Thermohemolysis of erythrocytes

Thermohemolysis kinetic curves of erythrocytes in the temperature interval of 37-75 degrees C in different media and in the course of blood storage were studied. Mechanism of thermohemolysis is proposed. The rate constants of hemolysis stages are introduced. It is shown that these parameters are sensitive to the changes of structural state of erythrocytes.     

Microencapsulation of erythrocytes

Human erythrocytes have been encapsulated in a polyacrylate membrane by a simple precipitation process. The encapsulated cells appeared to remain functional after encapsulation: the consumption of glucose and the ability to reversibly bind oxygen was unimpaired. Furthermore, storage at 4°C for almost 6 months had no effect on the P₅₀ and n₅₀ values. This is the first time to our knowledge that live mammalian cells have been encapsulated in a polymer other than alginate.     

Magneto-electro-fusion of human erythrocytes

In inhomogeneous (static) magnetic fields close contact between ‘magnetic’ human erythrocytes was established. The cells were made magnetic by incubating them in a medium containing small Fe₃O₄-particles which adsorbed to the outer membrane surface. Fusion was induced by applying two electric field pulses (field strength: 8.5 kV · cm^?1; duration: 60 μs) to the magnetically collected cells. This procedure allowed the use of electrically conductive media (3 · 10^?1 Ω^?1 · cm^?1). Fusion of red blood cells occured very often. If cell suspensions of high density were used fusion resulted in the formation of giant red blood cells with osmotically intact membranes.     

Ultrastructure of malaria-infected erythrocytes

Knobs, caveolae, caveola-vesicle complexes, cytoplasmic clefts, and electron-dense material are five major ultrastructural changes found in the membrane skeleton and cytoplasm of erythrocytes infected with species of primate malaria. Knobs are electron-dense, conical evaginations of the erythrocyte surface, which are believed to mediate cytoadherence and sequestration of Plasmodium falciparum-infected erythrocytes. Caveolae and caveola-vesicle complexes are flask-shaped invaginations of the membrane skeleton, which may be involved in the uptake or export of host- or parasite-derived substances. Cytoplasmic clefts are flattened or circular membranous structures found in the erythrocyte cytoplasm between the intracellular parasite and the host cell surface. The clefts are variable in length and bounded by two or more membranes. Fine, granular electron-dense material is often found on the cytoplasmic face of clefts or in amorphous packets in the erythrocyte cytoplasm. Immunocytochemistry has demonstrated that all of these ultrastructural changes are associated with the trafficking and interaction of specific malarial antigens with the host erythrocyte.     

Flicker spectroscopy of erythrocytes

Frequency analysis of thermally excited surface undulations of erythrocytes leading to the flicker phenomenon is applied to determine biochemically and physically induced modulations of the membrane curvature elasticity. Flicker spectra of individual cells fixed to the window of a flow chamber by polylysine are taken by phase contrast microscopy, enabling investigations of the reversibility of the structural modifications. The spectra may be approximated by Lorentzian lines in most cases. By measuring the amplitude (at zero frequency) and the line width, effects of the structural changes on the curvature elastic constant, K _c , and the wavelength distribution of the undulations may be studied separately. Effect of physically induced modifications: The temperature dependence of the flicker spectra are taken from 10°C to 37°C. Above 20°C, K _c decreases with increasing temperature whereas the reverse holds below this limit. The latter anomalous behaviour is explained in terms of a conformational change associated with protein and lipid lateral phase separation. The bending stiffness increases when the cells swell osmotically, owing to surface tension effects. The dependence of the flicker spectra on the viscosity of the suspension medium agrees with the theoretical prediction. Biochemically and drug induced modifications: 5 vol of ethanol leads to a pronounced and reversible suppression of the long wavelength undulations without altering the discoid cell shape and without affecting the bending stiffness appreciably. Adsorption of dextran to the glycocalix increases K _c by a factor of 1.6 at saturation. The bending stiffness is increased by a factor of 1.3 after cross-linking the proteins with the SH-oxidizing agent diamid. Injection of Ca⁺⁺ into the cell via ionophores evokes (within 10 min) the formation of fine — probably spectrin free — spicules. This leads to an increase in K _c by a factor of 1.3 which is explained in terms of a lateral condensation of the spectrin/actin network. The spicule formation and K _c change is completely reversible (within 2 min) after perfusion with Ca⁺⁺-free buffer. Cholesterol depletion leads first to a continuous increase in K _c without change of the cell shape whereas a sudden discocyte- to echinocyte transformation sets in below a critical steroid content. The latter transition is also observed in cell suspensions and is reminiscent of a phase transition. The anti-tumor drug actinomycin D evokes an increase in the bending stiffness K _c by a factor of two, suggesting that its effect is at least partially due to a modulation of the membrane structure. The -receptor agonist leads to a remarkable increase in K _c (by about 25%) at 10^-4 M but the effect is not reversed by the -antagonist prazosin, suggesting that the agonist exerts a non-specific effect.A new technique, dynamic reflection interference contrast microscopy, is introduced by which absolute values of the amplitudes of the surface undulations and therefore K _c can be determined. The value obtained: K _c =5·10^-13 erg is about a factor of two larger then the bending stiffness of pure lipid bilayers. We suggest that the surface undulations may also be determined by lateral fluctuations of the quasi-two-dimensional spectrin/actin network.     

Cytoadherence of malaria-infected erythrocytes

Malaria-infected erythrocytes express new antigenic structures on their surface. Some of these molecules are responsible for the cytoadherence of infected cells to endothelial cells which, because of the sequestration produced, may, in turn, be responsible for the pathogenesis of the severe forms of the disease in humans (e.g., cerebral malaria). This paper critically reviews the state of the art concerning cytoadherence, with particular emphasis on the different experimental approaches that have been used to study the phenomenon and the parasite molecules that may be involved.     

Sequestration of neuraminidase-treated erythrocytes

Summary Scintigraphic experiments and radioactivity measurements of tissues have shown that the radioactivity of ⁵¹Cr-labelled and neuraminidase-treated rabbit erythrocytes is rapidly accumulated in liver and spleen. Sequestration of these erythrocytes by liver and spleen was demonstrated by light and electron microscopy of these tissues after perfusion of the rabbits with solutions for tissue fixation. In liver the phagocytic activity of Kupffer cells was increased after injection of desialylated erythrocytes, while in spleen a significantly enhanced number of erythrocytes was found attached to the sinusoidal walls and in the reticulum of the red pulp.It was shown by scanning electron microscopy that neuraminidasetreatment did not influence the shape of erythrocytes.Desialylated and ⁵¹Cr-labelled erythrocytes from the cow are rapidly cleared from the blood-stream with a half-life time of about 3 h.It was shown in an in-vitro test that they adsorb to surviving slices from liver and spleen derived from the same animal. The amount of radioactivity adsorbed is appreciably enhanced in the presence of homologous serum when compared with buffer only.Human neuraminidase-treated erythrocytes are agglutinated in the direct and especially in the indirect Coombs-tests. The involvement of T-antigen in this phenomenon was largely excluded.The in vitro experiments and antibody consumption tests suggest that immunoglobulins (IgG) and complement from serum may be involved in recognition and sequestration of desialylated erythrocytes by macrophages in vivo.Part of this work has been reported at the 3. Int. Symposium on Glycoconjugates: Functions in Animals, Brighton, Great Britain, July 6–12, 1975Part of a doctoral thesis, Abteilung für Naturwissenschaftliche Medizin, Ruhr-Universität BochumWe are greatly indebted to Mrs. A. Chen Stute for her help with the Scintigraphic studies. We also thank Prof. Dr. W. Opferkuch, Universität Bochum, for discussion, Prof. Dr. W. Kaufmann, Institut für Milchforschung, Kiel-Schaedtbek, for valuable advices, and Mr. H.G. Richter, Universität Bochum, for the scanning electron microscopic studies of erythrocytes. A gift of human -globulin from Dr. W. Schneider from the Blutspendezentrale Hagen and a sample of desialylated N-blood-group substance from Prof. Dr. G. Uhlenbruck, Universität Köln, are gratefully acknowledged. This work was financially supported by the Deutsche Forschungsgemeinschaft (grants Scha 202/3 and 202/5) and by the Fonds der chemischen Industrie     

Capillary electrophoresis of erythrocytes

Capillary electrophoresis (CE) of erythrocytes from different sources under various conditions is reported in this paper. It was found that erythrocyte samples from sheep, duck, and human showed characteristic and reproducible elution peaks, and that the retention times of A-, B-, AB-, and O-type erythrocytes from human blood were distinctively different; even subtle differences, among individuals with the same blood type could be detected by CE. A strictly linear correlation was obtained between the peak area and the amount of human erythrocyte over a range of 4.8 x 10(2)-1.9 x 10(4) cells (r=0.999), indicating that CE could be used for rapid and accurate quantification of erythrocytes. Using this CE protocol, the decrease of the surface electrical charge of erythrocyte during storage was confirmed. Therefore, this work demonstrated that CE could be a useful alternative for characterizing and quantifying erythrocytes or other cells.