A combined artificial chromosome-stem cell therapy method in a model experiment aimed at the treatment of Krabbe's disease in the Twitcher mouse.

Mammalian artificial chromosomes (MACs) are safe, stable, non-integrating genetic vectors with almost unlimited therapeutic transgene-carrying capacity. The combination of MAC and stem cell technologies offers a new strategy for stem cell-based therapy, the efficacy of which was confirmed and validated by using a mouse model of a devastating monogenic disease, galactocerebrosidase deficiency (Krabbe's disease). Therapeutic MACs were generated by sequence-specific loading of galactocerebrosidase transgenes into a platform MAC, and stable, pluripotent mouse embryonic stem cell lines were established with these chromosomes. The transgenic stem cells were thoroughly characterized and used to produce chimeric mice on the mutant genetic background. The lifespan of these chimeras was increased twofold, verifying the feasibility of the development of MAC-stem cell systems for the delivery of therapeutic genes in stem cells to treat genetic diseases and cancers, and to produce cell types for cell replacement therapies.

The use of pregnancy serum to obtain trophoblastic cell cultures.

A method for obtaining trophoblastic cell cultures from first-trimester human placental villi is described. The essential feature of the method is the use of serum from a pregnant woman in the culture medium. Using this technique, pure cultures of trophoblastic cells are produced in 88 per cent of cases.

Endothelial cells cultured from human brain microvessels produce complement proteins factor H, factor B, C1 inhibitor, and C4.

The inflammatory mediators, cytokines and complement proteins are believed to regulate the sequential events during the development of lesions secondary to ischaemia and reperfusion. The endothelial cell monolayer of the brain microvasculature is the critical interface between the blood-borne mediators and brain tissue. The involvement of these cells in complement production and regulation has not been well documented. In the present study, expression of complement proteins (C1 inhibitor, factor H, factor B, C4) by cultured endothelial cells obtained from human brain microvessels has been characterized. Interferon gamma upregulates the production of all the complement factors studied. Serine proteases, plasmin and miniplasmin induce the expression of C4, decrease the level of ELISA detectable C1 inhibitor, and do not affect the production of factors H and B. These data indicate that complement proteins are expressed locally by the brain microvessels, and may modulate the inflammatory responses of brain tissue.

Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-1,2-benzopyrone (INH2BP).

Bovine aortic endothelial cells were converted to a highly tumorigenic cell line by transfection with Ha-ras and stimulation with thrombin. Sustained pretreatment with a non-cytotoxic concentration (600 mu M) of 5-iodo-6-amino-1,2-benzopyrone (INH2BP), a lipophilic ligand of poly(ADP-ribose) polymerase, abrogated in vivo tumorigenicity, of 10(5) cells per inoculum an effect which developed progressively during 2 to 6 weeks of drug treatment. The initial action of the drug was cytostasis, consisting of an arrest in prophase, extreme cell enlargement consistent with cytoplasmic hypertrophy, as seen by EM, and dramatic morphologic changes. Although neither DNA, RNA or protein syntheses are directly affected by INH2BP, apparently newly synthesized cellular DNA is degraded by endonucleases, which are upregulated by the inhibition of their poly-ADP-ribosylation. The drug treated cells exhibited greatly increased respiration and aerobic glycolysis, due to an augmentation of,glycolytic and respiratory enzymes in enlarged cells. These responses to the drug were reversible in cell cultures following drug removal, within 5-10 days drug exposure but the progressive loss of tumorigenicity in nude mice that developed after 3-6 weeks of drug exposure of cells, prior to inoculation to nude mice, was not reversible in vivo. Drug treatment produced a sustained 70-80% inhibition of pADPRT in intact cells at 600 mu M extracellular concentration of INH2BP. The prerequisite for the abrogation of tumorigenicity was the maintenance of pADPRT inhibition. The arrest of cell multiplication and a large decrease of Topo I, especially of Topo II and MAP kinase activities occurred without loss of enzyme protein as assayed in cell extracts of drug-treated cells. However INH2BP had no direct effect on these enzymes. Drug treatment down-regulated DNA-methyltransferase, PKC, ODC proteins, diminished cyclin A protein, but the hypophosphorylated form of Rb protein was significantly augmented. None of the enzymatic components of signal pathways so far studied, were directly affected by INH2BP. The inhibition of pADPRT by INH2BP coincided with an induction or activation of alkaline phosphatase and leucyl and glutamyl peptidase. The pADPRT content or the expression of pADPRT gene were not influenced by drug treatment, but the expression of ras gene was completely absent in nontumorigenic drug-treated cells, without a loss of ras gene from genomic DNA. Telomerase activity was not directly influenced by INH2BP treatment when assayed in diluted cell extracts, but the addition of homogeneous pADPRT to cell extracts, to approach physiological concentration of this protein in the cell, inhibited telomerase activity by binding of the polymer-free pADPRT to telomer templates. We conclude that inhibition of pADPRT indirectly down-regulates growth stimulatory signal pathways and sustains growth-arrested cells in culture at a pre-apoptotic threshold which explains the absence of tumorigenicity in vivo.

Interaction of antithrombin III and thrombin-antithrombin III complex with cultured aortic endothelial cells.

The binding of antithrombin III, thrombin, thrombin-antithrombin III complex to endothelial cells was investigated. While the rate of the binding of thrombin to these cells was very rapid, that of antithrombin III was relatively slow and the thrombin-antithrombin III complex was intermediate. Binding kinetics indicated that antithrombin III, like thrombin, showed high affinity to endothelial cells; with a Kd of 3 X 10(-8) M and with 5 X 10(4) binding sites per cell. The dissociation of the inhibitor molecule was also rapid, i.e., approximately 70% bound antithrombin III was released in 2 minutes. Heparin, in a 100-fold molar excess to antithrombin III, or the modification of lysine residues of the inhibitor involved in the interaction with heparin, did not influence the association of antithrombin III with endothelial cells. In addition, antithrombin III did not compete with thrombin blocked in its active center for binding to endothelial cells. It is suggested that the binding sites of endothelial cells are different for thrombin and antithrombin III, and antithrombin III does not bind to these cells through its heparin binding domain.

Perturbation of the integrity of the blood-brain barrier by fibrinolytic enzymes.

The action of fibrinolytic enzymes (plasmin, miniplasmin, neutrophil leukocyte elastase) on the blood-brain barrier is investigated. The binding and the effects of the fibrinolytic enzymes are studied in the first subcultivation of human brain capillary endothelial cells. 125I-labeled plasmin, miniplasmin and neutrophil leukocyte elastase bind to confluent monolayers of cultured endothelial cells with dissociation constants of 1 x 10(-8) mol/l, 4.8 x 10(-7) mol/l and 1.8 x 10(-8) mol/l, respectively, and the number of binding sites varies between 2.3 x 10(5) and 7.5 x 10(6) per cell. Following treatment of the cultured cells with purified and active-site titrated proteases, the changes in morphology of individual cells are analyzed with computerized morphometry. At low concentrations (in nanomolar range) all studied fibrinolytic proteases induce reduction of the cell area; the minimal size is achieved in 20-80 min after the application of an enzyme and the effect is completely reversed in 15 min after its removal. A possible in-vivo consequence of these in-vitro findings is studied in an organ-perfusion model: rat hemisphere is perfused with a protease solution followed by a circulating phase-borne tracer (horse-radish peroxidase). In perfused rat hemisphere, the fibrinolytic enzymes open the blood-brain barrier to the circulation-borne tracer. These results support the concept that fibrinolytic enzymes interact with the brain microvascular endothelium and thus affect the integrity of the blood-brain barrier through active cell contraction.

Imidazole in organs of inbred chickens selected for resistance or susceptibility to lymphoid leukosis or Marek's disease.

Total and soluble imidazole was determined on samples of liver and kidney and soluble imidazole was determined on blood from eight strains of chickens selected at three locations for resistance or susceptibility to lymphoid leukosis and/or Marek's disease. Liver and kidney total and soluble imidazole was not different between strains classed as resistant or susceptible to lymphoid leukosis and/or Marek's disease. The weighted average of soluble blood imidazole was higher (P smaller than 0.01) in strains classed as resistant to lymphoid leukosis. A similar difference (P smaller than 0.01) was observed with strains classed as resistant or susceptible to Marek's disease when the selections from one location were not included in the weighted average.

Circulatory consequences of reduced endogenous nitric oxide production during small-volume resuscitation.

Hypertonic small-volume resuscitation transiently restores the cardiovascular function during various circulatory disturbances. Nitric oxide (NO) is an important mediator of flow-induced peripheral and central hemodynamic changes, and therefore, we hypothesized that a decreased endogenous NO production could influence the consequences and the effectiveness of hypertonic fluid therapy. The main goal of this study was to outline and compare the circulatory effects small volume hypertonic saline-dextran (HSD, 7.5% NaCl-10% dextran; 4 ml/kg iv) infusion with (n=7) or without (n=7) artificially diminished NO production in normovolemic anesthetized dogs. HSD administration significantly increased cardiac index (CI), coronary flow (CF) and myocardial contractility, and elevated plasma nitrite/nitrate (NOx) and endothelin-1 (ET-1) levels. However, the late (2 h) postinfusion period was characterized by significantly decreased myocardial NO synthase (NOS) and enhanced myeloperoxidase activities. Pre-treatment with the non-selective NOS inhibitor N-nitro-L-arginine (NNA, 4 mg/kg) immediately increased cardiac contractility, and the HSD-induced CI and CF elevations and the positive inotropy were absent. Additionally, plasma ET-1 levels increased and NOx levels were significantly decreased. In conclusion, our results demonstrate that HSD infusion leads to preponderant vasoconstriction when endogenous NO synthesis is diminished, and this could explain the loss of effectiveness of HSD resuscitation in NO-deficient states.