Cryopreservation of sheep red blood cells. 3. Complement titrations with frozen sensitized cells

The effect of different suspending and washing procedures for recovery of sensitized sheep erythrocytes (EA) after freezing at −196°C was investigated. Best results were obtained using gelatin-veronal-buffered saline-sucrose containing 0.15 mM-Ca and 1 mM-Mg (GVBSM⁺⁺-sucrose) as the suspending and first washing buffer. The cryoprotective agents tested were polyvinylpyrrolidone (PVP), neutralized PVP, purified PVP and a gum product, Avelex 1030. All PVP preparations tested gave good results as cryoprotectants in terms of cell recovery after thawing whereas Avelex 1030 was less satisfactory. The EA cells frozen in the presence of untreated PVP showed, however, increased susceptibility to the hemolytic action of complement, whereas cells frozen with purified or neutralized PVP gave titers similar to that obtained with fresh cells. Good results were also obtained with Avelex 1030. Complement titrations with frozen EA cells were more reproducible than titrations with fresh cells.     

Exit of Anaplasma marginale from bovine red blood cells.

Incubation of Anaplasma marginale-infected bovine red blood cells in a flow culture system resulted in a decrease of observable marginal bodies. The decrease was twice the degree of hemolysis, indicating that marginal bodies may leave red blood cells without concomitant lysis of the host cell.     

The life span of glutathione-deficient red cells in Tasmanian Merino sheep.

59FeCl3 was used to measure red cell life span in GSH-low type sheep to the Tasmanian Merino breed, whose GSH deficiency is due to a diminished activity of gamma-glutamyl cysteine synthetase. The 59Fe survival slopes fell within normal limits, indicating that red cells with this type of GSH deficiency do not have a shortened life span.     

Osmotic stress in red blood cells from beagles with hemolytic anemia.

Red blood cell populations separated by density centrifugation were compared in a dynamic assay of osmotic stress. Red blood cells from Beagles genotypically normal and nonanemic (nonaffected), Beagles with inherited hemolytic anemia (anemic), and Beagles presumed to be carriers of the anemia trait (trait carriers) were examined for rate and extent of swelling after exposure to the ionophore A23187 in a medium containing calcium and potassium chloride. Comparisons were made between RBC populations separated on the basis of density. Significant differences were observed in the rates of cell swelling in RBC populations separated by density between nonaffected and anemic Beagles. The response of RBC from Beagles presumed to carry the anemia trait was similar to that of RBC from nonaffected dogs. One phenotypic expression of this inherited abnormality of RBC in Beagles was an accelerated rate of RBC swelling under osmotic stress, and this swelling response diminished with increasing RBC density.     

Cryopreservation of sheep red blood cells. 1. Experiments with polyvinylpyrrolidone and other protective agents

The protection of sheep erythrocytes at freezing temperatures was investigated using glycerol, dimethylsulfoxide (DMSO), glucose and four different types of polyvinylpyrrolidone (PVP) as cryoprotective agents. Depending on type (molecular weight) and concentration good protection was obtained with PVP, whereas glycerol, DMSO and glucose were unsatisfactory. Recovery of cells after thawing was most successful when the cells had been frozen at a concentration of 1–2 × 10⁹ cells/ml. No cells tolerated freezing at −20 °G. Best results were obtained when the cells were frozen directly in liquid nitrogen (−196°G).     

Lactate transport in canine red blood cells

OBJECTIVE: To detect monocarboxylate transporters (MCTs) in canine RBC membranes and to determine the distribution of lactate between plasma and RBCs. SAMPLE POPULATION: Blood samples obtained from 6 purpose-bred Beagles. PROCEDURES: Monocarboxylate transporter isoforms 1, 2, 4, 6, 7, and 8 and CD147 were evaluated in canine RBCs by use of western blot analysis. Lactate influx into RBCs was measured as incorporation of radioactive lactate. RESULTS: 2 MCT isoforms, MCT1 and MCT7, were detected in canine RBC membranes on western blot analysis, whereas anti-MCT2, anti-MCT4, anti-MCT6, and anti-MCT8 antibodies resulted in no signal. No correlation was found between the amount of MCT1 or MCT7 and lactate transport activity, but the ancillary protein CD147 that is needed for the activity of MCT1 had a positive linear correlation with the rate of lactate influx. The apparent Michael is constant for the lactate influx in canine RBCs was 8.8 +/- 0.9mM. Results of in vitro incubation studies revealed that at lactate concentrations of 5 to 15mM, equilibrium of lactate was rapidly obtained between plasma and RBCs. CONCLUSIONS AND CLINICAL RELEVANCE: These results indicated that at least half of the lactate transport in canine RBCs occurs via MCT1, whereas MCT7 may be responsible for the rest, although an additional transporter was not ruled out. For practical purposes, the rapid equilibration of lactate between plasma and RBCs indicated that blood lactate concentrations may be estimated from plasma lactate concentrations.     

Cryopreservation of sheep red blood cells. 2. Purified polyvinylpyrrolidone and hydrolyzed starch as protective agents

The protection of sheep erythrocytes against damage at low temperature (−196°C) was investigated using purified polyvinylpyrrolidone (PVP) and hydrolyzed starch as cryoprotective agents. Identical results were obtained with untreated PVP, neutralized PVP and PVP purified by chromatography. With hydrolyzed starch the cryoprotection was dependent on the type and concentration of the starch used and on the extent of hydrolysis of the starch prior to use. Cell recovery with some starch types was the same (80–85 %) as that obtained with PVP.     

Heritable characters of the red blood cells of sheep

Extract

From the start I want to make it clear that the research work described here is the work of one of us (J.V.E.), and the reason why I am presenting his work is that Evans comes from New Zealand and presented a paper on this subject to the annual meeting of the British Association for the Advancement of Science last September in Sheffield, so that it seemed appropriate that his work should also be presented to this Society.     

Thiopurine methyltransferase activity in red blood cells of dogs

Thiopurine methyltransferase (TPMT) is an important enzyme in the metabolism of thiopurine medications such as azathioprine. In humans, activity varies widely among individuals, primarily because of genetic polymorphisms. Low TPMT activity increases the risk of myelosuppression from azathioprine and 6-mercaptopurine, whereas high TPMT activity is associated with poor drug efficacy. The purpose of this study was to determine whether dogs also show a wide range of TPMT activity. Heparinized blood samples were obtained from 177 dogs associated with a veterinary teaching hospital. Red blood cell (RBC) TPMT activity was measured by means of a modification of a radiochemical method as established for use in people. TPMT activity varied across a 9-fold range (7.9-71.8 U of RBC per milliliter; median, 21.7). Variation in TPMT activity was not associated with age, sex, or neutering status. Giant Schnauzers had much lower TPMT activity (7.9-20 U of RBC per milliliter; median, 13.1; P < .001) than did other breeds, and Alaskan Malamutes had much higher TPMT activity (22.7-71.8 U of RBC per milliliter; median, 36.0; P < .001) than did other breeds. Such variations in TPMT activity in the canine population and within groups of related dogs could affect thiopurine drug toxicity and efficacy in canine patients.     

Mycoplasma gallisepticum-induced alterations in chicken red blood cells

Incubation of Mycoplasma gallisepticum with washed chicken red blood cells for 1 hr or 5 hr resulted in altered red blood cell surface morphology and perforations of the cells.     

Storage of equine red blood cells as a concentrate

The study was undertaken to determine how equine red blood cells (RBCs) survive in storage bags designed for use with human RBCs. Separated RBCs were stored in a routine manner for 35 days and examined every 7 days for storage lesions. Measured parameters included haematology, haemolysis, pH, potassium, lactate, adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG). All tests were performed in vitro. Haematology did not change significantly. Haemolysis increased during storage but did not exceed human limits. pH and 2,3-DPG decreased, while lactate, potassium and ATP increased. RBCs deteriorated somewhat during storage, but when compared with human in vitro parameters, remained suitable for transfusion. It is concluded that equine erythrocytes can be stored for at least 35 days before transfusion.     

Cellular adhesive phenomena in apicomplexan parasites of red blood cells

The apicomplexan parasites Babesia and Plasmodium are related, yet phylogenetically distinct haemoprotozoa that infect red blood cells and cause severe diseases of major human and veterinary importance. A variety of cellular and molecular interactions are pivotal in many aspects of the pathogenicity of these two parasites. Comparison of the cellular and molecular mechanisms that culminate in accumulation of parasitised red blood cells in the microvasculature of cattle infected with Babesia bovis (babesiosis) and humans infected with Plasmodium falciparum (falciparum malaria) is particularly instructive given the striking similarities in the pathophysiology of these two important medical and veterinary diseases. While such adhesive phenomena have been studied extensively in malaria, they have received relatively little attention in babesiosis. In this review, we summarise the findings of more than 25 years of research into cellular adhesive phenomena in malaria and speculate on how this body of work can now be applied to Babesia parasites. Such information is fundamental if we are to learn more about the biology of Babesia parasites, the cellular and molecular mechanisms by which they cause infection and disease and how to develop novel therapeutic strategies or vaccines for both Babesia and malaria infections.