Generation of high-titer retroviral vectors following receptor-mediated, adenovirus-augmented transfection.

A new procedure is described for the generation of high-titer, helper-free retrovirus vectors employing receptor-mediated, adenovirus-augmented transfection into a standard packaging cell line. Viral titers are increased 30-fold to 100-fold in transiently (> 10(5) infectious units per mL) and stable (> 10(7) infectious units per mL) transfected cells as compared with either CaPO4-mediated transfection or retroviral infection of a packaging cell line. Further, expression of the transduced genes was drastically increased in the transfected cells, but, as expected, there was no difference in transduction efficiency and gene expression in the infected target cells.

Tumor vaccines: effects and fate of IL-2 transfected murine melanoma cells in vivo.

We have previously demonstrated the general usefulness of the adenovirus-enhanced transferrinfection (AVET) in the generation of IL-2 producing tumor vaccines. By optimizing different parameters of the transfection protocol we were able to transform the poorly immunogenic M-3 mouse melanoma cell line into a potent immunogen. A long-lasting immunity was demonstrated after administration of the IL-2 releasing vaccine, since immunized animals successfully rejected native M-3 melanoma cells even after a period of more than 6 months.

Bridging of peripheral nerve defects with exogenous laminin-fibrin matrix in silicone tubes in a rat model.

Fibrin matrix (FM) is a biological substance involved in the comprehensive wound healing process, and has been used in local applications as a carrier of nerve growth factor (NGF) to achieve an effective local neurotrophic concentration by slow release of the factor. In the present experiment, an exogenous fibrin matrix enriched with laminin (LM) and tubulized by a silicone conduit was used to improve the bridging effect of a peripheral nerve defect in a rat model. A 10 mm nerve defect was bridged with a 14 mm silicone conduit which was prefilled either with 25 μl fibrin matrix enriched without or with laminin (0.

[TSH receptor double mutation in functional autonomic thyroid nodule].

TSH receptor stimulating antibodies (TSAb) are well known in the pathogenesis of Graves' disease. Recently mutations in the DNA coding for the TSH-receptor (TSHR) have been revealed in autonomously functioning thyroid nodules (AFTN). In this study we looked for mutations in 9 patients with AFTN.

Alcoholic liver disease: molecular-pathologic aspects.

Mallory bodies (MBs) are characteristic morphologic features of alcoholic hepatitis but are also associated with non-alcoholic liver diseases including long lasting cholestasis, metabolic and neoplastic disorders. MBs contain in addition to keratins non-keratin components, including microtubule-associated (tau protein) and other not yet characterized proteins in an aggregated form. Aggregation of these components in the cell is promoted by posttranslational modifications, such as partial proteolysis, phosphorylation and cross-linking, and may result in functional and structural disturbances of the cell depending on the physiologic function of the components involved.

High-level expression of various apolipoprotein(a) isoforms by "transferrinfection": the role of kringle IV sequences in the extracellular association with low-density lipoprotein.

Characterization of the assembly of lipoprotein(a) [Lp(a)] is of fundamental importance to understanding the biosynthesis and metabolism of this atherogenic lipoprotein. Since no established cell lines exist that express Lp(a) or apolipoprotein(a) [apo(a)], a "transferrinfection" system for apo(a) was developed utilizing adenovirus receptor- and transferrin receptor-mediated DNA uptake into cells. Using this method, different apo(a) cDNA constructions of variable length, due to the presence of 3, 5, 7, 9, 15, or 18 internal kringle IV sequences, were expressed in cos-7 cells or CHO cells.

Expression of three- and four-repeat tau isoforms in mouse liver.

Tau protein is a member of the family of microtubule-associated proteins, which support microtubule polymerization and stability. Under pathological conditions, tau is a major constituent of neurofibrillary tangles in nerve cells of patients with Alzheimer's disease. Neurofibrillary tangles share some morphological, biochemical and immunological properties with cytoplasmic inclusions associated with other diseases, such as Mallory bodies in the livers of patients with alcoholic hepatitis and in corresponding mouse models.

Posttranslational events involved in griseofulvin-induced keratin cytoskeleton alterations.

Alcoholic hepatitis is a disease associated with profound alterations of the hepatocytic intermediate filament cytoskeleton. Similar cytoskeletal alterations can be induced in mice with prolonged feeding of the fungistatic drug griseofulvin. Murine hepatocytic intermediate filaments are composed of equimolar amounts of keratin polypeptides A (type II) and D (type I).

In vivo production of human factor VII in mice after intrasplenic implantation of primary fibroblasts transfected by receptor-mediated, adenovirus-augmented gene delivery.

Hemophilia A is caused by defects in the factor VIII gene. This results in life-threatening hemorrhages and severe arthropathies. Today, hemophiliacs are treated with human blood-derived factor VIII.

Genetic modification of cells by receptor-mediated adenovirus-augmented gene delivery: a new approach for immunotherapy of cancer.

Most concepts of gene therapy of cancer are based on the generation of an enhanced immune response against the cancer by means of vaccination with gene-modified cancer cells. We have investigated the applicability of a new gene transfer technique which uses the receptor-mediated endocytosis pathway and the endosome disruption activity of adenovirus for the generation of a cancer vaccine consisting of interleukin-2 (IL-2)-transfected, irradiated murine melanoma cells (clone M-3). This technique resulted in very high IL-2 expression (in the range of 30,000 Units IL-2/10(6) cells/24 hrs) in the transfected cells without the need to selection of stably expressing cell clones.

Somatic gene therapy for cancer: the utility of transferrinfection in generating 'tumor vaccines'.

The last few years have seen the development of a branch of somatic gene therapy which aims at strengthening the immune surveillance of the body, leading to eradication of disseminated cancer tumor cells and occult micrometastases after surgical removal of the primary tumor. Such a tumor vaccination protocol calls for cultivation of the primary tumor tissue and the insertion of one of three types of genes into the isolated cultured tumor cells followed by irradiation of the transfected or transduced cells to render them incapable of further proliferation. The cells so treated constitute the 'tumor vaccine'.

Disturbance of keratin homeostasis in griseofulvin-intoxicated mouse liver.

Background: Alterations of the hepatocytic intermediate filament (IF) cytoskeleton, i.e., derangement and diminution of the keratin network and appearance of cytoplasmic aggregates of keratin-containing material, termed Mallory bodies, are characteristic features of human alcoholic hepatitis.

A combined biopsy-plugging device based on the Menghini- or Trucut needle for percutaneous liver biopsy: clinical experience.

Impaired blood clotting precludes percutaneous liver biopsy for histologic examination of liver tissue. The transjugular or laparoscopic approach are ways to reduce the risk of bleeding. These techniques, however, are laborious and confined to specialized centers.

Chicken adenovirus (CELO virus) particles augment receptor-mediated DNA delivery to mammalian cells and yield exceptional levels of stable transformants.

Delivery of genes via receptor-mediated endocytosis is severely limited by the poor exit of endocytosed DNA from the endosome. A large enhancement in delivery efficiency has been obtained by including human adenovirus particles in the delivery system. This enhancement is probably a function of the natural adenovirus entry mechanism, which must include passage through or disruption of the endosomal membrane.

Immunohistochemical detection of tumor necrosis factor-alpha, other cytokines and adhesion molecules in human livers with alcoholic hepatitis.

This immunohistochemical study was designed to investigate the possible contribution to and topographical distribution of some important cytokines, such as tumour necrosis factor alpha (TNF alpha) and interleukins, in acute alcoholic hepatitis. The well-known inductive capacity of these cytokines with respect to the expression and/or up-regulation of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and endothelial leukocyte adhesion molecule-1 (ELAM-1), was a further point to be studied. Moreover, the proposed induction of adhesion molecules might also be associated with the activation and attraction of a special population of inflammatory cells characteristic for alcoholic hepatitis.

Involucrin expression in breast carcinomas: an immunohistochemical study.

The expression of involucrin, a structural component of the envelope of mature squamous epithelium, was studied in 166 paraffin-embedded breast carcinomas. In 41 cases (24.7%) involucrin-positive, light microscopically non squamous tumour cells were detected.

Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand.

We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide.

Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle.

Complexes containing plasmid DNA, transferrin-polylysine conjugates, and polylysine-conjugated peptides derived from the N-terminal sequence of the influenza virus hemagglutinin subunit HA-2 have been used for the transfer of luciferase or beta-galactosidase marker genes to K562 cells, HeLa cells, and BNL CL.2 hepatocytes. These DNA complexes mimic the entry of viruses into cells, as they contain functions for (i) the packaging of the nucleic acid with polylysine, (ii) the attachment to the cell and receptor-mediated endocytosis with transferrin as a ligand, and (iii) the release from endosomes by using membrane-disrupting influenza peptides.

Cryopreservation of cytological specimens for immunocytochemistry.

A method has been established for storage and preservation of cytological specimens in liquid nitrogen and further processing for immunocytochemistry as smears prepared from thawed cells or cryo-sections of frozen cell pellets. For the experiments cultured cells of a T-lymphoblastic leukemia cell line (ATCC CCL 119) and blood cells of the buffy coat of healthy humans were treated with a cryo-solution (fetal calf serum +5% dimethylsulfoxid) and after freezing stored in liquid nitrogen. Alternatively, cells preincubated with cryo-solution followed by suspension in fetal calf serum without cryo-additive were frozen and stored in liquid nitrogen for the production of cryo-sections.

Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes.

We are developing efficient methods for gene transfer into tissue culture cells. We have previously shown that coupling of a chimeric adenovirus with polylysine allowed the construction of an adenovirus-polylysine-reporter-gene complex that transferred the transporter gene with great efficiency into HeLa cells. We have now explored simpler, biochemical means for coupling adenovirus to DNA/polylysine complexes and show that such complexes yield virtually 100% transfection in tissue culture cell lines.

Alcoholic liver disease. Parenchyma to stroma relationship in fibrosis and cirrhosis as revealed by three-dimensional reconstruction and immunohistochemistry.

Severe ethanol-induced liver damage is characterized by fibrous dissociation of liver cell plates leading to many apparently isolated hepatocytes. Three-dimensional reconstruction, however, revealed hepatocytes that were surrounded by connective tissue as endpoints of "parenchymal pillars" or in association with liver cell plates and bile ductules. Double immunofluorescence studies displayed the expression of cytokeratin (CK) 7 in bile ducts, including bile ductules, but also in some hepatocytes still organized in liver cell plates.

High-efficiency receptor-mediated delivery of small and large (48 kilobase gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles.

One limit to successful receptor-mediated gene delivery is the exit of the endocytosed material from the endosome. We demonstrate here the delivery of marker genes to tissue culture cells using a modification of the receptor-mediated gene delivery technique that exploits the endosomolytic activity of defective adenovirus particles. In particular, greater than 90% of the transfected-cell population is found to express a beta-galactosidase gene, and, most importantly, this high level of expression can be obtained with psoralen-inactivated virus particles.

High amount of epsilon-(gamma-glutamyl)lysine cross-links in Mallory bodies.

Alcoholic hepatitis, the most severe form of alcoholic liver disease, is associated with inflammation, liver cell necrosis, and the appearance of Mallory bodies (MBs) in hepatocytes. Identical MBs can be experimentally induced in mouse livers by chronic griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine treatment. MBs are filamentous cytoplasmic inclusions containing insoluble high molecular weight protein material.

Modulation of protein composition of nuclear lamina. Reduction of lamins B1 and B2 in livers of griseofulvin-treated mice.

Chronic griseofulvin (GF) intoxication of mice leads to severe alterations of the hepatocytic intermediate filament cytoskeleton similar to that found in alcoholic hepatitis in humans (i.e., derangement and diminution of the keratin filament network and appearance of cytoplasmic aggregates of keratin-containing material, termed Mallory bodies).