David Danon

Use of cationized ferritin as a label of negative charges on cell surfaces.

A polycationic derivative of ferritin for labeling of negative charges on cell surfaces was prepared by coupling horse spleen ferritin with N, N dimethyl-1,3 propanediamine (DMPA) via carbodiimide activation of the protein carboxyl groups. Various derivatives of cationized ferritin prepared in the pH range between 5 and 7 were characterized and tested for their labeling capacity. The DMPA-ferritin prepared at pH 6.5 was chosen as the optimal preparation for the present study. Labeling with cationized ferritin is performed at physiological conditions with or without prefixation with glutaraldehyde. It is effective on a variety of cells, and its geometry permits relatively easy counting on the membrane surface. The difference in surface charge labeling between young, old, and receptor-destroying enzyme (RDE)-treated rabbit red cells is reflected in the number of cationized ferritin particles attached per unit area of membrane in tangentially sectioned red cells. This is in agreement with the values obtained by electrophoresis.

Interaction of basic polyelectrolytes with the red blood cell II. Agglutination of red blood cells by polymeric bases

Abstract 1. 1. Agglutination of red blood cells and ghosts of polybases was studied by electrophoresis and electron microscopy. 2. 2. A critical potential of agglutnation was found and shown to be dependent on the chemical nature and the molecular weight of the polybase. 3. 3. Electron-microscopical study of polybase-treated ghosts demonstrated marked changes in the membrane texture. 4. 4. The structure of the seam of agglutination was studied on ultra thin sections of red blood cell aggregates. The seam presumably consists of a polymer film, in which the polybase molecules are anchored at each end on the adjacent cell membrabes. The maximum width of the bonding polymeric film is nearly equal to the length of the fully stretched polybase molecules. 5. 5. The aggregates are not dispersed by washing with physiological saline. They can be dispersed, however, by polyacids in a certain range of molecular weights. 6. 6. The cells obtained after dispersion of the aggregates exhibit the same electrophoretic behaviour and the same appearance in electron micrographs as the untreated cells.

The natural anti-α-galactosyl IgG on human normal senescent red blood cells

Summary. A highly sensitive antiglobulin test based on rosette formation due to the interaction between IgG bearing red blood cells (RBC) and Fc receptors on K562 cells, was used to study the immunoglobulin molecules present on human senescent RBC. Normal human RBC were separated into young and senescent subpopulations on the basis of age‐dependent differences in density by centrifugation on a discontinuous density Percoll gradient, and by flotation on phthalate ester mixtures. The senescent but not the young RBC were found to bear membrane bound IgG. Most of the bound IgG molecules could be specifically eluted by galactose in its α‐anomeric form. Antigalactosyl (anti‐Gal) IgG antibodies with similar reactivity were found to be present in high titres in every one of the 400 normal human sera tested. The natural anti‐Gal antibodies isolated from normal sera by affinity chromatography could bind to IgG depleted senescent RBC but not to young RBC. Erythrophagocytosis experiments indicated that the anti‐Gal bound to the senescent RBC induced their destruction by macrophages. It is suggested that the natural anti‐Gal antibodies interact with cryptic α‐galactosyl residues which are exposed in the course of the RBC senescence and mediate the removal of these RBC from circulation by cells of the reticuloendothelial system.

Agglutination by polylysine of young and old red blood cells

Abstract The rate of agglutination by polylysine of young and old human erythrocytes was automatically recorded and correlated with measurements of the electrophoretic mobility of these cells. Old red cells have a lower electrophoretic mobility than young cells, as well as a higher rate of agglutination than young cells. Mild treatment of red cells with receptor destroying enzyme, which reduces their surface charge, results in an increased rate of agglutination. The results indicate that there is correlation between the rate of agglutination and the surface charge of the red cell.

Treatment of human ulcers by application of macrophages prepared from a blood unit.

Decubital ulcers contribute to morbidity and mortality in elderly patients. Macrophages play a major role in the process of wound healing. We compared the efficacy of local treatment of decubital ulcers in elderly patients using macrophages prepared from a blood unit, vs. conventional treatments. Patients with decubital ulcers (n = 199) hospitalized during one year in a Geriatric Hospital in Israel, were included in the study. The ulcers of 72 patients (average age 82), who provided informed consent, by themselves or by family, were treated by local injection of macrophages prepared from a blood unit in a closed sterile system. The remaining 127 patients (average age 79) were treated conventionally and served as controls. No exclusion criteria were applied. Only a completely healed ulcer was considered a positive outcome of treatment. In the macrophage-treated group 27% (36 out of 131 ulcers) were healed compared to 6% (15 out of 248) in the control group (p < 0.001). There was also a significantly faster healing in the experimental group (p < 0.02). No side effects were noted. We conclude that Macrophages prepared from a blood unit, in cost-effective, closed, sterile system, are significantly more effective than conventional methods for the treatment of ulcers in elderly patients.

Chronic treatment with human recombinant erythropoietin increases hematocrit and improves water maze performance in mice

Erythropoietin is a glycoprotein produced endogenously in the kidney, which stimulates red blood cell production. We evaluated the effects of chronic treatment with recombinant human erythropoietin (epoetin alfa: EPO) on the performance of 6-month-old male C57BL/6J mice in a spatial learning task, the Morris water maze. Mice were treated with either EPO (1.5 U injected SC every other day) or vehicle (PBS also injected SC every other day). Results indicated that the treatment had no effect on maze performance after 8 weeks, but after 19 weeks the EPO-treated mice showed better performance compared to controls as measured by mean distance (centimeters) to reach the goal platform. The improved performance in EPO-treated mice at 19 weeks was accompanied by an increased hematocrit. After 32 wk of EPO-treatment, the hematocrit returned to baseline levels even though the size and density of the red blood cells were increased.

Surface charge properties of the luminal front of blood vessel, walls: An electron microscopical analysis

Abstract The negative charge density and distribution on cell membranes of vascular endothelium and subendothelial components of Guinea pig and rabbit blood vessels were evaluated using cationized ferritin (CF) and positively charged colloidal iron (CI). Isolated segments of vena cava and aorta as well as washed RBCs were fixed with aldehydes, washed and labeled with CF at pH 7.2 or with CI at pH 1.8. The particles were evenly distributed on the luminal front of the vascular endothelium. CF was seen on perpendicular sections about 20 A from the plasmalemma. No significant difference was detected between the charge density of vascular endothelium of Guinea pig and rabbit, despite a striking difference in the charge density of the respective RBCs. Treatment with neuraminidase resulted in 50% reduction of anionic groups on endothelial cells and 80% reduction RBCs. Subendothelial components including basement membrane, microfibrils and collagen fibers were densely labeled with CF and CI before and after treatment with neuraminidase. The results suggest that specific receptors rather than reduction in surface charge play a role in adhesion of thrombocytes to subendothelial components.

Localization of sialyl residues on cell surfaces by affinity cytochemistry

The use of the avidin—biotin complex for the specific ultrastructural visualization and evaluation of cell surface sialyl residues was investigated. Circulating red blood cells of various mammalian species and bone marrow cells of the rabbit were treated with sodium meta-periodate and biotin hydrazide in succession, with or without prior treatment of the cells with neuraminidase. The results were compared with the distribution of cell surface anionic sites, estimated by the rate of agglutination with poly- l -lysine and the binding capacity of positively charged colloidal iron and cationized ferritin. A uniform labeling of the erythrocyte surface was obtained with variations between different animal species. Neuraminidase treatment drastically reduced the sensitivity of cells to periodate-induced biotinylation. This method represents a distinct improvement over past approaches, since it can be applied to unfixed cells at physiological pH. An additional feature of this system enabled the topographic resolution of sialic acid residues from the outer dense line of the erythrocyte membrane. A value of 50–70 A was obtained for human erythrocytes. Developmental and species-dependent modulations in the proximity of sialic acid to the membrane were also observed. The frequency of attached ferritin—avidin conjugates to biotinylated erythroid cells in rabbit bone marrow was found to vary according to the degree of maturation in the erythroid line, corresponding to the previously described variations in colloidal iron binding.

Distribution of the T-antigen on erythroid cell surfaces. Studies with peanut agglutinin, an anti-T specific lectin

Abstract The interaction of peanut agglutinin, a lectin with a specificity similar to that of serum T-agglutinin, with human, guinea pig and rabbit young and old red blood cells and bone marrow erythroid cells, both before and after neuraminidase treatment, was investigated. Distribution of the peanut agglutinin receptors was determined by electron microscopical analysis with the ferritin conjugated lectin. In addition, kinetics of agglutination by the lectin were examined with the aid of the Fragiligraph. Rabbit red blood cells of all ages bound peanut agglutinin without neuraminidase treatment. Untreated human and guinea pig red blood cells, as well as immature erythroid cells of guinea pig bone marrow were not agglutinated by the lectin, nor was binding of the lectin to these cells observed by electron microscopy. Treatment with neuraminidase resulted in exposure of peanut agglutinin binding sites on erythroid cells of all stages of maturation and aging. Our findings show that peanut agglutinin receptors are absent from the surface of old circulating red blood cells and extruded erythroid nuclei, in spite of a remarkable in vivo loss of surface sialyl residues. The use of neuraminidase-treated red blood cells, as a model for the study of the clearance of aged red blood cells from circulation seems, therefore, not to be entirely justified.

Electron Microscopical Analysis of Surface Charge Labelling Density at Various Stages of the Erythroid Line

SummaryWith use of the positively charged, colloidal ferric oxide labelling technique for electron microscopy of sections of rabbit marrow, a reduction in labelling density on the surface of differentiating red cell precursors was demonstrated. The number of iron particles per unit length of membrane was counted. A progressive diminution in labelling density follows cell division, reaching a minimum in the orthochromatic erythroblast, from which the nucleus is expelled. A slight increase in charge density is noted in the reticulocyte, and further increase is observed with its maturation to the erythrocyte. The results indicate that biosynthesis of n-acetyl neuraminic acid stops at the earliest recognizable stage of erythroid differentiation.