin-labile accessory proteins.     

Intermediate filaments: a historical perspective

Intracellular protein filaments intermediate in size between actin microfilaments and microtubules are composed of a surprising variety of tissue specific proteins commonly interconnected with other filamentous systems for mechanical stability and decorated by a variety of proteins that provide specialized functions. The sequence conservation of the coiled-coil, alpha-helical structure responsible for polymerization into individual 10 nm filaments defines the classification of intermediate filament proteins into a large gene family. Individual filaments further assemble into bundles and branched cytoskeletons visible in the light microscope. However, it is the diversity of the variable terminal domains that likely contributes most to different functions. The search for the functions of intermediate filament proteins has led to discoveries of roles in diseases of the skin, heart, muscle, liver, brain, adipose tissues and even premature aging. The diversity of uses of intermediate filaments as structural elements and scaffolds for organizing the distribution of decorating molecules contrasts with other cytoskeletal elements. This review is an attempt to provide some recollection of how such a diverse field emerged and changed over about 30 years.     

Immunochemical comparison of prekeratin and alcoholic hyalin intermediate filaments

Alcoholic hyalin is an hepatocellular aggregate composed of filaments apparently related to the prekeratin intermediate filament subclass. The relationship between these two filament preparations was determined immunochemically using guinea pig antisera derived against alcoholic hyalin, prekeratin, and major prekeratin polypeptides. Immunocrossreactivities were determined using sensitive solid-phase enzyme-immunoassays. These assays indicated that antisera derived against a given filament preparation reacted 10–1000 times better with that preparation than with the other system. The nature of crossreactive meterial was determined using antisera derived against the larger prekeratin polypeptides (M_r 61,000 and 51,000). When tested against these two antisera, alcoholic hyalin appeared to react better with the serum derived against the larger prekeratin component. Moreover, anti-alcoholic hyalin antiserum bound four to five times better to the 61,000 dalton component than to the 51,000 dalton polypeptide in the enzyme-immunoassay. Our results indicate that antigenic determinants related to prekeratin can be detected in alcoholic hyalin, but that these determinants are present in relatively low concentrations in purified alcoholic hyalin. In addition, it appears that the relative concentrations of prekeratin components in alcoholic hyalin do not reflect those in purified prekeratin.     

Assembly characteristics of plant keratin intermediate filaments in vitro

After selective extraction and purification, plant keratin intermediate filaments were reassembled in vitro. Scanning tunneling microscope (STM) and transmission electron microscope (TEM) micrographs showed that acidic keratins and basic keratins can assemble into dimers and further into 10 nm filaments in vitro. In higher magnification images, it can be seen that fully assembled plant keratin intermediate filaments consist of several thinner filaments of 3 nm in diameter, which indicates the formation of protofilaments in the assembly processes. One of the explicit features of plant keratin intermediate filaments is a 24—25 nm periodic structural repeat alone the axis of beth the 10 nm filaments and protofilaments. The periodic repeat is one of the fundamental characteristic of all intermediate filaments, and demonstrates the half staggered arrangement of keratin molecules within the filaments.     

Intermediate filament protein partnership in astrocytes.

Intermediate filaments are general constituents of the cytoskeleton. The function of these structures and the requirement for different types of intermediate filament proteins by individual cells are only partly understood. Here we have addressed the role of specific intermediate filament protein partnerships in the formation of intermediate filaments in astrocytes. Astrocytes may express three types of intermediate filament proteins: glial fibrillary acidic protein (GFAP), vimentin, and nestin. We used mice with targeted mutations in the GFAP or vimentin genes, or both, to study the impact of loss of either or both of these proteins on intermediate filament formation in cultured astrocytes and in normal or reactive astrocytes in vivo. We report that nestin cannot form intermediate filaments on its own, that vimentin may form intermediate filaments with either nestin or GFAP as obligatory partners, and that GFAP is the only intermediate filament protein of the three that may form filaments on its own. However, such filaments show abnormal organization. Aberrant intermediate filament formation is linked to diseases affecting epithelial, neuronal, and muscle cells. Here we present models by which the normal and pathogenic functions of intermediate filaments may be elucidated in astrocytes.