Cow red blood cells. I. Effect of purines, pyrimidines, and nucleosides in bovine red cell glycolysis.

Cow red cells, under in vitro incubation conditions, exhibit a comparatively low glycolytic rate of 0.56 +/- 0.05 micromol/(ml cells.h), with a ratio of lactate formed to glucose consumed of 1.58. It has been found that this low glycolytic rate can be stimulated 50--60% above the basal level in the presence of a variety of purine and pyrimidine compounds including adenosine, inosine, adenine, hypoxanthine, xanthine, and uracil. In contrast, calf red cells, which have a much higher glycolytic rate, display no discernible response to these agents. In attempts to elucidate the mechanism by which this stimulation takes place, both glucose transport and glycolytic enzyme activities were determined in the presence of these stimulators. Glucose influx in cow red cells, measured using the glucose analog 3-O-methyl-glucose, exhibits both a low Km of 117 microM and a Vmax of 0.38 micromol/(ml cells.min), and is unaltered in the presence of adenosine. On the other hand, hexokinase, which in normal hemolysates of cow red cells has an activity of 0.49 +/- 0.03 micromol/(g Hb.min). was found to be stimulated to 0.73 micromol/(g Hb.min) in the presence of adenine. Both pyruvate kinase and phosphofructokinase were unaffected by this compound. These data suggest that certain purines and pyrimidine compounds may exert their stimulatory effect on hexokinase activity, resulting in an augmentation of cow red cell glycolysis.     

Mast cell phagocytosis of red blood cells.

The prevalence of mast cells infiltrating bone marrow of different rats varied widely, as did the staining properties and size of their cytoplasmic granules. Bone marrow mast cells from several rats revealed large membrane-limited inclusions which stained metachromatically or orthochromatically and resembled inclusions in some macrophages. Ultrastructurally, mast cells varied widely in content of uniform dense granules or enlarged granules with less dense, fine grained content. Some of the large inclusions observed ultrastructurally in mast cells were heterophagic vacuoles which contained erythrocytes or reticulocytes, or remnants from other phagocytized cells, possibly neutrophils or unidentified homogeneous material. Smaller bodies, interpreted as fragments of erythrocytes, lay extracellularly near mast cells and occupied small, membrane-limited, heterophagic vacuoles in some mast cells. In other mast cells, communal vacuoles enclosed several specific cytoplasmic granules in various stages of disruption. The communal vacuoles occasionally opened to the extracellular space. A few large indeterminate vacuoles in mast cells contained amorphous flocculent matter which apparently derived either from coalescence of cytoplasmic granules through fusion of granule membranes or from endocytosis.     

Stimulation of human NK cell activity by cultured cells. II Ingestion of aspirin by blood donors suppresses induced NK activity.

Incubation of mononuclear cells with a melanoma tumour cell. M4, induces a rapid increase in NK cell activity in both normal and tumour patient cells. A marked individual variation among the patient population can be attributed to the prior ingestion of aspirin. Single therapeutic doses of aspirin (two tablets, 660 mg) taken 12 hr prior to donation of the blood sample cause an 80-100% reduction in the NK cell activity induced by M4. Since many patients coming to cancer clinics take aspirin regularly, it is essential that they be questioned about their recent usage of this drug if their cells are to be used in assays for NK activity.     

Perturbation of red blood cell flow in small tubes by white blood cells

The flow of blood in the microcirculation is facilitated by the dynamic reduction in viscosity (Fahraeus-Lindquist effect) resulting from the axial flow of deforming crythrocytes (RBCs) and from the decrease in the ratio of cell to vessel diameter. RBC velocity exceeds that of average fluid velocity; however the slower moving white blood cells (WBC) perturb flow velocity and the ratio of cell to vessel diameter by obstructing red cell flow through formation of trains of red cells collecting behind the white cell. This effect of white cells was studied quantitatively in a model in vitro tubes less than 10 m in diameter with the demonstration that flow resistance increases linearly with white cell numbers up to 1,000 WBC/mm³ at tube hematocrit of 17.7%. The increase in resistance exceeds the flow resistance of WBC and appears to relate directly to train formation. A mechanical model of train formation developed to predict WBC influence in flow resistance over the range of WBC studied reasonably fits observed WBC effects.     

Effect of red blood cells and red blood cell ghosts on reticuloendothelial system function

Previous studies have shown that hepatic phagocytosis of red blood cell (RBC) stroma can depress reticuloendothelial system (RES) phagocytic function and increase susceptibility to shock. Since the RBC stroma used in these experiments contained substantial amounts of adherent hemoglobin, the present study was carried out to evaluate the role of the hepatic uptake of RBC membrane material on RES phagocytic function and susceptibility to endotoxin shock in rats. Neuraminidase-treated RBC which contained normal amounts of hemoglobin and RBC ghosts which were hemoglobin-free were used. Both preparations were removed from the circulation primarily by the liver. RES phagocytic function was depressed following the hepatic uptake of 29 X 10(8) neuraminidase-treated RBC and 26 X 10(8) RBC ghosts. RES uptake of neuraminidase-treated RBC was associated with an increase in susceptibility to endotoxin shock, but RBC ghosts did not affect shock susceptibility. Thus, RBC ghosts and intact RBC are equally effective in depressing RES phagocytic function, but RBC ghosts did not affect susceptibility to endotoxin shock.     

Foamy cells associated with phagocytosis of glutaraldehyde-treated red blood cells and red cell membranes

In order to clarify the mechanism for the formation of foamy cells (macrophages with foamy appearance) associated with increased erythrophagocytosis, we tried to reproduce these cells in mice by subcutaneous injection of intact red blood cells (RBCs), OsO4-treated RBCs (Os-RBCs), glutaraldehyde-treated RBCs (G-RBCs), or isolated red cell membranes, and time-course observation was done by light and electron microscopy. Foamy cells were induced by the latter two methods. Within the macrophages, G-RBCs were fragmented into spherules by newly formed small vacuoles, and with time these spherules lost their hemoglobin content transforming into small vacuoles with translucent matrix. In most of these vacuoles, red cell membrane structure was discernible adjacent to the phagocytic vacuole. Such macrophages containing abundant small vacuoles appear foamy in light microscopy. Foamy cells induced by injection of red blood cell membranes were positive for lipid stains and contained abundant laminated membrane structures in electron microscopy. These results suggest that the foamy cells related with increased erythrophagocytosis are heterogeneous with respect to their pathogenesis and cellular inclusions, and proteinaceous constituents resistant to intracellular digestion are also responsible for the occurrence of foamy cells.     

A titrographic method for determining the glycolysis flow rate with Plasmodium berghei-infected red blood cells

A procedure is described for the elucidation of the glycolytic flux rate of red blood cells infected with the malarial parasite Plasmodium berghei. It is based on the titration of the protons originating from the glycolytic lactate accumulation. Compared with traditional methods of biochemical measurements of glucose consumption or accumulation of lactate the proposed procedure shows the following advantages: continuously measurement is possible; constancy of the pH-value during the measurement; lower amounts of biological material are necessary (increased sensitivity) increased accuracy; The method can also be applied to studies on other cells and species with normal or increased glycolytic flux rate.     

Stimulation of human peripheral blood polymorphonuclear cell iodination by lignin-related substances.

Lignin is a heterogenous natural product composed of phenylpropane units and is usually associated with hemicellulose in its native state. Until now little attention has been paid to the potential therapeutic utility of lignified products. Natural lignified products are demonstrated in the present study to stimulate iodination significantly (incorporation of radioactive iodine into an acid-insoluble fraction) of human peripheral blood polymorphonuclear cells (PMN). This stimulation was significantly inhibited in the presence of myeloperoxidase inhibitors. These materials were almost completely deprived of their stimulation capacity by treatment with NaCIO2, but this capacity was not affected by severe treatment with H2SO4 or trifluoroacetic acid. Similar stimulating activity by chemically defined tannin-related polyphenolic compounds was observed. Degradation products or component units of lignin, and natural antitumor polysaccharides and their chemically modified derivatives (introduced with negatively or positively charged groups) and polysialoglycoproteins had little or no activity. The results indicate the importance of a polymerized phenolic structure for the stimulation of PMN iodination. Possible physiological relevance of the stimulation of iodination by lignified substances is discussed.     

Beyond the red cell: pegylation of other blood cells and tissues.

Immunological recognition of allogeneic tissue is of critical concern in transfusion and transplantation medicine. While the major emphasis of our work on the immunocamouflage of cells has been focused on the erythrocyte, we have extended this research beyond the red blood cell (RBC) to other tissues. Our studies from blood transfusion (i.e., a specialized form of cellular transplantation) suggest that covalent modification of cells and tissues with methoxypoly(ethylene glycol) mPEG can significantly diminish immunologic recognition of other allogeneic tissues and, furthermore, may enhance the induction of tolerance. The mechanisms underlying the mPEG-mediated immunocamouflage of alloantigens is the global camouflaging of antigenic sites, membrane surface charge and the attenuation of receptor-ligand and cell-cell interactions. As a consequence of the immunocamouflage imparted by the grafted mPEG, weak costimulation of alloreactive T cells is observed which subsequently induces apoptosis of these reactive cells. As a result of this clonal deletion, a pro-tolerance state is induced. The potency of immunocamouflage is readily observed in in vivo murine models of transfusion-associated graft versus host disease. Furthermore, initial studies on the in vivo transplantation of pegylated rat and murine pancreatic islets have demonstrated that mPEG-derivatization does not impair the finely tuned signaling necessary for glucose homeostasis. Finally, in contrast to the pharmacological inhibition of the immune response by agents such as cyclosporine, mPEG-mediated immunocamouflage directly attenuates the inherent antigenicity and immunogenicity of the donor tissue itself while leaving the recipient a fully competent immune system.     

Transient increase in deformability of stressed red blood cells and role of plasma proteins.

In the context of possible fatigue and eventual destruction of red blood cells (RBCs) in the circulation, changes in deformability (filterability) were studied in RBCs which had just undergone deformation. Fresh human RBCs suspended in media of varying protein concentrations were stressed by causing them to pass through a 5-microns Nuclepore filter. Resultant changes in their deformability were assessed from their mean pore passage time in a subsequent filter passage. Contrary to the expectation, that deformability would be reduced, shortened passage times were observed for those stressed RBCs. The changes were, however, transient and, like the initial passage times, were an increasing function of the protein concentration in the suspending fluid. These results appeared to be consistently explicable by assuming release due to stresses and readsorption in time for plasma protein molecules normally adsorbed on cell surfaces. An analysis, furthermore, yielded acceptable estimates for magnitude of the cell-plasma protein interaction, number of protein molecules normally adsorbed on a cell, and also cell membrane viscosity and its apparent change due to the protein adsorption.     

A density method for determining plasma and red blood cell volume

We employed a highly sensitive density measuring system to measure the decrease in blood density and plasma density due to the infusion of a saline bolus into the rabbit's circulation. Based on a vasculature model with the Fahraeus effect, we deduced a set of equations to calculate from the density decreases the red-blood-cell volume and plasma volume of the rabbit. A value of 0.82±0.02 was obtained forF _cell, the ratio of the hematocrit calculated from the two volumes and the arterial hematocrit.     

Cholesterol exchange between the red blood cells and plasma of young and old rats.

The exchange of unesterified cholesterol between plasma and red blood cells of young (1.5 month) and old (1-2 years) rats was studied using [3H]-cholesterol as label. Using labeled plasma and unlabeled red blood cells, no equilibration of specific activity of unesterified cholesterol with the specific activity of cholesterol of red blood cells occurred in either young or old rats. Using labeled red blood cells and unlabeled plasma, equilibration of radioactivity of cholesterol was observed in both young and old rats. However, a more rapid exchange of free cholesterol was observed when labeled old red blood cells were incubated with unlabeled plasma. This apparent paradox may be explained by the existence of a larger, more readily exchangeable pool of free cholesterol in red blood cells of old rats than of young rats. Red blood cells and plasma of old rats contained significantly more free cholesterol than those of young rats but phospholipid levels were comparable.     

Cryopreservation of red blood cells: effect of freezing on red cell quality and residual lymphocyte immunogenicity.

AIMS--To investigate treatment with glycerol/washing as a potential substitute for freeze-thawing in the production of leucocyte depleted red cell concentrates for patients with a history of non-haemolytic reactions following transfusion. METHODS--The standard procedure of treatment with glycerol/-80 degrees C freezing/thawing/washing was compared with a similar procedure in which freezing was omitted. The quality of the resulting red cell products was assessed in relation to: (1) standard red cell biochemical parameters; (2) leucocyte and lymphocyte subset composition using flow cytometry with fluorescent labelled monoclonal antibodies; and (3) immunogenicity of the residual lymphocytes in mixed lymphocyte culture. RESULTS--Compared with red cells subjected to the standard freeze-thaw technique, red cells undergoing the non-freezing procedure and suspended in additive solutions had significantly better biochemical preservation after 21 days of storage (p < 0.001). Both procedures removed an average 98% of the initial leucocytes at the expense of 18-20% of the red cells. The non-freezing procedure resulted in higher residual concentrations of HLA class II bearing lymphocytes (p < 0.01), but not higher numbers of dendritic cells. Both procedures were equally effective in annulling the residual lymphocytes'' ability to act as stimulator cells in one-way mixed lymphocyte culture. CONCLUSIONS--The non-freezing procedure produces a superior product for the provision of red cells to patients with granulocyte antibodies. These products may also offer a lower risk of HLA alloimmunisation to previously unexposed patients.     

Stability of spiculated red blood cells induced by intercalation of amphiphiles in cell membrane

The stability of speculated red blood cells, induced by intercalation of amphiphilic molecules into the cell membrane, is studied. It is assumed that the stable red blood cell shape corresponds to the minimum of its membrane elastic energy, which consists of the local and non-local bilayer bending energies and of the skeleton shear elastic energy. The cell volume and the membrane area are kept constant. It is calculated that the number of spicules of the stable echinocytic shape is larger when the amphiphile concentration is higher, which is in agreement with experimental observations. Also, it is established that, in explaining the stability of the echinocytic shape of the red blood cell, it is necessary to include the membrane skeleton shear elasticity.