the mallory body: morphological, clinical and experimental studies (part 1 of a literature survey)

Special Article

The Mallory Body: Morphological, Clinical and Experimental Studies (Part 1 of a Literature Survey)

KENNETH JENSEN AND CHRISTIAN GLUUD Department of Medical Gastroenterology, Huidoure University Hospital, DK-2650 Huidoure, and Institute of Preventive

Medicine, Copenhagen Health Seruices, DK-1399 Copenhagen, Denmark

To aid understanding of markers of disease and predictors of outcome in alcohol-exposed systems, we undertook a literature survey of more than 700 articles to view the morphological characteristics and the clinical and experimental epidemiology of the Mallory body. Mallory bodies are filaments of intermediate diameter that contain intermediate filament components (e.g., cytokeratins) observable by conventional light microscopy or immunohistochemical methods, identical in structure regardless of initiating factors or putative pathogenesis. Although three morphological types can be identified under electron microscopy (with fibrillar structure parallel, random or absent), they remain stereotypical manifestations of hepatocyte injury. A summary of the conditions associated with Mallory bodies in the literature and their validity and potential etiological relationships is presented and discussed, including estimates on the combined light microscopic and immunohistochemical prevalences and kinetics. Emphasis is placed on proper confounder control (in particular, alcohol history), which is highly essential but often inadequate. These conditions include (mean prevalence of Mallory bodies in parentheses): Indian childhood cirrhosis (73%), alcoholic hepatitis (65%), alcoholic cirrhosis (51%), Wil- son’s disease (25%), primary biliary cirrhosis (24%), nonalcoholic cirrhosis (24%), hepatocellular carcinoma (23%), morbid obesity (8%) and intestinal bypass surgery (6%). Studies in alcoholic hepatitis strongly suggest a hit-and-run effect of alcohol, whereas other chronic liver diseases show evidence of gradual increase in prevalence of Mallory bodies with severity of hepatic pathology. Mallory bodies in cirrhosis do not imply alcoholic pathogenesis. Obesity, however, is associated with alcoholism and diabetes, and Mallory bodies are only present in diabetic patients if alcoholism or obesity complicates the condition. In addition, case studies on diseases in which Mallory bodies have been identified, along with phar-

Received May 25, 1993; accepted May 2, 1994. Other abbreviations used in the text: AH, alcoholic hepatitis; HCC, hepato-

cellular carcinoma; MB, Mallory body. Address reprint requests to: Christian Gluud, M.D., Dr. med. sci., Institute

of Preventive Medicine, Copenhagen Health Services, Copenhagen Municipal Hospital, DK-1399 Copenhagen K, Denmark.

Copyright 0 1994 by the American Association for the Study of Liver Diseases.

0270-9139/94 $3.00 + 0 31/1/58183

macological side effects and experimental induction of Mallory bodies by various antimitotic and oncogenic chemicals, are presented. Mallory bodies occur only sporadically in abetalipoproteinemia, von Gierke’s disease and focal nodular hyperplasia and during hepatitis due to calcium antagonists or perhexiline maleate. Other conditions and clinical drug side effects are still putative. Finally, a variety of experimental drugs have been developed that cause Mallory body formation, but markedly different cell dynamics and metabolic pathways may raise questions about the relevance of such animal models for human Mallory body formation. In conclusion, the Mallory body is indicative but not pathognomonic of alcohol involvement. A discussion on theories of development and pathological significance transcending the clinical frameworks will be presented in a future paper. (HEPATOLOGY 1994;20: 106 t - 1077.)

Matthew Baillie described a relation between alcohol consumption and liver disease almost 200 years ago (1). Since then, the interest in this psychoactive drug and its somatic effects has been equal to the increasing number of consumers and their major impact on health-related, psychological, social and economic problems of the community. However, many aspects of the effects of ethanol are still controversial. The lack of linear dose- response kinetics has made risk estimates too vague to be effectively implemented in public information and abuse prevention. Predicting in whom somatic disease resulting from alcohol abuse will develop has been an increasingly important question in recent years. Interest has therefore focused on intercurrent physiological and histological qualities of alcohol-exposed systems (cells, organs) to help secure the basic mathematics needed for outcome prediction. This paper is dedicated to one such feature - the Mallory body.

This characteristic abnormality seen on light microscopy in the cells of cirrhotic livers of alcoholic patients was first described by Mallory in 1911 (2). First regarded as pathognomonic of alcoholic liver disease, the specificity for this injury has since been questioned because of repeated demonstrations in disorders not related to alcohol consumption. The diversity of pathological processes associated with the MB complicates the search for a common factor responsible for these

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TAULE 1. Clinical studies of common MB-associated conditions References Condition Prevalence (5%) Range (70)

Indian childhood cirrhosis" 73 14-100 12, 137, 155, 157, 162

Cirrhosis, alcoholic" 51 14-100 4, 9, 76, 89, 92, 250 Wilson's disease" 25 0-100 12, 138, 149, 152, 251

Cirrhosis, nonalcoholicimixed origin 24 0-100 8, 89, 92, 137, 253, 254

Morbid obesity 8 0-24 3, 100, 103, 105, 108, 111 Jejunoileal bypass 6 2-80 98, 117, 130

The criteria for inclusion have been original studies that identify MBs by light microscopy or immunohistochemistry, with a sufficient number of cases (n > 10) to avoid severe type I1 error. Case studies, studies in which MB prevalence is not directly specified or where selection bias seriously questions the results are excluded. An estimate of the mean prevalence of MBs at time of diagnosis is given with the range of values encountered in the studies. The estimate is pooled by addition of the number of MB-positive specimens from each study and dividing them with the total number of liver specimens. Appropriate references are listed. See text for further details.

AHh 65 39-100 8, 9, 75, 76, 79, 83, 93, 137, 245-249, 257

PBC" 24 3-41 12, 137-139, 252

HCC 23 11-100 92, 94-96, 255, 256

"Increasing MB prevalence is seen with clinical or histological severity. "The estimate is based on AH without concurrent cirrhosis. Note that 95% of AH patients with cirrhosis have MBs. 'Cases of intercurrent AH have not been fully ruled out.

alterations. Since Mallory first described the entity that bears his name, more than 700 reports have been published on the subject. Although the chemical characteristics and structure of the MB have largely been clarified, their pathogenesis and biological significance in human hepatobiliary disease are still controversial. I t has been postulated that MBs are immunogens that initiate an autoimmune process leading to chronic disease. Alternatively, they are considered epiphenomena without pathogenic potential.

MATERIALS AND METHODS The literature was obtained by means of a MEDLINE

search of the years 1966 to 1993 using several primary search words (Mallory body, alcoholic hyalinle], Mallory's hyaline) and thereafter a cross-bibliographic check. With this method more than 700 publications were identified. In addition, studies on subjects in close relation to MBs have been included where appropriate to help explain some contextual arguments. To limit the number of articles selected and referenced we have deemed it necessary to exclude the following studies from the survey: (a) studies with imprecise patient selection, description or both; (b) studies with inconclusive methods of detection; (c) double publications; (d) most review literature; (e) most case histories, except when MBs were suspected in rare conditions; and (0 studies with imprecise descriptions of the prevalence of MBs. A subjective element cannot be dismissed on the basis of this method of citation selection, but to remedy this bias we have tried to put forward a pro et contra discussion on the basis of the literature whenever possible. Using these criteria, we present pooled estimates of prevalences of MBs in various conditions (Table l), but they must be evaluated with some caution; most studies employ light microscopy or staining techniques that have been found to be quantitatively inferior to electron microscopy or the highly sensitive ubiquitin stains (see below), and this poses a particular problem for the conditions in which MBs are rarely seen. However, the estimates reliably represent the prevalence of fully developed MBs (but not necessarily precursor stages).

ULTRASTRUCTURAL STUDIES Methods of Detection. The MB was first observed with

low-power light microscopy and hematowlin-and-eosin staining (21, which seems to be the staining technique of choice in conventional light microscopy. Immunohis- tochemical stains such as monoclonal or polyclonal antibody techniques, horseradish peroxidase and ubiquitin stains (3-7) are superior in identifying additional amounts of MBs, mostly by detection of minute amounts or precursor stages of the MBs (8,9). However, electron microscopy remains the gold standard (7, 13) and must be used if positive identification of MRs is required because it can dismiss a variety of well-known MB- lookalike structures such as megamitochondria, cytoplasmic crystals, rough endoplasmic reticulum, smooth endoplasmic reticulum hypertrophy, amyloid deposits and other hyaline inclusions, all of which have been described frequently in alcoholism and other conditions in which MBs are seen. With this in mind, it seems clear that such advanced methods of detection have in some comparative studies had dramatic influence on prevalence estimates; for instance, immunperoxidase studies have found a twofold or threefold higher prevalence compared with hematoxylin-and-eosin stains (4, 8, 12). Characteristically, these high prevalences may in part be statistical consequences of medium-probability events (such as MBs in fibrosis, cirrhosis and hepatocellular carcinoma), and the paradox remains that however exact these techniques may be in terms of ultrastructure and topography, for the most part they seem to add quantity more than quality in epidemiological studies and do not seriously contradict the results obtained with traditional light microscopy. In addition to the detection methods outlined above, additional techniques for electron microscopy (13-15) and morphometry (16) have been developed, each outlining different characteristics of the MB structure to advantage.

Morphology. Light microscopically, the MB is seen as an intracytoplasmic, homogenous, irregularly shaped

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eosinophilic structure forming a coarse network, often close to or surrounding the nucleus (10, 17, 18) but without clear-cut topographical relationships (19). Electron microscopic studies have revealed a characteristic filamentous ultrastructure (10, 11, 19-22), ap- pearing as an aggregate of branching tubular filaments (18), rangmg in thickness from 14 to 20 nm (19, 23-26), from which thinner filaments extend (11, 26). Isolation by centrifugation and incubation with various detergents yields filaments of 5 to 8 nm in diameter (16, 25). Furthermore, such electron microscopic studies have identified three morphological types according to the orientation of the fibrillar structures, which may be parallel (Yokoo type I, present only in human beings), random (Yokoo type 11) or absent (Yokoo type 111, amorphous and granular structure) (11). These morphological characteristics are basically the same in various conditions, although the pattern of injury to hepatocyte organelles may differ (10, 19-21, 27-29). There is no limiting membrane (10). A study using infrared spectroscopy on mice fed griseofulvin has shown that the molecular structure of cytoskeletal proteins of MB-containing cells is modified by an increase in a-helixes to p-sheets (30).

The ultrastructure of the MB has also been explored by means of high-resolution electron microscopy with a quick-freezing cryotechnique in both human beings and in mice (15). The MB filaments have a diameter of 11 to 19 nm and appear to be composed of pairs of filaments twisted in a helical manner. Each filament has a diameter of less than 10 nm. This intriguing helical structure has parallels among other filamentous inclusion bodies such as Alzheimer’s tangling and Lewy bodies in terms of morphology, solubility and stain- ability; we know these diseases possess elements of microtubules, tau protein (31-33) and high molecular weight microtubule-associated proteins (341, but whether MBs contain any of these is unclear. However, they are all ubiquinated (see below) (6, 7, 34, 35).

Biochemistry. Nonfilamentous coating covers the filament rods (26). MBs seem to be glycosylated (36,371, and additional polypeptides are present (26, 38). His- tochemically, MBs contain unconjugated basic protein, RNA, glycogen, nonacid carbohydrate and phospholipid (37). They show marked insolubility to a variety of proteases and extraction procedures with, among others, phospholipid or fat solvents (39), although limited digestion by trypsin is possible during prolonged incubation (40). A complex mixture of detergents has successfully removed the nonfilamentous coating of the MBs (16, 25). Biochemical and immunological data (41) support the concept of a close relationship to major protein components of cytokeratin filaments of hepatocytes and other epithelial cells (26, 41, 42). Such filaments resemble intermediate filaments, which form the cytoskeleton of many cells; they are not contractile and have been conceived as mechanical integrators of cellular space (43). The cytoskeletons of most cells, including hepatocytes, consist of three components - 4- to 6-nm microfilaments containing actin, 10-nm inter-

mediate filaments and 22-nm microtubules containing tubulin (44). MBs do not contain actin or tubulin (18, 261, and because morphological and structural differences suggest that MBs represent an altered organization of cytokeratin polypeptides, it seems likely that MBs represent a pathological expression of intermediate filaments. Hepatocytes from mice fed griseofulvin contain MBs similar to those found in human subjects. Western blotting, with antibodies against cytokeratins, has indicated that cytokeratin 55 (equal to human cytokeratin 8) and cytokeratin 49 (equal to human cytokeratin 18) are present (38, 45, 461, as well as a 65-kDa component (38, 47). However, although morphological, biochemical and immunological similarities exist, cytoplasmic intermediate filaments and MB filaments are structurally different because apart from biochemical differences, the MB does not always react with antibodies against cytokeratins 55 and 49 (45, 46, 48-53). This loss of antigenicity has been ascribed to a conformational change (phenotypic switch) in the cytokeratins or the presence of some entirely different component. Normally, apart from cytokeratins 55 and 49, minute amounts of a 97-kDa band can be found on gel electrophoresis; in griseofulvin-fed mice, this high molecular weight protein band is more pronounced, and its resistance to denaturation with SDS (54) has led some to believe it is formed by crosslinking of preexisting cytokeratin monomers in various combinations. The crosslinks are believed to be covalent bonds by epsilon- (y-glutamy1)-lysine, mediated by activated tissue trans- glutaminase (55, 56) and elevated cytosolic free calcium (56).

In fact, the MBs induced experimentally have been shown to contain intermediate filaments that are ubiquinated (6) (i.e., they are covered with a basic protein known as ubiquitin). This would also explain the alterations in cytokeratin electromobility and molecular weight seen in griseofulvin-treated mice, although the full nature of this change is yet undetermined (54). Ubiquitin, which has been shown to be associated with human MBs and other intracellular filamentous inclusions (7, 571, is present in the 97-kDa band (54). Ubiquitin is a peptide that covalently binds to various kinds of protein and insoluble elements of the cytoskeleton (6,58,59) and also forms conjugates with itself, producing polyubiquinated chains that increase the molecular weight of the protein to which it is bound (60). Immunofluorescence studies have shown that ubiquitin and cytokeratins colocalize in normal hepatocytes (6) and are associated with MBs in livers of griseofulvin-fed mice (6, 7, 58).

Immunology. Immunofluorescence methods may label MBs with antibodies specific for epidermal cytokeratins that do not react with normal liver cytokeratins (26, 42). Changes in cytokeratin conformation may occur during MB development (461, as implied by differences in staining between MB and cytokeratin antibodies in early stages of MB development. Those changes in cytokeratin organization and conformation, resulting from pathological conditions leading to MB

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TABLE 2. Clinical studies of rare MB-associated conditions epithelium to cells resembling hepatocytes, but only the bile duct proliferation to the periphery of the portal

Condition confounders? References tracts seems to correlate to MB presence (69). Induction MBs due to

Abetalipoproteinemia N 165

Focal nodular hyperplasia N 170, 171, 174 Weber-Christian disease Y 175, 176 Porphyria cutanea tarda Y 178 Congenital hepatic fibrosis Y 180

Von Gierke’s disease N 167-169

~~

Because most studies are case reports, prevalence statistics and possible dose-response relationships are unavailable. The criterion of inclusion has been the identification of MBs in a liver specimen of a rare disease, regardless of the method of detection. In this case, MB presence may be due to the effects of confounders and may not in itself be relevant to the disease process; where appropriate, this is mentioned, with appropriate references. See text for further details.

formation, are claimed to expose cytokeratin determinants that are hidden in normal hepatocytes (46). This suggests that a change in accessibility of antigenic determinants is responsible for the staining of MBs with antibodies that normally fail to stain the cytoplasmic intermediate filaments in hepatocytes (41). A different acidic and basic cytokeratin polypeptide coat may be equally responsible (48). Abnormal filaments are expressed in MB-containing hepatocytes, indicating that their phenotypical expression is changed during MB formation (61, 62). MBs react with antisera to preker- atins, but it is not possible to demonstrate the presence of epidermal prekeratin containing intermediate filaments in normal hepatocytes (63). Furthermore, antigens identified with polyclonal antibodies to MBs do not react with epidermal prekeratin (64). MB-specific monoclonal antibodies are not directed against glycosylated residues. In addition, they have unique antigenic determinants that do not cross-react with normal intermediate filaments, proteins or keratinocytes (65). Several subtypes may be histochemically or morphologically recognized, probably on the basis of their age (46, 62) but not by differences in MB isolates or patient subgroups (26, 36, 42, 66). MB-containing cells (of griseofulvin-fed mice) show loss of cytoplasmic staining (unlike normal hepatocytes) with MB antibody (67).

However, there is increasing evidence of a phenotypical switch of hepatocytes toward bile duct epithelium during MB-related liver diseases; for instance, de nouo expression of so-called adhesion molecule receptors in inflammatory and cholestatic liver disease suggests such structural changes (681, and the heterogenic reactions of MBs toward a variety of monocIonaI and poIyclona1 antibodies raised against cytokeratins of “bile duct type” imply developmental differences in the immunological properties of MBs (9), but whether such hepatocytes expressing bile duct-type cytokeratins are the precursors of MB-containing cells is unclear. An immunohistochemical study of bile duct changes in alcoholic liver disease has demonstrated bile duct proliferation to the periphery of portal tracts, appearance of bile duct cells in liver parenchyma and metaplasia of bile duct

of y-glutamyltransferase, an enzyme normally only expressed by bile ducts in mouse livers, accompanies MB formation in griseofulvin-fed mice (70). Studies using monoclonal antibodies against bile duct keratins (cytokeratins 7 and 19) and hepatocyte keratins (cytokeratins 8 and 18) have demonstrated that hepatocytes can acquire some characteristics of bile duct cells in various conditions, including alcoholic liver disease (fibrosis, hepatitis, cirrhosis), focal nodular hyperplasia and cholestasis (9, 68, 69, 71), but MB cells have hetero- genous stains and the presence of MBs does not correspond to the bile duct metaplasia (9). It is, however, suggestive that the phenotypical expression of cytokeratin intermediate filaments in hepatocytes is changed to resemble that of bile duct cells in conditions where MBs are also formed. An immunohistochemical study of MBs in 122 alcoholic hepatitis patients demonstrated that hepatocyte MBs, bile duct MBs and cholestasis correlate significantly with marginal bile ductular proliferation, but not metaplasia of bile ducts into hepatocytes or bile duct proliferation from inter- lobular to portal tracts; stronger correlations were found with fibrosis, cirrhosis and portal inflammation, whereas steatosis correlated against neither (69). Certain cytokeratins of bile ductular type are present in some MBs (8, 45).

Conclusion. MBs are filaments of intermediate diam- eters with intermediate filament components, with altered outer biochemical characteristics but with intact core structures. MB formation is thought to be due to altered antigenic determinants of the cytokeratin meshwork in the hepatocyte, although some authors suggest a pathological decrease in the amount of cytokeratin in MB-containing cells (72). A number of clinical conditions have been described in which MBs appear, and studies suggest that MBs consist of similar material (chemical composition and common antigenic determinants) regardless of initiating factors (12, 36). MBs therefore seem to be a stereotypical response to a series of pathological conditions. Furthermore, MBs may be seen in cells other than hepatocytes (see below). We still do not know what makes the hepatocyte react in this particular fashion, but evidence is accumulating.

CLINICAL STUDIES OF COMMON MB-ASSOCIATED DISEASES

It is a hallmark of MBs that they are never seen in normal, healthy subjects. Although present in some acute and several chronic liver diseases, in many frameworks they never occur: Acute viral hepatitis, most toxic liver diseases, chronic persistent or chronic aggressive hepatitis and liver disease due to rheumatic disease are prime examples. The various clinical conditions that include MBs are listed in Tables 1 and 2. Prevalence statistics have been gathered from available sources, some of which may contain considerable flaws in patient selection and equally impressive type I1

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errors. An average prevalence estimate has been drawn from pooled data, with the range of observed minimum- maximum values in the studies concerned. Notice must be taken of the differences in material (biopsy specimens taken regularly or at autopsy) and methods of detection, which are not always fully compatible. As such, this estimate should not be taken at face value as systematic scientific inquiry but as a basic guideline. These systematic differences will be further discussed in relation to each disease.

Confounder Control. Most studies on “nonalcoholic patients” have excluded well-known confounding variables - in particular, alcohol consumption and morbid obesity - by simple questioning, thereby employing an author’s evaluation that is difficult to appreciate. Some studies even neglect to mention confounders. This omission poses several methodological questions. First, it is not advisable to rule out a hidden alcohol problem by means of a method of simple questioning because this method is more reliable at detecting teetotallers than the high-risk group of heavy consumers. With this method, ethanol as the sole or contributing etiological factor may still remain in the realm of possibility. Second, the concern for the “hidden” alcoholic may not just be a far-fetched attempt to discard new findings in MB epidemiology but would in fact appear relevant to the proper perspective in which to evaluate these findings. In many studies, in particular case stories on rare conditions with hepatic involvement, the importance of confounder control is most striking because low-probability events such as MBs in nonalcoholic patients (and noncirrhotic patients) remain highly sensitive to even small uncertainties in patient selection. In a study of 61 presumed nonalcoholic, morbidly obese patients, simple hematoxylin-and-eosin staining failed to recognize any MBs. Immunohistochemical methods did, however, detect two subjects with MBs in their liver specimens, both of which were steatotic with early fibrosis; each later confessed to having an alcohol problem (3). Very few conditions are not influenced by known confounders, a case in point being Indian childhood cirrhosis, in which alcohol consumption and any long-term effects of morbid obesity may reliably be ruled out but may still be influenced by its progressive structural changes, of which some may be confounding variables. In general, it is proposed that alcohol problem-detecting questionnaires (73, 74) should be employed in future studies to secure the validity of results. AH. MBs were first recognized in AH patients and

remain a cardinal sign of this disease, although their prevalence seems less related to clinical severity or the presence of cirrhosis (75) than to patient selection. There is some controversy as to whether the number of MBs in a liver biopsy specimen correlates with prognosis in alcoholic liver disease, which is in particular due to the high prevalence in AH. In a recent prospective study on 281 alcoholic patients (761, the combined condition of cirrhosis complicated by AH had both the highest 4-yr mortality (35%) and MB prevalence (95%), but patients with cirrhosis or AH alone had equal mortality rates

(49% vs. 58%), despite significant differences in MB prevalence (19% vs. 76%). On backward stepwise regression analysis, MBs were not implicated as a factor in the “histological severity score” devised by the authors. Also, the prevalence of MBs in clinical AH with concurrent cirrhosis seems additive rather than multipli- cative to the MB prevalences when the conditions are viewed separately (76). In patients with alcoholic cirrhosis, the presence and severity of AH was not correlated with prognosis 11 yr after liver biopsy (Gluud C et al., Unpublished observations, 1990).

MBs may even occur in early stages of alcoholic liver disease, corresponding to Edmondson lesions without cirrhosis (77). Some methodological problems do exist in proper prevalence studies. Obligatory for the histological diagnosis of AH are signs of hepatocyte damage (focal degeneration, necrosis and balloon degeneration), infiltration with polymorphonuclear leukocytes and pericellular fibrosis (78), commonly augmented by signs of cholestasis, Edmondson lesions and fatty liver (75,79) and at times, cirrhosis. Some authors, however, include MBs in the diagnosis of this alcohol-induced injury (80). Topographically, the relationship between MBs and polymorphonuclear leukocytes is still equivocal (81,821; although direct hepatocyte-leukocyte interaction has been observed on electron microscopy, MB-containing cells are vital and do not undergo preferential necrosis (83). Reports that propose a correlation with infiltration with polymorphonuclear leukocytes may be biased by its prerequisite for the histological diagnosis of AH (82).

The mechanism by which acute hepatocellular damage is caused in patients with AH has been debated for a long time, although the short-term fate of the MB after AH is well known: After cessation of alcohol intake, fatty liver disappears in less than 1 mo, MBs in 1 to 3 mo regardless of hyperalimentation or other treatment regimens; and histological signs of AH disappear in 3 to 6 mo, with the most marked regression in MB content in the group of clinically severe AH (78, 81). Because premorbid liver biopsy specimens (for obvious reasons) have not been performed in human beings, we cannot judge the time perspective of MB development before or during AH; experimental studies in mice fed griseofulvin or colchicine suggest that the period of MB formation may be very short (weeks). The fact that AH develops suddenly and then gradually subsides also suggests a hit-and-run effect and not a gradual increase in hepatic pathology. Although hypoxia (84), a direct effect of ethanol or its metabolite acetaldehyde (851, microsomal enzyme induction (86) or the immunological reaction to MB (87) have been proposed as possible causes of liver cell damage, little is known about the essential process through which liver cell necrosis is caused. A variety of immunological and tinctorial changes have been found in liver biopsy specimens with AH; they are the subject of another paper, to which the reader is referred (88).

Cirrhosis, Alcoholic and Nonalcoholic. Several studies demonstrate a significantly higher MB prevalence in alcoholic than nonalcoholic liver disease (13, 89, 90 and this study). With similar alcohol consumption,

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cirrhotic patients with MBs display liver cell necrosis and parenchymal inflammation more frequently than do those without MBs (911, but survival rates are completely unaffected. Little is known about the debut of MB formation during development of cirrhosis, but MBs have been observed, although rarely, in fibrosis before manifest cirrhosis has developed (93). In conclusion, we have no visible evidence that cirrhosis with the presence of MBs suggests a direct pathogenic relationship to alcohol. When AH is concurrent with alcoholic cirrhosis, the frequency of MBs reaches almost 9596, suggesting an additive effect of two independent paths of development (76, 93).

HCC. HCC develops in 5% to 15% of all cirrhotic patients (78). HCC mostly arises in cirrhotic livers (94, 95), and the frequent MB content in HCC has raised the question of a pathogenic link between the two (92, 94-96). However, MB prevalence in HCC seems equal to that of cirrhosis. A topographical relationship between MBs and HCC is not enough evidence of a pathogenic link. One should find MBs and HCC together in the absence of MBs in the non-HCC liver tissue. Many studies neglect control biopsy specimens from nonneoplastic tissue to evaluate a potential topographical selectivity (94,95). In one study, an increased prevalence of HCC was found when MBs could be detected in “normal” cirrhotic tissue in a group of nonalcoholic patients, but not in its complementary group of alcoholic patients, leading to inconclusive results (92). From the studies that do extract control biopsy specimens, only a few (two case studies) failed to locate MBs in nonneoplastic tissue, neither refuting nor supporting a cause- and-effect relationship (92, 96). Some authors have shown that MBs were more numerous in small tumors than large ones, indicating that in progression of the neoplasm the abilities to express, produce or degrade MBs are changed. Apart from the fact that we do not know the true nature of this change, it clearly under- mines any morphometrical approach to the question of MBs and neoplasia (95, 96). On the overall data, assuming that selection bias is minimal from cirrhosis to HCC patients, development of cirrhosis into HCC does not seem to influence the frequency of MBs; the argument cannot, however, be reversed to suggest that MBs are innocent in HCC formation because HCC development is a nonlinear phenomenon and not subject to known dose-effect relationships.

Morbid Obesity and Diabetes. Morbid obesity is defined by an increase in the body mass index of more than 30%. Various morphological changes have been described in the livers of the morbidly obese, with the most common alteration being fatty liver (in 80%), but more severe structural changes (i.e. pericellular fibrosis, portal fibrosis and cirrhosis) are more rare (97, 98). Occasionally MBs are observed (3,97,99, 100, 1011, but exact prevalences are hard to obtain. As previously mentioned, the association between degree of obesity and alcohol misuse transcends the idea of morbid obesity as a setting for MB formation and deserves some discussion.

The risk factors for steatosis are well known and include obesity, alcohol and occupational exposure to hepatotoxins (102, 103). In biopsy specimens from nonalcoholic patients who are asymptomatic and clinically well but have been referred because of abnormal liver enzyme biochemistry or hepatomegaly (that is not attributable to other causes), combined steatosis, hepatitis and often MBs have been found in about 10% (103-106). Curiously, a significant proportion of these patients (almost half) have type I1 diabetes, but an even larger proportion are obese (105,106). In fact, in a study of 42 patients in whom alcohol seemed reliably ruled out, all except 2 (who had lipodystrophy) were morbidly obese, and 26 had hyperlipidemia; 4 patients had MBs on their initial biopsy, but not on follow-up biopsies after 2 to 6 yr, and the MBs did not correlate with type I1 diabetes or hepatic pathology (105). In a retrospective study of 39 nonalcoholic patients with steatohepatitis, 90% had MBs, but only 8% were neither obese nor diabetic; alcohol problems were not completely ruled out, and whether nonobese diabetic subjects were able to produce MBs was not described (106). In a similar study of nine middle-aged type I1 diabetic patients who could be reliably classed as nonalcoholic, all had MBs, but information on body mass index had, regrettably, been left out (104). For demonstration, a 63-yr-old diabetic, nonalcoholic woman with hepatomegaly and a liver biopsy showing steatohepatitis and MBs did, in fact, weigh 79 kg and measure 151 cm in height (107). In a recent study of 50 obese patients, only 3 showed MBs, and they were all classified as excessive drinkers (109).

Although the interaction of morbid obesity, diabetes and MBs is difficult to assess, it is not entirely random. To illustrate the point: Approximately half of an unse- lected obese population will have diabetes (107); in a selection of obese subjects with hepatomegaly, 2 1% had MBs and 72% of these subjects had diabetes (100). In a random selection of nonalcoholic patients with steatohepatitis, 90% were found to be obese, 25% had diabetes and 15% were cirrhotic (101). Many diabetic patients have fatty liver (1 lo), but simple fatty liver alone cannot warrant the presence of MBs. Two of three diabetic patients with steatonecrosis have MBs (111). In a study of long-term alcoholic subjects without clinical liver disease, MBs appeared in diabetic patients only if morbid obesity was also present (110). In a large study of obese nonalcoholic patients with signs of liver injury, all had fatty liver, half had diabetes and 15% had MBs (but only if other structural changes [hepatitis, fibrosis or cirrhosis] were present) (100). Similarly, 70% of nonalcoholic obese subjects with steatohepatitis had both MBs and fibrosis (101), supporting the observation that MBs usually develop in the obese patient with liver fibrosis regardless of whether he or she has concurrent diabetes.

Finally, the strong association between obesity, steatohepatitis and MBs has been confirmed in an autopsy study of 169 nonalcoholic obese patients, of which 22 (13%) had steatohepatitis and 17 (10%) MBs. Although steatohepatitis was more frequently seen in diabetic patients, it only correlated significantly with obesity


grade. Three nonobese patients did have MBs, but they all had sepsis syndrome at the time; 8% of nonobese diabetic subjects had steatohepatitis (and probably also MBs), but the selection of patients in that study was unclear with regard to specific causes of liver disease, and alcohol consumption was equally ill-defined. The finding that 3% of nonobese nondiabetic subjects had steatohepatitis otherwise defies explanation (103).

These conclusions are fully compatible with the low prevalences of both MBs and fibrosis in the morbidly obese, as well as the secondary development of decreased glucose tolerance. Physiologically, we know that obesity may increase insulin levels, favoring development of fatty liver, hypertriglyceridemia and insulin resistance in adipose tissue, thereby increasing free fatty acid levels sustaining insulin resistance in liver and muscle (112). Conversely, diabetes and fatty liver may be completely abolished by weight loss and decreased insulin levels (113), but we have yet to prove that MB production is sensitive to these consistent metabolic changes. In addition, nonalcoholic fatty liver, generally accepted to be caused by low protein intake or some other form of dietary deficiency, as observed in kwashiorkor and protein-poor diet in baboons (114, 1151, cannot alone cause MBs. The final verdict on these ambiguous results is that we still cannot visualize the nature of metabolic requirements that may induce MB formation; nor can we yet make intelligent estimates on epidemiology and risk.

Intestinal Bypass and Resection. Jejunoileal bypass surgery has been extensively used to treat morbid obesity beyond medicinal limits. A comprehensive met- analysis covering 156 original reports on jejunoileal bypass from 1963 to 1977 accounted for 80 cases of secondary liver injury out of 6,722, of which cirrhosis was present in about one fifth of all injuries (116). Similarly, other studies confirm the cumulative risk of post-intestinal bypass cirrhosis at 3% (117). Although speculative, several studies indicate that liver injury in experimental animals after intestinal bypass may be promoted by bacterial overgrowth, which allows production of potential hepatotoxins such as lithocholic acid and endogenous alcohols in the defunctionalized bowel (118-123). The reported small increase in serum litho- cholate in these patients may be significant because this substance is more hepatotoxic in a protein-deficient state (124). This bacterial overgrowth hypothesis is supported by alcohol-induced failure of the reticuloen- dothelial system (125) and by the observation that in both the human subject and in the dog, fatty liver and other signs of liver injury are reduced or prevented by oral antibiotic therapy (119, 126). The results of other studies, however, suggest that intestinal bypass-induced liver dysfunction is more likely to result from nutritional deficiency states produced by an inadequate absorptive surface (127, 128), emphasizing that severe malnutrition and weight loss after intestinal bypass are important in producing the hepatic injury in susceptible individuals. After intestinal bypass and kwashiorkor (the latter being frequent in alcoholic subjects), protein

deficiency in the face of intact insulin secretion seems to favor development of fatty liver that may be reversed by amino acid or protein supplements (1291, thereby establishing a potential pathogenic link between this kind of hepatic injury and malnutrition. Even in normal-weight patients, it is interesting to note that intestinal resection (without blind loops) may introduce progressive fibrosis, cirrhosis and MBs within 2 yr in the absence of alcohol, despite nutritional therapy (130).

In conclusion, the pathogenesis of liver involvement and MB development has been tied to alimentary deficits drawn from case stories of prolonged fasting in obesity (131, 1321, improved liver histology with nutritional repletion and reanastomosis in jejunoileal bypass (133), low amino acid profiles after intestinal bypass (134) and hepatic changes following partial pancreatic resection (1351, but results are ambiguous. It is highly troublesome that these hepatic alterations are seen both before and after intestinal bypass operation, with somewhat unaffected rate (136). The basic question still remains whether the occurrence of MBs can be fully explained by the acute and chronic structural changes (hepatitis, fibrosis, cirrhosis) that are known to com- plicate obesity and intestinal resections and which by themselves seem to be more than sufficient reasons for MB formation.

PBC. MBs are frequently found in patients with PBC. Epidemiological and morphometric data have shown with increasing stage, increasing amounts of MB content, both in the number of MB-positive patients in the population (36,137,138) and in total content in each MB-positive patient (139). In the broad perspective, though, MBs develop late in the natural course of the disease, most likely as a result of changes in other components of cell metabolism. One may aptly say that MBs begin to dominate a few years before terminal illness develops (139). Topographically, MBs are present in periportal areas in patients with advanced stages of disease, and they are not found in the absence of piecemeal necrosis or copper-associated protein (orcein stain) (139). Immunologically, patients with MBs show evidence of aberrant humoral immunity, with presence of hepatobiliary autoantibodies, hypergammaglobulin- emia and circulating immune complexes (53, 65, 140). Purified isolates of MB evoke a variety of immunological effects in PBC patients, including mitogenesis, cytotoxicity, chemotaxis, fibrogenesis and cholestasis (141-145). The importance of MB presence as an independent risk factor has been downplayed by the much larger relative risks of other histological and clinical variables (fibrosis, cholestasis, ascites, variceal bleeding, age) and reach at best only borderline significance (146, 147).

Wibon’s Disease. Some authors have determined MBs to be the hallmark of Wilson’s disease (148). As in PBC, there is no doubt that MBs increase with clinical and histological stage, but particularly high MB prevalences are not characteristic even in the cirrhotic stage. Highly characteristic, however, is the fact that in some studies - even in cirrhotic patients - MBs are altogether absent (149, 1501, whereas they are plentiful in others


(137), a point perhaps more indicative of patient selection, clinical stage differences, methods of identification and dynamics of pathogenesis than actual dis- agreements in prevalence. Also, some authors (137) have suggested cholestasis as a common ground for MB formation. Topographically, MBs are not related to polymorphonuclear leukocytes or local cholestasis (15 1, 1521, and because cirrhosis seems to be a prerequisite for cholestasis, it may sufficiently explain any MB presence.

Indian Childhood Cirrhosis. First diagnosed in 1887 (1531, this progressive liver disease of unknown cause leads to cirrhosis within months to years in the 1- to 5-yr-old child (154). MBs are frequently reported and are identical to MBs in other conditions (155, 1561, but despite easy access to tools of identification no mean- ingful data for prevalence statistics are currently available. In a sibling study, MB prevalence increased with clinical and histological stage (157), but in particular, with the histological lesions (fibrosis, among others) preceding MB formation. The fact that MBs develop concurrently with the disease process and not in its initiating stages would seem typical of an epiphe- nomenon. Similarly, a topographical relation to polymorphonuclear leukocytes has been reported (154,158) but remains unclear. Recent studies have demonstrated the presence of an excess of histochemical copper in hepatocytes, in addition to very high liver copper concentrations, in well-established Indian childhood cirrhosis (159-1611, even compared with fibrosis and long-standing alcoholic hepatitis. In one important study (162), 41 of 43 afflicted children had increased hepatic copper content as measured with selective staining (rhodanine and orcein); additionally, a clear correlation between copper content and MB content was shown, although the devil’s advocate might explain this association by other structural changes (fibrosis/cirrhosis). A unified theory on the cause of Indian childhood cirrhosis has yet to emerge, but certain studies indicate relationships to certain social and ethnic factors (154, 163). Simple protein or energy malnutrition, as frequently seen in alcoholic patients with liver disease, does not alone warrant development of cirrhosis. The children are seldom malnourished, and fatty liver as it occurs in kwashiorkor is never seen.

CLINICAL STUDIES OF RARE MB-ASSOCIATED CONDITIONS

Because of the rarity of the following conditions, most information derives from case stories or a handful of selected patients, often subjecting itself to sparse confounder control. In general, most of these tentative results are too thin a thread to support statements on MBs, calling for more systematic approaches and fewer inspired hunches.

Abetalipoproteinemia. Abetalipoproteinemia is a ge- netic disorder in which the major defect is absence of plasma p-lipoprotein and complete inability to produce chylomicrons and very low density lipoproteins, resulting in abnormal hepatic fat storage (164, 165). One infant underwent consecutive liver biopsies for 2 yr after

initiation of a medium-chain triglyceride diet (165). These biopsies revealed development of cirrhosis, with MBs that were absent in the first biopsy. The evidence supports MB presence in abetalipoproteinemia but does not reflect whether it is a result of the disease process, cirrhosis or increased hepatic vulnerability to noxious stimuli.

Von Gierke’s Disease. The glycogen storage diseases result from hereditary deficiencies of enzymes involved in the synthesis or sequential degradation of glycogen. Von Gierke’s disease develops from glucose-6-phos- phatase deficiency and is associated with glycogen accumulation in the liver and other organs (166); it is distinguished, among other things, by large concentrations of cytoplasmic lipid (166). Most patients are found to have the disease in childhood and may die soon afterward, but dietary management increases survival. Hepatic adenomas may then, for reasons unknown, be seen in the third decade of life (167). Although 35 cases of adenomas in von Gierke’s disease have been described, only three reports on MB involvement have appeared (167,168), suggesting a frequency of 9%. Two cases with polymorphonuclear leukocyte-infiltrated adenomas had MBs exclusively within these; surrounding tissue was normal. Alcohol habits were not mentioned (167), and two other case reports (patient ages 22 and 37 yr) also describe MBs but fail to offer additional information (168, 169). No MBs are found in the other nine types of glycogen storage disease, all of which have glycogenosis and to some extent affected liver morphology, sometimes in the form of cirrhosis.

Focal Nodular Hyperplasia. Focal nodular hyperplasia is a rare, supposedly premalignant condition characterized by cellular atypia and nuclear hyperchro- masia (170, 171) and strongly associated with the use of oral contraceptives (172,173). MBs are mostly described in case reports in relation to intercurrent hepatitis (174), cirrhosis (170, 171) or HCC (171). In normal surrounding tissue, MBs are either extinct (174) or scarce (in cirrhotic subjects) (1701, so it would seem that focal nodular hyperplasia in itself can give rise to MBs in the absence of alcohol. Four cases of focal nodular hyperplasia in cirrhosis (of mixed origin) disclosed nodules with MBs and cellular atypia or HCC, as well as HCC in surrounding tissue (170); only one in four demonstrated MBs in nonneoplastic tissue, suggesting that MBs were connected to the disease process. However, of 86 focal nodular hyperplasia cases, only 7 contained MBs; 6 non- steatotic cirrhotic patients had focal nodular hyperplasia with MBs; 3 had HCC, mostly of alcoholic origin (170).

Weber-Christian Disease. Relapsing febrile nodular nonsuppurative panniculitis (Weber-Christian disease) is a rare condition characterized by abnormal fat metabolism (175). Liver involvement is principally in the form of fatty liver, but even portal fibrosis and cirrhosis are seen in as many as 10% (176). The hepatic changes are assumed to be caused by overload of excess fat from peripheral adipose tissue in the process of panniculitis by increased free fatty acid levels and hepatic triglyceride synthesis and accumulation (175). In one case,


liver biopsy was performed 11 days after high-dosage prednisone therapy, revealing MBs in a mixed picture of fatty liver, hepatitis and moderate fibrosis (175). Alcohol Clinical drugs confounders References

TABLE 3. Clinical studies of drug side effects MBs due to

N 193-197 intake was reliably ruled out, but there was no pre- Amiodarone morbid biopsy sample or information regarding obesity. Diltiazem N 200,201 No other MBs have been encountered in Weber- Perhexiline maleate N 202, 203 Christian disease.

Porphyria Cutanea Tarda. Porphyria cutanea tarda is characterized by chronic skin lesions and liver damage due to an enzymatic deficiency in heme metabolism. It has been proposed as a setting for MB formation (177). Porphyria cutanea tarda is often observed in alcoholic and diabetic patients, making MB assessment difficult. A 62-yr-old man with Dubin-Johnson syndrome and HCC had MBs in tumor cells; there was some doubt as to whether he was alcoholic (178). In general, studies linking porphyria cutanea tarda to MB presence always have structural alterations (cirrhosis, carcinoma) in themselves sufficient settings for MBs, so to date no direct pathogenic links have been established.

Congenital Hepatic Fibrosis. Congenital hepatic fibrosis is rare. It has been described in fewer than 100 subjects, often associated with polycystic liver and kidney disease (179). A young male with congenital hepatic fibrosis (180) had signs of cholestasis and MBs on biopsy. Although Wilson’s disease was ruled out, he did in fact have an increased hepatic copper content, which is known to precede MB formation.

MBs in Nonhepatic Tissue. MBs have been encountered in lung tissue during the course of certain chronic lung diseases, in particular asbestosis, radiation pneu- monia and lung fibrosis (181,182), but only if squamous metaplasia was present. MBs have been morphologically and tinctorially identified in metastatic lung carcinoma to the brain (183). They have been seen in bone metastases of occult HCCs (184) and in renal cell carcinoma (185,186) and have been suggested in muscle fibers of three children with hereditary congenital neuromuscular disease, but, sadly, no other organ specimens were analyzed (187). Finally, the identical ultrastructural characteristics of MB-like inclusions in the caudate nucleus and possibly in other neurological tissue (188) support the concept of the MB as a generalized marker of impaired cell function.

CLINICAL DRUG SIDE EFFECTS CAUSING MBS Because of the universal functions of the hepatocyte in

the metabolism of biological and medical substances, pharmacological liver injury is not uncommon. Chronic injury of this kind may be classified into six main categories: chronic aggressive hepatitis, biliary cirrhosis, quiescent fibrosis/cirrhosis, vascular lesions, tumors and AH-like lesions with fibrosis/cirrhosis (189). The pro- found significance of these classifications is that MBs may only be induced by drugs that cause liver injury in the final category. This fact underlines both the ne- cessity of viewing MB formation as a deterministic event (occurring in specific frameworks) and the dismal conclusions that may be drawn from the broad gener- alizations of a “permissive factor” method. Table 3

Nifedipine N 200,201 Coralgil Y 198, 199 Prednisone Y 207 Methotrexate Y 205 Anabolic-androgenic steroid Y 174, 208

Because most studies are case reports, prevalence statistics and possible dose-response relationships are unavailable. The criterion of inclusion has been the identification of MBs in a liver specimen suspected of pharmacological injury, regardless of the method of detection. In this case, MB presence may be due to the effects of confounders and may not in itself be relevant to the disease process; where appropriate, this is mentioned, with appropriate references. See text for further details.

summarizes the clinical drugs that have so far been implicated in MB formation.

Drugs. Long-term treatment with the cardiac antia- rhythmic drug amiodarone may cause structural liver changes with MBs, although some studies fail to mention the possibility of liver damage due to ischemia or circulatory insufficiency during treatment (190). The question of MB formation in amiodarone treatment is, however, debatable. Known but rare side effects include hepatitis, fibrosis, increased blood sugar and hypertriglyceridemia (191, 192), but no MB cases have been known to occur without some other pathological factor present (cirrhosis, alcohol, obesity, diabetes) (193-197). For full evaluation, baseline biopsy specimens or ad- equate alcohol histories are rarely present. The cardiac vasodilator coralgil(4,4’-diethylamino-ethoxyhexestrol) was associated with MBs in three subjects, of whom two were considered nonalcoholic (198, 199). The calcium channel-blocking drugs nifedipine and diltiazem, used for chest pain prophylaxis and arterial hypertension, may cause liver injury consisting of fatty liver and centrilobular necrosis (200, 201). To illustrate: One 78-yr-old woman whose liver biopsy showed MBs and steatosis had taken nifedipine for several years. The drug was discontinued, and follow-up biopsies disclosed disappearance of MBs. However, she had also been treated for gout with the antimicrotubular agent colchicine some years earlier, and alcohol history was unknown (200). Perhexiline maleate may cause hepatitis, hypoglycemia and hypertriglyceridemia (202); MBs have been observed in a number of cases, but all these patients had cirrhosis, which is sufficient in itself to explain the presence of MBs (203). An interesting study of mice fed griseofulvin and perhexiline maleate did, however, suggest that perhexiline maleate may induce MBs in “primed” individuals. MBs have also been reported in three patients who were treated with the folic acid antagonist methotrexate; steatohepatitis was a common feature of their liver pathology, but although


TABLE 4. Experimental drugs commonly used for MB of alcohol for manv vears to baboons, which are induction in mice

Duration of exposure for

Experimental drug MB induction References ~~ ~

Griseofulvin with Weeks 204

Griseofulvin Months 13, 26, 70, 218, 219 3,5-Diethoxycarbonyl- Months 223

Colchicine Months 221, 231 Dieldrin Years 222, 224

perhexiline maleate

1,4-di hydrocollidine

Times of exposure necessary for induction of MBs are listed with appropriate references; as a rule of thumb, the potency of the drug to induce MBs in terms of number of afflicted animalshepatocytes seems inversely related to the duration of exposure. See text for further details.

they were termed nonalcoholic, other potential causes of MB formation were not discussed (205). The association must remain doubtful because a major study on 35 patients in long-term methotrexate treatment for psoriasis did not display any MBs in spite of severe histological changes in half the specimens (206). Finally, a woman with lupus erythematosus was given high-dose prednisone for 16 mo. She gained 25 pounds, and diabetes developed. A liver biopsy showed fatty liver with MBs and alcohol abuse was ruled out, but no premorbid biopsy specimen was available (207). An elderly alcoholic patient who received androgen a few months before biopsy had MBs, but cirrhosis was also present, thus signifying nothing (174). No effect of oral testosterone treatment on the prevalence of alcoholic hepatitis was observed in a placebo-controlled study of men with alcoholic cirrhosis (208).

Although much evidence dismisses most of these drugs from participation in MB formation, it seems more than coincidental that such correspondence exists in terms of clinical use (cardiovascular problems), calcium- related properties and general side effects profile (hepatitis, disturbances in sugar and lipid metabolism). As to potential hepatotoxicity, however, the risk of hepatic involvement seems minimal.

EXPERIMENTAL STUDIES WITH MB INDUCTION MBs and Alcohol. Alcohol provocation studies have

never been successful in rats in inducing the full spectrum of alcoholic liver injury including MBs (209, 2101, and the obvious differences in metabolic pathways cannot but raise the question of the usefulness of such frequently used animal models in the study of human liver disease. Long-term feeding of alcohol to ferrets has been shown to provoke “clusters of thread-like eosinophilic material” that the authors termed “MB-like,” amidst steatosis, ballooning degeneration and mild necrosis. No fibrosis could be induced in this animal model, and the findings are based on light microscopy, which is notorious for identifying MBs when in fact the cellular changes are something different (21 1). Feeding

phylogenetically closer t o human beings than are rodents, produces fatty liver, increasing fibrosis and, finally, cirrhosis, but never hepatitis or infiltration with polymorphonuclear leukocytes (212-214), and ultrastructural studies have never confirmed the presence of MBs in these conditions. Previous studies that have reported MB-like structures have misinterpreted their findings, mistaking megamitochondria, in particular, for MBs (215, 216). In human subjects, we know that MBs occur with increased frequency in alcoholism, but whether they are caused by alcohol or some competing hepatic pathology (AH, cirrhosis, among others) is unknown. Short-term experiments may induce fatty liver but fail to induce MBs with alcohol consumption alone (217).

MBs and Chemicals. So far, MBs have only been experimentally provoked in studies on mice (see Table 4 for complete listing). Long-term exposure to various antimitotic and oncogenic chemicals is employed to this effect, causing MBs that are ultrastructurally and tinctorially indistinguishable from other MBs (46, 50). First recognized was griseofulvin (2 lS), which induced HCC, hyperplastic nodules and MBs in 1 to 5 mo (13,26, 70,2 19). Griseofulvin is thought to inhibit microtubular assembly by preventing the association between tubulin and microtubule-associated proteins. It has been shown to bind to these proteins rather than to tubulin dimer (220). This has led to a hypothesis of tanglement of cytokeratins due to a primary microtubular disturbance, with subsequent trapping of microtubule-associated proteins so that cytoplasmic tubulin dimers cannot form a tubular structure (15). Other MB-inducing chemicals include colchicine (2211, dieldrin (222) and 3,5-diethoxycarbonyl-l,4-dihydrocollidine (223). Many such drugs are carcinogens. In mice fed dieldrin, a chlorated hydrocarbon compound, MBs and both benign and malignant hepatic tumors develop within 2 yr (224). 3,5-Diethoxycarbonyl-1,4-dihydrocollidine requires 2 to 6 mo to produce MBs in most cells (223). In discontinu- ation of these drugs, MBs often disappear, but some may persist for as long as 6 mo (70, 225). In terms of cell kinetics, it is interesting that griseofulvin induces MBs in all exposed animals within 5 mo, not just a few, as is the case in human pathological studies (161). The topographical relationships of MBs in griseofulvin-fed mouse hepatocytes are also different from those in humans; MBs mostly occur in acinar zone 1, with focal necroses in zone 3 of griseofulvin-fed mice (2261, whereas human MBs are mostly present in zone 3 concurrent with the fibrotic changes (2271, suggesting important differences in pathogenesis. Furthermore, griseofulvin does not induce HCC and MBs in other rodents such as hamsters (228). Diethylstilbestrol has been reported to induce HCC within 1 wk in Chinese and Armenian hamsters, with MBs present in half (229, 230); these findings are based on light microscopy and have not been verified by electron microscopy, but in defense it must be acknowledged that MBs are known to appear frequently in neoplasias such as HCC and focal


nodular hyperplasia. In conclusion though, these points stress that the importance of remembering dissimi- larities between human hepatic metabolism and that of animals commonly involved in alcohol studies (mice, rats, hamsters, baboons).

In certain hepatic tumor cell lines (such as 72/22) established in uitro from the liver tissue of diethylnitrosamine-treated rats, insoluble hyaline aggregates of intermediate-sized filaments appear in the juxtanuclear region (231-233). On electron microscopy, they appear as randomly oriented filaments not surrounded by a membrane (234). However, unlike the short and rela- tively thick (14 to 20 nm) rods that are fimbriated with a coat of lateral projections in hepatocytes of mice fed griseofulvin, the hyaline MB-like inclusions in diethylnitrosamine-fed rats comprise thinner (7 to 15 nm) filaments of both the cytokeratin and vimentin classes (232-235). These filaments seem much less altered than the filaments in true MB. Vimentin (cytokeratin 57) has been found to be the main protein of these aggregates (235-237) but is often expressed in cultured epithelial cells (233, 238), which do not contain MB inclusions, suggesting that other factors are involved in the development of the aggregate. Other components include actin (cytokeratin 431, cytokeratin D (cytokeratin 49), cytokeratin A (cytokeratin 55) and some unknowns (46 kDa, 65 kDa, 69 kDa, 71 kDa) (232,236,2371, and their aggregation seems to some extent to be influenced by butyrate (239). Although they are interesting examples of cytoskeletal disturbance, the aggregates are distinct from MBs, mainly because true MBs contain large amounts of (even high molecular weight) cytokeratin polypeptides and completely lack the vimentin content; therefore they should not be confused with MBs (38, 233).

However, recent provocation studies using metals have provided some controversial new information about rats’ ability to form MBs: In an electron microscopic study of rats fed copper for 16 wk, a hyaline cytoplasmic material appeared in hepatocytes and Kupffer cells. Although the authors used the term “MB-like structures” and speculated on possible type I11 MBs, the changes were found to have a limiting membrane and were not further described to distinguish them from well-known MB-like structures (megamitochondria, endoplasmic reticulum, among others) (240). The most promising experimental studies, however, concern the in uztro induction of largely insoluble, cytokeratin-containing juxtanuclear aggregates in response to nickel using a T51B cell line derived from rat liver (241-2431, which seems to selectively cause cytokeratin aggregation, leaving vimentin unaffected (243). With monoclonal antibody staining after incubation in a high-calcium environment, the cytokeratins identified are cytokeratin D (cytokeratin 491, cytokeratin A (cytokeratin 55) and two unknowns (39 kDa and 52 kDa), and the aggregates are irreversible after long-term exposure (i.e., 7 mo) to nickel (241). Although enticing, proper ultrastructural studies are needed to completely char- acterize the similarities to MBs.

CLOSING REMARKS MBs do not occur in the normal population (244).

They only seem to occur in diabetic fatty liver when obesity is also present, and they are never seen in acute viral hepatitis and liver injuries produced by most pharmacological agents. From a clinical viewpoint, it is important to know the conditions that may produce MBs in the assessment of a liver biopsy. The search for a common denominator in pathophysiology has been long and hard and has left researchers with more questions than answers. It seems, however, that MBs most often occur as a result of alcohol abuse, chronic nonalcoholic liver disease with fibrosis/cirrhosis or several other rare and diverse conditions. Only few longtudinal studies have been performed; morphometrical analyses are equally rare, representing a serious impediment in study design. Also, the situation is peculiar by the lack of theoretical groundwork needed to carry out an investi- gative study.

For the pathologist and hepatologist, the MB has long been considered more than just a marker of alcoholic liver disease; despite the many conditions in which MBs have been reported, they remain an important diagnostic clue for alcoholic liver disease in adults of Western countries; however, alternative conditions should always be ruled out - in particular, hidden alcohol abuse - when rare pathogeneses for MB formation are in question.

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the mallory body: morphological, clinical and experimental studies (part 1 of a literature survey)

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