[Congenital hypopituitarism and giant cell hepatitis in a three month old girl]. / Konnataler Panhypopituitarismus und Riesenzellhepatitis bei einem 3 Monate alten Säugling.

CASE REPORT: A three month old girl, with recurrent hypoglycemia and neonatal cholestasis, is reported. A metabolic disease could be excluded. The liver biopsy revealed giant cell hepatitis and intrahepatic bile duct hypoplasia. ACTH, Cortisol and hGH measured during hypoglycemia were low. Magnetic tomography (MR) of the brain showed an "empty sella". After beginning a replacement therapy with hydrocortisone, growth hormone and thyroxine there was no further episode of hypoglycemia. Transaminases and bilirubin levels normalized. The girl is in good condition, growth and development are normal. DISCUSSION: Hypoglycemia is often the first sign in childrens with neonatal hypopituitarism. The association of liver disease and hypopituitarism has been documented in a few reports. The pathophysiological mechanism leading to the liver dysfunction is not well understood. The prognosis of neonatal hypopituitarism as well as the concomitant liver disease is good under sufficient replacement therapy.

Prognostic impact of molecular analyses in adenoid cystic carcinomas of the salivary gland.

BACKGROUND: Adenoid cystic carcinoma (ACC) of the salivary gland is a generally slow-growing but highly malignant neoplasm with a remarkable capacity for recurrence. Prognosis is greatly influenced by the histological subtype (tubular, cribriform or solid), presence of tumour at the margins, anatomic size, and lymph node metastases. However, none of these parameters has proven to be an unequivocal predictor of disease activity. Therefore, the current study was undertaken to investigate the prognostic value of molecular markers. PATIENTS AND METHODS: Samples from 22 patients, including 4 patients with recurrent disease, were included in the study. By means of immunohistochemistry, the staining pattern of p53, bcl-2, P-glycoprotein, glutathione S-transferase, and topoisomerase as well as sequence analyses of p53 were performed. These molecules were chosen because of their proven association with poor prognosis and therapy resistance in other malignancies. RESULTS: Homozygous p53 mutations were found in all of the 4 recurrent tumors. The other proteins were detected in some tumors, but showed no correlation with histological subtype or recurrence of tumor. CONCLUSION: The results of the current study emphasize the prognostic value of a p53 alteration as an independent prognostic marker. Further, it could be demonstrated for the first time that proteins known for their association with radio- and chemotherapy resistance can be overexpressed in some ACCs suggesting that those molecules could influence the outcome of new therapeutical approaches.

Interferon-alpha and ribavirin in treating children and young adults with chronic hepatitis C after malignancy.

OBJECTIVE: Chronic hepatitis C is a major long-term problem for children who survive cancer. Interferon (IFN)-alpha has been shown to be effective in treating patients with chronic hepatitis C; however, the rate of sustained response is low. Combining IFN-alpha and ribavirin (RBV) has been shown to significantly improve the response in adult patients with chronic hepatitis C. The aim of this pilot study was to evaluate the efficacy and safety of a combined virostatic treatment with IFN-alpha and RBV in a small cohort of children and adolescents with chronic hepatitis C and previous malignancy. METHODS: Twelve patients with a history of a hematooncologic disease (median follow-up: 13.5 years; range: 7-14.7 years) and chronic hepatitis C were treated with recombinant IFN-alpha-2a (6 megaunits/m(2) body surface area, 3 times a week, subcutaneously) combined with RBV (15 mg/kg body weight/day, orally) for 12 months. They were tested monthly for blood counts and liver function, and for serum virus concentrations (hepatitis C virus RNA by polymerase chain reaction) every 3 months. RESULTS: At the end of the treatment, hepatitis C virus RNA could not be detected in the serum of 8 of the 12 patients; 2 of these patients relapsed soon after therapy withdrawal, whereas 6 patients maintained in sustained virologic and biochemical remission (follow-up: 12 months). Treatment-induced toxicity was moderate and reversible with influenza-like symptoms and a decrease in blood counts in all 12 patients, alopecia in 5 of the 12, hemolysis in 4 of the 12, and weight loss of >10% in 2 of the 12. CONCLUSIONS: As demonstrated in adults with chronic hepatitis C, treatment with IFN-alpha and RBV also seems to be an effective and safe therapeutic option for children and adolescents with chronic hepatitis C after malignancy.

Atypical ductular proliferation and its inhibition by transforming growth factor beta1 in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse model for chronic alcoholic liver disease.

Many acute and chronic liver diseases are often associated with atypical ductular proliferation (ADP). These ADPs have gained increasing interest since a number of recent observations suggest that ADPs may represent progenies of the putative liver stem cell compartment. In this study, we show that feeding mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) results in persistent proliferation of primitive ductules with poorly defined lumens. Similar to oval cell proliferation in other rodent models as well as in various human liver diseases, DDC-induced ADP originated from the portal tract, spread into the hepatic lobule, and was associated closely with appearance of hepatocytes harboring an antigen (A6), which normally is expressed in biliary epithelium. Furthermore, DDC treatment severely inhibited the regenerative capacity of mice after partial hepatectomy. The development of ADP was selectively blocked in DDC-fed TGF-beta1 transgenic mice producing active TGF-beta1 in the liver and no accumulation of new hepatocytes expressing the A6 antigen was observed. Moreover, the transforming growth factor beta1 (TGF-beta1) transgenic mice did not survive beyond 3 weeks from starting the DDC-containing diet. The results suggest that persistent activation of the hepatic stem cell compartment is essential for liver regeneration in the DDC model and that active TGF-beta1 may negatively control activation of stem cells in the liver. These data further emphasize the relevance of the DDC model as an experimental tool for studying chronic liver diseases.

Multiphasic helical CT in diagnosis and staging of hilar cholangiocarcinoma.

OBJECTIVE: The purpose of our study was to assess the potential of thin-section multiphasic helical CT in diagnosis and staging of hilar cholangiocarcinomas. SUBJECTS AND METHODS: Identically collimated helical CT studies were performed before and during the hepatic artery dominant phase and during the portal vein dominant phase of contrast enhancement in 29 consecutive patients with proven hilar cholangiocarcinomas. Differences in attenuation between the tumor and the liver were calculated in each case by subtracting the average attenuation of the tumor from that of the liver. A four-point scale termed a "lesion conspicuity score" was used to determine rates of tumor detection. CT findings were correlated with surgically assessed extent of tumor, histologic findings, or both in all cases. RESULTS: Ten (34%) of the 29 hilar cholangiocarcinomas were detected on unenhanced images. All hilar cholangiocarcinomas (100%) were seen on hepatic artery dominant phase scans, and 25 (86%) of 29 hilar cholangiocarcinomas were seen on portal vein dominant phase scans, regardless of the morphologic appearance. An infiltrating stenotic lesion was found in 17 (59%) of 29 patients, an exophytic hilar lesion was found in 11 patients (38%), and one patient (3%) had an intraluminal polypoid lesion. Mean differences in enhancement between infiltrating stenotic lesions and the liver were significantly greater on hepatic artery dominant phase scans (28 +/- 10 H) than on portal vein dominant phase scans (10 +/- 8 H), whereas the mean difference in enhancement between the exophytic lesions and the liver was statistically greater during the portal vein dominant phase (p < .01). Two of the hilar cholangiocarcinomas were resectable at surgery, and 18 were not. The overall accuracy of helical CT for assessing resectability was 60%. In 10 (56%) of 18 patients, unresectable disease was correctly diagnosed with helical CT (sensitivity, 56%). Eight (44%) of 18 patients considered to have resectable tumors with helical CT had unresectable tumors at surgery. A resectable tumor was correctly diagnosed in two patients with helical CT. CONCLUSION: Multiphasic helical CT can be used to detect and classify hilar cholangiocarcinomas. However, the exact proximal tumor extent along bile ducts tends to be underestimated with helical CT; therefore, helical CT is inaccurate for determining resectability.

Inhibition of neoplastic development in the liver by hepatocyte growth factor in a transgenic mouse model.

Overexpression of the c-myc oncogene is associated with a variety of both human and experimental tumors, and cooperation of other oncogenes and growth factors with the myc family are critical in the evolution of the malignant phenotype. The interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in a transgenic mouse model has been analyzed. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Furthermore, tumor promotion by phenobarbital was completely inhibited in the c-myc/HGF double transgenic mice, whereas phenobarbital was an effective tumor promoter in the c-myc single transgenic mice. The results indicate that HGF may function as a tumor suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine.

Experimental Mallory body formation is accompanied by modulation of the expression of multidrug-resistance genes and their products.

Mallory bodies (MBs) are characteristic morphological features of alcoholic hepatitis and are also found in other chronic liver disorders and hepatocellular neoplasms. MBs can be produced in mouse liver by chronic administration of the porphyrinogenic drugs griseofulvin (GF) and 3,5-diethoxy-carbonyl-1,4-dihydrocollidine (DDC). The mechanisms causing the formation of MBs are poorly understood, and the significance of MB formation during the course of liver disease remains unclear. We investigated the relationship between the mechanisms underlying the formation of MBs and the regulation of multidrug resistance (mdr) genes and their products, the P-glycoproteins (Pgp). Immunofluorescence microscopy using the monoclonal antibody C219 revealed an increase of Pgp expression in almost all hepatocytes after 3 to 8 days of feeding mice DDC- and GF-containing diets. However, after approximately 4 weeks of DDC and approximately 8 weeks of GF feeding, when the first small MBs appeared and loosening and diminution of keratin intermediate filament (KIF) cytoskeleton occurred in some hepatocytes, a decrease or loss of Pgp staining in affected hepatocytes was observed. After feeding mice DDC for 6 weeks and GF for 12 weeks, many hepatocytes contained MBs and displayed a disruption of the immunohistochemically demonstrable KIF meshwork. Double immunofluorescence microscopy with the keratin polyclonal antibody and the mab C219 at this time point revealed a complete loss of Pgp staining in affected cells, although remaining hepatocytes with unaltered KIF meshwork showed a strong reaction with the C219 antibody. Northern blot analyses revealed a significant increase of mdr2 mRNA and, to a lesser extent, of mdr1a mRNA in the livers of DDC- and GF-fed animals.

Regulation of mdr1b gene expression in Fischer, Wistar and Sprague-Dawley rats in vivo and in vitro.

Administration of 2-acetylaminofluorene (2-AAF) to rats increased mdr1b expression in Fischer, Wistar and Sprague-Dawley rat livers; however, the response was much smaller in Sprague-Dawley livers. To investigate the basis of this difference we further examined the regulation of the mdr1b gene in hepatocytes isolated from Fischer, Sprague-Dawley and Wistar rats. A time-dependent increase in basal expression of mdr1b but not mdr2 was observed in hepatocytes isolated from all three strains of rats. After 4 days in culture, a larger increase in mdr1b mRNA levels was observed in Fischer and Wistar rat hepatocytes (3.5- and 4.6-fold respectively) than Sprague-Dawley hepatocytes (2-fold). Treatment of primary hepatocytes with 2-AAF caused an induction of mdr1b expression that varied among the three strains. Notably, Sprague-Dawley hepatocytes were not responsive to 2-AAF. In contrast to the parent compound, the electrophilic metabolites N-hydroxy-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene caused a dose-dependent induction of mdr1b expression in both Fischer and Sprague-Dawley hepatocytes, indicating that differences in the metabolic activation of 2-AAF between the strains may account for the differences in mdr1b by 2-AAF. Hepatocytes isolated from all three strains of rats showed an equivalent induction of mdr1b after treatment with cycloheximide. Nuclear run-on assays demonstrated that the increases in mdr1b expression with time in culture and after xenobiotic treatment were due to increased transcription.

Alcoholic liver disease: molecular-pathologic aspects.

Mallory bodies (MBs) are characteristic morphologic features of alcoholic hepatitis but are also associated with non-alcoholic liver diseases including long lasting cholestasis, metabolic and neoplastic disorders. MBs contain in addition to keratins non-keratin components, including microtubule-associated (tau protein) and other not yet characterized proteins in an aggregated form. Aggregation of these components in the cell is promoted by posttranslational modifications, such as partial proteolysis, phosphorylation and cross-linking, and may result in functional and structural disturbances of the cell depending on the physiologic function of the components involved. Several enzymes responsible for these modifications are Ca(++)-dependent. Thus, disturbance of Ca(++)-homeostasis may play an essential role in the pathogenesis of MBs. In some structural aspects MBs closely resemble inclusions associated with degenerative disorders of the central nervous system, including Alzheimer's and Parkinson's disease. Studies on the pathogenesis of MBs, therefore, not only shed light on a peculiar type of liver cell injury but may also assist in the understanding of other chronic degenerative diseases, particularly those of the central nervous system.

Induction of mammary tumors in female Sprague-Dawley rats by the food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and effect of dietary fat.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogen found in cooked meat, was determined to be a mammary carcinogen in female Sprague-Dawley rats on a high fat diet. Forty-three-day-old female Sprague-Dawley rats received 10 doses of PhIP (75 mg/kg, p.o., days 1-5 and 8-12). Two days after the last dose of PhIP, animals were placed on a high polyunsaturated fat diet (23.5% corn oil) or a standard low fat diet (5% corn oil). After 25 weeks on the defined diet, mammary tumor incidence (average tumor mass +/- SE) was 53% (5.7 +/- 1.3 g) and 16% (2.4 +/- 0.9 g) in rats on a high fat and standard low fat diet, respectively. The histological differences in mammary gland tumors found in animals on the standard low fat diet and the high fat diet were striking. Mammary gland tumors found in PhIP-treated rats on the low fat diet were all histologically benign. The histopathological changes in these tumors included hypertrophic changes resembling the normal mammary gland, fibrocystic changes, and sclerosing adenosis. However, 80% of the mammary gland tumors found in PhIP-treated rats on a high fat diet were histologically malignant. These tumors had several malignant phenotypes including intraductal carcinoma (papillary, cribriform, and comedotype), tubular adenocarcinoma, and infiltrating duct carcinoma. The data indicate that a high fat diet in combination with a heterocyclic amine carcinogen derived from cooked meat may enhance the incidence and severity of mammary gland cancer.

Expression of three- and four-repeat tau isoforms in mouse liver.

Tau protein is a member of the family of microtubule-associated proteins, which support microtubule polymerization and stability. Under pathological conditions, tau is a major constituent of neurofibrillary tangles in nerve cells of patients with Alzheimer's disease. Neurofibrillary tangles share some morphological, biochemical and immunological properties with cytoplasmic inclusions associated with other diseases, such as Mallory bodies in the livers of patients with alcoholic hepatitis and in corresponding mouse models. Recently a Mallory body component was identified that in molecular mass and isoelectric point resembles the abnormally phosphorylated tau of neurofibrillary tangles. There has been, however, so far no report describing the occurrence of tau in normal liver. We now demonstrate the expression of two tau isoforms containing three and four repeats, respectively, of the microtubule-binding domains in normal mouse liver and kidney. This finding provides evidence for a physiological role of tau in the liver and, consequently, the basis for the involvement of tau in pathological situations.

Induction of multidrug resistance gene expression during cholestasis in rats and nonhuman primates.

P-glycoprotein, an energy-dependent plasma membrane drug-efflux pump capable of reducing the intracellular concentration of a variety of hydrophobic xenobiotics, is encoded by mdr1, a member of the multidrug-resistant (mdr) gene family. The physiological function of this protein is unknown. Because of its location on the bile canalicular domain of the hepatocyte, we and others have hypothesized that P-glycoprotein may have a physiological role as a biliary transporter of xenobiotics and endobiotics and that its expression may therefore be altered in cholestasis. Both obstructive and alpha-naphthylisothiocyanate-induced cholestasis increased mdr1a and 1b gene expression in rat liver. Hepatic P-glycoprotein levels were also increased, and the protein remained localized at the biliary hepatocyte domain. Induction of mdr1a and mdr1b gene expression in rat liver was accomplished by means of increased transcription. alpha-Naphthylisothiocyanate-induced cholestasis in cynomolgus monkeys increased hepatic expression of both the mdr1 and 2 genes. To investigate the possible role of P-glycoprotein as a biliary efflux transporter, biliary excretion of vinblastine, a representative substrate of P-glycoprotein, was studied in rats. Increased hepatic mdr messenger RNA and P-glycoprotein levels, mediated by the xenobiotic inducer 2-acetylaminofluorene, resulted in a significant increase in biliary excretion of vinblastine, which was antagonized by the P-glycoprotein inhibitor verapamil. These findings suggest that P-glycoprotein functions as a biliary efflux pump for xenobiotics and, possibly, for unidentified physiological inducers that may mediate increased transcription of the mdr gene observed during cholestasis.

Cryopreservation of cytological specimens for immunocytochemistry.

A method has been established for storage and preservation of cytological specimens in liquid nitrogen and further processing for immunocytochemistry as smears prepared from thawed cells or cryo-sections of frozen cell pellets. For the experiments cultured cells of a T-lymphoblastic leukemia cell line (ATCC CCL 119) and blood cells of the buffy coat of healthy humans were treated with a cryo-solution (fetal calf serum +5% dimethylsulfoxid) and after freezing stored in liquid nitrogen. Alternatively, cells preincubated with cryo-solution followed by suspension in fetal calf serum without cryo-additive were frozen and stored in liquid nitrogen for the production of cryo-sections. Indirect immunofluorescence and alkaline phosphatase--antialkaline phosphatase based immunoreactions were performed for the decoration of various surface antigens with a panel of monoclonal antibodies. All immunoreactions were repeated at least three times and the stored cell preparations were investigated after different periods of storage (up to four months). The immunoreactions of fresh cells in suspension (which were used as controls) were comparable with those of cryopreserved cells, e.g. cells on smears after thawing and on cryo-sections of cell pellets. The strongest immunoreactions were achieved on fixed cryo-sections. The maintenance of cell morphology of smears from cryopreserved cells was slightly better than of cells from cryo-sections. In our hands the preparation of cell pellets, which are suitable for the storage in liquid nitrogen and the production of cryosections, is a very useful method for immunocytochemical investigations of cytological specimens especially in situations where immunoreactions cannot be performed on fresh material.(ABSTRACT TRUNCATED AT 250 WORDS)

Common epitopes of human and murine Mallory bodies and Lewy bodies as revealed by a neurofilament antibody.

The antibody SMI 31, which is directed against a phosphorylated epitope, associated with neurofilaments and recognizes Lewy bodies in brains of patients with Parkinson's disease (Bancher C, Lassmann H, Budka H, Jellinger K, Grundgke-Iqbal I, Iqbal K, Wiche G, Seitelberger F, Wisniewski H: J Neuropathol Exp Neurol 1:81, 1989), decorated in immunofluorescence microscopy Mallory bodies (MBs) present in livers of mice chronically treated with griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine. In immunoblots it recognized very acidic MB components in a molecular weight range between 55 and 69.5 kilodaltons in addition to poorly soluble high molecular weight material. Moreover, an antibody to tau protein showed similar reactivities in immunofluorescence microscopy and immunoblotting experiments. Both antibodies also stained MBs in human liver with alcoholic hepatitis. These observations support and extend earlier findings which indicate that several intermediate filament-related cellular inclusion bodies, including MBs, share a variety of morphologic, structural and antigenic features. They also suggest the involvement of tau or tau-like proteins in MB formation.

Changes of cytokeratin filament organization in human and murine Mallory body-containing livers as revealed by a panel of monoclonal antibodies.

Mallory bodies (MBs) are characteristics morphologic features of alcoholic hepatitis and can be produced in mouse hepatocytes by chronic griseofulvin (GF) intoxication. The formation of MBs, which share some immunological, biochemical, and ultrastructural features with cytokeratin (CK) filaments of normal liver, is accompanied by derangement and even loss of the CK cytoskeleton of hepatocytes ("empty cells") as revealed by immunofluorescence microscopy. To clarify whether this diminution or lack of CK-related staining of MB-containing hepatocytes was due to loss of CK filaments or changes in antigenicity or accessibility of antigenic determinants immunohistochemical studies using a battery of monoclonal and polyclonal CK antibodies were performed. It could be shown that all these antibodies directed against different CK polypeptide components and antigenic determinants of CKs revealed a highly reduced or even undetectable cytoplasmic CK meshwork in most cells with fully developed large MBs. In the light of our present knowledge of the organization of CK intermediate filaments, these results indicate that the phenomenon of the "empty cells" reflects a diminution of CK meshwork rather than altered antigenic determinants.

High molecular weight component of Mallory bodies detected by a monoclonal antibody.

To identify Mallory body (MB) constituents, monoclonal antibodies to murine MBs induced by long-term griseofulvin (GF) feeding were produced. One of these, antibody MM 120-1, specifically reacted in immunofluorescence microscopy with MBs in all developmental stages but not with other cell structures of human and mouse liver and other organs. The MM 120-1 antigen was present in murine MBs induced by griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding and also in human MBs in livers of patients with alcoholic hepatitis. In immunoblots, the MM 120-1 antigen was detectable in the high molecular weight fraction of MB preparations, most of which remained in the well and at the interphase between stacking and resolving gel. No reactivity with cytokeratin polypeptides of different conformational states (i.e., isolated cytoker atin components A and D, heterotetramers A2D2, reconstituted intermediate filaments) was found. It is concluded that the antibody MM 120-1 is a highly specific and sensitive marker for murine and human MBs recognizing a high molecular weight nonkeratin component. This component could play a central role in the pathogenesis of MBs.

[Cytoskeleton--function and pathology: studies on the pathology of the intermediate filament cytoskeleton of liver cells]. / Cytoskelett--Funktion und Pathologie: Untersuchungen zur Pathologie des Intermediärfilament-Cytoskelettes der Leberzelle.

Microfilaments, microtubules and intermediate filaments (IF) are major filamentous components of the cytoskeleton and play a role in the modulation of cell shape, in cellular movements, cellular stability, intracellular organisation as well as cell-to-cell and cell-to-stroma interactions. Particular interest was concentrated in the last few years on IF because of their cell type-specificity and, consequently, their suitability as cell markers in diagnostic pathology. Despite their apparent stability, IF are dynamic structures which may be modified under pathologic conditions. In recent years, pathologic alterations related to the IF cytoskeleton have been described in a diversity of chronic and degenerative disorders, including alcoholic hepatitis and neurologic diseases (e.g. M. Alzheimer, M. Parkinson). Our studies were particularly devoted to the elucidation of the pathogenesis of severe alcoholic liver injury (alcoholic hepatitis), which is associated with inflammation, liver cell degeneration and necrosis and morphologically characterized by the appearance of cytoplasmic hyaline inclusions (i.e., Mallory bodies). In the present review morphologic, immunologic and biochemical studies on nature and pathogenesis of Mallory bodies are summarized. Moreover, similarities between Mallory bodies and other cytoskeleton-related inclusion bodies suggest common routes of pathogenesis. Consequently, studies along these lines may not only lead to the understanding of mechanisms involved in alcoholic injury but may also provide information on general principles of cell damage as well as on regulation and function of the IF cytoskeleton.

[Hepatitis delta virus infection: an immunohistochemical study]. / Hepatitis-Delta-Virus-Infektion: Eine immunhistochemische Studie.

Hepatitis delta virus (HDV) is a pathogenic and transmissible virus which requires the helper function of hepatitis B virus for its replication. Epidemiological studies have shown that HDV infection is distributed world-wide, with a higher prevalence in southern areas, especially in Italy. HDV may increase the severity of chronic hepatitis and may be responsible for the fulminant course of this disease. To assess the prevalence and pathogenic role of HDV infection in our geographic area we examined 118 biopsies from HBsAg-positive patients by immunofluorescence microscopy. HDV was detected in 3 biopsies with the histological features of chronic active hepatitis. Our findings show that HDV infection is a rare event among HBsAg carriers in our area.