Characterization of monoclonal antibodies against human red blood group cells antigens by laser backscattering

A problem in immunohematology is to define the antibody quality which is related to its affinity expressed by the equilibrium constant. The activity of an antibody can be measured by the strength of its interaction, related to the adhesive energy exchanged during RBC agglutination which depends on the antigen-antibody liaison strength. To estimate this adhesive energy, two methods are used in this paper. Firstly, the dissociation behaviour of suspended RBC agglutinates was analysed by laser backscattering intensity (r) in a Couette flow. Backscattered intensity issued from shear-induced mechanical dissociation is recorded and submitted to a numerical process to obtain the energy parameter (ED). Secondly, a modification of this technique is proposed for measuring specific binding energy. Samples were exposed to increasing shear stress, and backscattered intensity was recorded. A constant increase of this intensity with raising shear stress was observed, pointed to a progressive dissociation of RBC agglutinates into smaller ones. Considering that complete dissociation of agglutinates is only approached asymptotically it is assumed that the final break-up of doublets (two-cell agglutinates) is produced at a critical shear stress (tauC) reflecting the work done to breaking-up the molecular bridges between both adjacent cells. This shear stress is defined by the extrapolation of the linear part of the curves [r-log tau] to the backscattered signal (r0) corresponding to the complete dispersion of RBCs. These approaches permit to define the specific surface adhesive energy (Gamma) by using the Derjaguin relation and to assess the functional characterization of specific immunoglobulins. In conclusion, two parameters characterizing monoclonal antibody agglutination properties, ED and Gamma, were estimated by laser backscattering methods, which could be very useful for antibodies quality control.     

Metabolic pathway analysis of enzyme-deficient human red blood cells

Five enzymopathies (G6PDH, TPI, PGI, DPGM and PGK deficiencies) in the human red blood cells are investigated using a stoichiometric modeling approach, i.e., metabolic pathway analysis. Elementary flux modes (EFMs) corresponding to each enzyme deficiency case are analyzed in terms of functional capabilities. When available, experimental findings reported in literature related to metabolic behavior of the human red blood cells are compared with the results of EFM analysis. Control-effective flux (CEF) calculation, a novel approach which allows quantification and interpretation of determined EFMs, is performed for further analysis of enzymopathies. Glutathione reductase reaction is found to be the most effective reaction in terms of its CEF value in all enzymopathies in parallel with its known essential role for red blood cells. Efficiency profiles of the enzymatic reactions upon the degree of enzyme deficiency are obtained by the help of the CEF approach, as a basis for future experimental studies. CEF analysis, which is found to be promising in the analysis of erythrocyte enzymopathies, has the potential to be used in modeling efforts of human metabolism.     

Quantitative flow cytometric analysis of ABO red cell antigens.

A flow cytometry method has been employed to quantitatively compare the expression of A, B and H antigens on various red blood cells (RBC). The H substance was directly labelled by fluorescein-conjugated anti-H lectin and the A and B antigens by indirect staining first with monoclonal anti-A or anti-B antibodies followed by fluorescently, fluorescein (FITC) or phycoerythrin (PE), labelled anti-mouse immunoglobulin (Ig) antibodies. More than a ten-fold difference in cellular fluorescence intensity was found within each sample. Both the percentage and the mean fluorescence of the positive subpopulation for each antigen were determined. Each RBC population was characterized with respect to the expression of A, B or H antigen by a compound mean value that was the calculated product of these two parameters. The results demonstrated a reciprocal relationship between the compound means of A or B and H. The ratio of A/H or B/H was found to be most informative. Homozygotes for A or B had ratios of greater than 200 and greater than 30, respectively, while heterozygotes (AO or BO) had ratios of less than 5. This method could also distinguish between A1 and A2; RBC carrying the A1 phenotype (as determined by agglutination with anti-A1 lectin) showed a higher A/H ratio than those carrying A2. In contrast to the reciprocity in the expression of A (or B) and H found in RBC obtained from different individuals, a direct correlation was found in the expression of these antigens by individual cells within a given population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reexpression of blood group ABH antigens on the surface of human thyroid cells in culture

Using indirect immunofluorescence (IFL) on viable human thyroid cultures, it has been shown that, although adult follicular cells do not express blood group ABH antigens in vivo, they invariably reexpress the corresponding antigens on the cell surface when cultured in monolayers, even for very short periods. The absence of blood group antigens on noncultured thyroid cells was confirmed by negative IFL on cell suspensions obtained after enzymatic digestion of the glands, whereas these antigens were readily demonstrable on cell suspensions obtained by trypsinization of established monolayers. The quantitative expression of ABH antigens on individual thyroid cells was variable and the cell-surface IFL pattern due to binding of blood group isoantibodies was different from that given by organ-specific thyroid autoantibodies on viable cultures. Reexpression of blood group antigens by cultured thyroid cells could not be related to the secretor status of the donors, the presence of a particular source of serum in the culture medium or cell division in vitro. After 2-3 wk in culture, thyroid cells became morphologically dedifferentiated and no longer displayed blood group antigens, though they still expressed cell- surface beta 2-microglobulin. Fibroblasts present in the primary thyroid cultures were invariably negative for ABH antigens. These results demonstrate that the surface antigenic repertoire of cultured human cells is not necessarily identical to that present on the same cells in vivo. Furthermore, the possibility that blood group natural isoantibodies bind to the cell surface must be taken into account in experiments in which cultured thyroid cells are exposed to human sera.     

The role of variant surface antigens on malaria-infected red blood cells.

It has been proposed that the primary role of variant antigens appearing on the surface of red blood cells infected with malaria parasites is to mediate cytoadherence, and that the antigenic variation they display is an adaptation to avoid immune attack. Here, Allan Saul proposes that their role is the opposite: that their primary purpose is to generate an immune response, which regulates their growth and thereby establishes a chronic infection, and that the role of cytoadherence is to ensure that parasites failing to express this flag to the immune system are destroyed by the spleen.     

ATP and integrity of human red blood cells

In spite of the well known significance of ATP in the energy dependent life processes, the role of ATP in maintaining cellular integrity is poorly understood. A possible model for studying ATP dependent life processes is to monitor the kinetics of changes seen intra/extracellularly during ATP depletion. In our model system anticoagulated human whole blood was incubated at different temperatures to reduce intracellular ATP without addition of any chemicals. The red blood cells in their own plasma were incubated for several days at 4 degrees C or at 37 degrees C, and ATP, glucose, K+, Na+, hemoglobin, water content, mean corpuscular volume (MCV), pH and Ca2+ were analyzed in time-sequences. All the examined parameters remained practically unchanged at 4 degrees C, while at 37 degrees C total ATP and glucose decreased parallel and after a transient increase of MCV, the water content of red blood cells decreased. As the actual ATP fell below 10% of the initial ATP content (at 48 h), the release of potassium sharply increased. Release of hemoglobin started only after 96 hours of incubation. Maximums of changes of the examined parameters were found at different time intervals. The maximal speed of concentration changes for glucose was found at 12-24 hours of incubation and at 24-36 hours for ATP, at 48-60 hours for K+(-)Na+ and after 96 hours for hemoglobin.     

Hypertonic cryohemolysis of human red blood cells

Summary Hypertonic cryohemolysis of human erythrocytes is caused by incubation of the cells in hypertonic medium at a temperature of 20–50°C (stage 1), with subsequent cooling to 0°C (stage 2). In 0.86m sucrose hemolysis increases, with increasing stage 1 temperature, whereas in 1m NaCl cryohemolysis has a temperature optimum at a stage 1 temperature of about 30°C.Cryohemolysis is inhibited by preceding ATP depletion of the cells and bypreincubation of the cells in hypertonic medium at 0°C. In general, anesthetics inhibit cryohemolysis strongly. Only in 1m NaCl at stage 1 temperatures in the range of 40–50°C is cryohemolysis stimulated by these drugs, if present during the entire incubation period. This effect is abolished, however, when the anesthetic is added after piror incubation of the cells at 40–50°C in 1m NaCl.Ghost-bound ANS fluorescence indicates complicated conformation changes in the membrane structure during the various experimental stages leading to cryohemolysis.Some of the experimental results can be considered as examples of molecular hysteresis, thus indicating several different metastable structures of the membrane, under various experimental conditions.The described results support the working hypothesis of Green and Jung that the experimental procedure results in membrane protein damage, preventing normal adaptation of the membrane during cooling.     

Shape memory of human red blood cells

The human red cell can be deformed by external forces but returns to the biconcave resting shape after removal of the forces. If after such shape excursions the rim is always formed by the same part of the membrane, the cell is said to have a memory of its biconcave shape. If the rim can form anywhere on the membrane, the cell would have no shape memory. The shape memory was probed by an experiment called go-and-stop. Locations on the membrane were marked by spontaneously adhering latex spheres. Shape excursions were induced by shear flow. In virtually all red cells, a shape memory was found. After stop of flow and during the return of the latex spheres to the original location, the red cell shape was biconcave. The return occurred by a tank-tread motion of the membrane. The memory could not be eliminated by deforming the red cells in shear flow up to 4 h at room temperature as well as at 37 degrees C. It is suggested that 1). the characteristic time of stress relaxation is >80 min and 2). red cells in vivo also have a shape memory.     

Structural and functional analysis of native peroxiredoxin 2 in human red blood cells

Peroxiredoxin 2, a typical 2-Cys peroxiredoxin, is the third most abundant protein in erythrocytes. It is understood that the physiologically functional state of peroxiredoxin 2 is the monomer, and that its role in scavenging low levels of H(2)O(2) results in the formation of disulfide-linked dimers, which are reversibly reduced to monomers by the thioredoxin-thioredoxin reductase system. Additionally, peroxiredoxins are highly susceptible to sulfinic acid formation through reactions with various peroxides. This overoxidized form, which is thought to convert peroxiredoxins into molecular chaperones and to be accompanied by a transition to polymeric forms, can be reversed by sulfiredoxins. However, physiological conformational changes and the antioxidant role of erythrocyte peroxiredoxin 2 are still unclear because there is low sulfiredoxin and thioredoxin-thioredoxin reductase activity in erythrocytes. In this study, we examined the structural and redox states of peroxiredoxin 2 in fresh hemolysates and estimated the activities of native and overoxidized peroxiredoxin 2 purified from red blood cells to clear the physiological roles of peroxiredoxin 2 in erythrocyte. Our findings demonstrate that native peroxiredoxin 2 exists as high molecular weight (>160 kDa) oligomers and that decamers or higher order molecular weight oligomers (260-460 kDa) have peroxidase activity. We further showed that peroxiredoxin 2 oligomers, which were predominantly composed of monomers in the reduced form, exert a chaperone activity equal to that of overoxidized peroxiredoxin 2 polymers. These results provide the novel insight that redox-active peroxiredoxin 2 functions in human red blood cells as high molecular weight oligomers that possess peroxidase and chaperone activities.     

ABH blood group antigens in O-glycans of human glycophorin A

The major O-linked oligosaccharide structures attached to human glycophorin A (GPA) have been extensively characterized previously. Our own recent findings, obtained by immunochemical methods, suggested the presence of blood group A and B determinants in O-glycans of human glycophorin originating from blood group A or B erythrocytes, respectively. Here, we elucidate the structure of O-glycans, isolated from GPA of blood group A, B, and O individuals by reductive beta-elimination, carrying A, B or H blood group epitopes, respectively. Structural studies based on nanoflow electrospray-ionization tandem mass spectrometry and earlier reported data on the carbohydrate moiety of GPA and ABH antigens allowed us to conclude that these blood group epitopes are elongations of the beta-GlcNAc branch attached to C-6 of the reducing GalNAc. The galactose linked to C-3 of the reducing GalNAc carries NeuAcalpha2-3 linked residue. Identified here O-glycans were found in low amounts, their content estimated at about one percent of all GPA O-glycans. These O-glycans with type-2 core, carrying the blood group A, B or H determinants, have not been identified in GPA so far. Our results demonstrate the efficacy of nanoESI MS/MS in detecting minor oligosaccharide components present in a mixture with much more abundant structures.     

Potassium-activated phosphatase from human red blood cells

Summary The cell membrane K⁺-activated phosphatase activity was measured in reconstituted ghosts of human red cells having different ionic contents and incubated in solutions of varying ionic composition. When K⁺-free ghosts are suspended in K⁺-rich media, full activation of the phosphatase is obtained. Conversely, very little ouabainsensitive activity is detected in K⁺-rich ghosts suspended in K⁺-free media. These results, together with the fact that Na⁺ competitively inhibits the effects of K⁺ only when present externally, show that the K⁺ site of the membrane phosphatase is located at the outer surface of the cell membrane. The Mg⁺⁺ requirements for K⁺ activation of the membrane phosphatase are fulfilled by internal Mg⁺⁺. Addition of intracellular Na⁺ to ATP-containing ghosts raises the apparent affinity of the enzyme for K⁺, suggesting that the sites where ATP and Na⁺ produce this effect are located at the inner surface of the cell membrane. The asymmetrical features of the membrane phosphatase are those expected from the proposed role of this enzyme in the Na⁺–K⁺-ATPase system.The authors are established investigators of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

Phosphoglycolate phosphatase from human red blood cells

The nucleotide profile of rat liver Golgi vesicles isolated using sucrose gradients has been determined by high-pressure liquid chromatography. The nucleotide composition of this Golgi preparation, probably modified by osmotic shock, differs from that of liver supernatant fraction and from isolated rough microsomes. The major nucleotides present in the Golgi have been tentatively identified as uridine diphosphate and a peak containing uridine monophosphate plus cytidine monophosphate at 1.6 and 0.5 nmol/mg protein, respectively. In order to minimize osmotic shock, we have modified the isolation of Golgi using D₂O-sucrose gradients. Intact Golgi from these gradients were extracted directly and analyzed. Higher levels of nucleotides were found in the unshocked preparation, and the profile was also altered, although it was still distinct from that of liver supernatant. Four major peaks were found, tentatively identified as uridine monophosphate plus cytidine monophosphate, adenosine monophosphate, UDP, and uridine diphosphogalactose plus uridine diphosphoglucose, at 6.4, 6.4, 6.1, and 3.3 nmol/mg protein. These results indicate that the membrane of the Golgi apparatus is not freely permeable to nucleotides but that selective mechanisms exist for the uptake or exclusion of specific nucleotides from this organelle. The fact that UDP is selectively retained in shocked Golgi vesicles may indicate the presence of a binding protein which would prevent interference of Golgi function by UDP, a highly inhibitory product of galactosyltransferase.     

Electrical hemolysis of human and bovine red blood cells

Summary The external electric field strength required for electrical hemolysis of human red blood cells depends sensitively on the composition of the external medium. In isotonic NaCl und KCl solutions the onset of electrical hemolysis is observed at 4 kV per cm and 50% hemolysis at 6 kV per cm, whereas increasing concentrations of phosphate, sulphate, sucrose, inulin and EDTA shift the onset and the 50% hemolysis-value to higher field strengths. The most pronounced effect is observed for inulin and EDTA. In the presence of these substances the threshold value of the electric field strength is shifted to 14 kV per cm. This is in contrast to the dielectric breakdown voltage of human red blood cells which is unaltered by these substances and was measured to be 1 V corresponding in the electrolytical discharge chamber to an external electric field strength of 2 to 3 kV per cm. On the other hand, dielectric breakdown of bovine red blood cell membranes occurs in NaCl solution at 4 to 5 kV per cm and is coupled directly with hemoglobin release. The electrical hemolysis of cells of this species is unaffected by the above substances with exception of inulin. Inulin suppressed the electrical hemolysis up to 15 kV per cm. The data can be explained by the assumption that the reflection coefficients of the membranes of these two species to bivalent anions and uncharged molecules are field-dependent to a different extent. This explanation implies that electrical hemolysis is a secondary process of osmotic nature induced by the reversible permeability change of the membrane (dielectric breakdown) in response to an electric field. This view is supported by the observation that the mean volumes of ghost cells obtained by electrical hemolysis can be changed by changing the external phosphate concentration during hemolysis and resealing, or by subjecting the cells to a transient osmotic stress immediately after the electrical hemolysis step. An interesting finding is that the breakdown voltage, although constant throughout each normally distributed ghost size distribution, increases with increasing mean volume of the ghost populations.     

NMR water-proton spin-lattice relaxation time of human red blood cells and red blood cell suspensions

NMR water-proton spin-lattice relaxation times were studied as probes of water structure in human red blood cells and red blood cell suspensions. Normal saline had a relaxation time of about 3000 ms while packed red blood cells had a relaxation time of about 500 ms. The relaxation time of a red cell suspension at 50% hematocrit was about 750 ms showing that surface charges and polar groups of the red cell membrane effectively structure extracellular water. Incubation of red cells in hypotonic saline increases relaxation time whereas hypertonic saline decreases relaxation time. Relaxation times varied independently of mean corpuscular volume and mean corpuscular hemoglobin concentration in a sample population. Studies with lysates and resealed membrane ghosts show that hemoglobin is very effective in lowering water-proton relaxation time whereas resealed membrane ghosts in the absence of hemoglobin are less effective than intact red cells.