Cell Survival Kinetics Research Articles

Measurement of post-transfusion red blood cell survival kinetics in sickle cell disease and β-Thalassemia: A biotin label approach.

Red blood cell (RBC) transfusions are an important treatment modality for patients with sickle cell disease (SCD) and β-thalassemia. A subgroup of these patients relies on a chronic RBC transfusion regimen. Little is known about RBC survival (RCS) of the transfused allogeneic RBCs. In this study, we aimed to study the RCS kinetics of transfused RBCs in SCD and β-thalassemia and to investigate factors that determine RCS. We performed a prospective cohort study on fourteen adults with SCD and β-thalassemia disease receiving a chronic transfusion regimen. RCS and the influence of donor and patient characteristics on RCS were assessed by simultaneous transfusion of two allogeneic RBCs using RBC biotinylation. Phenotyping of well-known RBC markers over time was performed using flow cytometry. RCS of the two transfused RBC units was similar in most patients. Although intra-individual variation was small, inter-individual variation in RCS kinetics was observed. Most patients demonstrated a non-linear trend in RCS that was different from the observed linear RCS kinetics in healthy volunteers. After an initial slight increase in the proportion of biotinylated RBCs during the first 24 h, a rapid decrease within the first 10-12 days was followed by a slower clearance rate. These are the first data to demonstrate that patient-related factors largely determine post-transfusion RCS behavior of donor RBC in SCD and β-thalassemia, while donor factors exert a negligible effect. Further assessment and modeling of RCS kinetics and its determinants in SCD and β-thalassemia patients may ultimately improve transfusion therapy.

LFA-1 signaling plays an important role in effector and memory CD8+ T cell survival via TNF receptor/ligand family-activated Bcl-2 apoptosis pathway and Caspase cascade (46.19)

Abstract LFA-1 signaling is required for CD8+ T cell priming and cytolytic activity. However, its role for effector and memory CD8+ T cell survival in transition and maintenance phase is elusive. We adoptively transferred ovalbumin (OVA)-pulsed dendritic cell (DCOVA)-stimulated effector CD8+ T cells or purified OVA-specific memory CD8+ T cells into wild-type C57BL/6 or CD54 gene knockout (CD54-/-) mice. We assessed CD8+ T cell survival kinetics and molecular mechanisms responsible for LFA-1 signaling defect-induced T cell apoptosis by flow cytometric and Western-blotting analysis. We found that transferred OVA-specific effector and memory CD8+ T cell survival significantly decreased (p<0.05) in transition and maintenance phase in CD54-/- mice due to increased T cell apoptosis, compared to C57BL/6 mice. Increased CD8+ T cell apoptosis is initiated via altered gene expression in TNF receptor/ligand (TNFR/L) (up-regulation of Fas and down-regulation of CD27, CD30 and 41BB), Bcl-2 (down-regulation of proapoptotic Bid, anti-apoptotic Bcl-2 and Bcl-XL and up-regulation of apoptotic Bax) and Caspase (down-regulation of pro-Caspase 8 and up-regulation of cleaved Caspase 3 and 7) families, and in survival signals (down-regulation of IL-15R). Therefore, LFA-1 signaling plays an important role in effector and memory CD8+ T cell survival via TNFR/L family-activated Bcl-2 apoptosis pathway and Caspase cascade.

Comparison of Optical Bioluminescence Reporter Gene and Superparamagnetic Iron Oxide MR Contrast Agent as Cell Markers for Noninvasive Imaging of Cardiac Cell Transplantation

In this study, we compared firefly luciferase (Fluc) reporter gene and superparamagnetic iron oxide (Feridex) as cell markers for longitudinal monitoring of cardiomyoblast graft survival using optical bioluminescence imaging (BLI) and magnetic resonance imaging (MRI), respectively. Rats (n = 31) underwent an intramyocardial injection of cardiomyoblasts (2 x 10(6)) labeled with Fluc, Feridex, or no marker (control) or an injection of Feridex alone (75 microg). Afterward, rats were serially imaged with BLI or MRI and killed at different time points for histological analysis. BLI revealed a drastically different cell survival kinetics (half-life = 2.65 days over 6 days) than that revealed by MRI (half-life = 16.8 days over 80 days). Injection of Feridex alone led to prolonged tissue retention of Feridex (> or =16 days) and persistent MR signal (> or =42 days). Fluc BLI reporter gene imaging is a more accurate gauge of transplanted cell survival as compared to MRI of Feridex-labeled cells.

Fibrin microbeads (FMB) as a 3D platform for kidney gene and cell therapy

Cell and gene therapy may alter the outcome of renal diseases, such as hereditary nephropathies, acute and chronic glomerulonephritis and allograft nephropathy. However, owing to blockade of many viral and cellular vehicles by the complex glomerular architecture, the exact nature of gene and cell delivery into specific renal compartments remains currently unknown. To study the interaction of viral vectors with a variety of renal cells and mesenchymal stem cells (MSCs), we employed a novel biological three-dimensional (3D) matrix comprised of fibrin microbeads (FMB) in comparison to monolayer cell culture. Our studies showed that renal cells of both established and primary lines can grow efficiently on FMB and differentiate into epithelial structures, as shown by electron microscopy. Gene delivery into renal cells in 3D was observed for several viral vectors and growth in 3D on FMB conferred resistance to renal cancer cells in the context of oncolytic adenoviruses. Finally, MSCs from various rodent species attached to FMB, grew robustly, survived for several weeks and could efficiently be transduced on FMB. Thus, on the basis of growth, differentiation and transduction of renal cells in 3D, FMB emerge as a novel 3D cellular microenvironment that differs substantially from monolayer cell cultures.

In Vivo Persistence of Donor Cells following Adoptive Transfer of Allogeneic Dendritic Cells in HIV-Infected Patients.

Peripheral blood samples from HIV-seropositive individuals enrolled in a pilot clinical trial investigating the use of allogeneic dendritic cell therapy were evaluated for mixed chimerism. In this study, dendritic cells from HLA-identical, HIV-seronegative siblings were used. Patients received an infusion of dendritic cells pulsed with HIV MN gp160 protein or with peptides from HLA-A2 restricted epitopes of env, gag, and pol proteins every month for 6-9 months. Of the five allogeneic dendritic cell recipients, two showed increases in HIV antigen-specific immune responses. Allele-specific polymorphisms were identified in three sib-pairs that allowed infused donor cells to be detected using sensitive PCR-based molecular methods. Analysis of blood samples from patients showed similar patterns of donor cell persistence after the first infusion, in that cells were detectable for at least 1 week. Also, differences were observed in the kinetics of cell survival between the first and subsequent infusion cycles in all three patients. This suggests variation in HIV-specific immune responses detected among these three patients was not due to differences in persistence of infused donor cells.

Hyperfractionation versus single dose irradiation in human acute lymphocytic leukemia cells: application to TBI for marrow transplantation

A major purpose of total body irradiation (TBI) for bone marrow transplantation in leukemia patients is to help eradicate all leukemia cells; the ideal regimen has not yet been determined. To answer basic questions regarding leukemic cell survival kinetics, a human acute lymphoblastic leukemia (ALL) cell line (Reh), with the common ALL antigen (CALLA-positive), has been used to assess in vitro the efficacy of one widely used hyperfractionated TBI (HTBI) regimen versus single dose TBI (SDTBI). The regimen studied in this model was 1.2–1.25 Gy/fraction, 3 fractions/day, 5 h apart each day, for 5 days (11–12 fractions) for a total dose of 13.2–15.0 Gy. It was found that: (i) cell survival was consistent with the linear-quadratic model for early responding tissues ( α β = 7.0 Gy ). (ii) The change in shape of the ‘effective’ cell survival curve for three fractions/day was consistent with the hypothesis that there was complete repair between fractions. (iii) Cell regrowth between fractions was minimal (≤ 5%). (iv) Division delay between fractions (2.9 h/Gy) could explain the small contribution to the survival curve of regrowth between fractions.(v) For a full HTBI course to 15 Gy, cell survival was predicted to be ~5 × 10 −5, compared with ~ 10 −3 for a low dose rate (0.04–0.07 Gy/min) SDTBI to 10 Gy; the latter projected from the initial slope of the high dose rate, single dose survival curve.

Hyperfractionation versus single dose irradiation in human acute lymphocytic leukemia cells: Application to TBI for marrow transplantation

Abstract A major purpose of total body irradiation (TBI) for bone marrow transplantation in leukemia patients is to help eradicate all leukemia cells; the ideal regimen has not yet been determined. To answer basic questions regarding leukemic cell survival kinetics, a human acute lymphoblastic leukemia (ALL) cell line (Reh), with the common ALL antigen (CALLA-positive), has been used to assess in vitro the efficacy of one widely used hyperfractionated TBI (HTBI) regimen versus single dose TBI (SDTBI). The regimen studied in this model was 1.2–1.25 Gy/fraction, 3 fractions/day, 5 h apart each day, for 5 days (11–12 fractions) for a total dose of 13.2–15.0 Gy. It was found that: (i) cell survival was consistent with the linear-quadratic model for early responding tissues ( α β = 7.0 Gy ). (ii) The change in shape of the ‘effective’ cell survival curve for three fractions/day was consistent with the hypothesis that there was complete repair between fractions. (iii) Cell regrowth between fractions was minimal (≤ 5%). (iv) Division delay between fractions (2.9 h/Gy) could explain the small contribution to the survival curve of regrowth between fractions.(v) For a full HTBI course to 15 Gy, cell survival was predicted to be ~5 × 10 −5, compared with ~ 10−3 for a low dose rate (0.04–0.07 Gy/min) SDTBI to 10 Gy; the latter projected from the initial slope of the high dose rate, single dose survival curve.

Cell-density Dependency in the Survival Kinetics of Neocarzinostatin-treated HeLa Cells in Monolayer Cultures and in Spheroids.

Cell survival kinetics of HeLa cells grown exponentially in monolayers or grown in multicellular spheroids following neocarzinostatin (NCS) treatment was analyzed in vitro. The effect of NCS was markedly reduced when a large number of cells were plated as compared to a small number per dish. Cell survival kinetics for various combinations of factors: the cell number per dish, concentration of NCS and volume of the medium containing NCS showed that the extent of cell killing by NCS was solely determined by the relative relation of cell density expressed as cells per unit volume of medium to the effective concentration of NCS; the effective total NCS available to each cell. The theoretical formulation in a controlled situation indicates that C37, the concentration of NCS required to reduce the surviving fraction to e-1, is directly proportional to the cell density. The experimental data points derived from all the combinations of factors essentially followed a linear regression line of the theoretical equation. Therefore, the cell-density dependency of drug-induced cell killing may not be due to a difference in cellular sensitivity or recovery capacity. The effect of NCS on cells in spheroids with central necroses can not be explained only on the basis of the cell-density dependency, but is likely to be reduced by hypoxic conditions, decreased reactivation and the limited penetration of NCS to the deeper layers.

Unusual Survival Kinetics of Neocarzinostatin-treated HeLa Cells: Its Relation to the Drug-inactivation.

Cell survival kinetics of exponentially growing HeLa S3 cells as a function of the period of treatment with neocarzinostatin (NCS) and its relation to drug inactivation were analyzed in vitro with various concentrations of NCS. NCS activity was rapidly lost during the treatment period in a simple exponential manner with a half-life of 2 to 4 minutes depending on the treatment conditions. A novel type of time-survival curve was observed; the number of surviving cells decreased within a few minutes of treatment, then showed a transient increase after which there was again a decrease then an increase which reached a constant level in 3 hours. The increase in the surviving fraction of cells during the treatment period is interpreted as being due to recovery from the potentially lethal NCS damage manifested on the decrease of NCS activity in the medium. The subsequent fluctuation in the surviving fraction is not readily explainable, but may be due to reactivation and re-inactivation of NCS in the cells.

Cell survival kinetics in peripheral blood and bone marrow during total body irradiation for marrow transplantation

Cell survival kinetics in both peripheral blood and in bone marrow have been studied over the time course of hyperfractionated total body irradiation (TBI) for bone marrow transplantation. Our unique TBI regimen allows the study of the in vivo radiation effect uncomplicated by prior cyclophosphamide, since this agent is given after TBI in our cytoreduction scheme. Peripheral blood cell concentrations were monitored with conventional laboratory cell counts and differentials. Absolute bone marrow cell concentrations were monitored by measuring cell concentrations in an aspirate sample and correcting for dilution with blood by a cell cycle kinetic method using cytofluorometry. In the entire group of patients, time to engraftment with donor marrow was found to be 16.6 × 4.4 days and more rapid when a nucleated donor cell dose of ≥4.0 × 10 8 cells/kg was given. For lymphocytes in peripheral blood in patients in remission, the effective D 0 ranged from 373 rad in 10 children ≤10 y old, to 536 rad in the four patients between 11–17 y old, while n = 1.0 in all groups. There was no trend observed according to age. Granulocytes had a much higher effective D 0, approximately 1000 rad in vivo. Absolute nucleated cell concentration in marrow dropped slowly initially, due to an increased lymphocyte concentration in marrow during a concurrent drop in lymphocyte concentration in peripheral blood, but eventually fell on the last day of TBI ranging from 7–44 % of the initial marrow nucleated cell concentration. Marrow myeloid elements, however, dropped continuously throughout the course of TBI.