Mallory bodies revisited Helmut Denk, Cornelia Stumptner, Kurt Zatloukal  Journal of Hepatology  Volume 32, Issue 4, Pages 689-702 (April 2000) DOI: 10.1016/S0168-8278(00)80233-0

Fig. 1 Liver biopsy with alcoholic hepatitis. Note enlarged (hydropic) hepatocytes containing MBs. In addition, there is pronounced pericellular fibrosis as revealed by connective tissue stain (blue; chromotrope aniline blue stain). The inset shows an enlarged (hydropic) hepatocyte with an MB. Note the large nucleus containing a large nucleolus (H-E). Bars, 100 μm. Journal of Hepatology 2000 32, 689-702DOI: (10.1016/S0168-8278(00)80233-0)

Fig. 2 Electron microscopy of MBs in human alcoholic hepatitis (a; Hu - AH), 2 months DDC (b; 2 m DDC-ML)- and 4 months GF (c; 4 m GF-ML)-fed mouse livers. The figures show the filamentous ultrastructure of MBs with filaments in irregular arrangement (type II). Note identical morphology of human and murine MBs. Bar, 500 nm. Journal of Hepatology 2000 32, 689-702DOI: (10.1016/S0168-8278(00)80233-0)

Fig. 3 Immunofluorescence microscopy of MBs in human liver with alcoholic hepatitis (Hu - AH; a, d, g, j, m) and in 2 months DDC (2 m DDC-ML; b, e, h, k, n)- and 4 months GF (4 m GF-ML; c, f, i, l, o)-fed mouse livers with antibodies against CKs8 and 18 (CK8/18), antibody 5B3 against phosphorylated CK8 (pCK8), antibodies against ubiquitin, and the MM120-1 and the SMI 31 antibodies. Note the pronounced immunostaining of MBs in enlarged hepatocytes with all these antibodies. With CK8/18 in addition to MBs a cytoplasmic CK filament network in hepatocytes is decorated which is clearly diminished in most enlarged MB-containing hepatocytes. With antibodies against phosphorylated CK8 (pCK8) in addition to MBs of different sizes (small granular to large irregular inclusions) the cytoskeleton with cell-peripheral accentuation of some hepatocytes is immunostained. Ubiquitin antibodies and the MM120-1 and SMI 31 antibodies exclusively react with MBs. Bar, 20 μm. Journal of Hepatology 2000 32, 689-702DOI: (10.1016/S0168-8278(00)80233-0)

Fig. 4 One-dimensional SDS-polyacrylamide gel electrophoresis of MBs isolated and semipurified from the livers of 2 months DDC-fed mice. Note the presence of three major polypeptide bands (I, II, III). Bands II and III react in immunoblotting with CK antibodies and thus are of CK nature. Band II corresponds to CK8 and band III to CK18. Band I is a non-CK component. In addition, poorly soluble high molecular material remains at the interphase between stacking and resolving gels. Journal of Hepatology 2000 32, 689-702DOI: (10.1016/S0168-8278(00)80233-0)

Fig. 5 The upper panel shows relative amounts of CKs 8 (K8) and 18 (K18) protein as revealed in mouse liver homogenates by immunoblotting with a polyclonal antibody directed to CKs 8 and 18. Note that in mice fed a normal diet CKs 8 and 18 are present in a 1:1 ratio. Already after 1 week of DDC feeding (1 w DDC) CK8 clearly prevails over CK18 and this situation is maintained after a 2.5-month (2.5 m DDC) DDC feeding period. The lower CK protein levels after prolonged DDC intoxication (2.5 m DDC) may reflect the higher number of hepatocytes with diminished intermediate filament cytoskeleton. The increased amount of CK8 resembles unassembled polypeptide. The lower panel shows mRNA levels in mouse liver homogenates. DDC intoxication (for 1 week and 2.5 months) leads to a conspicuous increase of CKs 8 (K8) and 18 (K18) mRNA levels. A peak level is already reached after 1 week (1 w DDC) of DDC feeding. Journal of Hepatology 2000 32, 689-702DOI: (10.1016/S0168-8278(00)80233-0)

Fig. 6 Chinese hamster ovary cells (CHOK 1) (a) and embryonal mouse hepatocytes (TIB 73) (b) were transfected with human CK18 gene constructs (using the receptor-mediated adenovirus-augmented gene delivery system). Double immunofluorescence was performed 24 h post-transfection with MM120-1 (green) and polyclonal CK8/18 (CK8+18) antibodies (red). Confocal images of both stainings were superimposed. Note that in transfected cells the foreign CK18 accumulates together with the MM120-1 antigen as small aggregates. Bar, 10 μm. Journal of Hepatology 2000 32, 689-702DOI: (10.1016/S0168-8278(00)80233-0)

Fig. 7 Double-label immunofluorescence microscopy of livers of 2-month DDC-intoxicated (2 m DDC-ML) mice using monoclonal antibodies (a. 5B3; b. LJ 4; see 75) to different phosphoepitopes of CK8 (green) and polyclonal CK antibodies (CK8+18; red). Note liver cells without MBs containing phosphorylated CK8 associated with the CK cytoplasmic network (asterisks). MB-containing hepatocytes lack a phosphorylated CK network and phosphorylated CK8 is only associated with MBs (arrow heads). This suggests that phosphorylated CK8 preferentially aggregates in MBs. Bar, 10 μm. Journal of Hepatology 2000 32, 689-702DOI: (10.1016/S0168-8278(00)80233-0)

Fig. 8 Immunofluorescence microscopy of DDC-intoxicated (2 months of DDC-feeding) wild-type (a; CK8/18 - WT), CK18 knockout (b; CK18−/−) and CK8 knockout (c; CK8−/−) mouse livers, using polyclonal CK8/18 antibodies. In livers of wild-type mice CK-positive MBs of different sizes are immunostained with the CK antibodies (a). The MB-containing hepatocytes are enlarged and show a reduced CK network mostly present only as a peripheral rim of filament bundles. In CK18−/− and CK8−/− mice no CK network is visible. Whereas CK8−/− animals do not develop MBs (c) CK18−/− mice (b) clearly develop small MBs (also positive with MM120-1) mostly situated at the cell periphery upon DDC intoxication. Bar, 10 μm. Journal of Hepatology 2000 32, 689-702DOI: (10.1016/S0168-8278(00)80233-0)