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Biomedical subjects

M L Schilsky

Publications and source records attributed to M L Schilsky.

At least 19 recordsLinked to original sources
PubMed2001

Correction of liver disease following transplantation of normal rat hepatocytes into Long-Evans Cinnamon rats modeling Wilson's disease.

To establish the efficacy of cell therapy in Wilson's disease, we used the Long-Evans Cinnamon (LEC) rat model with atp7b gene mutation and copper toxicosis. Several groups of LEC rats were established, including animals pretreated with retrorsine to exacerbate copper toxicosis and inhibit proliferation in native hepatocytes followed by partial hepatectomy to promote liver repopulation. Hepatocytes from normal, syngeneic LEA rats were transplanted intrasplenically. Animal survival, biliary copper excretion, and hepatic copper were determined. The magnitude of liver repopulation was demonstrated by measuring serum ceruloplasmin and hepatic atp7b mRNA. Long-term survival in LEC rats treated with retrorsine, partial hepatectomy, and cell transplantation was up to 90%, whereas fewer than 10% of animals pretreated with retrorsine, without cell therapy, survived, P < 0.001. Liver repopulation occurred gradually after cell transplantation, ranging from <25% at 6 weeks, 26 to 40% at 4 months, and 74 to 100% at 6 months or beyond. Liver repopulation restored biliary copper excretion capacity and lowered liver copper levels. Remarkably, liver histology was completely normal in LEC rats with extensive liver repopulation, compared with widespread megalocytosis, apoptosis, oval cell proliferation, and cholangiofibrosis in untreated animals. These data indicate that liver repopulation with functionally intact cells can reverse pathophysiological perturbations and cure Wilson's disease.

Adenosine Triphosphatases↗
PubMed2001

Treatment of Wilson's disease: what are the relative roles of penicillamine, trientine, and zinc supplementation?

New options are available for the medical treatment of patients with Wilson's disease. Penicillamine is no longer the treatment of choice, as there is a growing experience with safer and more effective alternatives. Trientine may be the best choice for initial therapy in symptomatic patients requiring chelation therapy, and it may be even more effective when used in combination with zinc, which is recommended for maintenance therapy. Further studies are needed to determine the best therapy for pregnant patients with Wilson's disease, and whether combination therapy using trientine and zinc will be the next treatment of choice for all symptomatic patients with liver or neurologic disease.

Adenosine Triphosphatases↗
PubMed2001

Metabolic liver disease.

The discovery of novel metabolic pathways and the genetic basis for diseases of the liver continues to yield new insights into the pathogenesis of inherited metabolic diseases of the liver, whereas the application of new technologies to their treatment continues to advance therapeutic options. This review of selected articles covers a wide range of subjects, from the identification of novel proteins and transport pathways to disease diagnosis and treatment of acute liver failure. Four selected topics, Wilson disease, hemochromatosis and iron overload disorders, alpha-1 antitrypsin disease, and exciting new therapeutic options for lysosomal storage diseases are the focus of this review.

Journal Article↗
PubMed2000

Biliary copper excretion capacity in intact animals: correlation between ATP7B function, hepatic mass, and biliary copper excretion.

Copper toxicosis can occur in the absence of biliary copper excretion. To demonstrate whether biliary copper excretion capacity is correlated with hepatic mass and ATP7B function, we undertook studies in intact animals. Copper-histidine was injected intrasplenically after baseline bile collection, followed by measurement of copper excretion in Long-Evans Cinnamon (LEC) rats lacking atp7b function and in normal, syngeneic Long-Evans Agouti (LEA) rats. The basal biliary copper excretion was very low in LEC rats compared with LEA rats, 8+/-4 and 37+/-18 ng copper/min, respectively; p<0.05. After addition of copper, copper excretion increased significantly (by two- to five-fold) in LEA rats during the 30 minute study period, whereas LEC rats showed only a slight and transient increase in copper excretion. After one-third and two-thirds partial hepatectomy immediately before copper loading, copper excretion decreased progressively. The studies indicate that biliary copper excretion depends on hepatocyte mass and ATP7B gene function. Analysis of copper excretion with our non-radioactive method will facilitate testing of novel therapies and pathophysiological mechanisms in copper toxicity.

Adenosine Triphosphatases↗
PubMed2000

Inherited metabolic disease.

This review focuses on two genetic disorders of metal metabolism, genetic hemochromatosis and Wilson disease, and on the most common lysosomal storage disorder, Gaucher disease, for which recombinant enzyme replacement therapy is available. The discovery of the genes for these disorders has led to an explosion of new information about the function of these gene products and the identification of other proteins involved in their metabolism. These discoveries have altered our current diagnostic and therapeutic approaches to these disorders and have furthered our understanding of disease pathophysiology. New modalities being developed for future use include cell transplant and genetic replacement therapies.

Journal Article↗
PubMed1999

Inherited metabolic disease.

Our understanding of the pathophysiology and of new treatments for inherited metabolic diseases that affect the liver continues to grow through the study of gene mutations and their functional effect on the proteins they encode. For genetic hemochromatosis and Wilson's disease, studies focused on the function of their respective gene products provide new insights into metal metabolism. For Crigler-Najjar syndrome, an inherited disorder that results in failure of proper bilirubin glucuronidation, the once futuristic idea of treatment by transplantation of donor hepatocytes has now proven successful in a human recipient. With continued study and experimentation, our diagnostic and therapeutic capabilities will continue to expand for these and other inherited metabolic disorders. Although this increase in new information has sparked numerous reviews of these subjects, the following are highlights from the past year that include information relative to disease diagnosis and treatment, as well as new insights into pathogenesis.

Journal Article↗
PubMed1999

New knowledge of genetic pathogenesis of hemochromatosis and Wilson's disease.

Discovery of the gene for WD has greatly enhanced our understanding of this disorder at the cellular level and has set the stage for future testing of new modes of therapy. Improvements in analytic methods for detecting mutations in genomic DNA will someday enable a rapid and cost-effective method of screening for this disorder. Until then, the time-tested clinical and biochemical evaluation, including measurement of ceruloplasmin oxidase activity, slit-lamp examination for Kayser-Fleischer rings, and measurement of hepatic copper content, will continue to remain the standard for establishing the diagnosis of WD.

Adenosine Triphosphatases↗
PubMed1998

Fractionation of rat hepatocyte subpopulations with varying metabolic potential, proliferative capacity, and retroviral gene transfer efficiency.

The liver contains hepatocytes with varying ploidy and gene expression. To isolate cells on the basis of ploidy for analyzing mechanisms concerning cell proliferation and differentiation, we used Percoll gradients to separate F344 rat hepatocyte subpopulations. Specific fractions were enriched in polyploid (H2 fraction) or diploid (H3 and H4 fractions) hepatocytes containing glycogen and glucose-6-phosphatase. H4 cells were relatively smaller with greater nuclear/cytoplasmic ratios, less complex cytoplasm, and higher serum albumin or ceruloplasmin biosynthetic rates. H2 fraction cells were larger with lesser nuclear/cytoplasmic ratio, more complex cytoplasm, and more cytochrome P450 activity. Phenotypic marking showed that H4 cells originated in zone one and H2 cells in zones two or three of the liver lobule. H4 cells showed much greater mitogenic responsiveness to human hepatocyte growth factor. Retroviral gene transfer, which requires both viral receptors and cellular DNA synthesis, was significantly more efficient in H4 cells. The findings indicated that small diploid and large polyploid hepatocytes show unique biological differences. The ability to isolate hepatocytes of varying maturity is relevant for mechanisms concerning liver growth control and hepatic gene expression.

Animals↗
PubMed1998

Identification of B10, an alkaline phosphodiesterase of the apical plasma membrane of hepatocytes and biliary cells, in rat serum: increased levels following bile duct ligation and during the development of cholangiocarcinoma.

Alkaline phosphodiesterase (APDE) is associated with the cellular plasma membrane of many organs. Several isoforms are also detected in normal human serum and their respective amounts vary in liver diseases but their significance is unknown. The aims of this study were: 1) to identify a serum form of B10, an APDE exclusively localized at the apical pole of the plasma membrane of rat hepatocytes and biliary cells; 2) to gain insight into its origin; and 3) to investigate its behavior, in two liver diseases in which an abnormal membrane expression of B10 has been reported, namely cholestasis and cholangiocarcinoma. A soluble form of B10 was immunoprecipitated from normal rat serum, which amounted to 13% of total serum APDE activity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the size of the serum enzyme was 125 kd, which is slightly lower than that found in the plasma membrane (130 kd). In bile, a 120-kd and a 130-kd form was found. A sixfold and fivefold increase of B10 APDE activity was observed in the serum of bile duct-ligated rats and in the Long-Evans Cinnamon (LEC) rats which spontaneously develop cholangiocarcinoma. The molecular size of the form present in serum was unchanged. A threefold increase was also observed in LEC rats which had not yet developed a cholangiocarcinoma. In conclusion, we identified a soluble form of B10 in normal rat serum. The increase in serum B10 in the experimental and pathological conditions investigated does not seem to result from passage of the biliary form to the serum but seems to be caused by increased cleavage of the membrane form. Its rise early during the onset of cholangiocarcinoma suggests that B10 in the serum might be a marker of carcinogenesis and/or be involved in the development of cholangiocarcinoma.

Animals↗
PubMed1998

Copper resistant human hepatoblastoma mutant cell lines without metallothionein induction overexpress ATP7B.

Mutant human hepatoblastoma cell lines resistant to copper toxicity were isolated from mutagenized HuH7. Two copper resistant cell lines (CuR), CuR 23 and CuR 27, had reduced basal expression of metallothionein (MT) messenger RNA (mRNA) and exhibited minimal or no increase in resistance to cadmium or zinc toxicity. Copper uptake, efflux of newly transported copper, glutathione content, and efflux rate were comparable with HuH7, whereas holoceruloplasmin synthesis and secretion were slightly decreased. Subcellular distribution of copper at steady-state showed an increase in organelle and membrane fractions with a reduction in cytosol. Expression of ATP7B mRNA was fivefold increased, and ATP7B protein approximately threefold increased in both CuR 23 and 27. Another cell line, CuR 41, showed increased basal expression of MT and ATP7B mRNA but not ATP7B protein, and resistance to cadmium and zinc toxicity. Copper uptake in CuR 41 was comparable with HuH7, but initial rates of efflux of copper and glutathione were reduced. The synthesis of holoceruloplasmin but not ceruloplasmin peptide was markedly diminished in CuR 41. Subcellular distribution of copper showed an increase in cytosolic and decreased organelle and membrane-associated copper. These data suggest that cellular resistance to copper toxicity was achieved in two independent cell lines without MT induction and that the induction of ATP7B may lead to the enhanced intracellular sequestration of copper by organelles.

Adenosine Triphosphatases↗
PubMed1998

ATP7B (WND) protein.

Wilson's disease is a genetic disorder of copper metabolism characterized by the excessive accumulation of this metal in the liver. The gene for Wilson's disease, designated ATP7B, encodes a copper transporting P-type ATPase expressed predominantly in the liver. Over 60 disease specific mutations of ATP7B have now been reported in patients with Wilson's disease. The gene for ATP7B is approximately 80 kb and contains 21 exons that encode an approximately 7.5 kb transcript. Recent studies that focus on the structure and expression of the ATP7B protein support its role as a copper transporter involved in the intracellular trafficking of copper in hepatocytes. The introduction of functional ATP7B protein by recombinant adenovirus mediated gene delivery will be a potential approach for correcting Wilson's disease.

Adenosine Triphosphatases↗
PubMed1998

Spontaneous cholangiofibrosis in Long-Evans Cinnamon rats: a rodent model for Wilson's disease.

The Long-Evans Cinnamon (LEC) rat is a rodent model of Wilson's disease characterized by ceruloplasmin deficiency, hepatic copper accumulation, and hepatocellular injury. So far, the LEC rat appears to be the only strain in which cholangiofibrosis develops spontaneously. The aim of the study reported here was to characterize the time course of development and investigate the structural and ultrastructural features of cholangiofibrosis and their possible relationship to hepatic copper and iron content. The livers of 54 rats (22 males), ages 5 to 113 weeks, were examined by light microscopy and graded for statistical analysis, with respect to extent of replacement of liver tissue by cholangiofibrosis. The study was complemented by electron microscopy, and by measurements of copper and iron contents by atomic absorption spectroscopy. Cholangiofibrosis was present in LEC rats by 20 weeks of age. The hyperplastic biliary epithelial cells varied markedly in size and shape, ranging from flat to cuboidal or elongated. Epithelial cells did not exhibit characteristics of intestinal cells. Some basement membranes had splits, duplications, or multiplications. Cytoplasmic organelles within hyperplastic biliary cells appeared unremarkable in contrast to the characteristic mitochondrial abnormalities present in neighboring hepatocytes. There was a positive correlation between histologic grades of cholangiofibrosis and ages of the animals (r = 0.68, P < 0.001), but no significant correlation between histologic grade and hepatic copper or iron content. We conclude that cholangiofibrosis is the predominant pathologic response to chronic liver injury induced by excess copper in LEC rats. The pathogenic role of copper in the development of cholangiofibrosis requires clearer definition.

Adenosine Triphosphatases↗
PubMed1996

Wilson disease: genetic basis of copper toxicity and natural history.

The discovery that the gene for Wilson disease encodes a copper-transporting ATPase has greatly improved our understanding of the pathophysiology of this disorder and of copper metabolism in humans. The abundance of disease-specific mutations and their location at multiple sites across the genome have limited molecular genetic diagnosis to kindred of known patients, and confirm the necessity for de novo screening by well-proven clinical and biochemical means. It is uncertain whether the variety of specific mutations will account for the wide range of presenting clinical signs and symptoms of Wilson disease, and environmental and extragenic factors are likely to be important contributing factors. Chelation therapy with penicillamine and trientine remain effective treatment for most symptomatic hepatic and neurologic Wilson disease. Zinc salts may be used for some asymptomatic patients, and OLT for fulminant hepatitis and patients for whom pharmacotherapy is ineffective. The chelating agent tetrathiomolybdate is under investigation for the treatment of neurologic Wilson disease. Gene therapy is the new horizon for treatment of Wilson disease. However, the ability to treat this disorder effectively by this means awaits further characterization of the gene product and more efficient methods for gene delivery to all hepatocytes in the liver.

Adenosine Triphosphatases↗
PubMed1995

Secretion, surface localization, turnover, and steady state expression of protein disulfide isomerase in rat hepatocytes.

Protein disulfide isomerase in isolated rat hepatocytes was present at a concentration of 7 micrograms/mg cell protein, representing a approximately 2-fold enrichment compared to isolated hepatic non-parenchymal cells. Though localized mainly in microsomal fractions of hepatocytes, direct immunofluorescence and cell surface radioiodination followed by immunoprecipitation revealed the presence of M(r) 57,000 disulfide isomerase at the cell surface. Electrostatic interaction of the protein with the cell surface was suggested by susceptibility to carbonate washing. Metabolic radiolabeling and immunoprecipitation studies also indicated that some of the newly synthesized M(r) 57,000 disulfide isomerase was secreted. Treatment of cells with colchicine markedly reduced the recovery of disulfide isomerase from the media, indicating microtubular-directed secretion of the protein. Partial staphlococcal V8 proteolytic digestion of the secreted protein revealed a peptide pattern similar to that of the cellular protein. Immunoprecipitation with antibody specific to the -KDEL peptide retention sequence confirmed the presence of this sequence in the secreted protein. Studies of the turnover of disulfide isomerase revealed a half-life of approximately 96 h. Treatment of cells with tunicamycin or heat shock resulted in an increased recovery of newly synthesized disulfide isomerase from cell lysates but diminished recovery from the media. The secretion and cell surface distribution of disulfide isomerase in hepatocytes may be important for the pathogenesis of immune mediated liver injury.

Animals↗