Canine Red Blood Cells Research Articles

In Vitro Lysis and Acute Transfusion Reactions with Hemolysis Caused by Inappropriate Storage of Canine Red Blood Cell Products

Transfusion of red blood cell (RBC) products carries considerable risk for adverse reactions, including life-threatening hemolytic reactions. To report the occurrence and investigation of life-threatening acute transfusion reactions with hemolysis in dogs likely related to inappropriate blood product storage. Four dogs with acute transfusion reactions and other recipients of blood products. Medical records were reviewed from 4 dogs with suspected acute hemolytic transfusion reactions after receiving RBC products at a veterinary clinic over a 1-month period. Medical records of other animals receiving blood products in the same time period also were reviewed. Blood compatibility and product quality were assessed, subsequent transfusions were closely monitored, and products were diligently audited. During or immediately after RBC product transfusion, 4 dogs developed hemolysis, hemoglobinuria, or both. Two dogs died and 1 was euthanized because of progressive clinical signs compatible with an acute hemolytic transfusion reaction. Blood type and blood compatibility were confirmed. RBC units from 2 blood banks were found to be hemolyzed after storage in the clinic's refrigerator; no bacterial contamination was identified. After obtaining a new refrigerator dedicated to blood product storage, the problem of hemolyzed units and acute transfusion reactions with hemolysis completely resolved. Acute life-threatening transfusion reactions can be caused by inappropriate storage of RBC products. In addition to infectious disease screening and ensuring blood-type compatibility, quality assessment of blood products, appropriate collection, processing, and storage techniques as well as recipient monitoring are critical to provide safe, effective transfusions.

Detection of anti-Babesia gibsoni heat shock protein 70 antibody and anti-canine heat shock protein 70 antibody in sera from Babesia gibsoni-infected dogs

Antibodies that recognized either Babesia gibsoni or canine red blood cell (RBC) 70-kilodalton (kDa) protein were detected in serum from acutely and chronically B. gibsoni-infected. In those sera, antibodies that reacted with recombinant B. gibsoni and canine heat shock protein 70 (rBgHsp70 and rcHsp70) were detected; therefore, B. gibsoni and canine RBC 70-kDa proteins seemed to be BgHsp70 and cHsp70, respectively. In infected dogs, the amounts of these antibodies increased after infection. Interestingly, polyclonal antibody raised against rBgHsp70 in two rabbits reacted not only with rBgHsp70 but also with rcHsp70 and native cHsp70 from canine RBCs. Because BgHsp70 showed high homology with cHsp70 (70.8%), anti-rBgHsp70 antibody might cross-react with cHsp70. Additionally, the localizations of both BgHsp70 and cHsp70 were observed by indirect fluorescence assay. As a result, cHsp70 was not found on the membrane surface of erythrocytes, suggesting that erythrocytes would not be targets of anti-cHsp70 antibody. Meanwhile, only exoerythrocytic parasites were stained by anti-rBgHsp70 antibody. This result showed that BgHsp70 would be expressed on the surface of parasites during the exoerythrocytic stage. These results indicated that BgHsp70 was a highly immunogenic protein in canine B. gibsoni infection, and that exoerythrocytic parasites might be targets of anti-BgHsp70 antibody.

Aquaporin-1 Expression in Canine Peripheral Erythrocytes and Its Relation to Cell Volume

To evaluate the relationship between aquaporin-1 (AQP1) expression and the cell volume of red blood cells (RBCs), canine peripheral RBCs were separated according to specific gravity, and expression of the AQP1 protein on the membrane of RBCs was compared using anti-dog AQP1 polypeptide serum. Western blot analysis indicated that there was no significant difference in AQP1 expression between large and small cell fractions. In addition, the AQP1 expression of inherited high K/low Na RBCs which are known to be 20% larger than normal RBCs, was comparable to that of normal RBCs. These results suggest that AQP1, the major water channel in RBCs, does not determine the cell volume of peripheral canine RBCs.

Lack of Evidence of Pregnancy‐Induced Alloantibodies in Dogs

It is controversial whether or not pregnant bitches become sensitized to red blood cell (RBC) antigens. Bitches do not develop alloantibodies to RBC antigens during gestation and can be used safely as blood donors. The study group included 35 healthy female dogs with a prior history of 1 (n = 12), 2 (n = 14), or >or= 3 (n = 9) pregnancies. The control group consisted of 15 healthy female dogs without any history of pregnancy. All dogs were blood typed for dog erythrocyte antigens (DEA) 1.1, 1.2, 3, 4, 5, and 7 using ethylenediaminetetraacetic acid blood samples and polyclonal antisera. Antibody screening was performed with serum and canine RBC panels of known blood type. An autocontrol and direct antiglobulin test were performed to rule out the presence of autoantibodies. The only alloantibodies identified were those against DEA 7 and the prevalence of anti-DEA 7 alloantibodies was similar in dogs with known history of pregnancy (11.4%) and in the control group (13.3%). These results confirm previous studies and clinical transfusion medicine experience. Naturally occurring anti-DEA 7 alloantibodies have been reported but their clinical relevance has not been shown. Pregnancy does not appear to sensitize dogs to RBC antigens. Consequently, dogs with prior history of pregnancy can be used safely as blood donors. Conversely, no additional pretransfusion compatibility studies would be required should these dogs themselves need to be transfused.

Lactate transport in canine red blood cells

To detect monocarboxylate transporters (MCTs) in canine RBC membranes and to determine the distribution of lactate between plasma and RBCs. Blood samples obtained from 6 purpose-bred Beagles. 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. 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. 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.

Heinz body anaemia in two dogs after Catalan spring onion ("calcot") ingestion: a case report

Catalan spring onions, or “calcots”, are a very popular kind of vegetables obtained by special agronomic practices that avoid photosynthesis and subsequent metabolism by the <i>Allium cepa</i> plants. They have been considered for more than a century harmless for pets, as pleasant odour and sweet taste differ so largely from any other onion that can be ingested by children and adults in huge quantities, up to ~3% body weight, without problems being observed. However, the organosulphur chemicals responsible for oxidative damage to canine red blood cells found in this plant species seem to remain present, as two dogs were confirmed to be poisoned after eating left-over “calcots”. Both patients presented haemolytic anaemia with Heinz body formation, and eccentrocytosis. Veterinarians and owners should be aware of the dangers associated with this particular onion variety and type, as it is highly palatable to pets and no effective treatment is currently available.

Influence of Pre-freeze Treatment and Cryo-storageTemperature on the Post-thaw Stability of Canine Red Blood Cells Cryopreserved in the Presence of Hydroxyethyl Starch

Influence of Pre-freeze Treatment and Cryo-storageTemperature on the Post-thaw Stability of Canine Red Blood Cells Cryopreserved in the Presence of Hydroxyethyl Starch

In vivorecovery and survival of red cells after photodynamic treatment with thiopyrylium and red light using a canine model

Recently, we have shown that thiopyrylium has robust inactivation capabilities against a broad spectrum of pathogens in the human red blood cell (RBC) suspensions while retaining key RBC in vitro properties. The 24-h recovery and survival of canine red cells were measured upon autologus reinfusion of control and phototreated units. The 24-h recovery of control and phototreated RBCs was 75.7 +/- 6.4% and 87.5 +/- 8.5%, respectively. The time for 50% survival of labelled control RBCs was similar to that of phototreated group (206 +/- 58 h vs. 255 +/- 63 h, respectively). Results suggest that thiopyrylium phototreatment does not negatively affect canine RBC in vivo recovery.

Phage display antibodies to allelic determinants of canine blood cells

Techniques were developed to select useful allele-specific antibodies directed to canine red blood cell alloantigens from a phage display library in which antibody single chain variable fragments (scFv) were expressed on filamentous bacteriophage. First, techniques were developed to detect specific antigens displayed on red blood cells using flow cytometry. Next, techniques permitting the efficient selection of red blood cell binders from a large phage library were developed. Finally, the amplified library was depleted using the red blood cells of one animal and the remainder enriched using cells from a genetically different animal. A high frequency of clones derived from this population bound antigen(s) of the second animal but not the first. Sequence analysis of these clones revealed that at least 11 clonally distinct isolates were present within the selected population. The procedure used to obtain these reagents is simple and inexpensive and the techniques developed should find applications in canine transfusion medicine and parentage assignment.

INHIBITION OF Na,K-ATPASE ACTIVITY REDUCES BABESIA GIBSONI INFECTION OF CANINE ERYTHROCYTES WITH INHERITED HIGH K, LOW Na CONCENTRATIONS

Babesia gibsoni multiplies well in canine red blood cells (RBCs) containing high concentrations of potassium (HK), reduced glutathione, and free amino acids as a result of an inherited high Na,K-ATPase activity, i.e., HK RBCs. To determine the role of Na,K-ATPase in the multiplication of B. gibsoni, the effect of ouabain on the proliferation of the parasites in HK RBCs was investigated. To determine the direct effect of ouabain on the parasites, the proliferation of the parasites in normal canine RBCs containing low potassium (LK) and high sodium concentrations, i.e., LK RBCs, which completely lack Na,K-ATPase activity, was observed. Ouabain at 0.1 mM significantly suppressed the multiplication of B. gibsoni in HK RBCs in vitro, whereas it had no effect on the parasites in LK RBCs. The results suggest that the multiplication of B. gibsoni in HK RBCs depends mainly on the presence of Na,K-ATPase in the cells. Therefore, the effects of ouabain on the intracellular cation and free amino acid composition of the HK RBCs were examined. In HK RBCs incubated with ouabain, a marked decrease in the concentration of potassium and an increase in sodium were observed, together with a decrease in the number of parasitized cells. These results suggest that the intracellular cation composition maintained by Na,K-ATPase might be advantageous to the parasites. Moreover, the concentrations of some free amino acids, i.e., asparagine, aspartate, glutamate, glutamine, glycine, and histidine, were markedly decreased in HK RBCs incubated with ouabain. Decreased concentrations of the free amino acids induced by inhibition of Na,K-ATPase seemed to affect the multiplication of B. gibsoni in HK RBCs. Based on these results, it is clear that the high Na,K-ATPase activity in HK RBCs contributes to the proliferation of B. gibsoni by maintaining high potassium and low sodium concentrations, as well as high concentrations of some free amino acids in the cells.

918. Autologous Canine Bone Marrow Stromal Cells Retrovirally Engineered with the Canine EPO Gene and Implanted In Vivo in a Collagen Matrix Produce and Release Systemically Functional EPO over a Prolonged Period

Many features of dogs make them suitable animal models to study new therapeutic approaches for human diseases: a large body mass, a long life span and spontaneous diseases such as chronic renal failure (CRF) that leads to anemia. This, and the longevity of canine red blood cells (RBC) comparable to that of humans, render dogs particularly suitable to develop new treatments of CRF-induced anemia. In contrast, rodents differ from humans by a much smaller body mass, shorter longevity of RBC (43 days) and the absence of spontaneous diseases resembling those of people. Current treatment of CRF-induced anemia in people (and dogs) consists of repeated injections of recombinant human EPO (rhEPO), which incurs high costs and specialized personnel. Our hypothesis is that the implantation of an “organoid” composed of autologous cells embedded in an organic matrix and engineered to produce and systemically release EPO can correct CRF-induced anemia in dogs for a long period, replacing the need for repeated injections of rhEPO. As a first step towards this goal, we demonstrated the feasibility of this approach in normal dogs. We used bone marrow stromal cells (BMSCs) for the sustained production of EPO in vivo because these cells are easily accessible, transduceable and expandable in vitro. We isolated and transduced canine BMSCs with the canine EPO gene using a bicistronic retrovector. Using a MOI of 60, the transduction efficiency was 60 to 90% as analysed by FACS; the in vitro production and release of canine EPO was verified by Western blot and measured by ELISA (2 U of EPO/106 cells/24 hrs). The incorporation of engineered cells in a biocompatible solid matrix makes it possible to remove the implant in case of over expression of the transgene or other complications. To demonstrate the functionality of the secreted protein, the canine EPO-engineered BMSCs were embedded in bovine type I collagen (Inamed™), and the resulting organoid was implanted in 5 NOD-SCID mice (7 × 106cells/500 μl collagen/mouse). Four control mice were treated identically except that the implanted BMSCs (from the same dog) were transduced with empty retroparticles. The hematocrit (Ht) of treated mice increased from 53% to 88% in 33 days and remained elevated over 3 months following implantation. The canine EPO-engineered BMSC were embedded within the same matrix and implanted s.c. in the respective donor dogs. The number of embedded BMSCs and volume of implants were optimized: 32 implants were given in 11 injections (3 implants per injection, each implant containing 4 × 106 BMSCs). This protocol has been implemented in 3 dogs whose Ht has increased from 42 (SD: ±6.9) to 55 (SD: ±2) within 12 days and has remained elevated for 4 weeks. These results clearly demonstrate the feasibility of cellular therapy in large immunocompetent mammals, a crucial step towards its application in people. We will next carry out the optimized protocol on canine and feline patients affected by CRF-induced anemia.

Inhibitory effect of antiserum to surface antigen P50 of Babesia gibsoni on growth of parasites in severe combined immunodeficiency mice given canine red blood cells.

The inhibitory effect of an antiserum to surface protein P50 of Babesia gibsoni on the growth of the parasite was determined with severe combined immunodeficiency mice given canine red blood cells. The antiserum to the recombinant P50 protein significantly inhibited the parasite growth, indicating that P50 might be a useful vaccine candidate.

A Comparative Study of the Effects of Glycerol and Hydroxyethyl Starch in Canine Red Blood Cell Cryopreservation

Hydroxyethyl starch (HES) is a nonpenetrating extracellular cryoprotectant. In contrast to glycerol, it does not require labor-intensive removal from thawed red blood cells (RBCs) prior to transfusion. In this study, we compared glycerol and HES, and assessed HES as a substitute for glycerol in cryopreserved canine RBCs. The RBCs were preserved for 2 months in liquid nitrogen using a 20% (w/v) glycerol solution, and variable concentrations of HES solution. We evaluated the two cryoprotectants by the percentage of post-thaw hemolysis from the total free hemoglobin, saline stability, osmotic fragility, and by observing the erythrocyte morphology using a scanning electron microscope after thawing. The optimal concentration of HES was 12.5% (w/v) for the cryopreservation of canine RBCs. The thaw hemolysis, saline stability, and osmotic fragility index were 25.6 +/- 4.7%, 87.8 +/- 6.9%, and 0.445 +/- 0.024% NaCl respectively. These parameters resemble the results of RBCs frozen in a 20% (w/v) glycerol solution, which are 24.7 +/- 5.2%, 99.2 +/- 0.1%, and 0.485 +/- 0.023% NaCl respectively. From a morphological point of view, 12.5% (w/v) HES showed the best cryoprotection of RBCs compared to the other concentrations of HES. These results suggest that HES could be a possible substitute for glycerol for the cryopreservation of canine RBCs.

Canine RBC osmotic tolerance and membrane permeability

The objective of this study was to determine the cryobiological characteristics of canine red blood cells (RBC). These included the hydraulic conductivity ( L p), the permeability coefficients ( P s) of common cryoprotectant agents (CPAs), the associated reflection coefficient ( σ), the activation energies ( E a) of L p and P s and the osmotic tolerance limits. By using a stopped-flow apparatus, the changes of fluorescence intensity emitted by intracellularly entrapped 5-carboxyfluorescein diacetate (CFDA) were recorded when cells were experiencing osmotic volume changes. After the determination of the relationship between fluorescence intensity and cell volume, cell volume changes were calculated. These volume changes were used in three-parameter fitting calculations to determine the values of L p, P s, and σ for common CPAs. These volume measurements and data analyses were repeated at three different temperatures (22, 14, 7 °C). Using the Arrhenius equation, the activation energies of L p and P s in the presence of CPAs were determined. The osmotic tolerance limits for canine RBC were determined by measuring the percentage of free hemoglobin in NaCl solutions with various osmolalities compared to that released by RBC incubated in double distilled water. The upper and lower osmotic tolerance limits were found to be 150 mOsm (1.67 V iso) and 1200 mOsm (0.45 V iso), respectively. These parameters were then used to calculate the amount of non-permeating solute needed to keep cell volume excursions within the osmotic tolerance limits during CPA addition and removal.

Use of canine red blood cell with high concentrations of potassium, reduced glutathione, and free amino acid as host cells for in vitro cultivation of Babesia gibsoni.

To determine the usefulness of canine RBC with high concentrations of potassium, reduced glutathione (GSH), and amino acid(i.e., HK cells) for in vitro cultivation of Babesia gibsoni. RBC were obtained from 3 dogs that had inherited HK cells and from 3 genetically unaffected dogs that, therefore, had RBC with lower potassium (LK) concentrations (i.e., LK cells). First, B. gibsoni were cultivated using HK or LK cells in alpha-modification of Eagle medium, consisting of Earle salts with glutamine and without ribosides, deoxyribosides, and sodium bicarbonate under a humidified atmosphere containing 5% CO2 at 37 C. Second, parasites were cultivated with LK- or HK-cell lysates. Finally, HK cells were separated into 3 fractions (bottom, middle, top layers) by density gradient centrifugation, and B. gibsoni were cultivated with each of the HK-cell fractions. In addition, the concentrations of free amino acids and reduced glutathione (GSH) in each HK-cell fraction were measured. B. gibsoni preferentially multiplied in HK-cell cultures rather than in LK-cell cultures. Furthermore, the addition of HK-cell lysate to the culture medium resulted in enhanced multiplication of the parasites. Higher multiplication of the parasites was observed in HK cells from the top layer, compared with HK cells from the middle and bottom layers. The HK cells from the top layer had higher concentrations of glutamate, aspartate, and GSH, compared with HK cells from the middle and bottom layer. Canine HK cells are useful host cells for in vitro cultivation of B. gibsoni, and the high concentrations of glutamate, aspartate, and GSH may result in enhancement of multiplication of the parasites in HK cells.

Use of a Prestorage Leukoreduction Filter Effectively Removes Leukocytes from Canine Whole Blood While Preserving Red Blood Cell Viability

Leukoreduction of blood products is a technique used to prevent leukocyte‐induced transfusion reactions. Filters currently used for human blood products achieve at least a 99.9% reduction in leukocyte numbers per unit (450 mL) of blood. Goals of this study were to determine if a prestorage leukoreduction filter could effectively achieve leukoreduction of canine blood and to determine if viability of the leukoreduced red blood cell (RBC) product could be maintained after 35 days of storage. Blood collected from each dog was filtered through a leukoreduction filter at either room temperature or after cooling (4°C) for 4 hours. Filtration efficacy was determined by measurement of pre‐ and postfiltration leukocyte counts. In vitro viability of RBCs was determined by comparing RBC adenosine triphosphate concentration and percent hemolysis before and after the storage period. In vivo viability of stored cells was determined using a biotin‐streptavidin‐phycoerythrin labeling technique and flow cytometry. Blood filtered within 30 minutes of collection versus blood filtered after cooling had mean reductions in leukocyte numbers of 88.90 and 99.99%, respectively. The mean ATP and hemoglobin concentrations from the in vitro analysis were comparable to those obtained in previously for canine RBC adequately stored for 35 days. The mean in vivo 24‐hour survival of the stored RBC was 84.7%. The leukoreduction filter used did not adversely affect in vitro or in vivo viability of canine RBCs. The filter effectively removed leukocytes from blood, with maximal efficiency of filtration achieved with use of cooled blood.

Use of a prestorage leukoreduction filter effectively removes leukocytes from canine whole blood while preserving red blood cell viability.

Leukoreduction of blood products is a technique used to prevent leukocyte-induced transfusion reactions. Filters currently used for human blood products achieve at least a 99.9% reduction in leukocyte numbers per unit (450 mL) of blood. Goals of this study were to determine if a prestorage leukoreduction filter could effectively achieve leukoreduction of canine blood and to determine if viability of the leukoreduced red blood cell (RBC) product could be maintained after 35 days of storage. Blood collected from each dog was filtered through a leukoreduction filter at either room temperature or after cooling (4 degrees C) for 4 hours. Filtration efficacy was determined by measurement of pre- and postfiltration leukocyte counts. In vitro viability of RBCs was determined by comparing RBC adenosine triphosphate concentration and percent hemolysis before and after the storage period. In vivo viability of stored cells was determined using a biotin-streptavidin-phycoerythrin labeling technique and flow cytometry. Blood filtered within 30 minutes of collection versus blood filtered after cooling had mean reductions in leukocyte numbers of 88.90 and 99.99%, respectively. The mean ATP and hemoglobin concentrations from the in vitro analysis were comparable to those obtained in previously for canine RBC adequately stored for 35 days. The mean in vivo 24-hour survival of the stored RBC was 84.7%. The leukoreduction filter used did not adversely affect in vitro or in vivo viability of canine RBCs. The filter effectively removed leukocytes from blood, with maximal efficiency of filtration achieved with use of cooled blood.

Evaluation of Biochemical Changes During In Vivo Erythrocyte Senescence in the Dog

One hypothesis to explain the age-dependent clearance of red blood cells (RBCs) from circulation proposes that denatured/oxidized hemoglobin (hemichromes) arising late during an RBC’s life span induces clustering of the integral membrane protein, band 3. In turn, band 3 clustering generates an epitope on the senescent cell surface leading to autologous IgG binding and consequent phagocytosis. Because dog RBCs have survival characteristics that closely resemble those of human RBCs (ie, low random RBC loss, ≈115-day life span), we decided to test several aspects of the above hypothesis in the canine model, where in vivo aged cells of defined age could be evaluated for biochemical changes. For this purpose, dog RBCs were biotinylated in vivo and retrieved for biochemical analysis at various later dates using avidin-coated magnetic beads. Consistent with the above hypothesis, senescent dog RBCs were found to contain measurably elevated membrane-bound (denatured) globin and a sevenfold enhancement of surface-associated autologous IgG. Interestingly, dog RBCs that were allowed to senesce for 115 days in vivo also suffered from compromised intracellular reducing power, containing only 30% of the reduced glutathione found in unfractionated cells. Although the small quantity of cells of age ≥110 days did not allow direct quantitation of band 3 clustering, it was nevertheless possible to exploit single-cell microdeformation methods to evaluate the fraction of band 3 molecules that had lost their normal skeletal linkages and were free to cluster in response to hemichrome binding. Importantly, band 3 in RBCs ≥112 days old was found to be 25% less restrained by skeletal interactions than band 3 in control cells, indicating that the normal linkages between band 3 and the membrane skeleton had been substantially disrupted. Interestingly, the protein 4.1a/protein 4.1b ratio, commonly assumed to reflect RBC age, was found to be maximal in RBCs isolated only 58 days after labeling, implying that while this marker is useful for identifying very young populations of RBCs, it is not a very sensitive marker for canine senescent RBCs. Taken together, these data argue that several of the readily testable elements of the above hypothesis implicating band 3 in human RBC senescence can be validated in an appropriate canine model.

Evaluation of Biochemical Changes During In Vivo Erythrocyte Senescence in the Dog

AbstractOne hypothesis to explain the age-dependent clearance of red blood cells (RBCs) from circulation proposes that denatured/oxidized hemoglobin (hemichromes) arising late during an RBC's life span induces clustering of the integral membrane protein, band 3. In turn, band 3 clustering generates an epitope on the senescent cell surface leading to autologous IgG binding and consequent phagocytosis. Because dog RBCs have survival characteristics that closely resemble those of human RBCs (ie, low random RBC loss, ≈115-day life span), we decided to test several aspects of the above hypothesis in the canine model, where in vivo aged cells of defined age could be evaluated for biochemical changes. For this purpose, dog RBCs were biotinylated in vivo and retrieved for biochemical analysis at various later dates using avidin-coated magnetic beads. Consistent with the above hypothesis, senescent dog RBCs were found to contain measurably elevated membrane-bound (denatured) globin and a sevenfold enhancement of surface-associated autologous IgG. Interestingly, dog RBCs that were allowed to senesce for 115 days in vivo also suffered from compromised intracellular reducing power, containing only 30% of the reduced glutathione found in unfractionated cells. Although the small quantity of cells of age ≥110 days did not allow direct quantitation of band 3 clustering, it was nevertheless possible to exploit single-cell microdeformation methods to evaluate the fraction of band 3 molecules that had lost their normal skeletal linkages and were free to cluster in response to hemichrome binding. Importantly, band 3 in RBCs ≥112 days old was found to be 25% less restrained by skeletal interactions than band 3 in control cells, indicating that the normal linkages between band 3 and the membrane skeleton had been substantially disrupted. Interestingly, the protein 4.1a/protein 4.1b ratio, commonly assumed to reflect RBC age, was found to be maximal in RBCs isolated only 58 days after labeling, implying that while this marker is useful for identifying very young populations of RBCs, it is not a very sensitive marker for canine senescent RBCs. Taken together, these data argue that several of the readily testable elements of the above hypothesis implicating band 3 in human RBC senescence can be validated in an appropriate canine model.

Babesia canis infection in canine-red blood cell-substituted SCID mice

We have developed a mouse model for Babesia canis infection using severe combined immunodeficiency (SCID) mice whose circulating red blood cells had been substituted with canine red blood cells. Substitution of red blood cells in SCID mice was achieved by repetitive transfusions of canine red blood cells, together with administration of an anti-mouse red blood cell monoclonal antibody. Following inoculation of canine-red blood cell-SCID mice with B. canis, parasites proliferated in the canine red blood cells that had been transfused into the SCID mice, resulting in much higher parasitaemia than that observed in dogs. In an attempt to demonstrate the utility of this mouse model, three anti-protozoal drugs, diminazene diaceturate, clindamycin and oxytetracycline, were examined for their efficacy to inhibit the growth of B. canis in canine-red blood cell-SCID mice. The mouse model clearly showed that diminazene diaceturate and oxytetracycline were capable of eliminating B. canis from the canine-red blood cell-SCID mice, whereas clindamycin exhibited only a static effect as parasitaemia relapsed upon cessation of drug administration.