Biomedical subjects

S Andreadis

Publications and source records attributed to S Andreadis.

6 recordsLinked to original sources

PubMed2000

Toward a more accurate quantitation of the activity of recombinant retroviruses: alternatives to titer and multiplicity of infection.

In this paper, we present a mathematical model with experimental support of how several key parameters govern the adsorption of active retrovirus particles onto the surface of adherent cells. These parameters, including time of adsorption, volume of virus, and the number, size, and type of target cells, as well as the intrinsic properties of the virus, diffusion coefficient, and half-life (t(1/2)), have been incorporated into a mathematical expression that describes the rate at which active virus particles adsorb to the cell surface. From this expression, we have obtained estimates of C(vo), the starting concentration of active retrovirus particles. In contrast to titer, C(vo) is independent of the specific conditions of the assay. The relatively slow diffusion (D = 2 x 10(-8) cm(2)/s) and rapid decay (t(1/2) = 6 to 7 h) of retrovirus particles explain why C(vo) values are significantly higher than titer values. Values of C(vo) also indicate that the number of defective particles in a retrovirus stock is much lower than previously thought, which has implications especially for the use of retroviruses for in vivo gene therapy. With this expression, we have also computed AVC (active viruses/cell), the number of active retrovirus particles that would adsorb per cell during a given adsorption time. In contrast to multiplicity of infection, which is based on titer and is subject to the same inaccuracies, AVC is based on the physicochemical parameters of the transduction assay and so is a more reliable alternative.

3T3 Cells↗

PubMed2000

Toward a more accurate quantitation of the activity of recombinant retroviruses: alternatives to titer and multiplicity of infection.

In this paper, we present a mathematical model with experimental support of how several key parameters govern the adsorption of active retrovirus particles onto the surface of adherent cells. These parameters, including time of adsorption, volume of virus, and the number, size, and type of target cells, as well as the intrinsic properties of the virus, diffusion coefficient, and half-life (t1/2), have been incorporated into a mathematical expression that describes the rate at which active virus particles adsorb to the cell surface. From this expression, we have obtained estimates of Cvo, the starting concentration of active retrovirus particles. In contrast to titer, Cvo is independent of the specific conditions of the assay. The relatively slow diffusion (D = 2 x 10(-8) cm2/s) and rapid decay (t1/2 = 6 to 7 h) of retrovirus particles explain why Cvo values are significantly higher than titer values. Values of Cvo also indicate that the number of defective particles in a retrovirus stock is much lower than previously thought, which has implications especially for the use of retroviruses for in vivo gene therapy. With this expression, we have also computed AVC (active viruses/cell), the number of active retrovirus particles that would adsorb per cell during a given adsorption time. In contrast to multiplicity of infection, which is based on titer and is subject to the same inaccuracies, AVC is based on the physicochemical parameters of the transduction assay and so is a more reliable alternative.

Animals↗

PubMed1997

Coupled effects of polybrene and calf serum on the efficiency of retroviral transduction and the stability of retroviral vectors.

The relative concentrations of Polybrene (PB) and calf serum (CS) in retroviral supernatant have considerable effects on the efficiency of retrovirus-mediated gene transfer and the stability of retroviral vectors. The effect of PB on the efficiency of transduction of Moloney murine leukemia virus (MMuLV)-derived vectors is strongly dependent on CS. At a fixed CS concentration, the efficiency of transduction shows a maximum as a function of PB concentration. Increasing the CS concentration shifted this maximum to higher PB concentrations, but the value of the maximum remained the same. Therefore, there were optimal combinations of PB and CS concentrations that maximized the efficiency of gene transfer: 4.4, 8.8, 13.2, and 22 micrograms/ml of PB for 1%, 2.5%, 5%, and 10% (vol/vol) CS, respectively. Moreover, the presence of PB affected significantly the kinetics of retroviral decay. The loss of retroviral activity did not follow simple exponential decay in the absence of PB during the decay period of the viral supernatant. The dynamics of viral inactivation showed an initial phase during which the transduction efficiency remained constant followed by exponential decay. However, in the presence of high PB concentrations (13.2 micrograms/ml) during the decay period of retroviral vectors, the initial delay was lost and the decay was exponential right from the outset. The present results suggest that in addition to virus-cell interactions that occur on the target cell surface, other physico-chemical processes may occur in solution that have profound effect on retroviral activity and therefore they are of particular importance for gene therapy.

3T3 Cells↗

PubMed1997

The physico-chemical factors that govern retrovirus-mediated gene transfer.

The most commonly used vehicle for gene transfer into human target cells is a replication incompetent retroviral vector. The efficiency of gene transfer with this type of vector has proven to be too low to implement effective gene therapy. To date much effort has gone into engineering the genetic and biochemical functionalities of retroviral vectors. Although progress has been achieved, high-efficiency reproducible gene transfer into human cells remains elusive. There are many important physico-chemical and systemic kinetic factors that govern the process of retrovirus-mediated gene transfer. These factors have gone mostly unrecognized to date. The former include the nature of the random Brownian motion of the retrovirus and the physico-chemical forces that determine the binding of the retroviral vector to the target cell. The latter arise from the kinetics of virus binding and entry into the target cell, as well as the kinetic interplay between cell-cycle and retroviral life-cycle events that determine the intracellular fate of the virus. This review describes these processes and how they constrain the efficiency of the gene transfer process.

Animals↗

PubMedDate not supplied

Cell cycle dependence of retroviral transduction: An issue of overlapping time scales.

Recombinant retroviruses are currently used as gene delivery vehicles for the purpose of gene therapy. It is generally believed that the efficiency of retroviral transduction depends on the cell cycle status of the target cells. However, it has been reported that this is not the case for the transduction of human and murine fibroblasts, in contrast to other cell types such as lymphocytes. The predictions of a mathematical model that we constructed, offer an explanation of this contradiction, based on the dynamics of the underlying processes of target cell growth and the intracellular decay of retroviral vectors. The model suggests that the utility of synchronization experiments, that are usually employed to study cell cycle specificity, is severely limited when the time scales of the above kinetic events are comparable to each other. The predictions of the model also suggest the use of retroviral vectors as cell cycle markers, as an alternative way to detect cell cycle dependence of retroviral transduction. This method obviates the need for cell synchronization and therefore, it does not perturb the cell cycle or interfere with the life cycle of retroviral vectors. Moreover, it does not depend on the intracellular stability of retroviral vectors. Our results show that in contrast to previously reported results, transduction of murine fibroblasts is cell cycle dependent, and they are consistent with the current notion that mitosis is the phase that confers transduction susceptibility.

3T3 Cells↗

PubMedDate not supplied

Immunological parameters in patients with epilepsy.

Various immunological parameters (IgA, IgG, IgM serum levels, C3 and C4 complement components and T and B lymphocytes and T-lymphocyte subsets) were determined in 50 patients with epilepsy (20 without medication, 15 on carbamazepine and 15 on valproate) and in 20 controls. The epileptic group had fewer CD4 and more CD8 lymphocytes, while the CD4/CD8 ratio was lower; the levels of IgG and IgM were increased and C4 complement component was also decreased. Patients without medication showed all the above mentioned abnormalities. Patients on carbamazepine had a lower CD4/CD8 ratio, increased IgG and IgM levels and decreased C4 complement component while patients on valproate had more CD8 lymphocytes, a lower CD4/CD8 ratio, increased levels of IgM and decreased C4 complement component. Our results indicate that there is a defective immune mechanism in epileptic patients modified by treatment.

Adolescent↗