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The sensitivity of current surveillance methods for detecting early-stage hepatocellular carcinoma (HCC) is suboptimal. Extracellular vesicles (EVs) are promising circulating biomarkers for early cancer detection. In this study, we aim to develop an HCC EV-based surface protein assay for early detection of HCC.Tissue microarray was used to evaluate four potential HCC-associated protein markers. An HCC EV surface protein assay, composed of covalent chemistry-mediated HCC EV purification and real-time immuno-polymerase chain reaction readouts, was developed and optimized for quantifying subpopulations of EVs. An HCC EV ECG score, calculated from the readouts of three HCC EV subpopulations (EpCAMHCC EV ECG score demonstrated great potential for detecting early-stage HCC. It could augment current surveillance methods and improve patients' outcomes.

Prohibitin 1 (PHB1) is a highly conserved, ubiquitously expressed protein that participates in diverse processes including mitochondrial chaperone, growth and apoptosis. The role of PHB1 in vivo is unclear and whether it is a tumor suppressor is controversial. Mice lacking methionine adenosyltransferase 1A (MAT1A) have reduced PHB1 expression, impaired mitochondrial function, and spontaneously develop hepatocellular carcinoma (HCC). To see if reduced PHB1 expression contributes to the Mat1a knockout (KO) phenotype, we generated liver-specific Phb1 KO mice. Expression was determined at the messenger RNA and protein levels. PHB1 expression in cells was varied by small interfering RNA or overexpression. At 3 weeks, KO mice exhibit biochemical and histologic liver injury. Immunohistochemistry revealed apoptosis, proliferation, oxidative stress, fibrosis, bile duct epithelial metaplasia, hepatocyte dysplasia, and increased staining for stem cell and preneoplastic markers. Mitochondria are swollen and many have no discernible cristae. Differential gene expression revealed that genes associated with proliferation, malignant transformation, and liver fibrosis are highly up-regulated. From 20 weeks on, KO mice have multiple liver nodules and from 35 to 46 weeks, 38% have multifocal HCC. PHB1 protein levels were higher in normal human hepatocytes compared to human HCC cell lines Huh-7 and HepG2. Knockdown of PHB1 in murine nontransformed AML12 cells (normal mouse hepatocyte cell line) raised cyclin D1 expression, increased E2F transcription factor binding to cyclin D1 promoter, and proliferation. The opposite occurred with PHB1 overexpression. Knockdown or overexpression of PHB1 in Huh-7 cells did not affect proliferation significantly or sensitize cells to sorafenib-induced apoptosis. Conclusion: Hepatocyte-specific PHB1 deficiency results in marked liver injury, oxidative stress, and fibrosis with development of HCC by 8 months. These results support PHB1 as a tumor suppressor in hepatocytes. (HEPATOLOGY 2010.)

The hepatitis C viral (HCV) genome is translated through an internal ribosome entry site (IRES) as a single polyprotein precursor that is subsequently cleaved into individual mature viral proteins. Non-structural protein 5A (NS5A) is one of these proteins that has been implicated in regulation of viral genome replication, translation from the viral IRES and viral packaging. We sought to identify cellular proteins that interact with NS5A and determine whether these interactions may play a role in viral production. Mass spectrometric analysis of coimmunoprecipitated NS5A complexes from cell extracts identified heat shock proteins (HSPs) 40 and 70.Weconfirmed anNS5A/HSPinteraction by confocal microscopy demonstrating colocalization of NS5A with HSP40 and with HSP70. Western analysis of coimmunoprecipitated NS5A complexes further confirmed interaction of HSP40 and HSP70 with NS5A.Atransient transfection, luciferase-based, tissue culture IRES assay demonstrated NS5A augmentation of HCV IRES-mediated translation, and small interfering RNA (siRNA)-mediated knockdown of HSP70 reduced this augmentation. Treatment with an inhibitor of HSP synthesis, Quercetin, markedly reduced baseline IRES activity and its augmentation by NS5A. HSP70 knockdown also modestly reduced viral protein accumulation, whereas HSP40 and HSP70 knockdown both reduced infectious viral particle production in an HCV cell culture system using the J6/JFH virus fused to the Renilla luciferase reporter. Treatment with Quercetin reduced infectious particle production at nontoxic concentrations. The marked inhibition of virus production by Quercetin may partially be related to reduction of HSP40 and HSP70 and their potential involvement in IRES translation, as well as viral morphogenesis or secretion. Conclusion Quercetin may allow for dissection of the viral life cycle and has potential therapeutic use to reduce virus production with low associated toxicity.

The effect of chronic ethanol feeding was determined on parameters of hepatic collagen metabolism in the monkey. Four monkeys of the species Macaca radiata received a nutritionally adequate diet containing 50% of the calories as ethanol, while four others were pair-fed a diet in which ethanol was isocalorically substituted by carbohydrate. Liver biopsies were obtained at 3, 12, and 24 months, and the animals were killed between 40 and 48 months after initiation of the diets. The ethanol-fed animals developed various degrees of fatty infiltration, but no necrosis, inflammation, or fibrosis. The amount and distribution of collagen Types I, III, and IV demonstrated by immunohistochemical techniques was not altered after ethanol feeding. No changes were found in hepatic protein-bound hydroxyproline or in collagen prolyl hydroxylase activity at the various time intervals. Liver free proline and the incorporation of labeled proline into protein-bound hydroxyproline by liver slices were not altered after 40 to 48 months of ethanol feeding. This study shows that prolonged feeding of ethanol together with an adequate diet results in fatty infiltration but not deposition or alteration of hepatic collagen metabolism.

The effect of chronic ethanol feeding on hepatic mitochondrial morphology and histochemically measured succinic dehydrogenase activity was assessed. Five monkeys of the species Macaca radiata received a nutritionally adequate diet containing 50% of the calories as ethanol, while five others were pair-fed the same diet except that ethanol was isocalorically substituted by carbohydrate. Liver morphology was assessed at 12 and 24 months and at sacrifice after 40 to 48 months of ethanol feeding. The ethanol-fed animals developed mild to moderate fatty liver as did some of the controls. No necrosis or fibrosis developed. All ethanol-fed animals developed centrilobolar mega-mitochondria and centrilobular “shift” in histochemically assayed succinic dehydrogenase activity characteristic of animals fed ethanol for prolonged periods. These mitochondrial changes persisted throughout the 48-month test period without progressive increase in severity or accompanying pathology. It is concluded that the morphologic and histochemically assessed mitochondrial changes do not necessarily represent a progressive destructive effect of ethanol.

Nonalcoholic steatohepatitis is prevalent among obese individuals with excessive caloric intake, insulin resistance, and type II diabetes. However, no animal models exist that recapitulate this important association. This study produced and characterized steatohepatitis (SH) caused by intragastric overfeeding in mice. C57BL/6, tumor necrosis factor (TNF) type I receptor-deficient, and genetically matched wild type mice were fed via an implanted gastrostomy tube a high-fat diet for 9 weeks in the increasing amount up to 85% in excess of the standard intake. Animals were examined for weight gain, insulin sensitivity, and histology and biochemistry of liver and white adipose tissue (WAT). Overfed C57BL/6 mice progressively became obese, with 71% larger final body weights. They had increased visceral WAT, hyperglycemia, hyperinsulinemia, hyperleptinemia, glucose intolerance, and insulin resistance. Of these mice, 46% developed SH with increased plasma alanine aminotransferase (121 +/- 27 vs. 13 +/- 1 U/L), neutrophilic infiltration, and sinusoidal and pericellular fibrosis. Obese WAT showed increased TNFalpha and leptin expression and reciprocally reduced adiponectin expression. The expression of lipogenic transcription factors (SREBP-1c, PPARgamma, LXRalpha) was increased, whereas that of a lipolytic nuclear factor PPARalpha was reduced in SH. SH was associated with reduced cytochrome P450 (Cyp)2e1 but increased Cyp4a. TNF type I receptor deficiency did not prevent obesity and SH. In conclusion, forced overfeeding with a high-fat diet in mice induces obesity, insulin resistance, and SH in the absence of TNF signaling or Cyp2e1 induction.

Hepatic metabolism of ethanol by cytochrome P450 2E1 (CYP2E1) is believed to contribute to alcoholic liver damage. To further evaluate CYP2E1 in alcoholic liver disease, we created a transgenic mouse containing human CYP2E1 complementary DNA (cDNA) under the control of mouse albumin enhancer-promoter. Two experiments were performed. In the first experiment, transgenic and nontransgenic mice were fed normal chow. In the second experiment, transgenic and nontransgenic mice were pair fed a nutritionally complete liquid diet for 16 weeks. The liquid diet contained 30% of calories as ethanol (or dextrose) and 25% of calories as corn oil. Liver damage was assessed by measuring serum alanine aminotransferase (ALT) levels and examining liver histology. Transgenic animals reproduced and were phenotypically normal. Hepatic levels of CYP2E1 messenger RNA (mRNA), protein, and enzyme activity did not differ between chow-fed transgenic and nontransgenic mice. Livers from transgenic mice fed the alcohol diet contained significantly more CYP2E1 protein and enzyme activity than livers from nontransgenic mice fed the same diet. Transgenic mice receiving the alcohol diet had significantly higher serum ALT levels than nontransgenic mice. Histologic examination of the livers showed higher histologic scores in transgenic mice fed ethanol compared with nontransgenic mice fed ethanol. Ballooning hepatocytes were seen in livers from transgenic mice fed ethanol. Apoptosis, as determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay, did not differ between groups. In conclusion, we have produced a transgenic mouse that expresses human CYP2E1 in the liver. When fed a nutritionally complete alcohol diet, transgenic mice develop more liver damage than nontransgenic mice.

We previously reported that ethanol elicits an increased protein oxidation in the liver of rats receiving chronic ethanol by continuous intragastric infusion (Tsukamoto-French method). This accumulation of oxidized proteins could result from a decrease in the cytosolic proteolysis, related specifically to alkaline protease and its major components, the proteasomes. Because several studies suggest that intracellular proteolysis depends on the severity of oxidative stress, we investigated the cytosolic proteolytic activity under two chronic ethanol treatment paradigms associated with varying degrees of oxidative stress. For 4 weeks, male rats received chronic ethanol by continuous intragastric infusion or by oral administration (10% ethanol ad libitum as sole drinking fluid). A significant decrease was evident for alkaline protease activity as well as for sodium dodecyl sulfate (SDS)-activated latent 20S proteasome (chymotrypsine-like [ChT-L] and peptidylglutamyl peptide hydrolase [PGPH] activities) in the liver of rats receiving ethanol by continuous intragastric infusion. Free radical production and related processes appeared to be contributing events in proteolysis inhibition, because phenethyl isothiocyanate (PIC), an inhibitor of cytochrome P4502E1 (CYP2E1), reduced the inhibition of the ethanol-related ChT-L activity. Moreover, the lipid peroxidation level was inversely correlated with ChT-L activity. In contrast, no such changes were observed in ChT-L and PGPH activities or in cellular free radical targets following the oral ad libitum consumption of 10% ethanol. It appears, thus, that only the alcohol treatment paradigm associated with an overt oxidative stress produced a significant inhibition of the proteasome activity. The mechanisms of proteasome inhibition could involve the formation of an endogenous inhibitor such as protein aggregates or aldehyde-derivative peptides. Whatever the mechanism, the inhibition of cytosolic proteolysis and the subsequent accumulation of damaged proteins may be involved in the oxidatively challenged alcoholic livers and play a pathogenic role in experimental alcoholic liver disease.

Ethanol consumption slows down the rate of hepatic protein catabolism. The present study was conducted to determine whether ethanol consumption, given by voluntary (pair) feeding or by intragastric administration, affected the peptidase activities of the proteasome in rat liver. Rats were pair-fed liquid diets containing either ethanol or isocaloric maltose-dextrin. A separate group of animals was intragastrically infused continuously with similar liquid diets containing either ethanol or isocaloric dextrose. Crude liver homogenates and their cytosolic fractions were assayed for their chymotrypsin-like (Cht-L), trypsin-like (T-L), and peptidyl-glutamyl-peptide hydrolase (PGPH) activities, using specific fluorogenic peptides as substrates. Voluntary ethanol feeding did not affect the three peptidase activities of the proteasome. However, intragastric ethanol administration caused a 35% to 40% decline in the Cht-L and the T-L activities, but did not significantly change the PGPH activity. The lower peptidase activities in cytosol samples from intragastrically ethanol-fed rats were not restored to control levels by overnight dialysis, nor by the inclusion of low levels of sodium dodecyl sulfate (SDS) or of 0.5 mmol/L adenosine triphosphate (ATP) in the proteasome assay mixture. Immunoblot analyses using anti-rat liver proteaseome exhibited equal levels of immunoreactive proteasome subunits in livers of control and ethanol-fed rats. Similar results were obtained when blots were probed with antibody made specifically against the proteasome subunit, LMP-7. The results indicate that intragastric, but not voluntary, ethanol consumption differentially affects the separate catalytic activities of the proteasome without affecting its steady-state levels. Such changes may be related to the degree of ethanol-induced oxidative stress.

Drug-primed mice, created by a 5-month feeding of diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC), followed by a 1-month withdrawal, were refed ethanol or isocaloric dextrose (control) diets intragastrically for 7 days. The formation of Mallory bodies (MBs) was monitored by immunofluorescence and immunoperoxidase microscopy using antibodies to cytokeratin and ubiquitin, and also by electron microscopy. The changes in cytokeratin 55 (CK55), ubiquitin conjugate, nuclear factor kappaB (NFkappaB) p65, NFkappaB p50, inhibitor kappaB alpha, c-myc, tumor necrosis factor alpha, and cytochrome P450 2E1 (CYP2E1) contents were determined by Western blotting using appropriate antibodies. The messenger RNA (mRNA) for CYP2E1, cytokeratin, ubiquitin, hepatocyte growth factor activator, and tissue transglutaminase was quantitated. MBs were present at 5 to 7 days' postfeeding with ethanol, but not with dextrose. They developed in clusters of "empty hepatocytes," where the cytokeratin antibody failed to recognize the typical filament structures seen in normal hepatocytes. MBs were larger and more numerous in the subcapsular region. Northern blots showed that CK55 mRNA was decreased by the ethanol treatment, but protein levels were increased, suggesting a decreased turnover of the cytokeratin. Likewise, the increase in CYP2E1 protein in the face of a lack of an increase in mRNA for CYP2E1 could be explained by a decreased turnover of this cytochrome. This is the first report of MB formation induced by ethanol ingestion in an experimental model.

Liver lipid peroxidation, nonheme iron, antioxidants, and protein oxidation were investigated in experimental alcohol-induced liver disease in the rat. Wistar male rats were intragastrically and continuously infused for 4 weeks with a high-fat diet plus an ethanol or an isocaloric amount of dextrose, maintaining a high blood alcohol level (200-300 mg%). This model induced fatty liver, spotty necrosis, and focal inflammation. This pathology was associated with an enhanced lipid peroxidation and a decrease in the major antioxidant factors. Hepatic alpha-tocopherol and glutathione concentrations were significantly decreased in ethanol-fed rats. Glutathione peroxidase (GPx) was also decreased, whereas glutathione S-transferase (GST) was unaffected. The nonheme iron level was significantly decreased. Protein oxidation was assessed through three parameters: protein thiols, protein carbonyl groups, and the activity of glutamine synthetase (GS), a centrilobular enzyme particularly susceptible to free-radical-mediated damage. Ethanol-fed rats had decreased protein thiol concentrations and reduced GS activity, together with increased protein carbonyls. A significant correlation between GS activity and the pathological score was observed. This study confirms the ethanol-related increase in lipid peroxidation and shows that ethanol impairs the hepatic antioxidant potential. Furthermore, evidence of oxidative protein damage is given, including decreased activity of a key enzyme of ammonia metabolism. These protein disturbances may contribute to the pathogenesis of the observed liver damage.

The aim of this study was to determine the various factors that are involved in the induction of Mallory body (MB) formation. A model was developed where MB formation was induced by refeeding either of the drugs griseofulvin or diethyl 1,4-dihydro-1,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC). Mice were fed the drugs for 5 months, followed by withdrawal of the drugs for 1 month (drug-primed livers). The drugs were refed for 1,3,5,7, or 11 days. Early MBs first appeared as small, enlarged aggregates of filaments in the perinuclear or pericanalicular location on the third day of refeeding. Mature MBs appeared on the fifth day of refeeding. MBs reached maximum concentration on day 5 of refeeding. Western blots showed a progressive increase in the cytokeratin proteins (CK49 and CK55) and actin while refeeding the drugs. Liver cell regeneration, as indicated by the percent of proliferating cell nuclear antigen (PCNA)-positive nuclei, increased on the third day of refeeding. However, there was no correlation between the frequency of MBs and the percent of PCNA-positive nuclei. It is concluded that MB formation is not related to the liver cell regeneration response to injury but rather involves a separate regulation pathway. The MBs were heavily ubiquitinated and were associated with increased ubiquitin-protein conjugates as indicated by Western blotting, suggesting that ubiquitinization of cytokeratin protein are involved in the formation of MB aggregation.

The cell of origin of intrahepatic bile ducts during fetal development remains a subject of controversy, although there has been recent evidence that they form from hepatocytes. However, the origin of neoductules and ducts in the setting of liver disease has not been extensively investigated in humans. Using anticytokeratins characteristic of hepatocytes and bile ducts, we repeated earlier studies of fetal development to compare ductule formation in normal developing and newborn livers with the ductules formed during extrahepatic biliary atresia. We utilized an antibody to proliferating cell nuclear antigen (PCNA) staining to determine which cells were in active DNA synthesis (S phase) during fetal development and liver disease progression. The results indicated that hepatocytes undergo a phenotypic switch (metaplasia) to form ductular cells during fetal development. There was no ductular cell replication in the fetal livers. In contrast, both bile ductular metaplasia and proliferation were observed in biliary atresia. Therefore, both a limiting plate phenotypic switch to ductules and replication of ductular cells play a role in the increase in the ductules seen in the progression to biliary cirrhosis. Bile ductular proliferation in biliary atresia, however, was less than that seen in hepatocytes, whereas the number of bile ductules increased and the relative proportion of hepatocytes diminished as the accompanying periductular fibrosis progressed to cirrhosis.

This study was done to determine if a relationship exists between CYP2E1 induction by ethanol, lipid peroxidation, and liver pathology in experimental alcohol-induced liver disease in the rat. Rats were fed ethanol with or without diallyl sulfide (DAS) or phenethyl isothiocyanate (PIC) intragastrically for 1 month. CYP2E1 induction by ethanol was correlated with lipid peroxidation, liver microsomal CYP2E1 hydroxylation of paranitrophenol, and the liver pathology score using the data from the PIC-fed rats. Some of the data from the ethanol and DAS-fed rats were not included here because they have been reported elsewhere. Microsomal CYP2E1 protein levels induction by ethanol was decreased by PIC ingestion. Similarly, PIC reduced the increase microsomal reduced form of nicotinamide-adenine dinucleotide (NADPH)-dependent lipid peroxidation and p-nitrophenol hydroxylase (PNPH) activity, induced by ethanol feeding. The lipid peroxidation was reduced to below control levels; however, the pathology score was partially but not significantly reduced by isothiocyanate feeding. CYP2E1 messenger RNA (mRNA) was decreased by both inhibitors of CYP2E1. Immunohistochemical staining of liver for CYP2E1 protein showed that the lobular distribution of the isozyme changed from the centrilobular to a diffuse pattern, with an increase in the periportal region when the CYP2E1 inhibitors were fed with ethanol, and that this change correlated with the change in the distribution of fat in the lobule. The data support the idea that there is a link between CYP2E1 induction by ethanol and the early phase of ethanol-induced liver injury in this rat model. This link may involve lipid peroxidation, but other factors related to CYP2E1 induction must also be involved.

A Veterans Affairs cooperative study involving 273 male patients was performed to evaluate efficacy of oxandrolone in combination with an enteral food supplement in severe alcoholic hepatitis. All patients had some degree of protein calorie malnutrition. On an intention-to-treat basis, only minimal changes in mortality were observed. However, in patients with moderate malnutrition mortality on active treatment at 1 mo was 9.4% compared with 20.9% in patients receiving placebo. This beneficial effect was maintained so that after 6 mo on active treatment 79.7% of patients were still alive, compared with 62.7% of placebo-treated patients (p = 0.037). Improvements in both the severity of the liver injury (p = 0.03) and malnutrition (p = 0.05) also occurred. No significant improvement was observed with severe malnutrition. To better determine the effect on therapeutic efficacy, we compared results with those from a nearly identical population (cooperative study 119) treated with oxandrolone but not given the food supplement. Patients were stratified according to their caloric intake (greater than 2,500 kcal/day was considered adequate to supply energy needs and promote anabolism). For patients with moderate malnutrition and adequate caloric intake, oxandrolone treatment reduced 6-mo mortality (4% active treatment vs. 28% placebo [p = 0.002]). For patients with moderate malnutrition and inadequate calorie intake, oxandrolone had no effect on mortality (30% active treatment vs. 33% placebo). In cases of severe malnutrition, oxandrolone had no effect on survival. However, adequate caloric intake was associated with 19% mortality, whereas patients with inadequate intake exhibited 51% mortality (p = 0.0001). These results indicate that nutritional status should be evaluated in patients with alcoholic hepatitis. When malnutrition is present, vigorous nutrition therapy should be provided, and in patients with moderate malnutrition oxandrolone should be added to the regimen. (HEPATOLOGY 1993;17:564–576.)

Patients with overt alcoholic liver disease who had participated in a multicenter therapeutic trial and subgroups of controls (i.e., alcoholic patients without liver disease and patients with neither alcoholism nor liver disease) were tested for hepatitis B virus and hepatitis C virus antibodies to determine the prevalence of these antibodies and any clinical association in the progression and outcome of alcoholic liver disease. Antibodies to hepatitis B (anti–HBs and/or anti-HBc) were found in 29.2% of patients with alcoholic liver disease, in 26.1% of hospitalized alcoholic patients without liver disease and in 24.2% of hospitalized nonalcoholic patients without liver disease; frequencies were not significantly different from one another. HBsAg was not evaluated because HBsAg+ patients had been excluded from the original trial. The presence of these antibody markers correlated with ethnic origin of and immunoglobulin levels in the patients. In contrast, antibody to hepatitis C, as detected by enzyme immunoassay, was positive in 27.1%, 4.8% and 3.0% of the three groups, respectively, the first differing significantly from the other two. Antibody to hepatitis C virus positivity correlated significantly with clinical severity of the disease and with the presence of histological features that imply chronic viral infection (periportal inflammation, cirrhosis), despite the fact that the supplementary assay for antibody to hepatitis C virus, using recombinant immunoblot assay, reduced the positive rate by 79%. Although the presence of hepatitis B antibodies did not correlate with patient survival, some categories of patients with antibody to hepatitis C virus did; the survival of those with antibody to hepatitis C virus and recombinant immunoblot assay reactivity was 67%; for those with antibody to hepatitis C virus positivity but recombinant immunoblot assay negativity the survival was 29% (p < 0.01). The reason for the poor prognosis for those with antibody to hepatitis C virus positivity and recombinant immunoblot assay negativity is not clear, although some theories are offered. (HEPATOLOGY 1991;14:581–589.)

A new method of visualizing the three-dimensional architecture of the cytokeratin filaments of the intact rat hepatocyte in situ has been achieved. Frozen sections of liver cut 10 micron thick were serially extracted to remove all elements of the cells except the intermediate filaments. Parallel sections were stained with monoclonal antibodies to the two main cytokeratins found in bile duct and liver cells. Immunofluorescent antibody and immunogold electron microscopy techniques were used to identify the proteins morphologically. Several new observations resulted from these studies. The pericanalicular sheath of intermediate filaments was visualized using steropairs as an uninterrupted branching tubular structure composed of cytokeratins located in the cell cortex of adjacent hepatocytes. Intermediate filaments in the cell cortex formed a distinct sheet of matted filaments which enveloped the entire hepatocyte. The cortical intermediate filaments were in continuity with the pericanalicular sheath and the filaments located within the cytoplasm. The intermediate filaments are attached to the centrioles and appeared to tent the nuclear lamina-pore complex at points of contact. Monoclonal antibodies to rat liver intermediate filament cytokeratins (CK49 and CK55) each stained intermediate filaments located in the cell cortex, within the cytoplasm and at the nucleus. By immunogold staining, some of the intermediate filament filaments were shown to contain both cytokeratins. Filaments which did not stain were thought to be either actin at the cell periphery or nuclear lamins around the nucleus. It is concluded that the cytokeratins form a specialized framework for the cell cortex, canaliculus, centrioles and the nucleus of hepatocytes. The filaments run continuously throughout the cytoplasm without terminating.

Mouse and human extracted liver tissue were examined by indirect immunofluorescent staining and transmission electron microscopy in order to study the alteration of cytokeratin intermediate filaments associated with Mallory body formation. Frozen sections of griseofulvin-fed mouse liver and human liver of primary biliary cirrhosis and primary sclerosing cholangitis were extracted by Triton X-100 and nuclease. Indirect immunofluorescent staining was performed by using anticow hoof keratin antibody for mouse liver and anti-human epidermal keratin antibody (AE1 and AE3) for human liver. Transmission electron microscopy was also performed on extracted and critical point-dried samples. The griseofulvin-fed mouse hepatoma cells showed four different types of altered staining pattern based on the indirect immunofluorescent staining of the cytoplasm and Mallory bodies: Type I—cytoplasm(+), Mallory body(−); Type II—cytoplasm(+), Mallory body(+); Type III—cytoplasm(−), Mallory body(+), and Type IV—cytoplasm(−), Mallory body(−). Types I and III were predominant, however, some hepatoma cells which contain Mallory bodies revealed bright cytoplasmic staining (Type II). The nuclear rims were strongly stained. In human liver, AE1 stained Mallory bodies and the bile duct epithelium intensely, but did not stain normal hepatocytes. AE3 mainly stained Mallory bodies and normal hepatocytes, but also stained bile duct epithelium weakly. Indirect immunofluorescent staining for human liver showed the same staining patterns as found in mouse liver, except that Type IV was not observed. Although many hepatocytes which contained Mallory bodies did not react with either of these two antibodies (Type III), some of the hepatocytes were stained, not only with AE3, but also with AE1 (Type II). Transmission electron microscopy revealed that Mallory bodies were often surrounded by numerous intermediate filaments, which is equivalent to the Type II pattern in indirect immunofluorescent staining. This was true of mouse liver frozen sections and primary cultures, as well as human liver studied. The results clearly show that cells associated with Mallory bodies show changes in the expression of cytokeratins as well as rearrangement of intermediate filaments within the cell. Hepatocytes forming Mallory bodies often contain a variable density of intermediate filaments which fail to stain with cytokeratin antibodies, indicating that the antigenic determinants of the intermediate filaments were lost or masked.

The clinical and histologic significance of cytokeratin antigen expression in various intrahepatic locations was assessed in 57 patients with alcoholic liver disease as part of a large Veterans Administration Cooperative Study of Alcoholic Hepatitis. Cytokeratin antigen was demonstrated in fixed, paraffin-embedded liver tissue by an avidin-biotin peroxidase method using a mixture of two different monoclonal antibodies, AE1 (acidic; 48, 50 and 56.5 kD) and AE3 (basic; 52, 56, 58 and 65 to 67 kD). In contrast to the normal liver, in which only bile duct epithelium was positive, this antibody mixture stained both bile ducts and hepatocytes in pathologic livers. Serum levels of vitamin A showed a significant inverse correlation with the amount of cytokeratin antigen (scale: 0 to 3) in hepatocytes without Mallory bodies (p = 0.001), in Mallory body-containing hepatocytes (p < 0.0001) and in bile ducts (p = 0.0074). Increased amount of cytokeratin antigen in each of these locations, in turn, correlated directly with the histologic severity of the liver disease. Histologic severity (fibrosis, parenchymal degeneration/necrosis, hepatocyte regeneration and inflammation) was significantly higher in patients when either Mallory bodies (p < 0.0001) or cytokeratin antigen (p = 0.0021) was present in hepatocytes. Demonstration of cytokeratin antigen in hepatocytes which contained Mallory bodies correlated positively (p = 0.03) with clinical severity of the liver disease as determined by high serum bilirubin and prolonged prothrombin time (Maddrey's discriminant function). This study suggests that (i) vitamin A deficiency may be a triggering factor in aggregation of cytokeratin filaments, and (ii) the degree of aggregation of cytokeratin may determine the histologic severity and thereby the prognosis of patients with alcoholic liver disease.

Blood alcohol levels (BAL) were maintained at high levels (overall mean +/- S.D. achieved in 14 alcoholic rats was 216.0 +/- 120.1 mg%) in male Wistar rats for 15 to 85 days by continuous intragastric infusion of ethanol and nutritionally defined low fat liquid diet. The ethanol intake was progressively increased from 32% of total calories up to 41.4% in order to maintain high BAL. Pair-fed animals received isocaloric glucose solution and the liquid diet. Despite the low level of dietary fat (4.9% of total calories), histopathological evaluation of the liver revealed severe and progressive fatty infiltration in the alcoholic rats. In addition, following 30 days of intoxication, one third of the animals showed focal necrosis with mononuclear cell infiltration in centrilobular areas of the livers. This was correlated with the markedly elevated levels of SGOT and SGPT in these animals. Pair-fed controls showed no abnormality in the morphology of liver or blood chemistry. Chemical quantitation of liver triglycerides confirmed the histological observation, with triglyceride levels of 61.51 +/- 16.45 and 89.61 +/- 5.94 mg per gm at 30 and 85 days, respectively. Most importantly, the degree of steatosis was tightly and significantly correlated with the mean BAL achieved (r = 0.80, p less than 0.001). These data represent the first confirmation of the hypothesis that continuously high BAL correlate with the severity of alcohol-induced liver pathology.

Evidence has been accumulating that Mallory body (MB) is a heritable phenotype of hepatocellular carcinoma (HCC) and is closely related to hepatocarcinogenesis in some carcinogen-fed animals. This prompted us to try to evaluate the MB phenomenon in human HCCs, in which its significance remains unclear to date. Whole liver slices were obtained from 28 autopsied livers in which MB-positive HCC was found, and distribution of MBs within the HCC was examined. There were three distribution patterns: clustering, diffuse, and sparse types. The clustering type was the most frequent and was found in 21 of the 28 cases. In this type, there was a formation of groups of the MB-containing cells within the HCC, and the cluster of MB-positive cells was variable in number, size, and shape. There was often a sharp border line between MB-positive clusters and the surrounding MB-negative HCC cell areas where a collision line could occasionally be seen. Some of the MB-containing HCC cells were positive for γ-glutamyl transpeptidase and α-fetoprotein. These data led the authors to speculate that MB-positive HCC cells in human livers are capable of proliferation to form the clusters and that therefore, MBs are expressed as a kind of heritable phenotype in such clusters.