Human and rat hepatic stellate cells express neurotrophins and neurotrophin receptors.

The expression of neurotrophins and neurotrophin receptors in non-neural tissue is related to tissue remodeling, differentiation, proliferation and migration of target cells. The literature yields contradictory results on neurotrophin and neurotrophin receptor expression in the liver. We show immunoreactivity to antibodies to nerve growth factor (NGF), brain-derived neurotrophin (BDNF), neurotrophin 3 (NT-3), neurotrophin 4/5 (NT-4/5), the low-affinity nerve growth factor receptor p75 and the high-affinity tyrosine kinase receptors (Trk) B and C in hepatic stellate cells and weak reactivity for BDNF, NT-3, and NT-4/5 in hepatocytes, in cryosections of human and rat liver, in normal and varying pathologic conditions. Immunoreactivity is unequivocally localized to hepatic stellate cells by double staining with alpha-smooth muscle actin (alpha-SMA) and desmin, studied by confocal laser scanning microscopy. Finally, the presence of mRNA transcripts for the different neurotrophins and neurotrophin receptors, with the exception of Trk-B, is shown by reverse transcription polymerase chain reaction (RT-PCR) on RNA extracted from freshly isolated rat hepatic stellate cells, compared with hepatocyte RNA. Hepatocyte RNA was found to contain BDNF, NT-3, NT-4/5 mRNA (which is compatible with the immunohistochemical findings) and Trk-A mRNA. In conclusion, hepatic stellate cells are a source of several neurotrophins in the liver and they express neurotrophin receptors. These findings correspond with the known involvement of hepatic stellate cells in tissue remodeling, their production of extracellular matrix components and their proliferation in acute necrotizing liver pathology. In analogy with findings in other organs and systems, neurotrophins are hypothesized to play a role in the pathophysiology of liver disease.

Expression of cytokeratin 20 in developing rat liver and in experimental models of ductular and oval cell proliferation.

BACKGROUND/AIMS: Recently, a novel type of cytokeratin (CK) has been added to the classical catalog of CKs as CK20. The aim of the present study was to examine the immunoreactivity for CK20 in normal and developing rat liver and in experimental models of bile ductular and oval cell proliferation. METHODS: Eighty-five Fischer rats, subdivided into five groups, were utilized in this study: fetal rats, ranging from day 10 to day 21 of gestation; newborn-neonatal rats, from 2 h to 10 days of age; bile duct ligated (BDL) rats; alpha-naphthyl-isothiocyanate (ANIT)-treated rats; and rats fed a choline-deficient diet containing N-2 Fluorenylacetamide (CD-AAF rats). Frozen sections from each liver were stained with the CK20 specific monoclonal antibody IT-Ks20.10. RESULTS: The present study shows that CK20 is a "bile duct type" CK. In the fetal rat, CK20 appears late during intrahepatic bile duct development, at day 20 of gestation. A marked increase in CK20 expression occurs after birth, suggesting that intrahepatic bile duct maturation continues after birth and that CK20 may be considered as a "maturation" marker of the biliary tree. In BDL rats and in ANIT-treated animals, immunoreactivity of bile ductules for CK20 was strikingly heterogeneous. A variable number of proliferating biliary cells were weakly positive or negative for CK20 and their number increased with the duration of the obstruction or ANIT treatment. In CD-AAF-treated rats, we found a uniform staining of proliferating oval cells for CK20. This finding is in contrast with the observation in BDL and in ANIT groups, and suggests the existence of different mechanisms regulating the proliferation and differentiation of biliary cells under those conditions. CONCLUSIONS: In rat liver, CK20 may be added to the list of "bile duct type" cytokeratins. During development, CK20 expression may be related to the maturation stage of the biliary tree. Typical ductular proliferation induced by BDL or ANIT feeding clearly differs from the oval cell proliferation in the CD-AAF model in terms of immunoreactivity for CK20.

Ludwig symposium on biliary disorders--part I. Pathogenesis of ductal plate abnormalities.

Intrahepatic bile ducts (IHBDs) develop from bipotential liver progenitor cells in contact with the mesenchyme of the portal vein and thus form the "ductal plates." The ductal plates are remodeled into mature tubular ducts. Lack of remodeling results in the persistence of periportal epithelial sleeves or "ductal plate malformation" (DPM). A proposal is that virtually all congenital diseases of IHBDs represent examples of DPM. Some early, severe types of extrahepatic bile duct atresia are characterized by DPM, a suggestion of a prenatal beginning of the disease. Several congenital diseases are characterized by dilatation of segments of IHBDs and variable degrees of fibrosis. Such "fibrocystic diseases" represent DPM at different levels of the biliary tree. Autosomal recessive polycystic kidney disease represents DPM of interlobular bile ducts, associated with tubular dilatation of collecting renal tubules. Congenital hepatic fibrosis may derive from the same type of liver lesion, through a superimposed destructive type of cholangiopathy associated with scarring fibrosis. Caroli's disease represents DPM of the larger IHBDs, whereas Caroli's syndrome combines the lesions of Caroli's disease and congenital hepatic fibrosis. von Meyenburg complexes represent DPM of smaller interlobular ducts; their dilatation gives rise to the liver cysts in autosomal dominant polycystic kidney disease. Finally, DPM is a component of the tissue abnormalities in so-called mesenchymal hamartoma.

Histopathology of chronic cholestasis and adult ductopenic syndrome.

This article reviews the histopathologic liver alterations in chronic cholestatic diseases and parenchymal changes associated with periportal ductular reaction and fibrosis, which may result in end-stage biliary cirrhosis. Adult ductopenic diseases are possible causes of such chronic cholestasis. Ductopenia is defined, and guidelines are given for its quantitation. The last section summarizes the specific histologic lesions of primary biliary cirrhosis, primary sclerosing cholangitis, their histologic mimics, and the differential diagnosis with drug-induced cholestasis and ductopenia.

Histological classification of chronic hepatitis.

The 1968 classification of chronic hepatitis distinguished cirrhotic and non-cirrhotic stages, and classified the disease according to the histological degree of disease activity into chronic persistent and chronic aggressive (active) varieties. This seemed appropriate at a time when the aetiology of chronic hepatitis was unknown, and presumed to be auto-immune. Immunosuppressive treatment was reserved for more severe variant of the disease, thus validating the usefulness of the classification. Over the past thirty years, several aetiologies were discovered for chronic hepatitis: the hepatitis viruses B, D, C, and G, side-effects of several therapeutic drugs, and autoimmune hepatitis. This progress created a growing need for a new or adapted classification of chronic hepatitis, since different aetiologies require divergent therapeutic approaches. In 1994, proposals for a new classification of chronic hepatitis came from two international organizations: the International Association for the Study of the Liver and the World Congresses of Gastroenterology. The essence of the proposals includes: primary classification according to aetiology, and determination of disease severity (grading) and stage of progression (staging). Several semiquantitative scoring systems for histological grading and staging of liver biopsies from patients with chronic hepatitis are available. Semiquantitative scoring is useful in the evaluation of new treatment regimens, and in comparing pre- and posttreatment biopsies. It is not indicated in the routine reporting of liver biopsies.

Zinc content and distribution in the newborn liver.

The newborn liver is a proven model for the study of liver storage of copper and iron. We analyzed zinc concentration and distribution in the livers of newborns and infants using a systematic tissue-sampling technique. We studied 14 newborns of 26-41 weeks of gestation (WG). One stillborn, and three infants (52-90 days old). At autopsy, a longitudinal liver slice extending from the right to the left lobe was subdivided into 10 samples that were analyzed for zinc concentration by atomic absorption spectroscopy. The mean zinc concentration in the newborn liver was 639 micrograms/g of dry tissue (dt). A striking interindividual variability in zinc liver stores was observed; the hepatic concentration of the metal ranged from 300 to 1,400 micrograms/g dt. We found a correlation between zinc liver content and gestational age. In newborns of 27-32 WG, the hepatic zinc concentration was significantly higher (p < 0.01) than in newborns of 34-41 WG. Zinc stores decreased in the postnatal period; in the infant group, the mean liver zinc concentration was 148 micrograms/g dt. The analysis of zinc concentration in 10 blocks from each liver revealed a regular distribution of the metal, without significant differences between liver lobes. Our data show that the newborn liver can be considered an interesting model for the study of zinc storage, which appears to correlate inversely with gestational age. From a practical point of view, the observed regular distribution of zinc implies that, at least in this model, zinc content determined in a small liver sample is representative of zinc content in the whole liver.

Desmin expressing nonhematopoietic liver cells during rat liver development: an immunohistochemical and morphometric study.

The expression and cellular distribution of desmin, alpha-smooth muscle actin (A-SMA) and cytokeratin no. 8 (CK-8) and no. 18 (CK-18) in normal adult, neonatal and fetal rat liver were examined immunohistochemically on cryostat sections. At days 14 and 15 of gestation, nonhematopoietic cells in embryonic liver were strongly desmin-positive, and some of the cells, mainly located in the periphery, were also stained with anti-A-SMA. Desmin immunoreactivity gradually decreased from day 16 of gestation. A close association of desmin-positive cell processes with hematopoietic cells was observed during fetal and early neonatal development. From day 16 of gestation the pre-hepatocytes became desmin-negative, remained CK-8 and CK-18 positive. Desmin-expressing cells were numerous in the liver from the embryonic period to the neonatal age. However, their absolute number per unit area, as well as their number relative to hepatocytes, decreased with age. We suggest that desmin-positive cells in embryonic liver may act as stromal cells in the hepatic hematopoietic microenvironment and support hepatocyte development.

Histopathology of cholestasis.

Cholestasis may be extrahepatic or intrahepatic in origin. The block in bile secretion may be complete or incomplete to variable extent. Complete cholestasis occurs in case of primary parenchymal disease (intrahepatic cholestasis) or total obstruction of extrahepatic bile ducts (extrahepatic cholestasis). Incomplete block in bile secretion is due to incomplete obstruction of intra- or extrahepatic bile ducts (intra- or extrahepatic cholestasis or both). Histologically, it is useful to distinguish between bilirubionstasis and cholate-stasis. Complete secretory block causes as early changers: bilirubinostasis (in hepatocytes, canaliculi and Kupffer cells) in acinar zone 3, and "ductular reaction" in acinar zone 1. The latter refers to an increase in periportal ductular profiles, associated with neutrophil infiltration. With longer duration of cholestasis, further lesions ensue: feathery degeneration of hepatocytes due to retention of detergent bile acids, cholestatic liver cell rosettes representing a shift from hepatocellular to biliary differentiation, xanthomatous cells reflecting hyperlipidemia, cholate stasis in acinar zone 1 due to overload of membrane-damaging bile acids, eventually paraportal bile infarcts, and progressive ductular reaction. The latter may be due to multiplication of pre-existing ductules, to metaplasia of periportal hepatocytes, or to activation of progenitor cells. It is invariably associated with periductular fibrosis: the pacemaker for increasing matrix deposition, resulting in biliary fibrosis and eventually in true biliary cirrhosis. Incomplete cholestasis (e.g. PBC, PSC) is characterized by absence of bilirubinostasis during long periods of time, whereas the afore mentioned features of chronic cholestasis do appear. Hence follows that the most reliable markers of chronic incomplete cholestasis are cholate stasis, cholestatic rosettes and ductular reaction. Bilirubinostasis is only a late and often ominous sign.

Flucloxacillin-associated hepatic injury.

Eleven cases of hepatic injury attributed to the intake of flucloxacillin were reported to the Netherlands Center for Monitoring of Adverse Reactions to Drugs between 1982 and 1992. They concerned four men and seven women, with a mean age of 57 years, treated for 2-28 days with an oral dose varying from 1500-4000 mg per day. Symptoms mostly appeared 10 to 30 days after starting treatment with flucloxacillin. Biochemically, the pattern was compatible with cholestatic hepatitis in seven cases, with a mixed cholestatic-hepatocellular type of injury in one case, a hepatocellular pattern in two cases, and mild liver enzyme elevations in one patient. Two patients died, one due to fatal bleeding from the liver after biopsy, and the second patient to a combination of hepatic and cardiac failure. The other patients recovered, on average 72 days after peaking of serum aminotransferase values. Histology in seven cases showed cholestatic hepatitis in five, with cholangitis or cholangiolitis in four of these patients. In the other two patients, there was centrilobular cholestasis with extensive bridging fibrosis and portal-central bridging necrosis, respectively.

Whipple's disease: a histological, immunocytochemical, and electron microscopic study of the small intestinal epithelium.

At endoscopy, the duodenum in Whipple's disease frequently appears abnormal and some clinical features such as gastrointestinal blood loss and anaemia suggest epithelial damage. However, the intestinal epithelial cells themselves appear to be normal on light and electron microscopy. The aims of this study were to analyse in detail the cytological changes in epithelial cells over time and in response to therapy in biopsies obtained from 20 patients, to investigate the functional repercussion on digestive enzymes such as lactase, and to assess the expression by the epithelial cells of MHC antigens. Cytological changes were minimal at both the light- and the electron-microscopic level and MHC class I expression was preserved. However, changes indicative of functional deficits were demonstrated. Lactase and MHC class II expression were reduced or even absent. Antibiotic therapy resulted in normalization within 3-6 months. These findings are consistent with the clinical evolution and are of interest with regard to the importance of the immune response in aetiopathogenesis.

Parathyroid hormone-related peptide expression in primary and metastatic liver tumours.

Parathyroid hormone-related peptide (PTHrP) is a major factor in the pathophysiology of hypercalcaemia of malignancy. Recent evidence suggests that PTHrP may play an important role in the growth and differentiation of neoplastic as well as non-neoplastic cells. PTHrP was originally detected in normal fetal, but not adult, liver. We have used immunocytochemistry to show that reactive human bile ductules expressing a neuroendocrine phenotype contain immunoreactive PTHrP. These observations raised the possibility that PTHrP immunoreactivity may be useful in the differential diagnosis of primary liver tumours and metastases of adenocarcinoma. A total of 24 primary liver tumours and 22 metastases of adenocarcinoma were studied. All cholangiocarcinomas showed immunopositivity for PTHrP and chromogranin A, while all hepatocellular carcinomas were negative for PTHrP and showed only focal and weak positivity for chromogranin A. Mixed types of primary liver tumour contained PTHrP immunoreactivity only in the areas of cholangiocellular differentiation. Moreover, all metastatic adenocarcinomas were negative for PTHrP and chromogranin A except for two out of five metastatic breast adenocarcinomas. These two patients had bone metastases and hypercalcaemia and thus did not yield differential diagnostic problems with cholangiocarcinoma. None of the patients with cholangiocarcinoma and hepatocellular carcinoma had hypercalcaemia. We conclude that PTHrP is a useful marker for primary cholangiocarcinoma, especially in the differential diagnosis of hepatocellular carcinoma and metastatic adenocarcinoma.

Alveolar soft-part sarcoma: evidence for its myogenic origin and for the involvement of 17q25.

A typical case of alveolar soft-part sarcoma was examined using ultrastructural, immunohistochemical and cytogenetic methods. Immunohistochemical stains were performed on frozen sections and showed strong desmin expression with the three anti-desmin antibodies used. In addition, the tumour cells were weakly positive for vimentin and myosin. Neural markers were negative. Chromosomal analysis showed consistent involvement of 17q25--an abnormality which has been reported in another alveolar soft-part sarcoma. The histogenesis of alveolar soft-part sarcoma is still debatable but our findings support a myogenic origin. The finding of an apparently identical chromosomal abnormality in two of three thus far examined cases of alveolar soft-part sarcoma is of interest and must await further confirmation, but it may result in the identification of a chromosomal marker for this enigmatic tumour and thus pave the way for further molecular elucidation.