Adhesion Of Erythrocytes Research Articles

Targeted Disruption of a Ring-infected Erythrocyte Surface Antigen (RESA)-like Export Protein Gene in Plasmodium falciparum Confers Stable Chondroitin 4-Sulfate Cytoadherence Capacity

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family proteins mediate the adherence of infected erythrocytes to microvascular endothelia of various organs, including the placenta, thereby contributing to cerebral, placental, and other severe malaria pathogenesis. Several parasite proteins, including KAHRP and PfEMP3, play important roles in the cytoadherence by mediating the clustering of PfEMP1 in rigid knoblike structures on the infected erythrocyte surface. The lack of a subtelomeric region of chromosome 2 that contains kahrp and pfemp3 causes reduced cytoadherence. In this study, microarray transcriptome analysis showed that the absence of a gene cluster, comprising kahrp, pfemp3, and four other genes, results in the loss of parasitized erythrocytes adhering to chondroitin 4-sulfate (C4S). The role of one of these genes, PF3D7_0201600/PFB0080c, which encodes PHISTb (Plasmodium helical interspersed subtelomeric b) domain-containing RESA-like protein 1 expressed on the infected erythrocyte surface, was investigated. Disruption of PFB0080c resulted in increased var2csa transcription and VAR2CSA surface expression, leading to higher C4S-binding capacity of infected erythrocytes. Further, PFB0080c-knock-out parasites stably maintained the C4S adherence through many generations of growth. Although the majority of PFB0080c-knock-out parasites bound to C4S even after culturing for 6 months, a minor population bound to both C4S and CD36. These results strongly suggest that the loss of PFB0080c markedly compromises the var gene switching process, leading to a marked reduction in the switching rate and additional PfEMP1 expression by a minor population of parasites. PFB0080c interacts with VAR2CSA and modulates knob-associated Hsp40 expression. Thus, PFB0080c may regulate VAR2CSA expression through these processes. Overall, we conclude that PFB0080c regulates PfEMP1 expression and the parasite's cytoadherence.

Antigen reversal identifies targets of opsonizing IgGs against pregnancy-associated malaria.

Clinical immunity to pregnancy associated-malaria (PAM) in multigravida women has been attributed to antibodies that recognize VAR2CSA on the infected erythrocyte (IE) surface. The size and complexity of VAR2CSA have focused efforts on selecting one or more of its six Duffy binding-like (DBL) domains for vaccine development. Presently, however, there is no consensus as to which DBL domain(s) would be most effective in eliciting immunity. This is because antibodies to a number of the DBL domains have been found to block the adhesion of VAR2CSA-expressing erythrocytes to chondroitin sulfate A (CSA)-a major criterion for evaluating vaccine candidacy. Opsonization of IEs by cytophilic antibodies that recognize VAR2CSA represents an important yet understudied effector mechanism in acquired immunity to PAM. To date, no studies have sought to determine the targets of those antibodies. In this study, we found that IgGs from multigravida Malian women showed (i) higher reactivity to recombinant DBL domains by enzyme-linked immunosorbent assay (ELISA), (ii) more binding to VAR2CSA-expressing IEs, and (iii) greater opsonization of these IEs by human monocytic cells than IgGs from malaria-exposed Malian men and malaria-naive American adults. Preincubation of IgGs from multigravida women with recombinant DBL2χ, DBL3χ, or DBL5ε domains significantly diminished opsonization of VAR2CSA-expressing IEs by human monocytes. These data identify the DBL2χ, DBL3χ, and DBL5ε domains as the primary targets of opsonizing IgGs for the first time. Our study introduces a new approach to determining the antigenic targets of opsonizing IgGs in phagocytosis assays.

Effects of Phyllanthus sellowianus Müll Arg. extracts on the rheological properties of human erythrocytes.

Phyllanthus sellowianus extracts have been used in Argentina since colonial times in the treatment of diabetes. The in vitro biorheological and hemoagglutinant action of different extracts of P. sellowianus bark on human erythrocytes (RBC) were studied. RBCs were incubated in vitro with four aqueous extracts: Maceration; Controlled Digestion (PD); Decoction; and Infusion. Biorheological parameters (deformability, membrane surface viscosity, elastic modulus, and dynamic viscolelasticity) were determined with an Erythrodeformeter, and erythrocyte adhesion was characterized by image digital analysis. Immunohematological assays in RBC incubated with all the extracts showed large globular aggregates and agglutination in human ABO blood group system. Isolated cell coefficient showed the increase of cell adhesion. Aggregated shape parameters were significantly higher than normal and they changed with the concentration, particularly of PD extracts. Rheological results showed that the extract biorheological action varies with the temperature used in the extract preparations. The results obtained are useful to study the action mechanism of extracts from P. sellowianus bark in order to evaluate its use as therapeutic agent in diabetes. Immunohematological Tests using ABO system showed its agglutinant power, which is of special interest in Immunohematology to be used as hemoclassifier.

Plasmodium falciparumantigenic variation: relationships between widespread endothelial activation, parasite PfEMP1 expression and severe malaria

BackgroundPlasmodium falciparum erythrocyte membrane protein 1(PfEMP1) is a family of variant surface antigens (VSA) that mediate the adhesion of parasite infected erythrocytes to capillary endothelial cells within host tissues. Opinion is divided over the role of PfEMP1 in the widespread endothelial activation associated with severe malaria. In a previous study we found evidence for differential associations between defined VSA subsets and specific syndromes of severe malaria: group A-like PfEMP1 expression and the “rosetting” phenotype were associated with impaired consciousness and respiratory distress, respectively. This study explores the involvement of widespread endothelial activation in these associations.MethodsWe used plasma angiopoietin-2 as a marker of widespread endothelial activation. Using logistic regression analysis, we explored the relationships between plasma angiopoietin-2 levels, parasite VSA expression and the two syndromes of severe malaria, impaired consciousness and respiratory distress.ResultsPlasma angiopoietin-2 was associated with both syndromes. The rosetting phenotype did not show an independent association with respiratory distress when adjusted for angiopoietin-2, consistent with a single pathogenic mechanism involving widespread endothelial activation. In contrast, group A-like PfEMP1 expression and angiopoietin-2 maintained independent associations with impaired consciousness when adjusted for each other.ConclusionThe results are consistent with multiple pathogenic mechanisms leading to severe malaria and heterogeneity in the pathophysiology of impaired consciousness. The observed association between group A-like PfEMP1 and impaired consciousness does not appear to involve widespread endothelial activation.

Increased adhesive properties of neutrophils and inflammatory markers in venous thromboembolism patients with residual vein occlusion and high D-dimer levels

Venous thromboembolism (VTE) develops via a multicellular process on the endothelial surface. Although widely recognized, the relationship between inflammation and thrombosis, this relationship has been mostly explored in clinical studies by measuring circulating levels of inflammatory cytokines. However, the role of inflammatory cells, such as neutrophils, in the pathogenesis of VTE is not clear in humans. To evaluate the adhesive properties of neutrophils, erythrocytes and platelets in VTE patients and to correlate findings with inflammatory and hypercoagulability marker levels. Study group consisted of twenty-nine VTE patients and controls matched according to age, gender and ethnic background. Adhesive properties of neutrophils, erythrocytes and platelets were determined using a static adhesion assay. Neutrophil adhesion molecules expressions were evaluated by flow cytometry. Inflammatory and hypercoagulability marker levels were evaluated by standard methods. Residual vein occlusion (RVO) was evaluated by Doppler ultrasound. No significant difference could be observed in platelet and erythrocyte adhesion between VTE patients and controls. Interestingly, VTE patients with high levels of D-dimer and RVO, demonstrated a significant increase in neutrophil adhesion, compared to controls and remaining patients. Inflammatory markers (IL-6, IL-8, TNF-α) were also significantly elevated in this subgroup, compared to other VTE patients. Adhesive properties of neutrophils correlated with IL-6 and D-dimer levels. Neutrophils adhesion molecules (CD11a, CD11b and CD18) were not altered in any of the groups. These findings not only support the hypothesis of an association between inflammation and hypercoagulability, but more importantly, highlight the role of neutrophils in this process.

Dynamic adhesion of eryptotic erythrocytes to immobilized platelets via platelet phosphatidylserine receptors

Glucose depletion of erythrocytes triggers suicidal erythrocyte death or eryptosis, which leads to cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to endothelial cells by a mechanism involving phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 at the endothelial cell membrane. Nothing has hitherto been known about an interaction between eryptotic erythrocytes and platelets, the decisive cells in primary hemostasis and major players in thrombotic vascular occlusion. The present study thus explored whether and how glucose-depleted erythrocytes adhere to platelets. To this end, adhesion of phosphatidylserine-exposing erythrocytes to platelets under flow conditions was examined in a flow chamber model at arterial shear rates. Platelets were immobilized on collagen and further stimulated with adenosine diphosphate (ADP, 10 μM) or thrombin (0.1 U/ml). As a result, a 48-h glucose depletion triggered phosphatidylserine translocation to the erythrocyte surface and augmented the adhesion of erythrocytes to immobilized platelets, an effect significantly increased upon platelet stimulation. Adherence of erythrocytes to platelets was blunted by coating of erythrocytic phosphatidylserine with annexin V or by neutralization of platelet phosphatidylserine receptors CXCL16 and CD36 with respective antibodies. In conclusion, glucose-depleted erythrocytes adhere to platelets. The adhesive properties of platelets are augmented by platelet activation. Erythrocyte adhesion to immobilized platelets requires phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 expression on platelets. Thus platelet-mediated erythrocyte adhesion may foster thromboocclusive complications in diseases with stimulated phosphatidylserine exposure of erythrocytes.

Heme-Induced Neutrophil Extracellular Traps (NETs) Formation Contributes To Sickle Cell Disease Pathogenesis

Sickle cell disease (SCD) is characterized by chronic inflammation, continuous oxidative stress and severe hemolysis. Activation of endothelial cells, adhesion of sickle erythrocytes and polymorphonuclear neutrophils (PMNs) have been reported to mediate SCD vaso-occlusion. PMNs from SCD patients produce significantly higher basal levels of reactive oxygen species (ROS) and contain less intracellular ROS scavengers than normal individuals. The formation of neutrophil extracellular traps (NETs), which requires the upregulation of intracellular ROS, has been described as a critical innate mechanism to contain infectious insults and to participate in thromboinflammatory injury. In addition, histones and granular proteins released during NET formation can directly damage lung microvascular endothelial cells. Based on these results, we explored the possibility that neutrophils contribute to SCD pathophysiology through NETs. To investigate the presence of NETs in vivo, we used a humanized mouse model of SCD that expresses human sickle hemoglobin exclusively and shares many features of human SCD. Immunofluorescence images of lung samples revealed a significantly higher number of NET DNA fibers in SCD mice (22.6 ± 2.8 NETs/field of view) compared to control mice (5.96 ± 0.86 NETs/field of view, p<0.0001) following proinflammatory cytokine (TNF-α) challenge. We confirmed that these DNA fibers were not resulting from section artifacts or remnants of apoptotic or necrotic cells by co-staining these fibers with antibodies against citrullinated histone H3 and neutrophil elastase (NE), both of which have been identified as markers of NETs. Citrullinated histone H3+ NE+ NETs were identified in the lungs of SCD mice and were found within blood vessels. Concurrently, the soluble NET component plasma DNA was significantly higher in the peripheral blood of SCD mice compared to control mice (∼524 versus ∼48 ng/ml, p<0.001). Further, plasma nucleosomes were detectable only in SCD mice (∼0.11 U/ml). We also found that SCD mice experienced body temperature decline (a reduction of 5.3 ± 1.0°C) following TNF-α challenge whereas the body temperature of control mice was unaffected. A significant positive correlation was found between numbers of NETs and reduction of body temperature in these mice (r=0.72, p<0.0001). DNase I treatment reduced NETs by 38% in SCD mice (12.2 ± 0.9 versus 7.5 ± 0.7 NETs/field of view, for vehicle- and DNase I-treated, respectively, p<0.001), protected these mice from NET-associated hypothermia and significantly prolonged their survival (p<0.05) compared to vehicle-treated SCD mice. As a result of chronic hemolysis, SCD is associated with increased plasma heme. Like SCD patients, SCD mice have significantly higher plasma heme concentration compared to control mice (50.8 ± 5.2 versus 23.8 ± 2.8 μM, p<0.001). We hypothesized that heme is the plasma factor that stimulates PMNs to produce NETs in SCD mice. In vitro study showed that heme activated PMNs to produce citrullinated histone H3+ NE+ NETs in a ROS- and heme-iron dependent manner. Heme injection significantly increased NET formation in control mice (4.5 ± 0.7 versus 9.6 ± 1.7 NETs/field of view for vehicle- and heme-treated, respectively, p<0.01) and accounted for the acute body temperature decline in these mice (3.1 ± 0.8°C, p<0.01). On the other hand, hemopexin administration, which decreased total plasma heme concentration in SCD mice, reduced NET formation by 41% (11.6 ± 0.8 versus 6.9 ± 0.7 NETs/field of view, for vehicle- and hemopexin-treated, respectively, p<0.0001) and protected treated mice from NET-associated hypothermia. Our study thus demonstrates that NETs may contribute to the pathogenesis of SCD and that heme released during hemolysis promotes NET generation in vivo. Targeting heme-mediated NET formation may represent a useful strategy in managing SCD and other pathological states involving severe hemolysis. Disclosures:No relevant conflicts of interest to declare.

Anemia, Shortened Erythrocyte Lifespan and Stomatocytosis In a Flippase Mutant Mouse Strain

BackgroundMammalian erythrocytes are anucleate biconcave discs with marked flexibility and deformability, features necessary for efficient function. The cell membrane consists of a lipid bilayer containing proteins and an asymmetric distribution of phospholipids (PL). Phosphatidylcholine and sphingomyelin are predominantly concentrated in the outer layer and phosphatidylserine (PS) and phosphatidylethanolamine are mainly confined to the inner layer of the erythrocyte membrane. Maintenance of PL asymmetry is essential for erythrocyte survival and function as increased externalization of PS results in adherence of erythrocytes to vascular endothelium, activation of plasma blood clotting factors and premature removal from the circulation. Flippases, floppases and scramblases are the enzymes responsible for the establishment and maintenance of PL distribution.A mouse deficient in the flippase ATP11C was generated through ENU mutagenesis and was found to have reduced hemoglobin in comparison to wild type (WT) littermates. This study was performed to characterize the etiology of anemia in these mice. ResultsATP11C-deficient mice had significant reductions in erythrocyte numbers and hematocrit compared to WT but higher MCH and MCV. Reticulocyte numbers were comparable to WT as were serum iron parameters. Bone marrow and splenic erythropoiesis was normal in ATP11C mutant mice, however, erythrocyte lifespan was reduced by 35%. A marked increase in the frequency of PS+ erythrocytes was demonstrated in ATP11C mutant mice and was shown to increase with erythrocyte age. Bone marrow and splenic erythroblasts from ATP11C-deficient animals displayed a lower rate of PS translocation in vitro compared to WT confirming defective flippase activity.Erythrocytes and late-stage splenic erythroblasts in ATP11C mutants were significantly larger than WT based on flow cytometry. SEM revealed the majority of mutant erythrocytes displayed stomatocyte-like morphology, as confirmed on peripheral blood smears. The osmotic fragility of the ATP11C-deficient erythrocytes was comparable to WT erythrocytes as was Na+ and K+ homeostasis. DiscussionThese studies reveal the important role of flippases in maintaining normal erythrocyte function through the maintenance of the asymmetric distribution of PS within the membrane. Perturbation of this process, as seen in the ATP11C-deficient mice, results in (i) increased exposure of PS on the erythrocyte outer membrane, (ii) increased size of late-stage erythroblasts and erythrocytes with stomatocyte formation (iii) reduced erythrocyte lifespan and (iv) normochromic anemia. This study, therefore, reveals a new mechanism for stomatocytosis and raises the question of whether mutations in ATP11C may serve as a previously unclassified cause of anemia in humans. Disclosures:No relevant conflicts of interest to declare.

Non-adsorbing macromolecules promote endothelial adhesion of erythrocytes with reduced sialic acids

BackgroundAbnormal adhesion of red blood cells (RBCs) to vascular endothelium is often associated with reduced levels of sialic acids on RBC membranes and with elevated levels of pro-adhesive plasma proteins. However, the synergistic effects of these two factors on the adhesion are not clear. In this work, we tested the hypothesis that macromolecular depletion interaction originating from non-adsorbing macromolecules can promote the adhesion of RBCs with reduced sialic acid content to the endothelium. MethodsRBCs are treated with neuraminidase to specifically remove sialic acids from their surface followed by the evaluation of their deformability, zeta potential and membrane proteins. The adhesion of these enzyme-treated RBCs to cultured human umbilical vein endothelial cells (ECs) is studied in the presence of 70 or 500kDa dextran with a flow chamber assay. ResultsOur results demonstrate that removal of sialic acids from RBC surface can induce erythrocyte adhesion to endothelial cells and that such adhesion is significantly enhanced in the presence of high-molecular weight dextran. The adhesion-promoting effect of dextran exhibits a strong dependence on dextran concentration and molecular mass, and it is concluded to originate from macromolecular depletion interaction. ConclusionThese results suggest that elevated levels of non-adsorbing macromolecules in plasma might play a significant role in promoting endothelial adhesion of erythrocytes with reduced sialic acids. General significanceOur findings should therefore be of great value in understanding abnormal RBC–EC interactions in pathophysiological conditions (e.g., sickle cell disease and diabetes) and after blood transfusions.

Poloxamer 188 reduces normal and phosphatidylserine-exposing erythrocyte adhesion to endothelial cells in dextran solutions

Abnormal red blood cell (RBC) adhesion to endothelial cells (ECs) has been correlated with vascular complications in diseases such as sickle cell anemia and diabetes. Poloxamer 188 (P188) has been clinically tested to treat vaso-occlusion. However, the underlying mechanism(s) have not been clarified, making a methodical application difficult. In this study, we investigate how and to what extent P188 reduces RBC adhesion to ECs in plasma-like solutions. RBC adhesion to ECs is studied in solutions containing dextran, which is known to induce adhesion via macromolecular depletion interaction. It is demonstrated that P188 itself does not induce adhesion of normal RBCs to ECs but significantly reduces the adhesion in solutions containing high molecular mass-dextran. In addition, it is shown that P188 can reduce the adhesion of RBCs with enhanced exposure of phosphatidylserine (PS). Measurements of the electrophoretic mobility indicate that P188 increases the local viscosity inside the electric double layer of RBCs. Based on these results this study suggests that P188 reduces macromolecular depletion interaction, via penetrating into the depletion layer. Taking into consideration that dextran mimics the effects of pro-adhesive non-adsorbing plasma proteins and macromolecules, our study therefore suggests a mechanism for the adhesion reducing effect of P188 and should thus be of potential value for a detailed understanding of how cell–cell interactions in pathological conditions can be reduced.

CD36 Recruits α5β1 Integrin to Promote Cytoadherence of P. falciparum-Infected Erythrocytes

The adhesion of Plasmodium falciparum-infected erythrocytes (IRBC) to receptors on different host cells plays a divergent yet critical role in determining the progression and outcome of the infection. Based on our ex vivo studies with clinical parasite isolates from adult Thai patients, we have previously proposed a paradigm for IRBC cytoadherence under physiological shear stress that consists of a recruitment cascade mediated largely by P-selectin, ICAM-1 and CD36 on primary human dermal microvascular endothelium (HDMEC). In addition, we detected post-adhesion signaling events involving Src family kinases and the adaptor protein p130CAS in endothelial cells that lead to CD36 clustering and cytoskeletal rearrangement which enhance the magnitude of the adhesive strength, allowing adherent IRBC to withstand shear stress of up to 20 dynes/cm2. In this study, we addressed whether CD36 supports IRBC adhesion as part of an assembly of membrane receptors. Using a combination of flow chamber assay, atomic force and confocal microscopy, we showed for the first time by loss- and gain-of function assays that in the resting state, the integrin α5β1 does not support adhesive interactions between IRBC and HDMEC. Upon IRBC adhesion to CD36, the integrin is recruited either passively as part of a molecular complex with CD36, or actively to the site of IRBC attachment through phosphorylation of Src family kinases, a process that is Ca2+-dependent. Clustering of β1 integrin is associated with an increase in IRBC recruitment as well as in adhesive strength after attachment (∼40% in both cases). The adhesion of IRBC to a multimolecular complex on the surface of endothelial cells could be of critical importance in enabling adherent IRBC to withstand the high shear stress in the microcirculations. Targeting integrins may provide a novel approach to decrease IRBC cytoadherence to microvascular endothelium.

High antioxidant activity of sargassum Muticum and Padina pavonica collected from Peniche coast (Portugal)

Peptidoglycans, bacterial wall components, have previously been shown to trigger eryptosis, the suicidal erythrocyte death, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Phosphatidylserine exposing erythrocytes adhere to the vascular wall at least partially by interaction of erythrocytic phosphatidylserine with endothelial CXC chemokine ligand 16 (CXCL16). The present study explored whether peptidoglycan exposure fosters the adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC). To this end, HUVEC were treated for 48 h with peptidoglycan (10 μg/ml) and CXCL16 abundance determined by confocal microscopy and FACS analysis. Moreover, human erythrocytes were exposed for 48 h to peptidoglycan (10 μg/ml) and phosphatidylserine exposure estimated from binding of fluorescent annexin-V, cell volume from forward scatter in FACS analysis and erythrocyte adhesion to human umbilical vein endothelial cells (HUVEC) from trapping of labeled erythrocytes in a flow chamber. As a result, bacterial peptidoglycan exposure was followed by increased CXCL16 expression in HUVEC as well as erythrocyte shrinkage, phosphatidylserine exposure and adhesion to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly attenuated but not abrogated in the presence of either, erythrocyte phosphatidylserine-coating annexin-V (5 μl/ml) or CXCL16 neutralizing antibody directed against endothelial CXCL16 (4 μg/ml). In conclusion, exposure to peptidoglycan increases endothelial CXCL16 expression and leads to eryptosis followed by phosphatidylserine- and CXCL16-mediated adhesion of eryptotic erythrocytes to vascular endothelial cells.

Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes

BackgroundThe peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). MethodsHuman erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin–luciferase based real-time luminometry. ResultsExposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%.Erythrocytes pre-exposure to 10μM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux.Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. ConclusionsMST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. General significanceKinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.

Thrombospondin‐1 and L‐selectin are associated with silent cerebral infarct in children with sickle cell anaemia

Thrombospondin‐1 and <scp>L</scp>‐selectin are associated with silent cerebral infarct in children with sickle cell anaemia

CD36 regulates the malaria-induced pro-inflammatory cytokine responses and activation of NK and T cell by dendritic cells (P3298)

Abstract In falciparum malaria, the scavenger receptor CD36 plays multiple roles: (i) mediates the sequestration of parasites in the microvascular endothelia through the adherence of parasite-infected erythrocytes, (ii) supports phagocytic clearance of parasites by macrophages, and (iii) modulates immune responses to parasites. While the function of CD36 in the parasite sequestration and clearance has been extensively studied, its role in malaria immunity remains poorly understood. We studied the role of CD36 in cytokine responses to malaria parasite, Plasmodiun falciparum, by DCs and in DC function. Blocking of CD36 with anti-CD36 antibodies resulted in the substantial decrease in the levels of infected erythrocyte internalization and pro-inflammatory cytokine responses by DCs. Consistent with these results, DCs deficient in CD36 internalized lower levels of infected erythrocytes and produced decreased levels of pro-inflammatory cytokines. The observed CD36-depedent internalization and cytokine responses by DCs are evident at relatively low doses of infected erythrocytes; at higher doses, the contribution of CD36 become masked due to the increased rate of infected erythrocyte uptake by various other receptor-mediated phagocytosis. Furthermore, IFN-γ production by NK and T cells incubated with IRBC-internalized DCs was dependent on CD36. Overall, our data demonstrate that CD36 plays a critical role in the development of immunity to malaria.

Histopathologies, immunolocalization, and a glycan binding screen provide insights into Plasmodium falciparum interactions with the human placenta.

During pregnancy, Plasmodium falciparum-infected erythrocytes cytoadhere to the placenta. Infection is likely initiated at two sites where placental trophoblasts contact maternal blood: 1) via syncytiotrophoblast (STB), a multicellular transporting and biosynthetic layer that forms the surface of chorionic villi and lines the intervillous space, and 2) through invasive cytotrophoblasts, which line uterine vessels that divert blood to the placenta. Here, we investigated mechanisms of infected erythrocyte sequestration in relationship to the microanatomy of the maternal-fetal interface. Histological analyses revealed STB denudation in placental malaria, which brought the stromal cores of villi in direct contact with maternal blood. STB denudation was associated with hemozoin deposition (P = 0.01) and leukocyte infiltration (P = 0.001) and appeared to be a feature of chronic placental malaria. Immunolocalization of infected red blood cell receptors (CD36, ICAM1/CD54, and chondroitin sulfate A) in placentas from uncomplicated pregnancies showed that STB did not stain, while the underlying villous stroma was immunopositive. Invasive cytotrophoblasts expressed ICAM1. In malaria, STB denudation exposed CD36 and chondroitin sulfate A in the villous cores to maternal blood, and STB expressed ICAM1. Finally, we investigated infected erythrocyte adherence to novel receptors by screening an array of 377 glycans. Infected erythrocytes bound Lewis antigens that immunolocalized to STB. Our results suggest that P. falciparum interactions with STB-associated Lewis antigens could initiate placental malaria. Subsequent pathologies, which expose CD36, ICAM1, and chondroitin sulfate A, might propagate the infection.

Effects of Gallid Herpesvirus 2 Marek's Disease Challenge Virus and Attenuated Vaccine Virus CVI988/Rispens on Immune Adhesion of Erythrocytes of Chickens

This study was designed to investigate the influence of Gallid herpesvirus 2 Marek's Disease challenge virus and different inoculated routes of attenuated vaccine virus CVI988/Rispens on immun e adhesion of erythrocytes in chickens. One day old chicks were randomly divided into a control group (C), an experimental group challenged with Jing-1 challenge strain (V), a second and third experimental group individually injected intra-abdominally (IA) or subcutaneously (SC) with vaccine strain, CVI988/Rispens. The erythrocyte immune adhesion was evaluated with yeast rosette-forming tests of red blood cell complement receptor type 1 (RBC-CR1) and red blood cell immune complex (RBC-IC). It was found that chickens from group V had fewer erythrocytes. During this time, the rate of RBC-IC rosette formation was higher in group V except on days 3 and 20. While the rate of RBC-CR1 rosette formation was lower in group V except on the 26th day when the number of RBC-CR1 rosettes was extremely higher. The rate of RBC-CR1 rosette formation and RBCs count was higher in group IA than that of SC. The results suggest that challenge virus lead to immune suppression, a decline in the immune adhesion of RBCs followed by a rebound during the later stages of infection; effects on RBC immunity were increased after inoculate attenuated vaccines strains; IA vaccination route may enhance the immune adhesion of RBCs comparing with SC route; and an improvement in the RBC immunity adhesion during the later stages of infection may have resulted from a compensatory immune response to immune organ atrophy and the decrease of RBCs.

Effect of bacterial peptidoglycan on erythrocyte death and adhesion to endothelial cells

Peptidoglycans, bacterial wall components, have previously been shown to trigger eryptosis, the suicidal erythrocyte death, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Phosphatidylserine exposing erythrocytes adhere to the vascular wall at least partially by interaction of erythrocytic phosphatidylserine with endothelial CXC chemokine ligand 16 (CXCL16). The present study explored whether peptidoglycan exposure fosters the adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC). To this end, HUVEC were treated for 48h with peptidoglycan (10μg/ml) and CXCL16 abundance determined by confocal microscopy and FACS analysis. Moreover, human erythrocytes were exposed for 48h to peptidoglycan (10μg/ml) and phosphatidylserine exposure estimated from binding of fluorescent annexin-V, cell volume from forward scatter in FACS analysis and erythrocyte adhesion to human umbilical vein endothelial cells (HUVEC) from trapping of labeled erythrocytes in a flow chamber. As a result, bacterial peptidoglycan exposure was followed by increased CXCL16 expression in HUVEC as well as erythrocyte shrinkage, phosphatidylserine exposure and adhesion to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly attenuated but not abrogated in the presence of either, erythrocyte phosphatidylserine-coating annexin-V (5μl/ml) or CXCL16 neutralizing antibody directed against endothelial CXCL16 (4μg/ml). In conclusion, exposure to peptidoglycan increases endothelial CXCL16 expression and leads to eryptosis followed by phosphatidylserine- and CXCL16-mediated adhesion of eryptotic erythrocytes to vascular endothelial cells.

Adhesion of Annexin 7 Deficient Erythrocytes to Endothelial Cells

Annexin 7 deficiency has previously been shown to foster suicidal death of erythrocytes or eryptosis, which is triggered by increase of intracellular Ca2+ concentration ([Ca2+]i) and characterized by cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface. Eryptosis following increase of [Ca2+]i by Ca2+ ionophore ionomycin, osmotic shock or energy depletion was more pronounced in erythrocytes from annexinA7-deficient mice (anxA7−/−) than in erythrocytes from wild type mice (anxA7+/+). As phosphatidylserine exposure is considered to mediate adhesion of erythrocytes to the vascular wall, the present study explored adhesion of erythrocytes from anx7−/− and anx7+/+-mice following increase of [Ca2+]i by Ca2+ ionophore ionomycin (1 µM for 30 min), hyperosmotic shock (addition of 550 mM sucrose for 2 hours) or energy depletion (removal of glucose for 12 hours). Phosphatidylserine exposing erythrocytes were identified by annexin V binding, cell volume estimated from forward scatter in FACS analysis and adhesion to human umbilical vein endothelial cells (HUVEC) utilizing a flow chamber. As a result, ionomycin, sucrose addition and glucose removal all triggered phosphatidylserine-exposure, decreased forward scatter and enhanced adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC), effects significantly more pronounced in anx7−/− than in anx7+/+-erythrocytes. Following ischemia, morphological renal injury was significantly higher in anx7−/− than in anx7+/+-mice. The present observations demonstrate that enhanced eryptosis of annexin7 deficient cells is paralleled by increased adhesion of erythrocytes to the vascular wall, an effect, which may impact on microcirculation during ischemia.

Molecular Architecture of a Complex between an Adhesion Protein from the Malaria Parasite and Intracellular Adhesion Molecule 1

The adhesion of Plasmodium falciparum-infected erythrocytes to human tissues or endothelium is central to the pathology caused by the parasite during malaria. It contributes to the avoidance of parasite clearance by the spleen and to the specific pathologies of cerebral and placental malaria. The PfEMP1 family of adhesive proteins is responsible for this sequestration by mediating interactions with diverse human ligands. In addition, as the primary targets of acquired, protective immunity, the PfEMP1s are potential vaccine candidates. PfEMP1s contain large extracellular ectodomains made from CIDR (cysteine-rich interdomain regions) and DBL (Duffy-binding-like) domains and show extensive variation in sequence, size, and domain organization. Here we use biophysical methods to characterize the entire ∼300-kDa ectodomain from IT4VAR13, a protein that interacts with the host receptor, intercellular adhesion molecule-1 (ICAM-1). We show through small angle x-ray scattering that IT4VAR13 is rigid, elongated, and monomeric. We also show that it interacts with ICAM-1 through the DBLβ domain alone, forming a 1:1 complex. These studies provide a first low resolution structural view of a PfEMP1 ectodomain in complex with its ligand. They show that it combines a modular domain arrangement consisting of individual ligand binding domains, with a defined higher order architecture that exposes the ICAM-1 binding surface to allow adhesion.