Intraerythrocytic parasites and red cell deformability: Plasmodium berghei and Babesia microti.

In the studies reported here, we examined the effects of two intraerythrocytic parasites (Plasmodium berghei and Babesia microti) on the deformability of their host red cells. Red cell deformability was assessed by three criteria: 1) the prevalence of tank-treading (the tank-tread-like movement of the red cell membrane around its cytoplasmic contents), 2) elongation under fluid shear stress (the steady-state length: width ratio), and 3) the time required for the red cell to reduce its steady-state elongation by 63.2% after the abrupt release of the shear stress (the characteristic shape-recovery time). Trophozoite-stage parasites of both species reduced the prevalence of tank-treading. Ring- and trophozoite-stage parasites of both species reduced steady-state elongation, and ring-stage P. berghei prolonged the shape-recovery time. These results suggest that altered red cell deformability is a common feature of infection with intraerythrocytic parasites.

Calcium and the malaria parasite: parasite maturation and the loss of red cell deformability.

In the studies reported here, we examined the role of calcium in the maturation of the human malaria parasite Plasmodium falciparum, and in the loss of red cell deformability associated with parasite maturation. P. falciparum alters the permeability of its host red cell, which normally maintains submicromolar cytoplasmic concentrations of calcium. Infection of the red cell and parasite maturation produce a 30-fold increase in calcium uptake. Both parasite maturation and the loss of red cell deformability are blocked by EGTA (by extracellular-free calcium concentrations less than or equal to 35 microM) and by other calcium antagonists. The loss of red cell deformability that occurs with parasite maturation is accompanied by alterations in the cytoskeletal proteins of parasitized red cells similar to those produced by the calcium ionophore A23187 (reductions in bands 2.1 [ankyrin], 4.1, and 5 [actin]). These results establish that parasite development and the loss of red cell deformability are calcium-dependent. They suggest that parasite-induced changes in the calcium permeability of the red cell activate endogenous transglutaminase activity by raising the free calcium concentration of the red cell cytoplasm.

Rheoscopic investigation of red cell deformability in sickle cell anemia.

Adequate erythrocyte deformability is crucial to microvascular function. In sickle cell anemia, a significant fraction of the circulating red cells lose deformability and assume highly abnormal shapes when exposed to low plasma oxygen tension (PO2). The loss of deformability is believed to induce blockage of flow in capillaries with consequent painful crisis or organ infarcts. The deformability of sickle erythrocytes at graded levels of PO2 were investigated in the rheoscope, a viscometric device consisting of transparent counter-rotating cone and plate. Quantitative indices of deformability obtained from still photographs and videotape recordings of cells subjected to shear flow were: fraction of all suspended cells capable of deformation, steady-state elongation, and time course of transient shape recovery following abrupt flow cessation. Suspensions of unfractionated cells were first equilibrated against gas mixtures (O2, N2, CO2) with PO2 = 160, 40 or 20 mm Hg at room temperature and then sheared under the same atmosphere. Results obtained with blood samples from ten pediatric patients being treated at St. Louis Children's Hospital show strong donor-to-donor variations and significant impairment of deformability in the unsickled members of the cell populations relative to normal controls.

Age-related changes in deformability of human erythrocytes.

The present study was designed to further the characterization of age-related changes in the deformability of human erythrocytes. The top (approximately young) and bottom (approximately old) 10% fractions of density-separated red cells from ten normal donors were subjected to graded levels of shear stress in a rheoscope. Measurements were made of steady-state elongation (cells tank treading in a state of dynamic equilibrium) and the time course of shape recovery following abrupt cessation of shear. In parallel with the rheologic experiments, several physical and chemical properties were assayed to determine correlates of mechanical properties. These included mean cell volume, mean corpuscular hemoglobin concentration, type A1 hemoglobin, glucosylation of membrane proteins, and membrane phospholipid and protein concentration. The microrheologic observations revealed that only about 90% of the old cells retained their capacity to tank tread. However, the tank-treading cells elongated less than their younger counterparts at corresponding levels of shear stress, thus demonstrating a reduced level of deformability. Further analysis of the data indicates that increases in membrane viscosity and elastic modulus along with a significant loss in excess surface area contribute to the limitation of the ability of the older cells to change shape.

Microrheologic investigation of erythrocyte deformability in diabetes mellitus.

This study was undertaken to determine whether diabetes alters the viscoelastic properties of erythrocytes. The oldest and youngest 10% fractions of circulating red cells were separated by centrifugation of freshly drawn blood obtained from ten diabetics with disease of one to 20 years' duration and from an equal number of age- and sex-matched control subjects. Cells from each fraction were suspended in phosphate-buffered saline, and their rheologic behavior was examined in a rheoscope. The elongation of cells, the percentage of cells that tank-treaded in response to shear stress, tank-treading frequency, and the rate of recovery of cell shape upon cessation of shear stress were determined in the oldest and youngest 10% of cells for diabetics as well as for controls. All four parameters were virtually identical for diabetics and controls. Additional aliquots of cells were taken for assessment of nonenzymatic glucosylation of hemoglobin and cell membrane protein. The absence of any measurable difference in rheologic behavior of cells from diabetic and control subjects, despite substantial differences in nonenzymatic glucosylation of hemoglobin and cell membrane proteins, suggests that the magnitude of glucosylation observed in these cellular constituents does not alter the viscoelastic properties of the cells. The implication of these observations is that microvascular complications of diabetes are not attributable to altered deformability of red cells.

Plasmodium falciparum maturation abolishes physiologic red cell deformability.

Normal red cells deform markedly as they pass through the spleen and the peripheral capillaries. In these studies, the effects of Plasmodium falciparum infection and maturation on the deformability of parasitized red cells exposed to fluid shear stress in vitro were examined by means of a rheoscope. Red cells containing the early (ring) erythrocytic stage of the parasite have impaired deformability at physiologic shear stresses, and recover their normal shape more slowly. Red cells containing more mature parasites (trophozoites or schizonts) exhibit no deformation under the same conditions. These results provide a mechanism to explain the ability of the spleen to remove parasitized red cells from the circulation of both immune and nonimmune hosts.

A study of variance in measurements of tank-treading frequency in populations of normal human red cells.

The statistical variance in rheoscopic measurements of tank-treading frequency ( TTF ) of normal human red cells has been investigated and the sources of this variance analyzed. Experiments were designed to expose the influence of donor, the stability of a donor's cells over a 2-month period, storage time from venipuncture to testing, cell age, applied shear rate and variations in the rheoscope gap due to trial-to-trial manipulation. The results show that the TTF varies linearly with applied shear rate within the range 20 to 180 s-1 and is strongly dependent on cell age. The slope of the TTF characteristic is not significantly different in cells taken from different, but hematologically normal adults nor does it change significantly over time in a particular donor. TTF measurements may be useful in distinguishing rheologically abnormal cell populations which differ from normal cells by at least 10%.