Activation energy, relative substrate specificity and optimum pH of guanase from humanrat, mouse and guinea pig sera and tissues.

Some properties of serum and tissue guanase from man, rat, mouse and guinea pig have been studied. Serum guanase differs from tissue guanase in activation energy in the human species and in guinea pig. The mouse and rat brain enzyme differs from the liver and kidney enzyme. The guanase of each species differs from the enzyme of the other species in one or more properties.     

Characterization of antibody against human liver guanase by immunoblotting and immunohistochemical staining of human liver guanase by a direct labeling antibody-enzyme method

Human liver guanase was purified and a specific antibody against it was raised in rabbits. The antiserum formed a single precipitin line with human liver extract, and also completely inhibited the activity of the liver enzyme. An immunoblotting study showed that the antibody bound specifically to one band of protein with guanase activity and not to other proteins. Therefore, we concluded that this antiserum against the liver enzyme was suitable for use in immunohistochemical demonstration of guanase. In tissue sections, the immunohistochemical reaction with this antibody was positive in the same locations as the histochemical guanase reaction with DAB (3,3'-diaminobenzidine tetrahydrochloride).     

Intracellular and extracellular activity of cathepsin D in the liver in cirrhosis and its involution

The distribution of cathepsin D in liver with CCl4 induced cirrhosis and its involution in rats was investigated by ultrastructural cytochemistry. Besides intracellular, it was revealed the extracellular activity of cathepsin D. The reaction product was on collagen fibers near the hepatocytes and connective tissue cells as well as on the hepatocytes microvilli and on the outside part of cellular membrane of connective tissue cells (macrophage, fibroblast, Ito cells). Hence the source of extracellular cathepsin D in liver are the parenchymatous as well as nonparenchymal cell elements. The results testify that under the cirrhosis and its involution, the cathepsin D takes part in intracellular proteolysis and is secreted by hepatocytes and connective tissue cells in the intracellular space; it also takes part in extracellular catabolism of connective tissue.     

Acid phosphatase distribution in the liver in cirrhosis

The distribution of acid phosphatase in liver cirrhosis, as well as in its reverse development, was investigated in mice using histochemistry and electron histochemistry methods. Histochemistry demonstrated a sharp activity increase of acid phosphatase (as compared with the same in the material of partial hepatectomy) in liver cells (especially hepatocytes) during liver cirrhosis regression 10 days after a partial hepatectomy. Electron histochemistry has shown the enzyme withdraw out of hepatocytes and connective tissue cells of fibrotic stratum in the extra-cell medium. The reaction product localized on the neighbouring collagen fibres giving evidence that during reverse development of liver cirrhosis the lisosomal enzyme release from specified cells by means of exocytosis and they are involved in the lysis of collagen.     

Immunohistochemical localisation of arginase in human liver using monoclonal antibodies against human liver arginase

Monoclonal antibodies against human liver arginase were raised in order to determine the exact distribution of arginase in human liver using a modified indirect unlabelled immunoperoxidase method. In normal human liver specific immunohistochemical staining was found in the cytoplasm of hepatocytes. Portal components (bile ducts and veins) and fibrous tissue were non-reactive, while erythrocytes were slightly positive. The specificity of the immunological reaction was confirmed by control tests. Spectrophotometry was used to quantitate the immunohistochemical reaction product, and the results indicated that arginase is homogeneously distributed in the liver lobule.     

Evaluation of serum guanase in hepatic diseases

Serum guanase activity was measured in 20 healthy adults and in 62 patients with acute viral hepatitis, chronic active hepatitis, chronic persistent hepatitis, liver cirrhosis and fatty liver. Guanase and gamma-GT in patients were elevated in 87 and 64%, respectively. Elevated guanase activities were found in most cases of acute viral hepatitis, as well as in chronic hepatopathies. In patients with acute viral hepatitis pathologic activities of guanase were found following partial or total normalization of other liver function tests.     

Cryopreservation of hepatocytes: a review of current methods for banking

Cryopreservation, the freezing of hepatocytes in liquid nitrogen for storage, has been investigated for many years, as a method of long-term storage for hepatocytes. Unfortunately an agreed acceptable protocol has been elusive, in part due to the susceptibility of hepatocytes to the freeze thaw process involved. A method for long-term storage (months, possibly years) of human hepatocytes, in particular, is desirable for the development of a clinically applicable bioartificial liver, hepatocyte transplantation and for pharmacotoxicological research. The sources of human liver tissue from which hepatocytes can be derived are limited. Many groups have modified and improved the process of cryopreservation and many new techniques have been published, including the incorporation of such cryopreserved cells in clinically based studies. Further evaluation is still required to develop a universally acceptable protocol. This article reviews the difficulties involved in cryopreserving hepatocytes for banking and examines recent technical advances within this field.     

Hepatitis B virus surface and core antigens in the liver of primary hepatocellular carcinoma cases in Virginia

Zenker-fixed paraffin-embedded sections of biopsy liver tissue from 64 cases of primary hepatocellular carcinoma (PHC) were stained for hepatitis B surface antigen (HBsAg) and for hepatitis B core antigen (HBcAg) by histochemical and/or immunohistochemical techniques in a retrospective study. PHC arose in livers with postnecrotic cirrhosis in 30 (46.9%) cases. Controls included liver biopsy sections from 123 miscellaneous liver disorders and from 67 randomly selected autopsy specimens, none of which were known to be associated with hepatitis B virus (HBV) infection. HBsAg was detected in tumorous hepatocytes in only one of the 64 cases of PHC. HBsAg was identified in nontumorous hepatocytes of 8 (20%) of 40 specimens that contained adequate nontumorous liver tissue. All of these HBsAg positive cases of PHC were associated with cirrhosis. Thus HBsAg was detected in 8 (33.3%) of 24 cases of PHC with cirrhosis, but in none of the remaining 16 cases without cirrhosis. HBcAg was not detected in the hepatocytes of those HBsAg positive PHC cases tested. Our results suggest that HBV infection may successively lead to chronic hepatitis, cirrhosis and ultimately PHC.     

Immunohistochemical localisation of arginase in human liver using monoclonal antibodies against human liver arginase

Summary Monoclonal antibodies against human liver arginase were raised in order to determine the exact distribution of arginase in human liver using a modified indirect unlabelled immunoperoxidase method. In normal human liver specific immunohistochemical staining was found in the cytoplasm of hepatocytes. Portal components (bile ducts and veins) and fibrous tissue were non-reactive, while erythrocytes were slightly positive. The specificity of the immunological reaction was confirmed by control tests. Spectrophotometry was used to quantitate the immunohistochemical reaction product, and the results indicated that arginase is homogeneously distributed in the liver lobule.Present address: Biologisches Institut der Universität Stuttgart, Ulmerstrasse 227, D-7000 Stuttgart 60, Federal Republic of Germany     

Extracellular breakdown of collagen as affected by lysosomal enzymes during the involution of liver cirrhosis

Electron microscopy and electron histochemistry (exposure to acid phosphatase) were used to study the mechanisms of extracellular degradation of collagen in the liver during involution of experimental cirrhosis. The following results were obtained: extracellular secretion of lysosomal enzymes from hepatocytes and connective tissue cells takes place in liver cirrhosis and its involution; partial hepatectomy during liver cirrhosis stimulates the activity of acid phosphatase in the liver cells; the lysosomal enzymes, excreted from hepatocytes and connective tissue cells by means of exocytosis take an active part in collagen extracellular degradation in vivo; at initial stages of cirrhosis involution extracellular degradation of collagen in the liver occurs at the expense of lysosomal enzymes from hepatocytes and connective tissue cells. Subsequently, as cirrhosis regresses, the principal role in the lysis of collagen gradually passes to lysosomal enzymes of hepatocytes.     

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen ¹.Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77±10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.     

Isolation and primary culture of adult human hepatocytes

Summary Biopsy tissue of adult human liver was gently dissociated with collagenase followed by Dispase. By repeated low g centrifugation, a large number of almost pure, viable hepatocytes was obtained. This is the first report of a successful procedure for obtaining adult human hepatocytes for study in tissue culture. The isolated cells have the typical morphology of liver parenchyma, and these characteristics persist throughout the period of culturing. Evidence of their function is indicated by albumin synthesis. This procedure is now being used to study human hepatocyte functions in vitro and the effects of a variety of agents including carcinogens and viruses.     

An improved technique for dissociating hepatocytes from fixed mouse liver for cytochemistry

A refined version of a method described previously for dissociating hepatocytes from fixed liver is described. The objective was to develop a procedure that would dispense with the postosmication of previously fixed tissue. In the new procedure fixed liver blocks are wrapped with a quadruple layer of nylon cloth, and, by squeezing them in a buffer solution, individually dissociated cells pass through the cloth without significant damage. The procedure makes it possible to dissociate liver tissue fixed with modified Karnovsky's fixative, Zamboni's fixative or cacodylate buffered glutaraldehyde. The subsequent compatibility of the single cells obtained with many cytochemical procedures has been confirmed.     

An Improved Technique for Dissociating Hepatocytes from Fixed Mouse Liver for Cytochemistry

A refined version of a method described previously for dissociating hepatocytes from fixed liver is described. The objective was to develop a procedure that would dispense with the postosmication of previously fixed tissue. In the new procedure fixed liver blocks are wrapped with a quadruple layer of nylon cloth, and, by squeezing them in a buffer solution, individually dissociated cells pass through the cloth without significant damage. The procedure makes it possible to dissociate liver tissue fixed with modified Karnovsky's fixative, Zamboni's fixative or cacodylate buffered glutaraldehyde. The subsequent compatibility of the single cells obtained with many cytochemical procedures has been confirmed.     

Dynamics of acid phosphatase activity in the liver in the process of involution of cirrhosis

Changes in the total activity of acid phosphatase in the liver as well as changes in the enzyme activity in hepatocytes and connective tissue cells of fibrosis layers were investigated, using quantitative histochemical method, in the process of mouse cirrhosis involution. After discontinuation of CCl4 injection, the animals with cirrhosis were divided into two groups. In the first group the resection of the left lobe of the liver was performed. The animals of the second group were not subject to operation. The results demonstrate that there is a close correlation between lysosomal hydrolase activity of hepatocytes and connective tissue cells of the liver and collagen resorption during cirrhosis involution. The most intensive lysis of collagen takes place within the first three weeks of cirrhosis involution in both experimental groups. Partial resection in cirrhosis has no significant effect on the changes and level of total activity of lysosomal hydrolase enzymes in the liver during cirrhosis involution.     

Differential regulated expression of keratinocyte growth factor and its receptor in experimental and human liver fibrosis

BACKGROUND AND AIM: Immunomodulatory and protective properties have been identified for the keratinocyte growth factor (KGF). For hepatocytes, pro-proliferative and anti-apoptotic effects of this growth factor have been reported in vitro. This study was designed to characterize a putative role of KGF in observed histomorphological changes in both, human and experimental liver fibrosis. METHODS: Liver fibrosis and cirrhosis was induced in rats by repetitive exposure to phenobarbitone and increasing doses of carbon tetrachloride. Human samples were obtained from patients undergoing surgery for partial hepatectomy or transplantation. Organ samples were scored for inflammation and morphological changes. Expression of KGF and its receptor (KGFR) mRNA was quantified by real-time RT-PCR. Protein expression and receptor phosphorylation was determined by Western blot analysis. In-situ hybridization and immunohistochemistry were utilized to determine distribution of KGF and KGFR in the liver. RESULTS: Expression of KGF was significantly increased in damaged liver tissue in correlation to the degree of fibrosis, whereas expression of the receptor was up-regulated in early stages of liver fibrosis and down-regulated in cirrhotic organs. Protein expression of this growth factor and its receptor correlated with the alterations in mRNA. KGF expression was restricted to mesenchymal cells, whereas expression of KGFR was detected on hepatocytes only. CONCLUSION: The expression of KGF and KGFR is differentially and significantly regulated in damaged liver tissue. This growth factor might therefore not only contribute to morphological alterations but also regeneration of liver parenchyma most likely mediated by indirect mechanisms of action.     

Lysosome participation in collagen resorption by hepatocytes during regression of liver cirrhosis

Electron microscopy and electron histochemistry employed in experiments on mice with liver cirrhosis regression have demonstrated the presence of vacuoles with collagen in the cytoplasm of hepatocytes as well as the presence in these vacuoles of a reaction product to acid phosphatase. Based on the data obtained it is concluded that during liver cirrhosis regression, there takes place an intracellular resorption of collagen by hepatocytes via phagocytosis with an active involvement in this process of lysosomal enzymes.     

Collagenase of hepatocytes and sinusoidal liver cells in the reversibility of experimental cirrhosis of the liver

In order to explore the cellular source(s) and the behaviour of the collagenolytic activity previously described in rat liver homogenates, in the reversibility of experimental cirrhosis of the liver, enriched suspensions of hepatocytes and of sinusoidal liver cells were obtained by a procedure which employs low EDTA concentrations and no bacterial collagenase. Cell suspensions were prepared from three different groups of animals: 1) normal controls, 2) rats with CCl4-induced cirrhosis of the liver, and 3) rats with swine serum-induced cirrhosis of the liver. Animals were sacrificed in each group upon completion of treatment and also after 3, 6 and 12 months. In each liver wet weight and collagen concentration were determined, and collagenolytic activity of both enriched cell suspensions was measured separately. In addition, histological studies of liver tissue and ultrastructural examination of cell suspensions were performed by standard procedures. Enriched suspensions of both normal hepatocytes and sinusoidal liver cells display Ca2(+)-dependent collagenolytic activities. Both cell suspensions obtained from each of the two types of cirrhotic livers show normal or slightly increased average levels of collagenase activity at the time of treatment discontinuation, when average liver collagen content ranges from 6 to 10-fold over normal, suggesting that the normal collagenase/collagen ratio is disturbed and that collagenolytic activity is deeply decreased in relation to the actual liver collagen load.(ABSTRACT TRUNCATED AT 250 WORDS)