The surface reactivity of heterologous immune sera with erythrocytes infected with Plasmodium falciparum has been difficult to assess in quantitative terms because of the restricted accessibility of surface epitopes and the lack of sensitive methodologies. In a previous study we showed that cryptic antigens can become exposed on the surface of intact trophozoites if the sterol content of the membranes is increased by means conservative of cell integrity (D. Baruch and Z. I. Cabantchik, Molecular and Biochemical Parasitology36, 127–138, 1990). In this work we introduce a novel and highly sensitive method of fluorescence cell ELISA for the quantitative estimation of immunoglobulin binding to the surface of P. falciparum-infected erythrocytes. We obtained that elevation of the membrane sterol content markedly increased the (external) surface accessibility of antigenic epitopes of trophozoites as well as rings of various strains of P. falciparum. This treatment induced exposure of similar epitope(s) on the surface of both rings and trophozoites insofar as preadsorption of sera on sterol-treated cells abolished immunoglobulin binding to either stage of infected erythrocytes (treated or not with sterol). These putative epitopes have relatively low but demonstrable accessibility on the surface of untreated rings but become virtually inaccessible at the trophozoite stage. Application of a large variety of sera (98) to sterol-treated infected cells revealed that almost 70% of the tested sera were found to give positive surface reactivity. Relatively higher intensity of binding was obtained with sera originating from clinically immune individuals. Binding of sera to cells infected with five different P. falciparum strains was essentially indistinguishable, strongly suggesting that elevation of membrane viscosity induces surface exposure of cryptic epitopes common to different parasite strains.

The membranes of Plasmodium falciparum-infected human red blood cells contain antigens of demonstrably cryptic character. We show here, by a cell surface radioimmunoassay using anti-human red cell membrane antisera, that raising the membrane microviscosity of intact cells leads to a marked increase in the cell surface antigen reactivity of normal cells, and even more so in cells infected in vitro with two strains of P. falciparum. A variety of sera from adults and children living in endemic areas and from malaria patients, all of which showed no detectable surface reactivity with either normal or infected red cells, were demonstrably surface-reactive to infected cells whose sterol membrane content has been raised by means conservative of cell integrity. New epitopes become exposed on the surface of infected cells after lipid modification. The present studies indicate that the reduced membrane viscosity reported in malaria-infected cells determines to a considerable extent the expression of cell surface antigens of both host and parasite, and could play a significant role in parasite immune evasion.

The sterol content of human erythrocyte membranes was modified by polyvinylpyrrolidone (PVP)-mediated enrichment or depletion of cholesterol (CHL) or incorporation of cholesteryl hemisuccinate (CHS). The effects of these modifications on osmotic fragility and anion exchange protein (AEP) disposition and function were evaluated. CHS enrichment was fast (1 hr, 37 degrees C) and led to a concentration-dependent crenation as well as a decrease in osmotic cell fragility, in parallel with increased membrane microviscosity. CHL caused similar but considerably less marked effects due to slower incorporation rates into membranes. CHS enrichment of cells induced susceptibility of AEP to trypsin, a protease which otherwise does not affect AEP in intact cells. Although transport rates of monosaccharides, nucleosides, and anions were markedly slowed down by CHS enrichment of cells in parallel with increased membrane viscosity, anion transport was the most affected. The temperature profile of anion transport in CHS-enriched cells revealed a 10-kcal/mol increase in the enthalpy of activation relative to normal cells. Anion transport measured in heteroexchange conditions (Cl in--pyruvate out) and (Cl in-sulfate out) was relatively more susceptible to CHS modification than when it was measured in homoexchange conditions (Cl in-Cl out). The results of these measurements indicate that CHS-mediated increase in membrane viscosity affects AEP translocation capacity and transmembrane disposition via changes in lipid compressibility. Specific effects of CHS on AEP function, however, could not be ruled out.

Human erythrocyte glycophorin, a putative receptor to Plasmodium falciparum malaria parasites, was studied in terms of its structural domains involved in mediating invasion. These domains were isolated from purified glycophorin A and from supernatants and membranes obtained from protease-treated erythrocytes. They were tested for invasion blocking capacity by using an in vitro assay system. The role of carbohydrate-rich domains was assessed with the following compounds: (i) sialoglycopeptides released by proteases either from whole cells or isolated glycophorin A; (ii) the sialoglycoproteins fetuin and alpha 1 acid glycoprotein and the N-acetylglucosamine-rich ovomucoid; and (iii) the saccharides N-acetylneuraminlactose, N-acetylglucosamine, and free sialic acid. With the exception of N-acetylglucosamine, all of the compounds failed to block invasion. The role of carbohydrate-poor domains of glycophorin was assessed with peptides isolated from membranes of proteolyzed cells and with the hydrophobic fragment of glycophorin A. Glycophorin and the derived hydrophobic peptides formed high-molecular-weight aggregates in physiological solutions. They all inhibited invasion to a comparable extent. The inhibitory potency of glycophorin A increased by sixfold after reconstitution into egg lecithin vesicles. The observations reported here underscore the role played by the hydrophobic domain in the glycophorin-mediated blockage of invasion. They also suggest that in the interactions between P. falciparum merozoites and the erythrocyte membrane, the exposed glycosylated domains of glycophorins provide the initial but rather weak binding sites, whereas the internal domains of the molecules provide the more stable attachment sites for merozoites.

During the intraerythrocytic growth of Plasmodium falciparum in culture, marked changes are observed in the permeability properties of the host cell membrane. Anionic substances otherwise impermeant to normal cells, become highly permeant to infected cells. These changes in permeability become apparent as rings mature into trophozoites and remain throughout schizogony. The permeability changes to anionic substances are not manifested as degradation of band 3, the purported erythrocyte anion transporter. They probably reflect alterations of a more general nature.

ECG tracings of 10,059 men aged 40 and over first participating in 1963 in the Israel Ischemic Heart Disease Project were interpreted by computer at the Instrumentation Field Station in Washington. The computer, using criteria developed by the Field Station, reported a high prevalence of ischemic heart disease. Computer interpretation of a sample group of ECGs was compared with that of a group of cardiologists using the same criteria. It was concluded that a computer diagnosis of normal could be accepted, but those readings denoted as abnormal by the computer had to be reinterpreted by cardiologists using uniform criteria that were modified as a result of their combined clinical experience. The final analysis of these tracings together with a sample of computer normals showed a much lower prevalence of ischemic heart disease. Differences between magnetic and standard ECG tracings are described and the advantages of computer interpretation indicated.