Rosetting of human T lymphocytes with goat red blood cells: Effect of treatment with 2-aminoethylisothiouronium bromide (AET) and comparison with AET treated sheep red blood cells

In vitro treatment of goat red blood cells (GRBCs) with the sulphydryl compound 2-aminoethylisothiouronium bromide (AET) increases their specific reactivity with human T lymphocytes without affecting the specificity of the reaction. AET-GRBCs bind to only part of T lymphocytes rosetting with AET-sheep red blood cells (SRBCs): the receptors for both types of RBCs are very similar if not identical, but display higher affinity for AET-SRBCs than for AET-GRBCs. Rosetting of T lymphocytes with AET-GRBCs may be useful to enumerate T lymphocyte subsets in patients with abnormality of the immune system and to fractionate T lymphocyte subpopulations.     

DMO-transport in human red blood cells

The DMO (5,5-dimethyloxazolidine-2,4-dione) transport was studied in human red blood cells by measuring the¹⁴C-DMO back exchange under self exchange conditions from¹⁴C-DMO labeled cells at 0°C. The unidirectional DMO-flux was linearly related to the DMO concentration up to approx. 100 mM. The unidirectional DMO-flux increases as pH is reduced. The DMO transport is not inhibited by anion transport inhibitors like DIDS, SITS, dipyridamole, phlorhizin, salicylate or butanol. A close correlation between the unidirectional DMO-flux and DMOH, the unionized form of DMO, has been observed suggesting that DMO is transported predominantly by nonionic diffusion. The permeability of DMOH is 9.5·10^–6 cm/s (0°C) while the permeability of DMO^– cannot be assessed from our data.     

Alternating current electrophoresis of human red blood cells

An a.c. electrophoretic technique in the frequency range between 2–10 Hz, using a rectangular chamber design, has been developed for measuring the surface electric charge density of human red blood cells. With the a.c. approach, mobility can be determined rapidly, electrode polarization is minimized and the construction of the chamber simplified. For frequencies under 10 Hz and a chamber thickness of 150 μm, a theoretical analysis of the electroosmotic flow profile shows that it is almost identical to the d.c. case. With the a.c. method and using a 0.145 N NaCl solution buffered with NaHCO₃, the mobility of red cells measured at the lower stationary level is found to be −1.07±0.02 (S.D.) μmsec⁻¹ V⁻¹ cm. The zeta potential and charge density calculated from the mobility are −14.03±0.26 (S.D.) mV and −3603±69 (S.D.) esu cm⁻², respectively.     

Adult and fetal galactokinases in human red blood cells

This paper reports the biochemical properties of galactokinase from fetal and adult human red blood cells. The specific activity of galactokinase is three times higher in the fetal red cells than in adult cells, shows a significant difference in the Michaelis constant toward galactose, and is more thermostable. On the other hand, no differences were found in molecular weight, electric charge, temperature and pH dependence between the two enzymes partly purified from fetal and adult erythrocytes.The possibility that these differences could be due to the shorter lifespan of the fetal erythrocytes (which could result in a higher proportion of young cells in the blood samples utilized) was investigated. Fetal and adult red blood cells were separated into fractions of different mean age by ultracentrifugation through density gradients. The kinetic properties and thermostability of galactokinase from fetal erythrocytes do not show any similarity with the same properties of the enzyme from young red blood cells.These results indicate that galactokinase from fetal erythrocytes show some biochemical properties that are typical signs distinguishing a fetal enzyme.     

Alternative pathway-mediated rebinding of immune complexes to human red blood cells.

Antigen-antibody complexes (Ag-Ab) prepared from 125I-bovine serum albumin (BSA) and guinea-pig anti-BSA were (i) incubated at 37 degrees for 4 min with undiluted normal human serum and autologous red blood cells (RBC) together, or (ii) incubated first at 37 degrees for 30 min with serum diluted optimally for binding and then with RBC. Reactions were stopped by dilution and cooling, and RBC bearing antigen-antibody-complement complexes (Ag-Ab-C) were washed and resuspended in either untreated normal human serum (undiluted or diluted), serum treated with zymosan (SZYM), ethylenediamine tetracetic acid (SEDTA) or ethyleneglycol tetracetic acid-Mg++ (SEGTA), serum heated at 56 degrees for 30 or 120 min (S delta 30 or S delta 120), or buffer alone. The mixtures were placed at 37 degrees and the percentage of Ag-Ab-C dissociated from RBC after progressively increasing times determined. (i) Ag:Ab:C bound to RBC with undiluted serum dissociated more rapidly following resuspension in SZYM, SEDTA, or S delta 30 than following resuspension in untreated serum. Rate of dissociation in SEGTA paralleled that in untreated serum. (ii) Ag-Ab-C bound to RBC with diluted serum dissociated following resuspension in SZYM, SEDTA, or S delta 30, but rebound and dissociated a second time following resuspension in untreated serum or SEGTA. Initial dissociation occurred in less than 1 min in undiluted serum, took place at 0 degrees as well as 37 degrees, and was diminished but detectable in greater than 8- and greater than 64-fold-diluted serum, respectively. Rebinding required 37 degrees, factors B and D and C3, and was maximal at 4-8 min. Subsequent dissociation had similar complement requirements to initial dissociation, but occurred only at 37 degrees and was 90% complete at 15 min. No dissociation of Ag-Ab-C bound to RBC in (i) or in (ii) occurred following resuspension in S delta 120 or in buffer. These findings suggest that after initial binding, release of experimental immune complexes from RBC in whole serum involves concurrent dissociation and alternative pathway-dependent rebinding.     

Generation of red blood cells from human induced pluripotent stem cells

Differentiation of human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) into the erythroid lineage of cells offers a novel opportunity to study erythroid development, regulation of globin switching, drug testing, and modeling of red blood cell (RBC) diseases in vitro. Here we describe an approach for the efficient generation of RBCs from hiPSC/hESCs using an OP9 coculture system to induce hematopoietic differentiation followed by selective expansion of erythroid cells in serum-free media with erythropoiesis-supporting cytokines. We showed that fibroblast-derived transgenic hiPSCs generated using lentivirus-based vectors and transgene-free hiPSCs generated using episomal vectors can be differentiated into RBCs with an efficiency similar to that of H1 hESCs. Erythroid cultures established with this approach consisted of an essentially pure population of CD235a(+)CD45(-) leukocyte-free RBCs with robust expansion potential and long life span (up to 90 days). Similar to hESCs, hiPSC-derived RBCs expressed predominately fetal γ and embryonic ? globins, indicating complete reprogramming of β-globin locus following transition of fibroblasts to the pluripotent state. Although β-globin expression was detected in hiPSC/hESC-derived erythroid cells, its expression was substantially lower than the embryonic and fetal globins. Overall, these results demonstrate the feasibility of large-scale production of erythroid cells from fibroblast-derived hiPSCs, as has been described for hESCs. Since RBCs generated from transgene-free hiPSCs lack genomic integration and background expression of reprogramming genes, they would be a preferable cell source for modeling of diseases and for gene function studies.     

Subpopulations of human lymphocytes differing in receptors to mouse and sheep red blood cells

A segregation procedure of human lymphocytes by successive rosetting with mouse and sheep red blood cells, and separation of rosettes by Ficoll Hypaque gradient centrifugation, is described. This method yields four lymphocyte subpopulations: lymphocytes with mouse and sheep receptors (M+E+), with mouse receptor only (M+E-), with sheep receptor only (M-E+), and without any receptors (M-E-). Presence of surface membrane receptors: E, C3, Fc, and DR on the cells in the subpopulations have indicated that M-E+ and M+E+ cells are T cells, M+E- cells are B cells and M-E- cells are nonhomogeneous, consisting of B cells, T cells, monocytes, and granulocytes.     

Reactions of monoclonal antibodies to human MN blood groups with red blood cells of subhuman primates

All 12 samples of chimpanzee erythrocytes tested were agglutinated by the commercial polyclonal anti-M reagent of rabbit origin, but none was agglutinated by monoclonal anti-M reagent of murine origin. This, as well as results of absorption experiments, showed that the polyclonal and monoclonal anti-M reagents detect different M epitopes. Polyclonal anti-N reagent of rabbit origin and monoclonal reagent of murine origin obtained by immunization with N blood group substance did not react with any chimpanzee erythrocyte samples. On the other hand, monoclonal anti-N reagent originating from immunization with M blood group substance agglutinated two of the 12 tested chimpanzee erythrocyte samples. This and other experimental results strongly suggested that the latter monoclonal anti-N reagent combined with "N" antigen, i.e., an antigen present on glycophorin B of human M as well as N erythrocytes; apparently erythrocytes of some chimpanzees contain also this antigen.     

Biological and mechanical quality of red blood cells cultured from human umbilical cord blood stem cells

Human umbilical cord blood (CB) has moved from the status of biological waste to that of a valuable source of haematopoietic stem (HS) cells. There are potentially three major clinical applications for HS cells andex vivo-expanded HS cells: reconstitution of haematopoiesis in patients undergoing chemotherapy; gene therapy (e.g. in thalassaemia, sickle cell anaemia); and large-scale production of mature blood cells. Erythropoiesis is accomplished by highly complex interactions of haematopoietic progenitor cells, stromal cells and cytokines in the bone marrow. Among them, erythropoietin is the principal regulator.Ex vivo cell culture experiments to obtain mature red blood cells were the focus of this study. Attempts to elucidate appropriate medium components and amounts of haematopoietic growth factors were successful: enucleated and haemoglobin-filled erythroid cells were obtained from primitive HS cells. Dimethylsulphoxide (DMSO) was found to be of particular importance as an efficient differentiation inducer. The differentiation process was followed microscopically and by fluorescence-activated cell sorting (FACS). Using the micropipette aspiration technique, the elastic properties of erythroid cells were evaluated as erythropoiesis progressed. Discocyte-like cells, comprising reticulocytes and finally differentiated red blood cells, showed an about ten-fold higher membrane shear modulus compared with control cells.     

Antibodies for trypsinized human red blood cells in human sera.

During studies of agglutination of trypsinized human red blood cells (RBC) by anti-Rh sera, it was noticed that some human sera without Rh antibodies agglutinated these erythrocytes. Of 933 normal and pathological sera subsequently screened, 116 produced such agglutination. The agglutinins were of IgM nature and were directed against an antigen exposed or created by tryptic digestion of RBC. They reacted equally well with autologous as with allogeneic trypsinized RBC and were different from T agglutinins and from Paul-Bunnell antibodies. The mode of formation of these antibodies remains unknown.     

Survival of lyophilized and reconstituted human red blood cells in vivo

To assess the viability of human red blood cells that have been lyophilized and reconstituted to the hydrated state, we phlebotomized a unit of whole blood from six healthy male volunteers. Their packed red blood cells were lyophilized at − 40 °C and stored at 4 °C. Upon rehydration, recovery of erythrocytes was 85.2 ± 2.79 %. Aliquots of 20 ml were labeled with ⁵¹Cr and re-infused into the original donors for red cell survival studies. The red cells retained ABO and Rh identity upon rehydration. There were no adverse clinical affects of re-infusion. The half time of ⁵¹Cr disappearance from the circulation was 31 ± 8.19 days, and there was no evidence of significant splenic sequestration on the day of reinfusion. Red cell indices of the rehydrated erythrocytes were normal, oxyhemoglobin content was 98.58 ± 1.46 %, and P₅₀ was 27.25 ± 1.84 mmHg. Although deformability was slightly decreased, the osmotic fragility and filterability of the red cells were normal. These data demonstrate that human erythrocytes can be lyophilized and reconstituted to the hydrated state And survive normally in the circulation. Metabolic, osmotic, hemotological and rheological function remains intact.     

Effects of preincubating human peripheral blood lymphocytes on their ability to bind sheep red blood cells

The effect of preincubating human peripheral lymphocytes at 37°C or 4°C for various lengths of time on their subsequent ability to form rosettes with sheep red blood cells (SRBC) was investigated. Lymphocytes suspended in either medium or medium containing fetal calf serum (FCS) and preincubated at 37°C for 30 min exhibited marked decrease in rosette formation with a return to normal values by 120 min. However, neither lymphocytes suspended in medium and preincubated at 4°C nor lymphocytes suspended in medium containing normal human serum (HS) and preincubated at 37°C exhibited this phenomenon.     

The nature of virus-like inclusions in human red blood cells

Summary The author was unable to discover any difference in the erythrocytes of cancer patients and healthy persons by electron microscopic investigation.Hemoglobin granules and slight defects similar to the formations revealed by Reagan in erythrocytes of patients suffering fron virus diseases were observed in the erythrocytic stroma of both healthy persons and cancer patients.Presented by Active Member AMN SSSR N. N. Zhukov-Verezhnikov