Large-scale production of CD4+ T cells from HIV-1-infected donors after CD3/CD28 costimulation.

We describe a procedure for large-scale enrichment, growth, and harvesting CD4+ T cells. This method may be effective for HIV-1 immunotherapy, as the mode of stimulation, with anti-CD3 plus anti-CD28 coated beads (CD3/CD28 beads) induces a potent antiviral effect. PBMC were obtained by density gradient centrifugation of an apheresis product. Monocytes/macrophages were removed by incubating PBMC with beads coated with IgG. The cells were then magnetically depleted of B cells and CD8+ cells with mouse anti-CD20 and anti-CD8 MAbs and sheep antimouse coated beads. The remaining cells were >80% CD4+ and were transferred to gas-permeable bags containing CD3/CD28 beads and cultured in a closed system. After 14 days, the cell number increased an average of 37-fold, and cells were nearly 100% CD4+. Viral load, assessed by DNA PCR for HIV-1 gag, decreased >10-fold during culture in the absence of antiretroviral agents. Removal of CD3/CD28 beads from the cell suspension was accomplished by passing cells plus beads (3-30 x 10(9) cells in 2-12 L) over a MaxSep magnetic separator using gravity-driven flow. The cells were then concentrated to 300 ml in an automated centrifuge. This process allows safe and efficient growth of large numbers of CD4+ T cells from HIV-1+ donors.

Design of large-scale separation systems for positive and negative immunomagnetic selection of cells using superparamagnetic microspheres.

The ex vivo selective separation of cells from bone marrow and peripheral blood stem cell preparations is increasingly used as an adjunct to hematopoietic rescue following high-dose therapy for refractory cancer. Immunomagnetic separation, in which the target cells are identified using monoclonal antibodies and separated by attachment to paramagnetic particles and passage through a magnetic field, is widely used for both negative and positive cell selection. In this paper, we discuss the factors that should be considered when developing a magnetic separation device for purging tumor cells and selecting stem cells from bone marrow using superparamagnetic microspheres.

A large-scale magnetic separator for selective cell separations with paramagnetic microbeads.

An improved magnetic separator has been developed for use in large-scale cell separations. This separation method uses paramagnetic microbeads coated with antibodies that selectively bind to target cells. The magnetic separator attracts the microbead-target cell aggregates and holds these aggregates at its surface while the suspending fluid and nontarget cells flow past. The optimum separator design was determined to be two magnetic assemblies in series along with a peristaltic pump. The assemblies consist of neodymium-iron-boron magnet bars sandwiched between steel bars (magnetic pole pieces). The size and pole spacing of the two magnetic assemblies are designed to be different, so that the first assembly, which captures greater than 99.99% of the microbeads, has good magnetic reach-out and a high magnetic holding force at its surface, while the second assembly has an even higher magnetic holding force at its surface. Studies show that the separator can remove 1 x 10(10) microbeads from a suspension of red blood cells processed at a flow rate of 9 ml/min, so that no microbeads are detected in the effluent.

Evaluation of membranes for plasmapheresis.

Commercial flat-sheet microporous membranes were evaluated for potential use in plasmapheresis with a specially designed filtration module. Significant differences in filtration rates were observed with different membranes. Saline filtration data were not useful in predicting the capacity of the membranes to filter plasma from whole blood. For all membranes studied, no rejection of plasma proteins was detected. No activation or deactivation of clotting factors was detected as a result of filtration. In addition, little or no hemolysis was caused by filtration with the various membranes. Saline, cell-free plasma, platelet-poor plasma, and whole blood were perfused over a track-etched membrane and the resulting filtration rates were compared. The cell-free plasma filtration rate decreased significantly with time, probably owing primarily to protein adsorption in the membrane pores. Cell-free plasma and saline filtration data were used to calculate an apparent adsorbed layer thickness in the membrane pores. Perfusion of platelet-poor plasma and whole blood resulted in time-dependent filtration rates that were much lower than those obtained when cell-free plasma was perfused. Results of the study support recent theoretical models that postulate that the rate-limiting process for blood filtration is the formation of a layer of blood cells (particle polarization) on the membrane surface.

The effect of PGI2 and theophylline on the response of platelets subjected to shear stress.

A specially designed rotational viscometer was used to investigate the effects of the antiplatelet agent PGI2 in combination with theophylline on the response of human platelets subjected to shear stress. Samples of citrated platelet-rich plasma (PRP) were exposed to shear stress in the viscometer for a period of 5 min at 23 degrees C. The levels of stress studied ranged from 50 to 300 dynes/sq cm. Pretreatment of the platelets with 0.01 microM PGI2 and 500 microM theophylline before exposure to shear stress caused a large reduction in shear-induced platelet aggregation. However, it was also observed that the PGI2--theophylline pretreatment concomitantly caused a large increase in shear-induced platelet lysis and serotonin release at stress levels equal to or greater than 150 dynes/sq cm. This observed increase in platelet fragility may have important implications for clinical applications of PGI2. The results are discussed and compared to those obtained in prior work in which platelets were pretreated with acetylsalicylic acid or with PGE1.

Prospective study of fetal heart rate and rhythm patterns.

The fetal heart beat, detected by ultrasound, was recorded for five minutes from 934 antenatal women--266 at 30 to 35 weeks' gestation and 718 at 36 to 41 weeks. Episodes of bradycardia < 100/minute ranging from 5-30 seconds' duration occurred in 3 (1.1%) of the former and in 9 (1.3%) of the latter group. Tachycardia > 180/minute ranging from 30-90 seconds' duration occurred in 1 (0.4%) of the former and in 4 (0.6%) of the latter. Premature beats were not detected between 30 and 35 weeks, but occurred in 12 (1.7%) of 720 at 36 to 41 weeks' gestation. 50 subjects were monitored at both 30 and 35 weeks and 36 to 41 weeks' gestation and in one woman premature beats were present in the latter but not in the former recording. The incidence (1.7% of premature beats in the fetus at 36 to 41 weeks' gestation was similar to that in the healthy neonate (0.8%). Recordings of arrhythmias or rates outside the range 100-180/minute were replayed through a standard antepartum fetal heart rate monitor. The monitor failed to detect premature beats, 2 to 4 episodes of tachycardia > 180/minute, and 9 of 12 episodes of bradycardia < 100/minute, stressing its unreliability for detecting rapidly changing rates.