Human erythrocytes as drug carriers: loading efficiency and side effects of hypotonic dialysis, chlorpromazine treatment and fusion with liposomes.

Human red blood cells (RBCs) are emerging as a highly biocompatible microparticulate drug delivery system. So far, drugs have commonly been loaded into freshly isolated RBCs using rather disruptive methods based on hypotonic shock, and assessment of damage was restricted to hemolysis. Here, we investigated loading of RBCs from blood bank units with enzymes of various molecular weights using hypotonic dialysis (HD), pretreatment with chlorpromazine (CPZ) and fusion with liposomes. The latter two techniques have received little attention in RBC loading so far. Along with loading efficiency, all methods were tested for the induction of side effects. Very importantly, next to hemolysis, we also addressed morphological changes and phosphatidyl serine (PS) exposure, which has been recognized as a critical parameter associated with premature RBC removal and induction of transfusion-related pathologies. The efficiency of loading using hypotonic dialysis decreased with the molecular weight of the enzyme. For liposomes and chlorpromazine, loading efficiencies were higher and independent of enzyme molecular weights. While hypotonic dialysis always induced a high degree of hemolysis, irreversible modifications in the morphology of the cells and PS exposure, the side effects that were induced by loading using CPZ and liposomes were limited. In particular, PS exposure, although high immediately after treatment, returned to physiological levels after recovery. Retention and deformability studies using a spleen-mimicking device showed that RBCs treated with CPZ and liposomes behave like physiological RBCs, while HD led to very fragile and poorly deformable RBCs.

Functional consequences of sphingomyelinase-induced changes in erythrocyte membrane structure.

Inflammation enhances the secretion of sphingomyelinases (SMases). SMases catalyze the hydrolysis of sphingomyelin into phosphocholine and ceramide. In erythrocytes, ceramide formation leads to exposure of the removal signal phosphatidylserine (PS), creating a potential link between SMase activity and anemia of inflammation. Therefore, we studied the effects of SMase on various pathophysiologically relevant parameters of erythrocyte homeostasis. Time-lapse confocal microscopy revealed a SMase-induced transition from the discoid to a spherical shape, followed by PS exposure, and finally loss of cytoplasmic content. Also, SMase treatment resulted in ceramide-associated alterations in membrane-cytoskeleton interactions and membrane organization, including microdomain formation. Furthermore, we observed increases in membrane fragility, vesiculation and invagination, and large protein clusters. These changes were associated with enhanced erythrocyte retention in a spleen-mimicking model. Erythrocyte storage under blood bank conditions and during physiological aging increased the sensitivity to SMase. A low SMase activity already induced morphological and structural changes, demonstrating the potential of SMase to disturb erythrocyte homeostasis. Our analyses provide a comprehensive picture in which ceramide-induced changes in membrane microdomain organization disrupt the membrane-cytoskeleton interaction and membrane integrity, leading to vesiculation, reduced deformability, and finally loss of erythrocyte content. Understanding these processes is highly relevant for understanding anemia during chronic inflammation, especially in critically ill patients receiving blood transfusions.

Comparative proteomics of erythrocyte aging in vivo and in vitro.

During aging in vivo and in vitro, erythrocytes display removal signals. Phagocytosis is triggered by binding of autologous IgG to a senescent cell antigen originating on band 3. Erythrocytes generate vesicles as an integral part of the aging process in vivo and in vitro, i.e. during storage. These vesicles display senescent cell antigens as well as phosphatidylserine, that is recognized by scavenger receptors. Recent comparative proteomic analyses of erythrocytes and their vesicles support the hypothesis that aging is accompanied by increased binding of modified hemoglobins to band 3, disruption of the band 3-mediated anchorage of the cytoskeleton to the lipid bilayer, vesicle formation, and antigenic changes in band 3 conformation. Proteomic data also suggest an, until then unknown, involvement of chaperones, stress proteins, and proteasomes. Thus, the presently available comparative proteomic analyses not only confirm previous immunochemical and functional data, but also (1) provide new clues to the mechanisms that maintain erythrocyte homeostasis; (2) open new roads to elucidate the processes that regulate physiological erythrocyte aging and removal, and thereby; (3) provide the foundation for rational interventions to prevent untimely erythrocyte removal, and unwanted interactions between the erythrocyte and the immune system, especially after transfusion.

Production of yeastolates for uniform stable isotope labelling in eukaryotic cell culture.

Preparation of stable isotope-labelled yeastolates opens up ways to establish more cost-effective stable isotope labelling of biomolecules in insect and mammalian cell lines and hence to employ higher eukaryotic cell lines for stable isotope labelling of complex recombinant proteins. Therefore, we evaluated several common yeast strains of the Saccharomycetoideae family as a source of high-quality, non-toxic yeastolates with the major aim to find a primary amino acid source for insect and mammalian cell culture that would allow cost-effective uniform stable isotope labelling (13C, 15N). Strains of the facultative methylotrophic yeasts Pichia pastoris and Hansenula polymorpha (Pichia angusta) as well as a strain of the baker's yeast Saccharomyces cerevisiae were compared as a source of yeastolate with respect to processing, recovery and ability to sustain growth of insect and mammalian cell lines. The best growth-supporting yeastolates were prepared via autolysis from yeast obtained from fed-batch cultures that were terminated at the end of the logarithmic growth phase. Yeastolates obtained from H. polymorpha performed well as a component of insect cell cultures, while yeastolates from S. cerevisiae and H. polymorpha both yielded good results in mammalian cell cultures. Growth of yeasts in Heine's medium without lactic acid allows relatively low concentrations of 13C and 15N sources, and this medium can be reused several times with supplementation of the 13C source only.

Erythrocyte ageing in vivo and in vitro: structural aspects and implications for transfusion.

Erythrocyte transfusion is essential in conditions of large blood loss, of inadequate bone marrow production and of increased erythrocyte breakdown. The structural and biochemical changes that erythrocytes go through during storage, probably associated with the disappearance of up to 30% of the erythrocytes within 24 h after transfusion, are likely to contribute to the transfusion side effects: iron overload, erythrocyte adhesion to the endothelial surface with proinflammatory consequences, autoantibody formation, endothelial damage by released erythrocyte constituents, a hampered microcirculation and oxygen delivery. In vivo, senescent erythrocytes are marked for removal by binding of autologous immunoglobulin G to ageing antigens, which arise by changes in the conformation of the membrane domain of band 3. Also, vesicle formation has been described as an integral part of the erythrocyte ageing process. Comparable changes occur during erythrocyte storage. This review describes the current state of knowledge of the mechanism of erythrocyte ageing in vivo, ageing-related changes occurring during erythrocyte storage in blood bank conditions and their possible relation with the transfusion side effects. In view of the key position of band 3 in the maintenance of erythrocyte structure and function, elucidation of the pathways that control posttranslational modification of band 3 during storage may lead to new approaches towards maintaining ATP concentration and cellular integrity. This review concludes with the challenge to further explore the underlying processes of erythrocyte ageing in order to provide physiologically relevant tools for assessing and predicting erythrocyte homeostasis in vitro and in vivo and thereby to contribute to the development of rational transfusion protocols for various patient categories.

Storage-related changes in erythrocyte band 3: not a case for the Diego blood group antigens.

Removal of erythrocytes from the circulation is mediated by the immune system. Changes in structure and function of band 3, a major membrane protein of the erythrocyte, trigger the binding of antibodies to a band 3-derived neoantigen, senescent cell antigen, on erythrocytes aged in vivo. This mechanism probably is also involved in determining the survival of erythrocytes after transfusion. Band 3 is the carrier of the Diego blood group system, and subtle changes in the three-dimensional conformation of the same extracellular loops of band 3 determine Diego blood group activity as well as senescent cell antigen activity. Therefore we used the Diego blood group system to probe these changes with a combination of serological and immunochemical methods. Our data indicate that changes in band 3 structure during storage under blood bank conditions, as shown by immunoblot analysis, are not detectable as changes in expression of Diego antigens in intact cells. This makes it unlikely that immunological removal of erythrocytes after transfusion is mediated by reactions involving the Diego blood group system.

The red cell revisited--matters of life and death.

An erythrocyte-fractionating method combining volume and subsequent density separation is described. Iron isotope (59Fe)-validation proved this combination of methods to be complementary. By deploying HbA1c as cell age marker, obtained fractions demonstrated that circulating erythrocytes lose 20% of hemoglobin and membrane by shedding vesicles. Vesiculation from older cells proved to be facilitated by the spleen. Animal studies revealed that such vesicles are rapidly removed from the circulation by scavenger receptors on Kupffer cells with phosphatidylserine acting as the principal ligand. These studies reveal the existence of an alternative pathway of erythrocyte breakdown. This means that the premortal substrate of 20% of any erythrocyte is at our disposal. As this kind of vesiculation takes place during the entire erythrocyte lifespan, loss and sometimes reutilisation of marker substances limits the usefulness of isotope studies to the first half of the erythrocyte lifespan, thereby putting the dogmatic lifespan of 120 days into question. Furthermore, these studies add to the understanding of hemoglobin A1c (HbA1c) metabolism and the origin of the wide variation of erythrocyte parameters in peripheral blood. Removal of old erythrocytes from the circulation and from donor blood may open new ways into the treatment of both bilirubin and secondary iron overload.

Survival of the fittest?--survival of stored red blood cells after transfusion.

During the last 90 years many developments have taken place in the world of blood transfusion. Several anticoagulants and storage solutions have been developed. Also the blood processing has undergone many changes. At the moment, in The Netherlands, red blood cell (RBC) concentrates (prepared from a whole blood donation and leukocyte-depleted by filtration) are stored for a maximum of 35 days at 4 degrees C in saline adenine glucose mannitol (SAGM). Most relevant studies show that approximately 20% of the RBCs is lost in the first 24 hr after transfusion. Even more remarkable is that the average life span is 94 days after a storage period of 42-49 days. Such observations create the need for a parameter to measure the biological age of RBCs as a possible predictor of the fate of RBCs after transfusion. The binding of IgG to RBCs can lead to recognition and subsequent phagocytosis by macrophages. This occurs during the final stages of the RBC life span in vivo. We determined the quantity of cell-bound IgG during storage, and found considerable variation between RBCs, but no significant storage-related change in the quantity of cell-bound IgG. The significance of this finding for predicting the survival of transfused RBCs in vivo remains to be established. Hereto we developed a flow cytometric determination with a sensitivity of 0.1% for the measurement of survival in vivo based on antigenic differences. This technique has various advantages compared with the 'classical' 51Cr survival method.

Erythrocyte aging in sickle cell disease.

Physiological removal of old erythrocytes from the circulation by macrophages is initiated by binding of autologous IgG to senescent cell antigen (SCA). SCA is generated from the anion exchanger band 3. This process is accompanied by a number of alterations in the function and structure of band 3. We measured these aging-related parameters in erythrocytes from individuals with sickle cell anemia. Most sickle erythrocytes have characteristics that are also found in senescent normal erythrocytes, such as an increased density and considerable concentrations of cell-bound IgG. Together with the concomitant changes in structure and function of band 3, these data suggest that most sickle erythrocytes have undergone a process of accelerated aging. Preliminary results indicate that this process is reversed upon vitamin E supplementation. These data show that the erythrocyte aging paradigm may provide a useful conceptual framework for the study of the pathophysiology and the evalution of therapeutic intervention in sickle cell disease, and support the view that oxidation can generate neoantigens that are recognized by autoantibodies.