Engineered human Tmpk fused with truncated cell-surface markers: versatile cell-fate control safety cassettes.

Cell-fate control gene therapy (CFCGT)-based strategies can augment existing gene therapy and cell transplantation approaches by providing a safety element in the event of deleterious outcomes. Previously, we described a novel enzyme/prodrug combination for CFCGT. Here, we present results employing novel lentiviral constructs harboring sequences for truncated surface molecules (CD19 or low-affinity nerve growth factor receptor) directly fused to that CFCGT cDNA (TmpkF105Y). This confers an enforced one-to-one correlation between cell marking and eradication functions. In-vitro analysis demonstrated the full functionality of the fusion product. Next, low-dose 3'-azido-3'-deoxythymidine (AZT) administration to non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice injected with transduced clonal K562 cells suppressed tumor growth; furthermore, one integrated vector on average was sufficient to mediate cytotoxicity. Further, in a murine xenogeneic leukemia-lymphoma model we also demonstrated in-vivo control over transduced Raji cells. Finally, in a proof-of-principle study to examine the utility of this cassette in combination with a therapeutic cDNA, we integrated this novel CFCGT fusion construct into a lentivector designed for treatment of Fabry disease. Transduction with this vector restored enzyme activity in Fabry cells and retained AZT sensitivity. In addition, human Fabry patient CD34(+) cells showed high transduction efficiencies and retained normal colony-generating capacity when compared with the non-transduced controls. These collective results demonstrated that this novel and broadly applicable fusion system may enhance general safety in gene- and cell-based therapies.

Role of plasminogen, plasmin, and plasminogen activators in the migration of fibroblasts into plasma clots.

Human diploid fibroblasts were seeded onto or into plasma clots and different aspects of cell adhesion and migration were measured. The roles of plasminogen activators and plasmin were studied by either the removal of plasminogen from plasma prior to clotting or by the addition of 10 mM epsilon-aminocaproic acid, which brings about an inhibition of plasmin in this system. When cells were seeded onto the surface of plasma clots, rates of attachment, spreading, and migration were unaffected by plasminogen depletion or plasmin inhibition. In contrast, when cells were seeded into plasma clots, then, although the rates of cells spreading were unaffected, cell migration was abolished by plasminogen depletion or by plasmin inhibition. When cells were seeded onto the surface of plasma clots and the rate of migration into the clots was measured, there was an absolute requirement for plasmin activity; while fibroblasts migrated rapidly into the fibrin lattice of control clots, in the case of plasminogen-depleted clots, cells failed to penetrate the lattice. Focussing through a plasma clot revealed that fibroblasts do not migrate through the fibrin lattice but instead, localized areas of fibrinolysis are generated and cells migrate over the surface of the area of lysis.

An in vitro cytotoxicity test to predict the ocular irritation potential of detergents and detergent products.

Two in vitro cytotoxicity procedures, the measurement of cell-membrane integrity using fluorescein diacetate and ethidium bromide, and the quantitation of the release of a cell-membrane-bound enzyme, alkaline phosphatase, were used to assess the cytotoxicity of a range of cationic, anionic and nonionic detergents. The in vitro results were compared with the in vivo irritancy of these compounds in the rabbit eye. Although in general the decreasing order of potency of cationic, anionic and nonionic detergents was similar in vivo and in vitro, there were some apparent anomalies which may be due to the differing penetration characteristics of the detergents, as indicated by electrical impedance measurements of the isolated cornea. The study was extended to an examination of the cytotoxicity of a range of completely soluble, detergent-based formulations in a suspension culture of mouse fibroblasts. In this case the in vitro results correlated more closely with those from the in vivo tests.

An investigation of detergent action on cells in vitro and possible correlations with in vivo data.

Synopsis A cell fluorescence method for quantifying the effects of detergents on cultured cell lines is described. The results are discussed in relation to other findings in the literature. These results are compared to data obtained with the same detergents in the rabbit eye test and the possible correlations and discrepancies are considered. Summary Details are given of the culture of two human epithelioid cell lines (HeLa and HEp2) and their utilization in a procedure to assess the cytotoxicity of detergents on cells in monolayer culture. The method is dependent on the ability of relatively intact cells to liberate and retain fluorescein from fluorescein diacetate. Ethidium bromide, a red fluorescent compound, is used to stain the nuclear material remaining in membrane-damaged cells. The results of the in vitro test are compared to the data obtained from the responses seen in the rabbit eye on instillation of the same detergents. For sodium and triethanolamine alkyl sulphates at comparable concentrations, an increase in chain length increases the in vitro cytotoxicity but decreases the effects seen in the conjunctivae, cornea and iris. For the C12, C14 and C16 alkyl trimethylammonium bromides, an increase in chain length increases in vitro cytotoxicity as well as the in vivo responses. Tweens 20 and 40 appeared more damaging in vivo than Tweens 60 and 80; this differentiation was not observed in vivo. The findings of both tests are discussed in terms of detergent solubilities, penetration of, and adsorption of tissues in vivo as well as the effects of detergents on enzymes.