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Anti-gs antisera were tested on embryo and adult Chick erythrocytes. Specific immunsera directed against Chick embryo or adult erythrocyte antigens have been tested on Hamster cell lines infected with the Rous virus. Results demonstrate that the erythrocyte and viral antigens are different.
Erythrocytes containing micronuclei serve as an indicator of genotoxic exposure in splenectomized individuals. Micronucleated erythrocytes, derived from cytogenetically damaged RBC precursors, are not selectively removed from peripheral blood in individuals who lack splenic function. The relationship between micronucleated cell frequencies and demographic, environmental, and dietary factors was examined in 44 subjects with previous splenectomy due to trauma. Their micronucleated cell counts fit a log-normal distribution, with geometric means of 3.3 micronucleus-containing cells/1000 reticulocytes and 2.7/1000 normochromatic erythrocytes. A multiple regression analysis showed that drinking five cups of coffee or tea/day (relative to none) was associated with an approximately 2-fold higher frequency of micronucleated cells. Weaker statistical associations were also noted with micronucleus frequency and the consumption of calcium supplements (associated with a higher frequency) and vitamins A, C, or E (lower frequency). An apparent trend of higher micronucleus counts with age was attenuated when other factors were considered in the regression. Cigarette smoking and decaffeinated coffee consumption were among the factors not associated with elevated micronucleated cell frequencies. Because the occurrence of micronuclei in reticulocytes reflects cytotoxic exposures within the past 3-8 days, it may be possible to test directly the relationship of these factors to micronucleus formation through intervention studies.
It has been found that erythrocytes of hemodialysed patients show significantly increased glucose-6-phosphate dehydrogenase (G-6-PD, E.C. 1.1.1.49) activity when compared with that in healthy subjects. Activity of that enzyme in erythrocytes from patients treated with hemodialyses incubated in 3 hours with hemodialysate (HD) or its peptide fractions (PF) showed no statistical difference when compared with controls. Similar effect of HD or PF on activity of the enzyme studied has been noted in erythrocytes from healthy subjects except fraction II having concentration equal to 14 micrograms/ml calculated against the protein content. This last fraction caused significant inhibition of the enzyme activity.
In experiments in vitro it proved possible to bind HLA antigens in serum on erythrocytes after 4 to 48 hours incubation at 20 degrees C and 4 degrees C. With the period of incubation the amount of bound HLA antigens increased. The absorption and neutralization test revealed that the HLA antigen content of erythrocytes, thrombocytes and serum in a given subject is the same. HLA antigens present on the erythrocytes of some subjects were not removed from their surface even after nursing 15 times with saline. Based on the results of the experiment it is not possible to decide whether HLA antigens detected on red blood cells are an integral part of their membrane or whether they are bound to the red cells from plasma.
The ESR data on the influence of membrane potential of the fusion of Sendai virus envelope with erythrocyte membrane are presented. The hyperpolarization of cell membrane takes place at low concentration of KCl (1-5 mM) in extracellular medium in the presence of valinomycin, while at high concentration of KCl (125-150 mM) its depolarization occurs. The hyperpolarization of erythrocyte plasma membrane is accompanied by the increase of its fusion with viral envelope and virus-induced hemolysis. At the same time depolarization of erythrocyte membrane leads to the decrease of virus fusion activity. This evidence together with previously obtained by patch-clamp method data on potential-dependence of virus-induced increase of cell membrane conductivity provide us an opportunity to make a proposal that the electric field membrane damage may be the initial stage of the virus-induced membrane fusion.
Adult patients with chronic autoimmune thrombocytopenic purpura (ATP), which proved refractory to various treatments, received a single dose of autologous in vitro opsonized erythrocytes with 100 micrograms of anti-D IgG. In 1983, 30 of these patients were treated with autologous erythrocytes that had been opsonized and labeled with 25 mCi (740 MBq) of technetium Tc 99m; this treatment was designated as the radioimmune method. Favorable responses were noted in 36% of patients so treated. In 1985, another group of 16 patients with refractory ATP received therapy with autologous opsonized erythrocytes (AOPE) and 55% of these patients showed favorable responses. Five (17%) of the patients treated using the radioimmune method attained a complete, long-term (greater than 35 months) remission of their ATP, and five (31%) of the patients treated using AOPE remained in complete remission over 270 days after cessation of therapy. Major complications were not seen. We concluded that the interaction of macrophages with low-dose AOPE is a successful therapeutic approach in ATP refractory to standard treatment.
The question of how often erythrocytes should be washed in order to obtain a protein-free and therapeutically efficient unit of washed red blood cells is still controversial. To obtain more information regarding this problem, 6 X 15 units of whole blood were washed 1-6 times with a saline solution immediately and after 7, 14, 21, 28 and 35 days of storage. After each washing procedure the content of total protein, of immunglobulins A, G, and M and of complement factor C3 was determined in the supernatants, as well as the osmotic fragility in the corresponding erythrocyte sediments. The statistically analyzed results enabled us to describe the average degree of influences as equations and to recommend a preparation prescription. The results show that the protein levels decrease precipitously after the first and second washing procedures. The following 4 procedures have no significant effect. On the other hand, with increasing time of storage more washing procedures are necessary to reach the same results than with younger units. That is also true for the concentrations of immunoglobulins and complement C3. The osmotic fragility of the erythrocytes increases with each washing procedure as well as each day of storage. According to these results, an optimal, protein-free and effective unit of washed red blood cells is obtained when a 1-2-week-old unit of whole blood is washed 2-3 times.
Risk factors for atherosclerosis are often associated with haemorheological changes. On this point, obesity (recently advocated as an independent risk factor) was not much studied and with not univocal results. We have studied 70 obese patients (BMI greater than 30) and 50 healthy subjects (BMI less than 25). Among obese 26 had no more pathologies, 29 had hypertension, 3 suffered from ischemic heart disease, 3 suffered from occlusive arteriopathy, 9 were hyperlipidemic, 10 were smokers. We determined plasma viscosity and whole blood viscosity (at haematocrit corrected to 45% too). Washed erythrocytes, poor in leucocytes and platelets and resuspended in phosphate-buffered saline, were used for study of erythrocyte viscosity and deformability. Obese patients showed raised mean blood viscosity values when compared to healthy controls (p less than 0.01); an even more significant increase (p less than 0.001) was found concerning plasma viscosity and fibrinogen. Erythrocyte viscosity and red blood cell filterability index did not show any significant difference. We found no significant correlation between viscosity values and presence of hypertension, hyperlipidemia and smoking habit among obese. In conclusion, the higher vasculopathy incidence might be caused by an increase in blood viscosity, mostly due to plasmatic component. This fact appears to be independent from the presence of atherosclerosis complications or other risk factors.
The number of erythrocytes adopting the sickled configuration in rabbit aqueous was determined with a masked counting technique in vitro and in vivo. The percentage of cells that became sickled in aqueous humor was greater than that observed in a control salt solution and in the donor blood itself. Aqueous humor, therefore, can be considered a particularly deleterious medium for erythrocytes having a propensity for sickling. Blood samples from six donors with systemically severe hemoglobinopathies (SC, Sthal, and SS) had relatively high levels of hemoglobin S and, in general, showed faster and more frequent sickling than did blood samples from donors with low levels of hemoglobin S (one SS patient, whose chronic blood transfusion regimen had replaced most of his hemoglobin S with normal hemoglobin, and two AS patients). Even erythrocytes from sickle cell trait cases, however, were capable of sickling when immersed in acqueous humor.
Erythrocytes can be made to entrap enzymes, lipids, drugs, pesticides, and large macromolecules by a variety of encapsulation procedures. Methods of encapsulation include hypotonic dilution and dialysis, lipid fusion, electrical hemolysis, and chemical perturbation. The survival of carrier erythrocytes is dependent on the method of encapsulation as is the encapsulation efficiency. Research has led to utilization of carrier cells for drug and enzyme-replacement therapies, for incorporation of DNA and antibodies into living cells, and for enhancement of the oxygen-carrying capacity of hemoglobin. Many of the possible uses of erythrocytes as carriers are explored.
A micromethod for measurement of erythrocyte filtrability is described. The time is recorded for the spreading of drops (25 microliter) of an erythrocyte suspension in filter papers which are calibrated with the suspension solution. The spreading time for the erythrocytes in relation to a reference calibration time correlates well with the filtration time of a modified filtration technique according to TEITEL.
Mice injected with rat red blood cells developed anti-erythrocyte autoantibodies detectable by a direct Coombs' test. Nude mice injected with rat red blood cells did not develop a Coombs-positive state, but nude mice injected with rat red blood cells plus the T cell helper factor, interleukin-2, produced autoantibodies to autologous mouse erythrocytes. The simultaneous injection of rat red blood cells and allogeneic spleen cells induced an early and vigorous autoantibody response in athymic nude mice as well as in euthymic control mice. These results are interpreted as indicating the possibility of an interleukin-2-stimulated in vivo differentiation (or clonal expansion) of helper T cells in nude mice in response to heterologous erythrocytes which could mediate an autoimmune B cell response.
Hemin hemolysis of human erythrocytes at 37 degrees C has been investigated by the kinetic method. It has been observed that hemin affects erythrocytes by two mechanisms, inducing "quick" and "slow" hemolysis. The "quick" hemolysis is completed after the first few minutes. The final level of "quick" hemolysis is a function of relative hemin concentration. It is equal to zero when the latter is smaller than the minimum concentration Cmin = 10(8) molecules of added hemin per cell. The final level of "slow" hemolysis is always 100%. It is suggested that the limiting step of autohemolysis is the membrane proteins oxidation catalyzed by endogenous hemin originating from methaemoglobin during erythrocyte incubation at 37 degrees C.
The depletion of 18O from CO2, caused by the exchange of oxygen between CO2 and water during the hydration-dehydration cycle, is catalyzed by carbonic anhydrase. This depletion process at chemical equilibrium in the presence of erythrocytes is biphasic, exhibiting a very rapid depletion rate immediately following the addition of cells to an isotonic solution containing 18O-enriched CO2, followed by a much slower depletion rate. It is hypothesized that these depletion characteristics are caused by the diffusion of labeled CO2 into erythrocytes where depletion occurs rapidly due to the large intracellular carbonic anhydrase content. Kinetic equations which describe this hypothesis are solved and a rate constant is obtained which represents the depletion of 18O in CO2 caused by the presence of red cells. These are equilibrium experiments with no net uptake or loss of CO2 in the cells. Consequently, depletion processes are not limited in rate by bicarbonate-chloride exchange or proton distribution across the membrane. The purpose of these measurements is to determine whether the rate of 18O depletion in red cell suspensions is determined by carbonic anhydrase activity in the cell or by the diffusion process by which CO2 enters the cell. This goal is achieved by partially inhibiting carbonic anhydrase with acetazolamide. The rate constant representing 18O depletion caused by the presence of red cells is unchanged, even though up to 90% of carbonic anhydrase is inhibited. From this rate constant the permeability constant of the membrane of rat erythrocytes to CO2 at 25 degrees and pH 7.4 is determined to be (7.6 +/- 1.2) X 10(-3) cm s-1 in the presence of 3.2 mM picrate, a passive anion diffusion inhibitor intended to block HCO3 -flux across the membrane. Using no picrate and allowing HCO3-flux to introduce an error in the measurements, the permeability constant is (1.6 +/- 0.4) X 10(-2) cm s-1. The permeability constants measured by this technique include the diffusion barrier to CO2 not only of the red cell membrane but also of a portion of the intracellular medium.
The increased levels of glycosylated hemoglobin and glycosylated erythrocyte membrane proteins in gestational diabetes could bring about alterations in osmotic fragility in erythrocytes of gestational diabetes during their first trimester. The glycosylated hemoglobin levels in erythrocytes were found to be significantly elevated with a mean of 5.42 +/- 0.77 mg/dl when compared to 3.45 +/- 0.62 mg/ dl in non diabetic pregnant control (where as in non pregnant women the value was 3.4 +/- 0.47 (n = 25)). The mean osmotic fragility (MOF) of control cells was 60.2 + 0.6 mMol/L Nacl and MOF of gestational diabetic cells was 63.93 +/- 0.6 mMol/L Nacl. The mean osmotic fragility, operationally defined as the Nacl concentration for 50% hemolysis, was found significantly higher by 3.9 + 0.01 mMol/L Nacl in gestational diabetes, than in normal cells. The total levels of glycosylated membrane proteins were increased from 50.60 +/- 8.0 in control to 69.14 + 0.47 in gestational diabetes. Therefore it is proposed that the increased levels of glycosylated Hb as well as glycosylated membrane proteins have a role in altering the membrane permeability resulting in increased osmotic fragility of erthrocytes in gestational diabetes.
The measurements of erythrocyte orientation, obtained through a spin labeling technique, are compared with a phenomenological model. Several rheological conditions are varied: hematocrit, suspending medium viscosity, blood age, artificial reversible aggregation. We found that the onset of orientation is very sensitive to any variation of these conditions, and that its measurement would be a good method to assess erythrocyte deformability. A critical shear rate for the orientation process is then determined and compared to the corresponding parameter obtained from viscosity measurements of identical suspensions. A close qualitative relationship is found between the two sets of values of the critical shear rate.
Heat altered erythrocytes are taken up by spleen macrophages without any signs of mechanical strain as revealed by light and scanning electron microscopy. There was no scintigraphic evidence for changed flow rates within the spleen after sequestration of erythrocytes.
We have compared the effects of two slightly different methods of erythrocyte ghost preparation on membrane autophosphorylation in Duchenne muscular dystrophy. Addition of 1 mmol/l EDTA to two of the washing steps altered the differences between patients and controls. These data support the known sensitive interrelationship of Ca2+ and erythrocyte membrane autophosphorylation, underline the importance of strict adherence to methodology when comparing data, and have suggested a new direction of investigation.