742 symptoms of ASH sometimes show only moderate histo- pathological alterations with few or no MDBs, whereas patients with pronounced histological alterations do not necessarily exhibit signiWcant clinical and laboratory abnormalities (Zatloukal et al. 2007). Despite that, con- trolled clinical-pathologic studies comparing NASH patients with ambulatory and hospitalized alcoholics revealed that hepatocellular damage, presence of MDBs, inXammation, and Wbrosis collectively correlated with disease severity (Cortez-Pinto et al. 2003). Also in other studies, hepatocellular ballooning and MDB formation was positively correlated with disease progression, development of Wbrosis, and cirrhosis and liver-related mortality (Orrego et al. 1987; Matteoni et al. 1999; Gramlich et al. 2004; Mendler et al. 2005). Morphology and composition of MDBs MDBs are irregularly shaped, usually dense cytoplasmic inclusions of diVerent sizes (Mallory 1911). Small MDBs arise in association with IF bundles throughout the cyto- plasm, whereas larger MDBs are often seen in the perinu- clear region (Denk et al. 2000; Zatloukal et al. 2007; Ku et al. 2007). Ultrastructurally, they usually consist of hap- hazardly arranged Wlamentous rods, approximately 10– 15 nm in diameter, covered by a fuzzy coat (designated as type II by Yokoo et al. 1972). In addition, MDBs with an electron dense granular to amorphous center (designated type 3) are seen predominantly in older inclusions, and Wla- ments in parallel arrangement were also described (desig- nated as type I) but seem to be exceedingly rare (Yokoo et al. 1972). Keratins 8 and 18, sequestosome 1/p62 (p62) and ubiquitin are major, and low and high molecular weight heat shock proteins (HSP 70, HSP 25), but also proteins of the protein degradation machinery, are minor constituents (Denk et al. 2000; Riley et al. 2002, 2003; Zatloukal et al. 2007; Ku et al. 2007; Figs. 1c, d; Fig. 2). Keratins 7, 19, and 20 have also occasionally been detected (Cadrin et al. 1990; Dinges et al. 1992). The K8/K18 within MDBs exhibit increased -sheet structure (Cadrin et al. 1991; Kachi et al. 1993), are hyperphosphorylated, partially degraded and cross-linked (Hazan et al. 1986; Zatloukal et al. 1992; Cadrin et al. 1995; Stumptner et al. 2000; Fig. 1d). The list of MDB components is likely to grow since, in addition to intrinsic components, proteins non-spe- ciWcally incorporated in the aggregate have to be expected. Pathogenesis of MDBs Studies on MDB formation and composition are greatly enhanced by the availability of animal models. MDBs can 123 Histochem Cell Biol (2008) 129:735–749 be induced under standardized conditions and their fate fol- lowed in mouse liver by chronic griseofulvin or 3,5-dieth- oxycarbonyl-1,4-dihydrocollidine (DDC) administration (Denk et al. 1975; Yokoo et al. 1982; Denk et al. 2000; Zat- loukal et al. 2004, 2007; Fig. 3c, d). In patients, MDB formation is usually a chronic process requiring several years of alcohol intoxication or metabolic imbalance, that is, in association with the metabolic syn- drome, Wilson disease, other metabolic disorders, and chronic cholestasis. An exception is idiopathic copper toxi- cosis of children in which end stage liver disease associated with MDB formation occurs at very early age (Müller et al. 2004; Zatloukal et al. 2007). Surprisingly, in alcoholics recovered from ASH, an alcohol excess may almost instan- taneously lead to MDB recurrence, a situation which has been compared to an immunologic response and termed “toxic memory” (Jensen and Gluud 1994; Denk et al. 2000). The animal model of MDB formation does not only reproduce the structural and morphological features seen in humans, but also the phenomenon of “toxic memory” (Fig. 3; Denk et al. 2000; Zatloukal et al. 2004, 2007). To induce MDBs in mice, griseofulvin or DDC is usually fed for 2–3 months. MDB formation in mice is reversible, since MDBs disappear after recovery on standard diet for 1 month. However, after rechallenge of the recovered (“primed”) animals, MDBs reappear within a few days (Denk et al. 2000; Zatloukal et al. 2004, 2007). Rapid MDBs formation in primed mice is rather non-speciWc, since it can be triggered by a variety of stress conditions including colchicine (but not by lumicolchicine), bile acids, bile duct ligation, several other toxins, and proteasome inhibitors which were unable to induce MDBs in the naïve animal (Zatloukal et al. 2007, and references therein). The reasons for this phenomenon are as yet unclear but point to a Wnal common pathway activated by the trigger. The availability of cellular and animal models of MDB formation led to valuable insights into the mechanism of MDB formation. Several pathogenic mechanisms were implicated in this process (Fig. 4; Dobson 2004): 1. Enhanced oxidative stress 2. Disproportional K8/K18 expression together with keratin modiWcations 3. Chaperone dysfunction 4. Elevated p62 levels 5. InsuYcient protein degradation Ad 1. The MDB-inducing conditions both in humans and mice cause elevated levels of oxidative stress (Tephly et al. 1981; Mehta et al. 2002; Dey and Cederbaum 2006; Farrell and Larter 2006) and MDBs themselves were shown to con- tain misfolded keratins with increased -sheet formation (Cadrin et al. 1991; Kachi et al. 1993). Recent animal studies highlight the importance of altered methyl group metabolism and mitochondrial stress in this process (Li et al. 2008;