Therapeutic liver repopulation for the treatment of metabolic liver diseases

Orthotopic liver transplantation is the treatment of choice for many inherited and acquired liver diseases. Unfortunately, the supply of donor organs is limiting and therefore many patients cannot benefit from this therapy. In contrast, hepatocyte suspensions can be isolated from a single donor liver can be transplanted into several hosts, and this procedure may help overcome the shortage in donor livers. In classic hepatocyte transplantation, however, only 1% of the liver mass or less can be replaced by donor cells. Recently though, we have used a mouse model of hereditary tyrosinemia to show that > 90% of host hepatocytes can be replaced by a small number of transplanted donor cells in a process we term "therapeutic liver repopulation". This phenomenon is analogous to repopulation of the hematopoietic system after bone marrow transplantation. Liver repopulation occurs when transplanted cells have a growth advantage in the setting of damage to recipient liver cells. Here we will review the current knowledge of this process and discuss the hopeful implications for treatment of liver diseases.     

Ultrastructural changes of liver parenchyma following digitonin-pulse perfusion of rat liver

Summary It has been shown that pulse perfusion of rat liver with a digitonin-containing medium results in a highly zonated hepatocyte permeabilization, allowing selective sampling of cytosolic constituents from periportal and perivenous (centrolobular) hepatocytes in situ. In the present paper we provide an ultrastructural evaluation of the perfusion method. Identical changes in hepatocytes from affected periportal and perivenous zones are found. Affected hepatocytes appear light (electron-lucent) in electron micrographs with a sharp transition to normal hepatocytes. The most conspicuous ultrastructural findings are: (1) transformation of the sinusoidal part of the light hepatocytes, the lipocyte processes and the endothelium of affected zones apparently unifying into a continuous layer dominated by disrupted plasma membranes and 7-nm filaments; (2) deposition of osmiophilic digitonin-cholesterol complexes along the sinusoidal plasma membranes of affected zones; and (3) reduction of the cytoplasmic matrix (cytosol) in the light hepatocytes, a dilation of the mitochondrial intermembrane space with a preserved mitochondrial matrix, and a dilation of cisternae of the granular endoplasmic reticulum. The ultrastructural findings are consistent with marker-enzyme activity measured in eluates from digitonin-perfused livers, except that lysosomes appear intact, apparently contrasting with the observed eluation of amyloglucosidase (Quistorff et al. 1985).     

Advances on liver cell-derived exosomes in liver diseases

Exosomes are extracellular vesicles with diameters ranging from 30 to 150 nm, which contain several donor cell-associated proteins as well as mRNA, miRNA, and lipids and coordinate multiple physiological and pathological functions through horizontal communication between cells. Almost all types of liver cells, such as hepatocytes and Kupffer cells, are exosome-releasing and/or exosome-targeted cells. Exosomes secreted by liver cells play an important role in regulating general physiological functions and also participate in the onset and development of liver diseases, including liver cancer, liver injury, liver fibrosis and viral hepatitis. Liver cell-derived exosomes carry liver cell-specific proteins and miRNAs, which can be used as diagnostic biomarkers and treatment targets of liver disease. This review discusses the functions of exosomes derived from different liver cells and provides novel insights based on the latest developments regarding the roles of exosomes in the diagnosis and treatment of liver diseases.     

清肝泄浊法治疗脂肪肝30例临床研究

目的:观察清肝泄浊法治疗脂肪肝的临床疗效.方法:治疗组30例口服自拟清肝泄浊汤,对照组24例口服复方益肝灵,观察肝功能及血脂变化.结果:治疗组30例,有效率90.0%,对照组30例,有效率36.6%.结论:清肝泄浊法治疗脂肪肝的疗效明显,适宜临床推广运用.     

Phosphorylation and inactivation of liver glycogen synthase by liver protein kinases

A rapid method for purifying glycogen synthase a from rat liver was developed and the enzyme was tested as a substrate for nine different protein kinases, six of which were isolated from rat liver. The enzyme was phosphorylated on a 17-kDa CNBr fragment to approximately 1 phosphate/87-kDa subunit by phosphorylase b kinase from muscle or liver with a decrease in the activity ratio (-Glc-6-P/+Glc-6-P) from 0.95 to 0.6. Calmodulin-dependent glycogen synthase kinase from rabbit liver produced a similar phosphorylation pattern, but a smaller activity change. The catalytic subunit of beef heart cAMP-dependent protein kinase incorporated greater than 1 phosphate/subunit initially into a 17-kDa CNBr peptide and then into a 27-30-kDa CNBr peptide, with an activity ratio decrease to 0.5. Glycogen synthase kinases 3, 4, and 5 and casein kinase 1 were purified from rat liver. Glycogen synthase kinase 3 rapidly phosphorylated liver glycogen synthase to 1.5 phosphate/subunit with incorporation of phosphate into 3 CNBr peptides and a decrease in the activity ratio to 0.3. Glycogen synthase kinase 4 produced a pattern of phosphorylation and inactivation of liver synthase which was very similar to that caused by phosphorylase b kinase. Glycogen synthase kinase 5 incorporated 1 phosphate/subunit into a 24-kDa CNBr peptide, but did not alter the activity of the synthase. Casein kinase 1 phosphorylated and inactivated liver synthase with incorporation of phosphate into a 24-kDa CNBr peptide. This kinase and glycogen synthase kinase 4 were more active against muscle glycogen synthase. Calcium-phospholipid-dependent protein kinase from brain phosphorylated liver and muscle glycogen synthase on 17- and 27-kDa CNBr peptides, respectively. However, there was no change in the activity ratio of either enzyme. The following conclusions are drawn. 1) Liver glycogen synthase a is subject to multiple site phosphorylation. 2) Phosphorylation of some sites does not per se control activity of the enzyme under the assay conditions used. 3) Liver contains most, if not all, of the protein kinases active on glycogen synthase previously identified in skeletal muscle.     

Expression of liver and placental alkaline phosphatases in Chang liver cells

This paper presents evidence that a protein characteristic of differentiated liver cells, liver alkaline phosphatase, is synthesized by the Chang liver cell line. Liver alkaline phosphatase was demonstrated by immumochemical assay, ³²P-labeling and polyacrylamide gel electrophoresis, immunofluorescence microscopy, and the fluorescence-activated cell sorter. The synthesis of the liver enzyme by the Chang liver cells is interpreted to indicate fidelity of the Chang cells to their origin from human liver tissue. Chang liver cells also synthesize a phosphatase which is similar if not indentical to the placental alkaline phosphatase. Since a placental-type alkaline phosphatase has been observed in a number of non-trophoblastic cell lines and also in some neoplasms, it does not seem reliable as an index of the origins of the cell line. Because of the claims that Chang liver cells are actually HeLa cells, HeLa cells were studied in tandem with the Chang cells. The results showed that the HeLa cells do not make the liver type phosphatase. The data are discussed in relation to the question of HeLa cell contamination of the Chang cell line and the validity of criteria normally used to identify cell lines.     

Cell-based therapy of acute liver failure: The extracorporeal bioartificial liver

The need for an alternative ttreatment to orthotopic liver transplantation for acute liver failure is a major issue, and systems capable of temporalily providing liver functions are being actively tested. Liver assist devices based on detoxication by dialysis or hemoperfusion through various membranes or cartridges proved to be inefficient because of their lack of metabolic function. An extracorporeal hybrid bioartificial liver might be an appropriate treatment, since it can provide liver-specific functions, maintain the patient alive, and allow spontaneous recovery of the patient's own liver or act as a bridge toward liver transplantation. Many devices have been proposed, including flat culture substrates, hollow-fiber bioreactors, or microcarriers, using xenogenic hepatocytes or hepatoma cell lines. Various drawbacks of these devices led us to attempt to develop a reliable extracorporeal bioartificial liver based on alginate bead-entrapped hepatocytes. This system was used successfully for the correction of the Gunn rat genetic defect, which results in lack of bilirubin conjugation. The development of this system for clinical purposes requires large yields of functional hepatocytes. We have isolated normal porcine hepatocytes by collagenase perfusion of the liver. Cells were immobilized in membrane-coated alginate gel beads, which were subsequently inoculated into a bioreactor. Porcine hepatocytes expreessed liver-specific functions at high levels, particularly protein neosysnthesis and enzymatic activities involved in detoxication and biotransformation processes. In addition, hepatocytes entrapped in coated alginate beads were isolated from immunoglobulins. This system represents a promising tool for the design of anoartificial liver in human beings.Abbreviations ALF acute liver failure - EBAL extracorporeal bioartificial liver - OLT orthotopic liver transplantation     

Needle biopsy of the liver for the diagnosis of nonneoplastic liver diseases

The results of 209 liver biopsy needle washings were compared with the corresponding histologic findings. An effort was made to distinguish major categories of liver disease on the basis of cytologic findings. Pigment identification and fat quantitation were also evaluated. It was found that it was often possible to determine whether a liver needle washing was normal or abnormal; however, it was rarely possible to provide specific diagnoses. Pure fatty change and primary cholestasis could be reliably differentiated from the steatosis and bile plugging seen in other liver disorders. Cirrhosis could be suggested in a limited number of cases, although in most cases the findings were nonspecific. Cytology was found to be less reliable than biopsy in the discrimination between the major pigments. Finally, cytology was an acceptable method for detecting and quantitating fatty change.