Retention of poorly deformable red blood cells (RBCs IntroductionRetention of poorly deformable red blood cells (RBCs) by the human spleen has been recognized as a critical determinant of pathogenesis of several RBC disorders, including hereditary spherocytosis and malaria. The severity of hereditary spherocytosis (HS), a syndrome resulting from mutations of RBC membrane proteins, 1 is related to the extent of decreased membrane surface area. Splenic retention is the dominant mechanism responsible for reduced life span of HS-RBCs, such that splenectomy is beneficial in reducing anemia in symptomatic HS patients. 1 Changes of RBC mechanical properties have also been observed in blood stored for 3 weeks or longer. 2 Prolonged storage influences the survival of RBCs after transfusion 3 and probably contributes to transfusion-related side effects, including respiratory distress and systemic sepsis. [3][4][5] Not least, the deformability of Plasmodium falciparum-infected red blood cells (Pf-RBCs) is also central in malaria pathogenesis. It progressively decreases as P falciparum matures in its host RBCs, from rings (ring-RBCs) 6 to trophozoites and then to schizonts. 7,8 When mature stages are sequestered in small vessels, they escape retention in the spleen. Ring-RBCs only rarely adhere to endothelial cells 9 and are found in the peripheral blood circulation. 10,11 We recently showed that a fraction of ring-RBCs is retained in isolated-perfused human spleens. 12 Ring-RBCs accumulated upstream from interendothelial slits, a spleen-specific microanatomic structure that retains RBCs with reduced deformability (rigid-RBCs). [13][14][15] In brief, in malaria as in several other RBC disorders, extensive RBC retention in the spleen is associated with anemia, 1,16,17 whereas impaired retention potentially favors adverse events, such as acute microvascular obstruction in malaria patients, 18,19 late vascular disorders in splenectomized patients with HS or thalassemia, 20,21 and acute organ dysfunction in critically ill patients transfused with "older" blood. 4 Exploration of how the human spleen senses poorly deformable RBCs would be facilitated by a physiologically relevant in vitro approach simpler than the ex vivo spleen perfusion system. To experimentally replicate the mechanical sensing of RBC by the splenic microcirculation, we first analyzed the fine structure of interendothelial slits in the human spleen. We realized that the geometry of narrow and short interendothelial slits would be accurately mimicked by spaces between microbeads. We then allowed RBCs to flow through a The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on April 8, 2019. by guest www.bloodjournal.org From mixture of 5-to 25-m-diameter microbeads. Heated RBCs, Pf-RBCs, and RBCs from patients with HS were retained in the microbead layer without hemolysis. The r...

Sequestration, the adherence of infected erythrocytes containing late developmental stages of the parasite (trophozoites and schizonts) to the endothelium of capillaries and venules, is characteristic of Plasmodium falciparum infections. We have studied two host factors, the spleen and antibody, that influence sequestration of P. falciparum in the squirrel monkey. Sequestration of trophozoite/schizont-infected erythrocytes that occurs in intact animals is reduced in splenectomized animals; in vitro, when infected blood is incubated with monolayers of human melanoma cells, trophozoite/schizont-infected erythrocytes from intact animals but not from splenectomized animals bind to the melanoma cells. The switch in cytoadherence characteristics of the infected erythrocytes from nonbinding to binding occurs with a cloned parasite. Immune serum can inhibit and reverse in vitro binding to melanoma cells of infected erythrocytes from intact animals. Similarly, antibody can reverse in vivo sequestration as shown by the appearance of trophozoite/schizont-infected erythrocytes in the peripheral blood of an intact animal after inoculation with immune serum. These results indicate that the spleen modulates the expression of parasite alterations of the infected erythrocyte membrane responsible for sequestration and suggest that the prevention and reversal of sequestration could be one of the effector mechanisms involved in antibody-mediated protection against P. faleiparum malaria.Among the four species of malaria infecting man, one distinguishing feature of Plasmodium falciparum is that only erythrocytes containing young forms of the parasite (rings) are found in the peripheral blood (1); erythrocytes containing more mature forms (trophozoites and schizonts) are sequestered in the postcapillary venules of various organs where they adhere to endothelial cells (2). The mechanisms of parasite sequestration remain unclear. Ultrastructural studies have suggested that adherence of parasitized erythrocytes to the vascular endothelium occurs by means of excrescences on the infected erythrocyte membrane that have been called knobs (3,4); antigens present on these knobs are recognized by immune serum of the host (5, 6). Cytoadherence of infected erythrocytes can be studied in vitro through the binding of infected erythrocytes to monolayers of human endothelial cells (7); infected erythrocytes also bind to monolayers of human melanoma cells, which can replace endothelial cells for in vitro studies of parasite sequestration (8).Sequestration favors the development of the parasite by protecting it from the filtering action of the spleen (9); it is also responsible for the severe clinical forms of cerebraL malaria in which brain capillaries are obstructed by sequestered parasites. Means of inhibiting sequestration may therefore hinder parasite development and alleviate the clinical severity of the disease.In the P. falciparum infection of the squirrel monkey, we have studied two host factors that influence both sequestration in vi...

AbstractAchievement of malaria elimination requires development of novel strategies interfering with parasite transmission, including targeting the parasite sexual stages (gametocytes). The formation of Plasmodium falciparum gametocytes in the human host takes several days during which immature gametocyte-infected erythrocytes (GIEs) sequester in host tissues. Only mature stage GIEs circulate in the peripheral blood, available to uptake by the Anopheles vector. Mechanisms underlying GIE sequestration and release in circulation are virtually unknown. We show here that mature GIEs are more deformable than immature stages using ektacytometry and microsphiltration methods, and that a switch in cellular deformability in the transition from immature to mature gametocytes is accompanied by the deassociation of parasite-derived STEVOR proteins from the infected erythrocyte membrane. We hypothesize that mechanical retention contributes to sequestration of immature GIEs and that regained deformability of mature gametocytes is associated with their release in the bloodstream and ability to circulate. These processes are proposed to play a key role in P falciparum gametocyte development in the host and to represent novel and unconventional targets for interfering with parasite transmission.