Danny Chiu

Increased adherence of sickled and phosphatidylserine-enriched human erythrocytes to cultured human peripheral blood monocytes.

The precise mechanism by which sickle erythrocytes (RBC) are removed from the circulation is controversial, although it is possible that enhanced recognition of these cells by circulating mononuclear phagocytes could contribute to this process. We investigated this possibility by interacting sickle cells with cultured human peripheral blood monocytes. Our results show that both irreversibly sickled cells (ISC) and deoxygenated reversibly sickled cells (RSC) had a higher avidity for adherence to monocytes than did oxygenated sickle and normal RBC. ISC were the most adherent cell type. Adherence of RSC to monocytes was found to be reversible; reoxygenation of deoxygenated RSC resulted in a significant decrease in RSC--monocyte adherence. Concomitant with alterations in sickle RBC adherence were alterations in the organization and bilayer distribution of membrane phospholipids in these cells. Specifically, enhanced adherence was associated with increased exposure of RBC membrane outer leaflet phosphatidylserine (PS) and phosphatidylethanolamine, whereas lack of adherence was associated with normal patterns of membrane phospholipid distribution. To investigate the possibility of whether the exposure of PS in the outer membrane leaflet of these cells might be responsible for their recognition by monocytes, the membranes of normal RBC were enriched with the fluorescent PS analogue 1-acyl-2[(N-4-nitro-benzo-2-oxa-1,3-diazole)aminocaproyl]-phosphatidy lse rine (NBD-PS) via transfer of the exogenous lipid from a population of donor phospholipid vesicles (liposomes). RBC enriched with NBD-PS exhibited enhanced adherence to monocytes, whereas adherence of RBC enriched with similar amounts of NBD-phosphatidylcholine (NBD-PC) was not increased. Furthermore, preincubation of monocytes with PS liposomes resulted in a approximately 60% inhibition of ISC adherence to monocytes, whereas no inhibition occurred when monocytes were preincubated with PC liposomes. These findings strongly suggest that erythrocyte surface PS may be a ligand recognized by receptors on human peripheral blood monocytes and that abnormal exposure of PS in the outer leaflet of the RBC membrane, as found in sickle RBC, might serve to trigger their recognition by circulating monocytes. Our results further suggest that abnormalities in the organization of erythrocyte membrane phospholipids may have significant pathophysiologic implications, possibly including shortened cell survival.

GROWTH RETARDATION IN SICKLE-CELL DISEASE TREATED BY NUTRITIONAL SUPPORT

The effect of increased nutritional intake was evaluated in 5 growth-retarded children with sickle-cell disease. Growth on recommended daily calorie and protein intakes had been inadequate in all 5. Fat absorption and intestinal mucosal morphology were normal in all 5. 2 children were given nutritional supplementation by nasogastric intubation, 1 received nightly oral formula supplements, and 2 were supplemented with zinc, iron, folate, and vitamin E only. Nutritional supplementation by the nasogastric route produced a rapid sustained increase in growth rate, associated with striking reductions in pain crises and infections which had previously necessitated many hospital admissions. Oral supplementation improved the clinical course but had no effect on growth rate. Mineral and vitamin supplements influenced neither the growth rate nor the clinical course. The observations indicate that nasogastric nutritional supplementation may accelerate growth and reduce the incidence and severity of complications in growth-retarded children with sickle-cell disease.

PEROXIDATION, VITAMIN E, AND SICKLE‐CELL ANEMIA*

In summary, we propose the following scheme (Figure 5) to describe the role of peroxidation in the pathophysiology of SCA. Sickle erythrocytes are more susceptible to peroxidation than are normal erythrocytes. This increased susceptibility to peroxidation is, in part, due to decreased blood vitamin E levels and abnormal membrane phospholipid organization induced by sickling. The peroxidative damage of sickle erythrocytes may accelerate or contribute to loss of cell deformability and to chronic hemolysis. Peroxidative damage can produce abnormal cellular properties, such as potassium leak and reduced filterability, and contribute to formation of ISCs. Increased red cell rigidity can initiate episodes of capillary obstruction, leading to vasoocclusive painful crises and to tissue infarction. Liver dysfunction as well as increased production of bilirubin secondary to hemolysis could result in bile sludging and decreased secretion of bile salts into the intestinal lumen. Reduced bile salt secretion leads to partial fat and vitamin E malabsorption. Vitamin E deficiency enhances red cell susceptibility to peroxidation and promotes a vicious cycle in SCA. Although we have not studied factors that might initiate peroxidative damage, sickle hemoglobin and excess body iron should be considered as potential sources. Our studies suggest that vitamin E supplementation to sickle-cell patients could be of clinical benefit.

Properties of Vitamin E-Deficient Erythrocytes following Peroxidant Injury

Summary: Several membrane properties of vitamin E-deficient and normal erythrocytes were studied after incubation of these cells with hydrogen peroxide. Measurements of mean corpuscular volume, cation permeability, membrane Na+, K+ ATPase activity, red cell filterability through 5 μ millipore filters, and membrane protein pattern on sodium dodecyle sulfate-gel electrophoresis revealed marked alterations before lysis. Vitamin E sufficient cells were unaffected by a similar incubation with hydrogen peroxide. We speculate that the changes in membrane function, which follow peroxidant injury, contribute to the shortened red cell survival in the vitamin E deficient state.

Alteration of membrane phospholipid bilayer organization in human erythrocytes during drug-induced endocytosis.

Our plan was to evaluate the potentially important role of phospholipids in erythrocyte shape alterations by determining if their orientation was altered during endocytosis. Stomatocytosis and endocytosis were induced in normal intact human erythrocytes by incubation with three agents: primaquine, vinblastine, and chlorpromazine, each of which has its own requirements and time course for producing endocytosis. The organization of the phospholipid bilayer was assessed by measuring the extent of degradation of phophatidylcholine (PC), phophatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM) produced by exposure of erythrocytes to a nonpenetrating protease-free phospholipase A2 alone or in combination with a purified sphingomyelinase as well. The induction of stomatocytosis did not change this orientation. However, correlating with the onset of endocytosis but not its extent, there was an increase in PE degradation, which could be detected regularly only by use of phospholipase A2 alone. Use of the combination of phospholipase A2 and sphingomyelinase showed that the extent and course of endocytosis was paralleled by an apparent movement of PC and SM from the outer to the inner half of the lipid bilayer. Since no further PE was hydrolyzed and because no PS was ever degraded, this inward movement of PC and SM did not represent the establishment of complete symmetry in the membrane. By adjusting the experimental design it was possible to implicate the endocytic process, and not insertion of drug in the membrane, as the cause of the alterations in phospholipid organization seen. Our findings indicate that the phospholipid orientation is very closely involved in the endocytosis process and that specific states of phospholipid asymmetry may be related to identifiable membrane events.

Recognition Signals for Phagocytic Removal of Favic Malaria-Infected and Sickled Erythrocytes

According to a recently published concept (1), immunologicallymediated erythrocyte (RBC) removal involves binding of autologous, naturally circulating antibodies directed against normally hidden epitopes of integral RBC membrane proteins. Prerequisite of antibody binding are modifications of specific RBC proteins, presumably band 3 and the glycophorins. The exact nature of the modifications resulting in exposure of hidden epitopes or generation of neoantigenic sites, is still unknown. Evidence that oxidant-elicited oligomerization of band 3 is a sufficient change to enhance anti-band 3 binding has been provided (2,3). The precise molecular composition of the opsonin complex, and the nature of the bonds which keep its components together, are poorly defined as yet. An essential characteristic of the anti-band 3-mediated removal is the precipitation of complement (1–3). Indeed, conditions that inactivate complement convertases and abrogate the formation of the active complement component C3b also extensively inhibit phagocytic removal of variously damaged RBC. The same inhibition of phagocytosis occurs after blockage of the C3b receptor (CR1, complement receptor type one) on the macrophage surface (3,4). Lutz et al.(2,3–5) hypothesized that anti-band 3 antibodies bivalently bound to aggregated band 3 stimulate generation and deposition of activated C3 on the RBC surface.

Interaction of Phosphatidylserine-Phosphatidylcholine Liposomes with Sickle Erythrocytes: EVIDENCE FOR ALTERED MEMBRANE SURFACE PROPERTIES

The sickle erythrocyte (RBC) is a pathologic RBC that contains multiple membrane abnormalities. Some of these abnormalities have been implicated in the pathophysiology of vasoocclusive crises characteristic of sickle cell disease; others have yet to be defined in terms of their clinical significance. Recent information has shown that sickle RBC adhere abnormally to cultured endothelial cells yet little is known about the ways in which sickle cells interact with model membranes of defined size and lipid composition. We investigated this phenomenon by interacting sickle RBC with artificial lipid vesicles (liposomes) containing acidic phospholipids. Our results demonstrate that sickle disease (hemoglobin SS) RBC bind more of these liposomes than do normal or sickle trait (hemoglobin AS) RBC and that these differences are accentuated by hypoxia-induced sickling. Binding of liposome phospholipid to sickled RBC was not attributable to phospholipid exchange between liposomes and RBC and was consistent with a mechanism involving both membrane fusion and a stable reversible adhesion of liposomes to the RBC membrane.Investigations into the mechanism(s) underlying increased liposome binding to sickled RBC suggested that the known reversible translocation of aminophospholipids, phosphatidylserine (PS) and phosphatidyl-ethanolamine (PE), from the inner to the outer leaflet of the reversibly sickled RBC (RSC) plasma membrane during sickling may be a component of increased liposome binding to RSC. This idea was supported from results of experiments in which normal RBC were treated with diamide resulting in the expression of outer leaflet PE and PS and a stimulation of liposome binding to these cells. However, sickle RBC separated according to cell density on stractan gradients showed that irreversibly sickled RBC (ISC) were less capable of liposome binding than were discoid RSC. Since ISC are known to contain elevated levels of outer leaflet aminophospholipids, such a result suggests that other changes in the plasma membrane of sickle cells, in addition to phospholipid reorganization, are probably involved in enhanced liposome binding to these cells. In other experiments, we showed that liposomes containing l-phenylalanine were capable of delivering this antisickling agent into intact sickle RBC as demonstrated by the partial inhibition of hypoxia-induced sickling in vitro. Our results suggest that liposomes can be used as sensitive probes for investigating changes in RBC membrane properties, especially those that affect intermembrane interactions, and that liposomal transport systems may have significant implications in the therapy of sickle cell disease.

Bioavailability of vitamin E in rats fed diets containing pectin

Abstract Weanling male Sprague-Dawley rats were fed: o (1)a basal diet without supplemental vitamin E (-E) (2)a basal diet supplemented with 39 IU vitamin E/kg of diet ( + E) or (3)an E supplemented basal diet to which 10 percent citrus pectin was added (+E+P) The animals were studied over a 56 day period. Growth rate as measured by body weight was noticeably slower in the +E+P group as compared to both the -E and the +E groups. The final average body weight of the +E+P group was 28 percent less (P

NUTRITION IN SICKLE CELL ANEMIA |[lpar]|HB SS|[rpar]|

To evaluate the role of nutrition in Hb SS we performed anthropometric and laboratory measurements on 95 Hb SS patients and determined the effects of caloric supplementation on these parameters and on clinical course. Height and weight were both below the 5th percentile in 17% of patients. Laboratory studies included vitamin C (ng/108 cells), vitamin E (mg/dl), zinc (μg/ml), selenium (ng/ml), and lipids (mg/dl).Immune status was measured as a reflection of nutritional adequacy. Total T cell (patient/control, 44%/78%), helper T cell (23%/55%), and suppressor T cells (17%/32%) as well as skin test reactivity were depressed. Two patients with marked growth retardation, multiple laboratory abnormalities and frequent sickle cell complications received dietary supplementation with nasal gastric hyperalimentation for 4-10 months. Improvements in growth, weight, skin test reactivity, T cell numbers, and clinical status were noted. We conclude that nutritional assessment and intervention may minimize complications associated with sickle cell disease.