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M A Zern

Publications and source records attributed to M A Zern.

At least 37 records · Page 2Linked to original sources
PubMed1998

The prospects of hepatic drug delivery and gene therapy.

Liver targeted therapy is designed to deliver a substance preferentially to the organ in order to increase the accumulation, improve the therapeutic effect and reduce toxicity to other organs. The aim of selective targeting is to deliver a substance to a specific cell type in the liver. A variety of vehicles have been designed and further modified for selective targeting of therapeutics to the liver. The targeting properties and strategies of commonly used agents, such as liposomes, microspheres and recombinant chylomicrons, are discussed. Viral and non-viral vectors, such as cationic liposomes, reconstituted chylomicron remnants, adenoviruses, adeno-associated viruses, retroviruses, and SV-40, are currently being evaluated for the delivery of DNA to the liver. New developments in improving the targeting efficiency of the available vectors while avoiding their disadvantages have made their use in clinical trials of various genetic disorders possible. For viral hepatitis, antisense and ribozyme techniques are being employed with selective targeting approaches. A commonly employed current strategy for targeting hepatocellular carcinoma cells is to make the tumour cells convert non-toxic 'prodrugs' to toxic metabolites in situ, achieving a high concentration of the toxic product in the local milieu, while avoiding systemic toxicity. Although gene therapy itself is in its infancy, some encouraging results have been developed in studies of familial hypercholesterolaemia, haemophilia, alpha1-antitrypsin deficiency and Crigler-Najjar syndrome. The potential strengths as well as the problems with these studies are discussed.

Journal Article↗
PubMed1997

Hepatic drug delivery and gene therapy.

On September 21-22, 1995, an international meeting entitled "Targeting of Novel Therapeutics to the Liver and GI Tract" was held at the Natcher Conference Center on the campus of the National Institutes of Health. The conference was sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases through the Division of Digestive Diseases and Nutrition and Digestive Diseases Interagency Coordinating Committee (DDICC). Section 440A of Public Law 94-562 in 1976 created the DDICC for the purpose of coordinating the digestive disease-related research activities of relevant federal health agencies into a coordinated program aimed at combating digestive diseases. As part of this federal effort, an assessment of the "state of the art" for targeted drug therapeutics to the liver and gene therapy was undertaken through the conference, cochaired by Dr. Mark Zern (Thomas Jefferson Medical College) and Dr. Flossie Wong-Staal (University of California, San Diego, CA). The conference was divided into four sessions: Session I was Vectors and Techniques; Session II was Liver and Metabolic Diseases; Session III was Hepatitis and GI Disease; and Session IV was Approaches for HIV Infection. This summary focuses on the new technologies and the studies directly pertaining to liver disease. Table 1 lists the techniques and their applications. Table 2 describes viral vectors that have been employed for the purpose of hepatic gene therapy. Table 3 summarizes the studies presented as posters at the conference.

DNA Repair↗
PubMed1997

A role for tissue transglutaminase in hepatic injury and fibrogenesis, and its regulation by NF-kappaB.

This study was undertaken to delineate a possible role for tissue transglutaminase (tTG), an enzyme that catalyzes protein cross-linking, in hepatic fibrogenesis. Rats were treated with CCl4 solution and then killed at different stages of liver injury and fibrogenesis. Liver tTG mRNA levels were markedly increased as early as 6 h after the first injection, peaked at 4 days and 1 wk, and remained increased for 8 wk. The enzymatic activity of tTG was increased in livers of rats treated with CCl4, in a fashion that paralleled the Northern blot results. Cell isolation experiments indicated that all hepatic cell types synthesize tTG mRNA. Increased binding to the nuclear factor-kappaB (NF-kappaB) motif of the tTG promoter was found in the nuclear extracts prepared from CCl4-treated samples. These data demonstrate an increase in tTG gene expression during hepatic injury and fibrosis, suggesting a possible role for this enzyme in stabilizing the fibrotic bands during hepatic fibrogenesis. Moreover, increased NF-kappaB binding to the tTG promoter may represent one of the mechanisms by which cell injury induces tTG transcription and thus potentiates the process of fibrogenesis.

Animals↗
PubMed1996

The antioxidant function of the physiological content of vitamin C.

The synthesis of vitamin C is substantially reduced in Osteogenic Disorder Shionogi (ODS) rats. Hepatocytes prepared from these rats contained approximately 12% of the wild-type content of this vitamin. In culture, the ascorbate content remained low in the absence of supplementation of the medium. Independent of their vitamin C status, cultured hepatocytes become depleted of vitamin E. Supplementation of the culture medium with 10C microM ascorbate and 1.2 microM alpha-tocopherol phosphate maintained the physiological content of both vitamins C and E in ODS hepatocytes. Thus, the antioxidant function of vitamins C and E could be evaluated in the presence of both or either vitamin or in the absence of both vitamins. Hepatocytes deficient in both vitamins were the most susceptible to lipid peroxidation (as measured by thiobarbituric acid) and to cell kllling within a 90-min exposure to 125-500 microM tert-butyl hydroperoxide (TBHP). Supplementation to achieve a physiological content of both vitamins C and E reduced the evidence of lipid peroxidation and abolished the cell killing. Supplementation with either vitamin alone resulted in an intermediate degree of both lipid peroxidation and cell killing. In ODS hepatocytes treated with TBHP, the decline in vitamin E preceded the decline in vitamin C. In ODS hepatocytes depleted of vitamin C, the loss of vitamin E after exposure to TBHP was greater than that in the presence of physiological levels of ascorbate. This greater loss of vitamin E in the face of a depletion of vitamin C was readily attributable to the increased peroxidation of lipids. Thus, the physiological level of vitamin C in cells does not seem to regenerate vitamin E. In contrast, the rate and extent of the depletion of vitamin C correlate with the degree of cell killing. These data document the antioxidant function of the physiological level of cellular vitamin C and relate this function to protection against peroxidative cell injury.

Animals↗
PubMed1995

Molecular biological aspects of alcohol-induced liver disease.

Molecular biological investigations have become a predominant methodology applied to the study of alcohol-induced liver disease. The enzymatic pathways responsible for ethanol metabolism, and their genetic as well as environmental control, have become the focus of detailed investigation. More recently, the significance of cytokines in the pathogenesis of alcohol-induced liver disease has also become a major area of speculation. This review focuses on the advances made in studies of two important enzymes responsible for alcohol metabolism, alcohol dehydrogenase and aldehyde dehydrogenase, as well as the investigation of the proinflammatory and profibrogenic cytokines involved in the process of hepatic fibrogenesis. The quality and quantity of new discoveries made in the field of alcohol-induced liver disease is impressive, especially when one realizes that molecular biological approaches have been employed in this area for only 15 years. However, in most cases the studies have been predominantly descriptive, with little direct relevance to the therapeutics of alcoholism and alcohol-induced organ injury. Because the groundwork has been laid, one hopes that the next 15 years will rectify this failure.

Alcohol Dehydrogenase↗
PubMed1995

Vitamin E therapy of acute CCl4-induced hepatic injury in mice is associated with inhibition of nuclear factor kappa B binding.

Oxidative stress, with reactive oxygen intermediate formation, may represent a common mechanism by which liver injury is induced by diverse etiologies. Oxidative stress enhances nuclear factor kappa B (NF-kappa B) activity, and NF-kappa B activity has been shown to enhance the expression of cytotoxic cytokines. Acute hepatic injury caused by reactive oxygen intermediate production was induced by an intraperitoneal injection of CCl4 in mice. This injury was significantly inhibited by intravenous pretreatment of the mice with a water-soluble emulsion of alpha-tocopherol. Alpha-tocopherol treatment of the mice given the CCl4 also reduced the NF-kappa B binding to levels approaching those found in normal mice. In vitro treatment of a monocyte/macrophage cell line with CCl4 led to enhanced NF-kappa B binding and an increase in tumor necrosis factor-alpha (TNF-alpha) messenger RNA levels. Liver specimens taken from patients with acute fulminant hepatitis had markedly increased NF-kappa B binding activity in comparison with the binding of normal livers. These data demonstrate that abolishing acute hepatic injury with alpha-tocopherol, a free radical scavenger, also eliminated increased NF-kappa B binding. It is tempting to speculate that enhanced NF-kappa B expression caused by free radical production/oxidative stress may modulate liver injury, perhaps through an effect on cytotoxic cytokine synthesis.

Animals↗
PubMed1995

Expression of SPARC in normal and fibrotic livers.

SPARC (secreted protein, acidic and rich in cysteine)--also known as osteonectin, BM-40, and 43K glycoprotein--is secreted by endothelial cells and fibroblasts in response to culture shock. SPARC has been found in association with tissues undergoing cell proliferation, migration, and extracellular matrix remodeling. We demonstrate that normal livers from humans, rats, and mice express substantial levels of SPARC messenger RNA (mRNA). Moreover, when compared with control specimens, significantly increased levels of SPARC mRNA were found in fibrotic livers from two animal models of liver disease: murine schistosomiasis and carbon tetrachloride-induced fibrosis in rats. Fibrotic human livers also had markedly increased levels of SPARC mRNA in comparison with normal livers. We also detected an increased production of SPARC protein in the liver of animals treated with carbon tetrachloride. By immunocytochemical analysis, SPARC protein was apparent in freshly isolated Ito cells. Hybridization studies showed Ito cells to be the main source of SPARC mRNA. Extracts from a Kupffer-endothelial cell fraction exhibited traces of SPARC transcript, but expression of SPARC mRNA was absent in extracts from freshly isolated hepatocytes. These studies demonstrate the increased expression of SPARC--a protein that modulates cell shape and disrupts cell-matrix interactions--during the initial stages of hepatic fibrosis.

Animals↗
PubMed1994

Gene expression of transforming growth factor beta 1 and extracellular matrix proteins in murine Schistosoma mansoni infection.

BACKGROUND/AIMS: The study of Schistosoma-induced hepatic fibrosis in murine Schistosoma mansoni infection has elucidated the nature of hepatic fibrosis in humans. In the present study, fibrogenic gene expression was determined in murine S. mansoni infection during primary infection, after chemotherapy with praziquantel, and during secondary infection. METHODS: Both histomorphometric analysis and Northern blot profiles were performed. RESULTS: Histomorphometric analysis of granulomatous inflammation showed smaller hepatic fibrotic granulomata after chemotherapy and during secondary infection. Albumin gene expression remained relatively constant throughout primary infection, chemotherapy, and secondary infection. Fibronectin gene expression in primary infection was comparable with the level observed in noninfected mice and was reduced by chemotherapy. Reinfection resulted in augmented expression levels equal to primary infection levels. Osteonectin gene expression was active in primary infection, was reduced by chemotherapy, and was actively reexpressed in secondary infection. Interstitial matrix macromolecules, types I and III collagen, and basement membrane collagen showed high levels of gene expression in primary infection, were virtually terminated by chemotherapy, and were reexpressed on reinfection. The gene expression of transforming growth factor beta 1, a major, fibrogenic cytokine, paralleled collagen expression. CONCLUSIONS: Chemotherapy of schistosomiasis initiated a dramatic decrease in steady-state messenger RNA levels of major proteins associated with fibrosis; reinfection resulted in a reexpression of these genes.

Animals↗
PubMed1994

FK506 enhances fibrogenesis in in vitro and in vivo models of liver fibrosis in rats.

BACKGROUND/AIMS: The immunosuppressant FK506 is undergoing clinical trials in transplantation and autoimmune diseases. FK506 modulates several cytokines and exerts hepatoprotection in acute models. This study was initiated to determine whether FK506 would be beneficial as an antifibrogenic agent. METHODS: Fibrosis was induced by carbon tetrachloride administration to rats. Half of those animals were treated with FK506. The rats were killed after 8 weeks of carbon tetrachloride treatment. The livers were evaluated by histology, Northern hybridizations, and collagen quantitation. Additionally, rat fibroblasts were incubated with and without FK506 and used for Northern hybridization analysis. RESULTS: Surprisingly, FK506 exacerbated hepatic inflammation and fibrosis. Increased hepatic collagen and higher messenger RNA levels of transforming growth factor beta 1 and collagens I, III, and IV were found in the FK506-treated group. Rat fibroblasts treated with FK506 expressed higher levels of collagens I and III, fibronectin, macrophage-colony stimulating factor, tissue inhibitor of metalloprotease, and transforming growth factor beta 1 messenger RNAs. CONCLUSIONS: These findings suggest that FK506 increases the expression of extracellular matrix genes and enhances fibrosis in the rat model. While further studies are needed to elucidate these profibrogenic mechanisms, caution is indicated for the unrestricted use of FK506 in patients subject to recurrent fibrogenic stimulation.

Animals↗
PubMed1994

Inhibition of carbon tetrachloride-induced liver injury by liposomes containing vitamin E.

We tested a variety of antioxidants as possible therapeutic agents in an acute CCl4 mouse model of hepatotoxicity. Liver damage, gauged by the amount of serum aminotransferase released into the blood, morphological changes, lethal dose response, and presence of thiobarbituric acid-reactive substances (TBARS), were significantly inhibited in a dose-dependent manner by liposomes containing vitamin E (LVE) or by Rocavit E, a water-soluble emulsion of alpha-tocopherol. Serum aminotransferase levels in LVE- or Rocavit E-treated animals were always > 10-fold lower than levels in corresponding CCl4 controls. Other liposome-associated antioxidants, butylated hydroxytoluene, vitamin E succinate, catalase, desferoxamine, superoxide dismutase, and ascorbic acid 6-palmitate, were also able to elicit a decrease in damage; however, they were substantially less effective. Intravenous therapy with LVE decreased mortality by nearly 90% when a lethal dose of CCl4 was given. When the biodistribution of the liposomes was examined, it was determined that the vast majority were localized in the Kupffer cell population. This approach of delivering nontoxic therapeutic agents selectively to the liver offers a variety of clinical applications in humans.

Animals↗
PubMed1994

Fish oil, alcohol, and liver pathology: role of cytochrome P450 2E1.

Rats were fed ethanol in combination with fish oil or a corn diet in order to evaluate the effect of fish oil feeding on liver injury, microsomal ethanol oxidation, and NADPH-dependent lipid peroxidation. The rats were maintained on the dietary regimen for 72 days, and for comparison, pair-fed controls were studied. The liver pathology score progressively worsened in rats fed alcohol, both in combination with fish oil and corn oil, but the severity of inflammation and focal fibrosis was greater in the ethanol fish oil fed rats as compared with the ethanol corn oil group, whereas the fatty change was greater in the ethanol corn oil fed rats. The alcohol treatment caused a 2-fold increase of the liver microsomal P450 content, and about a similar increase in the rate of microsomal NADPH oxidation. The amount of ethanol-inducible CYP2E1 was about 10-fold higher in alcohol-fed rats as compared with pair-fed controls. The NADPH-dependent lipid peroxidation in liver microsomes was about 10-fold higher in microsomes from alcohol-treated rats fed corn oil as compared with controls, but only 2- to 3-fold higher in alcohol-fed rats receiving fish oil than in pair-fed controls. This was due to a higher rate of NADPH-dependent lipid peroxidation in the control rats receiving fish oil. There was a pronounced correlation between the amount of CYP2E1 and the microsomal NADPH peroxidation in variously treated rats, and between the 2E1 levels and the pathology score. The data suggest that fish oil diet, like corn oil, supports ethanol-induced liver injury which is related to CYP2E1 induction and the presence of polyunsaturated fatty acids in the diet (i.e., either n-6 or n-3).

Alanine Transaminase↗
PubMed1993

A role for cytokines as regulators of hepatic fibrogenesis.

It is evident that hepatic fibrogenesis is a complex process involving a cascade of cytokines which interact to enhance the expression of ECM. Cytokines involved early in this cascade may serve as proinflammatory agents or as stimulators of macrophage and Ito cell activation and proliferation, while those cytokines involved later in this process may be directly fibrogenic. Furthermore, we speculate that a balance between profibrogenic and antifibrogenic cytokines normally exists but in the presence of hepatic insults, a relative super-abundance of the fibrogenic factors promotes the development of liver fibrosis. To date, most of the evidence supporting a role for cytokines in liver fibrosis has been obtained in in vitro systems or in animal models. We now need to extend these findings to man in order to determine whether a similar cascade of cytokines is important in the development of this pathologic process in man. Further delineation of these cytokines (as well as other profibrogenic soluble factors), and the mechanisms by which they act, are critical to our development of more rational forms of therapy for liver fibrosis.

Animals↗
PubMed1993

Hepatic fibrosis and gene expression changes induced by praziquantel treatment during immune modulation of Schistosoma japonicum infection.

In the present study fibrogenic gene expression was determined in murine Schistosoma japonicum infection during the progression of immune modulation of infection and following chemotherapy during the course of immune modulation. Histomorphometric analysis of granuloma size and collagen deposition revealed peak granuloma size in acute infection (5 weeks) and peak hepatic collagen content at 16 weeks of infection. Peak Type I collagen gene expression was concomitant with TGF-beta 1 gene expression at 8-11 weeks. Chemotherapy during either acute (9 weeks) or chronic (24, 28 weeks) infection resulted in increased collagen deposition and increased gene expression of Type I collagen and TGF-beta 1. However, chemotherapy at 14-16 weeks resulted in decreased levels of TGF-beta 1 gene expression and essentially minimal change in Type I collagen deposition and gene expression. These data indicate that chemotherapy of schistosomiasis japonica does not reverse hepatic fibrogenesis when administered in acute infection-when granuloma size is maximal-or in chronic infection. However, a beneficial effect on hepatic fibrogenesis is seen when chemotherapy is administered at 14-16 weeks post-infection, a time of decreasing granuloma size and maximal hepatic collagen content. Thus the ability to reverse schistosomal-induced hepatic fibrogenesis by chemotherapy may depend on disease stage.

Animals↗
PubMed1993

Role of the cyclic AMP response element in rat fibronectin gene expression.

Fibronectin expression is of considerable importance in normal and fibrotic liver. Plasma fibronectin levels are correlated with good prognosis in liver failure, and cellular fibronectin plays a crucial role in fibrogenesis. In this study, we observed that the H4II rat hepatoma cell line does not express fibronectin. Furthermore, a recombinant vector (pFGH) containing the promoter elements of the fibronectin gene showed no promoter function when transfected into this cell line. However, pFGH was actively expressed in L-cells and rat skin fibroblasts, cell types that express large amounts of endogenous fibronectin. To study the mechanisms regulating fibronectin expression, we evaluated the transcriptional regulatory elements of the rat fibronectin gene by mutational analysis and DNA-protein binding studies. Deletional mutation analysis showed that the sequences between positions -164 and -90 are essential for promoter activity. This region contains the consensus binding sites for CCAAT and the cyclic AMP-responsive element. Gel retardation assays demonstrated that although the binding activity to the CCAAT site at -140 was essentially the same as that in extracts from L-cells, hepatoma cells and rat livers, substantially greater amounts and different patterns of binding to the adjacent cyclic AMP-responsive element were observed in the extracts from the expressing L-cells and rat livers compared with those in the nonexpressing hepatoma cell nuclear extracts. Furthermore, mutagenesis of the cyclic AMP-responsive element site dramatically reduced promoter activity in transient transfection assays. The cyclic AMP-responsive element at position -160 appears to play an important role in the constitutive expression of the rat fibronectin gene.

Animals↗
PubMed1992

Increased transforming growth factor-beta 1 gene expression in human liver disease.

We recently demonstrated that transforming growth factor-beta 1 stimulates collagen synthesis in hepatic cells in vitro, and that the synthesis of this cytokine is markedly increased in two rodent models of hepatic fibrosis. In the present study, we investigated the association of transforming growth factor-beta 1 (TFG-beta 1) gene expression in human liver disease. Sixteen patients with active liver disease had percutaneous liver biopsies performed for diagnostic purposes. Total RNA was extracted from an unused portion of each biopsy and then subjected to hybridization analysis with the following human cDNA clones: albumin, pro alpha 1 (I) collagen, and TGF-beta 1. Surgical liver biopsy specimens from two patients without hepatic disease were used as controls. When compared to controls, the patients with active liver disease had a 19% decrease in albumin, a 97% increase in type I collagen, and a 120% increase in transforming growth factor-beta 1 mRNA levels. Moreover, steady-state levels of TGF-beta 1 and procollagen mRNAs were significantly correlated. Nuclear run-on assays showed that livers from two patients with fibrosis had TGF-beta 1 transcription rates that were more than 2-fold higher than rates in control livers. These findings indicate that transforming growth factor-beta 1 gene expression is significantly enhanced in man during active liver disease.

Actins↗
PubMed1992

The effects of hepatic fibrosis on Ito cell gene expression.

While Ito cells appear to be a major source of increased matrix synthesis during hepatic fibrogenesis, the cellular changes that occur in these cells during liver fibrosis have not been well delineated. In this study we examined Ito cell gene expression in isolated cells from normal rats, and rats with carbon tetrachloride-induced fibrosis, in order to better define the changes occurring in these cells during this pathologic process. Specifically, we addressed three questions: (1) which matrix genes are over expressed in Ito cells in fibrotic liver; (2) do these cells increase their expression of the fibrogenic cytokine transforming growth factor-beta 1 (TGF-beta 1); and (3) do Ito cells change their phenotype during hepatic fibrogenesis as reflected by alterations in the expression of their intermediate filament genes? Northern hybridization analysis revealed that Ito cells isolated from fibrotic livers had significant increases in mRNA levels of types I, III and IV procollagen compared to normal cells, while no increases were found in hepatocytes, and Kupffer/endothelial cells had only an increase in type I procollagen mRNA. Analysis of other matrix proteins which increase during hepatic fibrogenesis revealed elevations in laminin B and fibronectin mRNA levels only in Ito cells. Increased Ito cell matrix gene expression was also associated with a 4-fold increase in TGF-beta 1 levels in these cells. No increase in TGF-beta 1 mRNA was found in hepatocytes, and less than a 2-fold increase was found in Kupffer/endothelial cells isolated from fibrotic livers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗