Antibody formation to autologous erythrocytes following the immunization with rat erythrocytes of normal animals and mice tolerant to the immunizing antigen

(CBA X C57B1/6)F1 mice immunized three times with rat erythrocytes produced antibodies both to this antigen and to autologous erythrocytes. Most of the antibodies to rat erythrocytes belonged to IgM isotype while antibodies to autologous red cells were of IgG isotype. Combined injection of thymectomized (CBA X C57B1/6)F1 mice with a massive dose of rat spleen cells and cyclophosphamide induced in animals stable tolerance to rat cells. Inducibility of antibodies to autologous red cells in tolerant mice injected 3-5 times with rat erythrocytes was drastically reduced. Nonspecific suppression (thymectomy and cyclophosphamide) did not prevent production of autoantibodies.     

Adhesion,phagocytosis and cell surface energy. The binding of fixed human erythrocytes to rat macrophages and polymethylpentene

Fixed human erythrocytes were used as model particles for the study of adhesion and phagocytosis by rat peritoneal macrophages. Erythrocytes were fixed with various concentrations of glutaraldehyde or tannic acid, or were treated with neuraminidase. Adhesion and phagocytosis of these cells were measured. In addition, the surface energy of these erythrocytes and macrophages was estimated by the contact angle technique. Free energies of adhesion, based on the cell surface energies, were correlated with both adhesion and phagocytosis.     

Selective binding of specific rat epididymal secretory proteins to spermatozoa and erythrocytes

Tissue pieces from the caput epididymidis of the rat were incubated in vitro with (³⁵S) methionine to produce radioactive secretory proteins. The radioactive secretory proteins so formed were tested for their ability to bind to washed rat spermatozoa collected from the rete testis and cauda epididymidis, and to rat erythrocytes. The sperm and erythrocytes bound approximately 5% of the total radioactive protein. Binding was protein-specific in that only selected proteins became associated with the cells. Binding was not cell-specific, however, since testicular spermatozoa, caudal spermatozoa, and erythrocytes all bound the same proteins to a similar degree.     

A PAS reaction in erythrocytes of formalin-fixed tissue treated with Holt's hypertonic gum sucrose

Summary When paraffin sections of mouse and rat kidney, liver and spleen were fixed in a formalin-type fixative overnight, then preserved in Holt's hypertonic gum-sucrose solution, erythrocytes stained strongly by the McManus periodic acid-Schiff method. The same tissues processed without gum-sucrose did not exhibit such positive staining of red blood cells. Tissues treated with the individual gum-sucrose components, a solution of gum arabic or of sucrose, gave positive staining of erythrocytes only after gum arabic. Powdered gum arabic but not sucrose crystals stained positively. The erythrocyte reaction in formalinfixed tissue treated with gum-sucrose is therefore related to the presence of gum arabic in Holt's solution. Opposing results were encountered with two non-formalin fixatives, glutaraldehyde and acetone, in that staining was consistently positive throughout in the former and consistently negative in the latter, regardless of gum-sucrose treatment.     

Radioprotection of bovine erythrocytes to haemolysis

The effect of several free-radical scavengers on the radiation-induced haemolysis of bovine erythrocytes (50 krad, 60Co, cell concentration: 0.05 v/v) was studied. A two-phase effect of the additives was found with a maximum of the protective action in the millimolar concentration range. A correlation was established between the degree of protection at a fixed concentration (5mM) and the rate constants of reactions of respective compounds with OH. A radiosensitizing effect of diethyldithiocarbamate, an inhibitor of superoxide dismutase (SOD) was not confirmed and no specific protective effect of SOD was seen.     

Antigenicity of erythrocytes as analyzed in terms of their cross-reactivity.

The antigenicity of human erythrocytes of four different ABO blood groups and sheep erythrocytes of unknown blood type from different individual sheep were analyzed in terms of their cross-reactivity with antibody-producing cells (plaque-forming cells, PFC) and serum antibody in immunized C57BL/6 and C3H/He mice. The antigenicity of human erythrocytes of different ABO blood groups in the C57BL/6 mice, as determined by the number of specific PFC, was, in decreasing order, AB = A greater than B = O (p less than 0.005). The efficiency of immunogenicity of the human erythrocytes in terms of their cross-reactivity with PFC was, in order, AB = A = B greater than O, and the degree of reactinogenicity was, in order, AB greater than A greater than B greater than O. The order of antigenicity of sheep erythrocytes from different animals, SRBC No. 1 - No. 6, was No. 1 (= No. 2) greater than No. 3 = No. 4 = No. 5 greater than No. 6 in C57BL/6 mice and No. 1 = No. 2 = No. 3 = No. 4 = No. 6 greater than No. 5 in C3H/He mice, determined by the number of specific PFC (p less than 0.01). The cross-reactivity of SRBC No. 1 - No. 6 with PFC demonstrates that the order of immunogenicity of SRBC was No. 1 = No. 2 = No. 3 = No. 4 = No. 5 greater than No. 6 in C57BL/6 mice and No. 1 = No. 2 = No. 3 = No. 4 = No. 6 greater than No. 5 in C3H/He mice, and that of their reactinogenicity was No. 1 greater than No. 2 =No. 3 = No. 4 = No. 5 greater than No. 6 in C57BL/6 mice and No. 1 greater than No. 4 = No. 6 greater than No. 2 = No. 3 greater than No. 5 in C3H/He mice. The cross-reactivity at the antibody level was indicative of the immunologic characteristics of blood cells of low antigenicity (human group O erythrocytes and SRBC No. 5 and No. 6). SRBC No. 5 and No. 6 were somewhat opposed to each other regarding antigenicity in C57BL/6 and C3H/He mice. This signifies the presence of different immunogenic components on SRBC No. 5 and No. 6. The production of anti-SRBC No. 1 antibody reached its peak on the third day after secondary immunization. That of anti- SRBC No. 1, cross-reactive with SRBC No. 6, occurred after a longer latent period, reaching its peak on day 6. This indicates that SRBC No. 1 possesses more than one kind of immunogenic component or immunogenic determinant group on its surface.     

Aggregate formation of erythrocytes in postcapillary venules

The purpose of the present study was to obtain information on erythrocyte aggregate formation in vivo. The movements of erythrocytes in postcapillary venules of the rat spinotrapezius muscle at various flow rates were recorded with a high-speed video camera before and after infusion of dextran 500. To distinguish aggregates, the following criteria were used: 1) a fixed distance (4 microm) between the center points of two adjacent cells, 2) lack of visible separation between the adjacent cells, and 3) movement of the adjacent cells in the same direction. Without dextran 500 infusion, 11 and 5% of erythrocytes formed aggregates in low (33.2 +/- 28.3 s) and high pseudoshear (144.2 +/- 58.3 s) conditions, respectively, based on the above criteria. After dextran 500 infusion, 53% of erythrocytes satisfied the criteria in the low pseudoshear condition (26.5 +/- 17.0 s) and 13% of erythrocytes met the criteria in the high pseudoshear condition (240.0 +/- 85.9 s), indicating erythrocyte aggregation is strongly associated with shear rate. Approximately 90% of aggregate formation occurred in a short time period (0.15-0.30 s after entering the venule) in a region 15 to 30 microm from the entrance. The time delay may reflect rheological entrance conditions in the venule.     

Electron microscope study on the occurrence of electron-dense deposits at the cell membrane of chicken erythrocytes

Summary Electron-dense deposits were found along the inner side of the cell membrane of chicken erythrocytes when fixed in a CaCl₂-containing glutaraldehyde solution. Osteoblasts and osteoclasts were studied in an attempt to determine whether the deposits are related to calcium metabolism. No electron-dense deposits adjacent to the cell membrane were found in either these cells or in rat erythrocytes. Therefore, it is assumed that the deposits are not calcium-binding protein necessary for the cellular calcium uptake.Supported by the Deutsche Forschungsgemeinschaft.The author is indebted to Professor R. Schauer for valuable discussions.     

The interaction of phenyldichloroarsine with erythrocytes

The purpose of the study was to identify binding sites of organic arsenic in the erythrocyte and to explain species differences in binding. Washed erythrocytes were exposed to graded concentrations of [U-14C]phenyldichloroarsine (PDA) in phosphate-buffered saline containing 0.1% glucose and 0.1% bovine serum albumin. At low PDA concentrations, all cells bound the arsenical rapidly (within 10 min) and quantitatively. Human, pig, hamster, guinea pig, and mouse erythrocytes approached saturation at 0.02-0.3 mumol PDA/10(9) cells, depending on the species. Saturation points correlated well with each respective species' erythrocyte glutathione content. In contrast, rat erythrocytes showed no sign of saturation at PDA loads as high as 3.0 mumol/10(9) cells. Hemolysates of PDA-treated erythrocytes were subjected to Sephadex G-75 gel filtration chromatography. 14C from rat hemolysate was distributed between the hemoglobin and small molecular weight (glutathione-containing) fractions. In all other species, the 14C eluted almost exclusively with the glutathione-containing fractions. In equilibrium dialysis experiments, human hemoglobin did not bind PDA, whereas rat hemoglobin bound 2 PDA/mol with Kd approximately 5 microM. In conclusion, glutathione is the principal binding site of phenyldichloroarsine in erythrocytes. In most species, the arsenical does not bind to hemoglobin, even though it has free (titratable) sulfhydryls considerably in excess of the glutathione concentration. In rat erythrocytes, phenlydichloroarsine binds both to glutathione and to hemoglobin. Arsenical binding by rat hemoglobin is presumably due to the unique location of the extra titratable cysteine in that protein.     

Hypertension: cation transport and the physical properties of erythrocytes

In recent years, many reports have appeared describing altered Na+ and K+ transport in erythrocytes of individuals with essential hypertension. Collectively, the interpretation of these results has been unclear. Our studies revealed that the active ouabain-sensitive K+ influx, the furosemide-sensitive K+ influx and the residual passive K+ influx in both human and rat erythrocytes can vary considerably among individual persons or rats and that these measurements alone can not be used to distinguish normotensive from hypertensive individuals. The only consistent cation transport difference observed was an increased Na+ permeability in spontaneously hypertensive rat (SHR) erythrocytes. We have also examined certain physical properties (equilibrium density distribution and sedimentation velocity) of erythrocytes from normotensive Wistar-Kyoto (WKY) and SHR rats, since these characteristics may be altered in response to abnormalities of ion transport. It was found that the erythrocytes from geographically, environmentally, and age-matched littermates of WKY and SHR rats have identical equilibrium density distributions. It was also found that the density distribution of erythrocytes can vary among geographically dispersed colonies of the same strain of rat, and even among successive litters of the same rat colony. However, the sedimentation time required for erythrocytes to reach their equilibrium density was always shorter in the normotensive WKY samples than in the matched SHR. Utilizing a simple centrifugation method, we were able to clearly show that for any population of erythrocytes with the same upper limit of cell density, normotensive WKY cells always sediment at a faster rate than those of the hypertensive SHR.(ABSTRACT TRUNCATED AT 250 WORDS)     

An in vitro assay for sequestration: binding of Plasmodium falciparum-infected erythrocytes to formalin-fixed endothelial cells and amelanotic melanoma cells

Erythrocytes infected with Plasmodium falciparum bind specifically to cultured endothelial cells and to a line of amelanotic melanoma cells. We have fixed endothelial cells and amelanotic melanoma cells in various ways and determined whether the fixed cells were still able to bind infected erythrocytes. Only cells fixed with 1.0-2.5% formalin in phosphate-buffered saline continued to bind infected erythrocytes as well as unfixed cells. The mechanism of binding to fixed and unfixed cells appeared to be identical for the following reasons. First, erythrocytes infected by parasite strains that bound to unfixed cells also bound to fixed cells while those that did not bind to unfixed cells did not bind to fixed cells. Second, immune serum that inhibited binding to unfixed cells also inhibited binding to fixed cells. Third, electron microscopy showed that knobs were the points of attachment between infected erythrocytes and both fixed and unfixed melanoma cells. Fixed cells gave reproducible results over at least 2 months. Thus, we have developed a simplified, reproducible assay for measuring binding of P. falciparum-infected erythrocytes to target cells.     

Anomeric preference of glucose utilization in human erythrocytes loaded with glucokinase

Human erythrocytes were loaded with homogeneous rat liver glucokinase by an encapsulation method based on hypotonic hemolysis and isotonic resealing. As assayed at 10 mM glucose, glucokinase and hexokinase activities in glucokinase-loaded erythrocytes were 218 and 384 nmol/min/gHb, respectively; whereas hexokinase activity in both intact and unloaded red cells, which contain no glucokinase activity, was about 400 nmol/min/gHb. No difference in the rate of lactate production from glucose anomers between intact and unloaded erythrocytes suggested that the encapsulation procedure itself did not affect glucose utilization in red cells. Alpha-anomeric preference in lactate production from glucose was observed in glucokinase-loaded erythrocytes, whereas the beta anomer of glucose was more rapidly utilized than the alpha anomer in intact and unloaded erythrocytes. The results indicate that the step of glucose phosphorylation determines the anomeric preference in glucose utilization by human erythrocytes, since glucokinase and hexokinase are alpha- and beta-preferential, respectively, in glucose phosphorylation.     

The photodynamic action of riboflavine on erythrocytes

The photodynamic action of riboflavine and riboflavine phosphate on erythrocytes was studied. It was found that irradiation of rat erythrocyte suspension in the presence of riboflavine phosphate resulted in an enhancement in erythrocyte potassium losses. Also, in the presence of riboflavine and light the release of lactose isonicotinoyl-hydrazone entrapped in released mouse erythrocytes, was markedly increased. The delayed photosensitizing effect, after keeping the irradiated samples for 20 hours in the dark, was more pronounced. No significant photohemolysis of erythrocytes could be demonstrated. Products of lipid peroxidation were formed, and was assumed to be responsible for the alteration in membrane permeability.     

Specific binding of cholera toxin to rat erythrocytes revealed by analysis with a fluorescence-activated cell sorter

The direct binding of cholera toxin to the receptor on the native cell surface was analyzed with a fluorescence-activated cell sorter (FACS) by the direct membrane immunofluorescence technique using FITC-conjugated cholera toxin B subunit as a ligand and erythrocytes, but the binding was significantly affected by a change in pH, showing optimum pH of 7.2. The optimum conditions for analysis of the cholera toxin-binding with a FACS were reaction of the target cells with 0.2 M phosphate-buffer (pH 7.2) containing 0.025% of BSA and 0.175 M of NaCl at 4 degrees C for 40 min. The binding of cholera toxin B subunit to rat erythrocytes was linear in the range of 1.2 ng to 80 ng, which corresponded to 2,469 to 163,500 molecules of toxin per cell, and the latter was almost the saturated level of binding. although erythrocytes from different strains of rats possessed equal binding ability for the cholera toxin, no binding was observed with erythrocytes from mouse, guinea pig, cow, pig, man, or rabbit, indicating that the cholera-toxin binding occurs specifically on rat erythrocytes. This is in accord with our previous analytical deta on the absence of GM1 in erythrocytes of these animals except rat, of which erythrocytes contain GM1. Also, the structural specificity of the receptor for cholera toxin was assessed by a binding inhibition experiment using glycolipid-containing liposomes as inhibitors and GM1 was found to be the most potent inhibitor, showing complete inhibition of toxin (40 ng) binding to 5 x 10(6) erythrocytes at 505.6 pmol of GM1.     

Cryopreservation of cyanate-treated sickle erythrocytes

Metabolic features and in vivo recovery of cryopreserved cyanate-treated erythrocytes from patients with sickle cell anemia were studied. Red cells were treated with the anti-sickling agent sodium cyanate, glycerolized, and frozen at ?80 °C. Cyanate increased post-thaw hemolysis of both normal and sickle erythrocytes. The thawed carbamylated sickle erythrocytes maintained high oxygen affinity but lost more than half of their ATP content. Addition of the metabolic nutrients adenine, pyruvate, and inosine (rejuvenation) during cyanate incubation prevented ATP loss. Rejuvenation also increased red cell 2,3-DPG and opposed the cyanate effect by lowering oxygen affinity. Yet cyanate improved by nearly 50% the intravascular recovery of thawed rejuvenated sickle erythrocytes in a rat transfusion model. Cryopreservation of autologous cyanate-treated erythrocytes could lead to their use as an extracorporeal treatment of sickle cell disease.     

Failure to respond to the surface of Plasmodium falciparum infected erythrocytes predicts susceptibility to clinical malaria amongst African children

Following infection with Plasmodium falciparum malaria, children in endemic areas develop antibodies specific to antigens on the parasite-infected red cell surface of the infecting isolate, antibodies associated with protection against subsequent infection with that isolate. In some circumstances induction of antibodies to heterologous parasite isolates also occurs and this has been suggested as evidence for cross-reactivity of responses against the erythrocyte surface. The role of these relatively cross-reactive antibodies in protection from clinical malaria is currently unknown. We studied the incidence of clinical malaria amongst children living on the coast of Kenya through one high transmission season. By categorising individuals according to their pre-season parasite status and antibody response to the surface of erythrocytes infected with four parasite isolates we were able to identify a group of children, those who failed to make a concomitant antibody response in the presence of an asymptomatic parasitaemia, at increased susceptibility to clinical malaria in the subsequent 6 months. The fact that this susceptible group was identified regardless of the parasite isolate tested infers a cross-reactive or conserved target is present on the surface of infected erythrocytes. Identification of this target will significantly aid understanding of naturally acquired immunity to clinical malaria amongst children in endemic areas.     

Stomatocytosis of latex particles (0.26 micron) by rat erythrocytes by the electrical breakdown technique

The uptake of macromolecules by erythrocytes can be achieved with the electrical breakdown technique [2, 4]. In this technique the erythrocyte membranes are subjected to a high external electrical field pulse for a short period. Local, reversible breakdowns of the cell membrane occur above a critical field strength which lead to a time-dependent increase in the permeability of the membrane. By this means, human erythrocyte membranes can be made permeable to DNA, pharmaceutical compounds, and latex particles following an electrical field pulse [1, 3, 5]. Larger particles should also be taken up by erythrocytes using this method. Vienken et al. [5] demonstrated the entrapment of latex particles with a diameter of 0.091 micron in human erythrocyte ghosts, although this was shown with only a single electron micrograph which does not prove that the ghost membrane was intact. In our experiments in order to entrap latex particles with a diameter of 0.26 micron rat erythrocytes were subjected to an electrical field pulse of 12 kV/cm with a decay time of 60 microseconds. Experiments using the electron microscope show that after such an electrical field pulse the uptake of latex particles by rat erythrocytes follows the stomatocytotic pathway. We show further that using electron microscopic techniques, a single section cannot demonstrate the completed uptake of a latex particle by the erythrocyte.     

Differential resistance to calcium-induced bilirubin-dependent hemolysis in mammalian erythrocytes

Washed erythrocytes from human, buffalo, sheep and goat preincubated with different concentrations of calcium chloride (16.7–1830 μM) showed significantly different rates of hemolysis (up to 62%) after addition of bilirubin (72 μM). Goat erythrocytes displayed marked resistance to hemolysis with only 11% hemolysis observed at the highest calcium concentration. Similar trend in hemolysis was also observed when the concentration of CaCl₂ was fixed (330 μM) and bilirubin concentration varied (0–72 μM). (Ca²⁺–Mg²⁺)-ATPase levels were found significantly lower in goat and sheep erythrocyte membranes compared to human and buffalo erythrocyte membranes. This was correlated well with the observed hemolysis in various mammalian erythrocytes.     

Serological and immunological studies with a hexane extract of Babesia bovis-infected erythrocytes.

Antigenic and immunogenic activities of a hexane extract from Babesia bovis-infected erythrocytes were investigated. Positive ELISA and IFAT reactions were obtained with bovine antisera to B. bovis and B. bigemina produced by natural infection and rabbit antisera to the hexane extract, respectively. In contrast, negative ELISA reactions were obtained with Anaplasma marginale antisera indicating that the antigen(s) is specific for the genus Babesia. The IFAT clearly demonstrated that the antigen was associated with the parasite and the infected erythrocyte and not present in uninfected erythrocytes. Furthermore, cross-reactions with Babesia bigemina antisera suggested that serological cross-reactivity in bovine Babesia species is at least due in part to lipid or lipid-associated antigens.     

Comparative estimation of morphofunction characteristics alive and fixed erythrocytes

The method of power spectroscopy carries out the quantitative analysis of elastic properties of alive cells. It has been established that the highest indicators of elasticity nuclear (amphibious) and denuclearized (mammals) alive erythrocytes are registered in epi- and perinuclear space. When fixing with methanol and drying of cells the greatest values of the elastic modulus are displaced to the periphery of the cells. The revealed inversion of elasticity properties of erythrocytes must be considered when assessing the morphofunction characteristics of the fixed cells.