Oxygenation is a major determinant of the physiological state of cultured cells. 19F NMR can be used to determine the oxygen concentration available to cells immobilized in a gel matrix by measuring the relaxation rate (1/T1) of perfluorocarbons (PFC) incorporated into the gel matrix. In calcium alginate gel beads without cells the relaxation rate (1/T1) of the trifluoromethyl group of perfluorotripropylamine (FTPA) varies linearly with oxygen concentration, with a slope of 1.26 +/- 0.15 x 10(-3) s-1 microM-1 and an intercept of 0.50 +/- 0.04 s-1. During perfusion with medium equilibrated with 95%/5% O2/CO2, changes in PFC T1s indicate that the average oxygen concentration was reduced from 894 +/- 102 microM in the absence of cells to 476 +/- 65 microM and 475 +/- 50 microM in the presence of 0.7 x 10(8) EMT6/Ro and RIF-1 murine tumor cells per milliliter of gel, respectively. The presence of 0.2 microliters of FTPA/ml of gel had no effect on the energy status of the cells as indicated by 31P NMR spectra. To calculate oxygen gradients within the beads from the average PFC T1 of the sample, a mathematical model was used assuming that oxygen is the limiting nutrient for cell metabolism and that the cellular oxygen consumption rate is independent of oxygen concentration. Data for EMT6/Ro cells were fit using experimentally determined perfusion parameters together with literature values for cell volume and oxygen consumption rate.(ABSTRACT TRUNCATED AT 250 WORDS)
Progressive multifocal leukoencephalopathy (PML) is a rare demyelinating neurological disorder caused by JC virus. Immunocompromised patients such as those with chronic lymphocytic leukemia, AIDS and following organ transplantation are at increased risk. We report a patient with PML complicating longstanding Waldenström's macroglobulinaemia. Although PML is a rare occurrence, the newer highly immunosuppressive treatment approaches for patients with lymphoproliferative disorders necessitate a high index of clinical suspicion. The diagnosis should be considered in patients with compatible clinical features who have received long-term immunosuppressive treatments recognized to impair cellular immunity.
Summary Studies have been performed to investigate the radiosensitivity of human squamous carcinoma cells. A431 cells were grown in itro as exponential and fed-plateau monolayer cultures or as multicellular spheroids. Radiobiological studies of various cultures showed that fed-plateau phase cells were more sensitive (Do =1.3Gy) than exponentially growing cells (Do =1.5Gy). After a single dose of 12Gy or two doses of 6 Gy irradiation, A431 cultures exhibited a large capacity for potentially lethal damage (PLD) repair (PLD repair factor= 17), but a relatively small sublethal damage (SLD) repair. In order to measure the radiation sensitivity of proliferating (P) and quiescent (Q) cells, enriched populations of P-and Q-cells were isolated from A431 spheroids. Flow cytometric analysis with acridine orange (AO) staining demonstrated that there was a shift of the RNA histograms in fed-plateau and spheroid cultures towards lower values, suggesting the presence of a subpopulation of Q-cells. Centrifugal elutriation was used to isolate the Q-cells from dissociated spheroid cells. Coulter cell volume distributions and flow cytometric analysis showed that Q-cells had a small cell volume (-1380,um3), low RNA content and a G,-like DNA content. Continuous labelling experiments with tritiated thymidine confirmed the non-proliferating nature of the Q-cells. Irradiation of the Q-cells after isolation from spheroids with between 0 to lOGy showed that they were more radiosensitive (decreased Do) than the P-cells isolated from these spheroids. The latter were, however, similar in radiosensitivity to exponential G, cells.
The densities of viable EMT6 cells grown in vitro as monolayer exponential and plateau-phase cells or as multicell spheroids or in vivo as a solid tumor in BALB/c mice were determined with the use of isopyknic centrifugation in linear Ficoll gradients. Exponential cells banded at a density of 1.069 g/ml, whereas plateau-phase cells appeared at 1.073 g/ml. Cells grown as spheroids were more dense than the monolayer cells and were recovered mainly at 1.081. Solid-tumor cells, separated under the same conditions, banded at 1.080. Narrowing the range of the Ficoll gradient failed to resolve more than one band of cells in the solid-tumor separation. This provides evidence that the density of cells obtained from the spheroids is greater than that of the monolayer cells but agrees well with the density of the solid-tumor cells. Ficoll was demonstrated to be nontoxic to the cells, and plating efficiency assays showed similar cell viabilities between noncentrifuged and centrifuged cells. The plating efficiency of the peak fraction of exponential cells after centrifugation was 68%, and that of plateau-phase cells was 50%. Corresponding figures for the multicell spheroid and solid-tumor cells were 62 and 28%, respectively. The recovery of cells after centrifugation in the Ficoll gradients ranged from 62 to 83%. The effects of cell load and centrifugation time on the density distributions of the EMT6 cells were also investigated.
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