Cytoadherence of Plasmodium falciparum-infected erythrocytes in the human placenta

In Plasmodium falciparum-parasitized pregnant women, erythrocytes infected by mature stages of the parasite sequester into placental intervillous spaces. The presence of parasites in the placenta causes maternal anaemia and low birth weight of the infant. In-vitro studies suggest placental sequestration may involve the cytoadherence of infected erythrocytes to chondroitin sulphate A (CSA) and/or intercellular adhesion molecule 1 (ICAM-1) expressed by human placental syncytiotrophoblast. We identified P. falciparum receptors expressed on the surface of human syncytiotrophoblast using immunofluorescence of placental biopsies from Cameroon, a malaria-endemic area. In all placentas, a strongly positive staining was observed on the syncytiotrophoblast for CSA, but not for ICAM-1, vascular endothelium cell adhesion molecule-1, E-selectin, nor CD36. The cytoadherence ability of parasites from pregnant women and nonpregnant subjects was assessed on in-vitro cultured syncytiotrophoblast. Parasites from pregnant women bound to the trophoblast via CSA but not ICAM-1. Parasites from nonpregnant hosts either did not bind to the trophoblast culture or bound using ICAM-1. Our data support the idea that placental sequestration may result from cytoadherence to placental trophoblast and that pregnant women are parasitized by parasites that differ from parasites derived from nonpregnant host by their cytoadherence ability.     

Hexose permeation pathways in Plasmodium falciparum-infected erythrocytes

Plasmodium falciparum requires glucose as its energy source to multiply within erythrocytes but is separated from plasma by multiple membrane systems. The mechanism of delivery of substrates such as glucose to intraerythrocytic parasites is unclear. We have developed a system for robust functional expression in Xenopus oocytes of the P. falciparum asexual stage hexose permease, PfHT1, and have analyzed substrate specificities of PfHT1. We show that PfHT1 (a high-affinity glucose transporter, K(m) approximately 1.0 mM) also transports fructose (K(m) approximately 11.5 mM). Fructose can replace glucose as an energy source for intraerythrocytic parasites. PfHT1 binds fructose in a furanose conformation and glucose in a pyranose form. Fructose transport by PfHT1 is ablated by mutation of a single glutamine residue, Q169, which is predicted to lie within helix 5 of the hexose permeation pathway. Glucose transport in the Q169N mutant is preserved. Comparison in oocytes of transport properties of PfHT1 and human facilitative glucose transporter (GLUT)1, an archetypal mammalian hexose transporter, combined with studies on cultured P. falciparum, has clarified hexose permeation pathways in infected erythrocytes. Glucose and fructose enter erythrocytes through separate permeation pathways. Our studies suggest that both substrates enter parasites via PfHT1.     

Abnormal membrane phospholipid organization in Plasmodium falciparum-infected human erythrocytes

The membrane phospholipid organization in Plasmodium falciparum-infected human erythrocytes was analysed by employing phospholipase A2 and Merocyanine 540 as external membrane probes. Both bee venom and pancreatic phospholipases A2 failed to hydrolyse phosphatidylserine in uninfected human red cells isolated from in vitro P. falciparum cultures. However, these enzymes under identical conditions readily degraded this aminophospholipid in P. falciparum-infected erythrocytes. Phosphatidylethanolamine hydrolysis also increased in parasitized cells. The degree to which these aminophospholipids were cleaved by the enzymes in intact infected cells depended on the developmental stage of the intracellular parasite, and was maximum at the schizont stage. This was consistent with the finding that the 'fluid-sensing' fluorescent dye, Merocyanine 540, readily labelled both the schizont and trophozoite-infected cells but not the fresh, uninfected or ring-infected erythrocytes. These results demonstrate that P. falciparum produces stage-dependent changes in the membrane phospholipid organization of its host erythrocyte.     

In vitro and in vivo inhibition of activation induced T cell apoptosis by bucillamine

OBJECTIVE: To investigate the mechanism of autoimmune phenomena, occasionally seen in patients with rheumatoid arthritis treated with bucillamine (BUC) and D-penicillamine (D-Pen), by evaluating their effects on apoptosis of T cells induced by T cell receptor activation or dexamethasone. METHODS: In vitro apoptosis was induced in a T cell hybridoma (SSP3.7) and a B cell line (WEHI 231) by activation of respective receptors or dexamethasone, in the presence or absence of BUC or D-Pen. In vivo apoptosis was induced in BALB/c mice by staphylococcal enterotoxin B (SEB), with or without BUC or D-Pen, and thymocytes were examined for it by FACS. RESULTS: Stimulation with anti-CD3 and dexamethasone induced apoptosis in 72% and 71% of SSP3.7 cells, respectively. However, only 16% of SSP3.7 cells became apoptotic by anti-CD3 when BUC was added to the culture media. By contrast, 80% of SSP3.7 cells became apoptotic when stimulated by dexamethasone, even in the presence of BUC. BUC did not affect apoptosis of WEHI 231 cells induced by anti-IgM. Although SA981 (a metabolite of BUC) inhibited apoptosis of SSP3.7 cells induced by anti-CD3, D-Pen did not. BUC, SA981, or D-Pen did not significantly influence the level of interleukin 2 secretion stimulated by anti-CD3. In contrast, both BUC and D-Pen inhibited apoptosis of Vbeta8+ thymocytes induced in vivo by SEB superantigen. Neither BUC nor D-Pen significantly changed the number of CD4+CD8+ thymocytes in BALB/c mice injected with dexamethasone. CONCLUSION: BUC decreased, while D-Pen did not, the apoptosis of T cells stimulated by anti-CD3 in vitro, although they both inhibited the deletion of immature thymocytes reactive with SEB in vivo. This may explain autoimmune phenomena sometimes seen during the treatment of rheumatic patients with these drugs.     

Altered plasma membrane phospholipid organization in Plasmodium falciparum-infected human erythrocytes

The intraerythrocytic development of the malaria parasite is accompanied by distinct morphological and biochemical changes in the host cell membrane, yet little is known about development-related alterations in the transbilayer organization of membrane phospholipids in parasitized cells. This question was examined in human red cells infected with Plasmodium falciparum. Normal red cells were infected with strain FCR3 or with clonal derivatives that either produce (K+) or do not produce (K-) knobby protuberances on the infected red cells. Parasitized cells were harvested at various stages of parasite development, and the bilayer orientation of red cell membrane phospholipids was determined chemically using 2,4,6-trinitrobenzene sulphonic acid (TNBS) or enzymatically using bee venom phospholipase A2 (PLA2) and sphingomyelinase C (SMC). We found that parasite development was accompanied by distinct alterations in the red cell membrane transbilayer distribution of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Increases in the exoplasmic membrane leaflet exposure of PE and PS were larger in the late-stage parasitized cells than in the early-stage parasitized cells. Similar results were obtained for PE membrane distribution using either chemical (TNBS) or enzymatic (PLA2 plus SMC) methods, although changes in PS distribution were observed only with TNBS. Uninfected cohort cells derived from mixed populations of infected and uninfected cells exhibited normal patterns of membrane phospholipid organization. The observed alterations in P falciparum-infected red cell membrane phospholipid distribution, which is independent of the presence or absence of knobby protuberances, might be associated with the drastic changes in cell membrane permeability and susceptibility to early hemolysis observed in the late stages of parasite development.     

Increased nicotinamide adenine dinucleotide content and synthesis in Plasmodium falciparum-infected human erythrocytes

Plasmodium falciparum-infected red blood cells (RBCs) are characterized by increases in the activity of glycolytic enzymes. Because nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) are cofactors in the reactions of glycolysis and pentose phosphate shunt, we have examined NAD and NADP content in P. falciparum-infected RBCs. Although NADP content was not significantly altered, NAD content was increased approximately 10-fold in infected RBCs (66% parasitemia) compared with uninfected control RBCs. To determine the mechanism for the increase in NAD content, we examined the activity of several NAD biosynthetic enzymes. It is known that normal human RBCs make NAD exclusively from nicotinic acid and lack the capacity to make NAD from nicotinamide. We demonstrate that infected RBCs have readily detectable nicotinamide phosphoribosyltransferase (NPRT), the first enzyme in the NAD biosynthetic pathway that uses nicotinamide, and abundant nicotinamide deamidase, the enzyme that converts nicotinamide to nicotinic acid, thereby indicating that infected RBCs can make NAD from nicotinamide. In addition, infected RBCs have a threefold increase in nicotinic acid phosphoribosyltransferase (NAPRT), the first enzyme in the NAD biosynthetic pathway that uses nicotinic acid. Thus, the increase in NAD content in P falciparum-infected RBCs appears to be mediated by increases in NAD synthesis from both nicotinic acid and nicotinamide.     

Interactions between docetaxel (Taxotere) and Plasmodium falciparum-infected erythrocytes.

Taxotere (docetaxel) inhibits Plasmodium falciparum erythrocytic development in vitro at nanomolar concentrations, both in chloroquine-sensitive (F32/Tanzania) and chloroquine-resistant (FcB1/Colombia, FcR3/Gambia) strains. The dose-response assays performed on asynchronous cultures during 42 hr showed clear biphasic curves with a plateau from 50 microM to 10 nM and a single sigmoid curve with a concentration inhibiting 50% of growth (IC50) of 3-6 nM observed after a 72-hr incubation. Addition of Taxotere to different stages of FcB1 revealed two types of targets: one type on ring/trophozoite-infected erythrocytes (RBCs), at the micromolar level, and another type on schizont-infected RBCs with Taxotere at micromolar concentrations inhibited the merozoite invasion of erythrocytes and parasite growth. These Taxotere-RBC interactions were stable, at least for 1 day. Pulse experiments of 5 hr with Taxotere efficiently inhibit parasite development regardless of the period of the parasite's erythrocytic life cycle. However, different cellular effects were obtained depending upon periods of drug incubations. The inhibition of P. falciparum development by Taxotere should provide additional strategies to block parasite development.     

HMG-CoA reductase inhibitor attenuates experimental autoimmune myocarditis through inhibition of T cell activation

OBJECTIVE: This study tested the hypothesis that 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor affects T cell-mediated autoimmunity through inhibition of nuclear factor-kappaB (NFkappaB) and reduces the severity of experimental autoimmune myocarditis (EAM). METHODS: EAM was induced in Lewis rats by immunization with myosin. High-dose or low-dose fluvastatin or vehicle was administered orally for 3 weeks to rats with EAM. RESULTS: Fluvastatin reduced the pathophysiological severity of myocarditis. Fluvastatin inhibited expression of NFkappaB in the nuclei of myocardium in EAM. Fluvastatin reduced production of Th1-type cytokines, including interferon (IFN)-gamma and interleukin (IL)-2, and inhibited expression of inflammatory cytokine mRNAs in the myocardium. Infiltration of CD4-positive T cells into the myocardium and T cell proliferative responses were suppressed by fluvastatin. Plasma lipid levels did not differ between the groups. CONCLUSIONS: Fluvastatin ameliorates EAM by inhibiting T cell responses and suppressing Th1-type and inflammatory cytokines via inactivation of nuclear factor-kappaB, and this activity is independent of cholesterol reduction.     

Phosphorylation of protein 4.1 in Plasmodium falciparum-infected human red blood cells

The composition of the erythrocyte plasma membrane is extensively modified during the intracellular growth of the malaria parasite Plasmodium falciparum. It has been previously shown that an 80-kD phosphoprotein is associated with the plasma membrane of human red blood cells (RBCs) infected with trophozoite/schizont stage malaria parasites. However, the identity of this 80-kD phosphoprotein is controversial. One line of evidence suggests that this protein is a phosphorylated form of RBC protein 4.1 and that it forms a tight complex with the mature parasite-infected erythrocyte surface antigen. In contrast, evidence from another group indicates that the 80-kD protein is derived from the intracellular malaria parasite. To resolve whether the 80-kD protein is indeed RBC protein 4.1, we made use of RBCs obtained from a patient with homozygous 4.1(-) negative hereditary elliptocytosis. RBCs from this patient are completely devoid of protein 4.1. We report here that this lack of protein 4.1 is correlated with the absence of phosphorylation of the 80-kD protein in parasite- infected RBCs, a finding that provides conclusive evidence that the 80- kD phosphoprotein is indeed protein 4.1. In addition, we also identify and partially characterize a casein kinase that phosphorylates protein 4.1 in P falciparum-infected human RBCs. Based on these results, we suggest that the maturation of malaria parasites in human RBCs is accompanied by the phosphorylation of protein 4.1. This phosphorylation of RBC protein 4.1 may provide a mechanism by which the intracellular malaria parasite alters the mechanical properties of the host plasma membrane and modulates parasite growth and survival in vivo.     

CD4 T cell activation as a predictor for treatment failure in Ugandans with Plasmodium falciparum malaria

Host immunity plays an important role in response to antimalarial therapy but is poorly understood. To test whether T cell activation is a risk factor for antimalarial treatment failure, we studied CD4(+) and CD8(+) T cell activation in 31 human immunodeficiency virus-negative Ugandan patients 5-37 years of age who were treated for uncomplicated Plasmodium falciparum malaria. Increased CD4(+) T cell activation, as indicated by co-expression of HLA-DR and CD38, was an independent risk factor for treatment failure (hazard ratio = 2.45, 95% confidence interval = 1.02-5.89, P = 0.05) in multivariate analysis controlling for age, baseline temperature, and pre-treatment parasite density. The results provide insight into the role of cellular immunity in response to antimalarial therapy and underscore the need to investigate the mechanisms behind immune activation.     

Platelet-induced autoagglutination of Plasmodium falciparum-infected red blood cells and disease severity in Thailand

The relationship of the platelet-mediated autoagglutination of Plasmodium falciparum-infected red blood cells (IRBCs) to disease severity was investigated in 182 Thai patients with falciparum malaria; it was evident in 43% of uncomplicated malaria (n=63), 41% of severe malaria (n=104), and 100% of cerebral malaria (n=15; P=.001) isolates. The median (range) number of IRBCs in agglutinates per 1000 IRBCs was significantly higher in cerebral malaria (6 [3-42]) than in severe (0 [0-52]) and uncomplicated (0 [0-24]) malaria (P=.01). In multivariate analyses, high parasitemia and cerebral malaria were associated independently with parasite agglutination.     

Cooperative domains define a unique host cell-targeting signal in Plasmodium falciparum-infected erythrocytes

When the malaria parasite Plasmodium falciparum infects an erythrocyte, it resides in a parasitophorous vacuole and remarkably exports proteins into the periphery of its host cell. Two of these proteins, the histidine-rich proteins I and II (PfHRPI and PfHRPII), are exported to the erythrocyte cytoplasm. PfHRPI has been linked to cell-surface "knobby" protrusions that mediate cerebral malaria and are a frequent cause of death. PfHRPII has been implicated in (i) the production of hemozoin, the black pigment associated with disease, as well as (ii) interactions with the erythrocyte cytoskeleton. Here we show that a tripartite signal that is comprised of an endoplasmic reticulum-type signal sequence followed by a bipartite vacuolar translocation signal derived from HRPII and HRPI exports GFP from the parasitophorous vacuole to the host cytoplasm. The bipartite vacuolar translocation signal is comprised of unique, peptidic (approximately equal to 40-aa) sequences. A domain within it contains the signal for export to "cleft" transport intermediates in the host erythrocyte and may thereby regulate the pathway of export to the host cytoplasm. A signal for posttranslational, vacuolar exit of proteins has hitherto not been described in eukaryotic secretion.     

Monocyte activation in patients with Wegener's granulomatosis

OBJECTIVE: Wegener's granulomatosis (WG) is an inflammatory disorder characterised by granulomatous inflammation, vasculitis, and necrotising vasculitis and is strongly associated with anti-neutrophil cytoplasmic antibodies (ANCA). Activated monocytes/macrophages are present in renal biopsy specimens and participate in granuloma formation by synthesising and secreting a variety of chemoattractants, growth factors, and cytokines. In view of these findings, in vivo monocyte activation was evaluated in patients with WG and the findings related to parameters of clinical disease activity. METHODS: Monocyte activation was analysed by measuring plasma concentrations of soluble products of monocyte activation, that is neopterin and interleukin 6 (IL6), by ELISA, and by quantitating the surface expression of activation markers on circulating monocytes by flow cytometry. RESULTS: Twenty-four patients with active WG were included in this study. Ten of these patients were also analysed at the time of remission. Twelve patients with sepsis served as positive controls, and 10 healthy volunteers as negative controls for monocyte activation. Patients with active disease had increased monocyte activation compared with healthy controls as shown by increased concentrations of neopterin (p < 0.0001) and increased surface expression of CD11b (p < 0.05) and CD64 (p < 0.05). In those patients with increased concentrations of IL6 during active disease plasma concentrations of IL6 decreased during follow up when patients went into remission (p < 0.0001). In addition, neopterin (r = 0.37, r = 0.44), IL6 (r = 0.37, r = 0.60) and CD63 expression (r = 0.39, r = 0.45) correlated significantly with disease activity as measured by the Birmingham Vasculitis Activity Score and C reactive protein values, respectively. Compared with patients with sepsis, all markers of monocyte activation in patients with vasculitis were lower. CONCLUSION: It is concluded that disease activity in WG correlates with the extent of activation of monocytes, compatible with their role in the pathophysiology of this disease.     

Lack of a role for transforming growth factor-beta in cytotoxic T lymphocyte antigen-4-mediated inhibition of T cell activation

Similarities in the phenotypes of mice deficient for cytotoxic T lymphocyte antigen-4 (CTLA-4) or transforming growth factor-beta1 (TGF-beta1) and other observations have led to speculation that CTLA-4 mediates its inhibitory effect on T cell activation via costimulation of TGF-beta production. Here, we examine the role of TGF-beta in CTLA-4-mediated inhibition of T cell activation and of CTLA-4 in the regulation of TGF-beta production. Activation of AND TCR transgenic mouse T cells with costimulatory receptor-specific antigen presenting cells results in efficient costimulation of proliferation by CD28 ligation and inhibition by CTLA-4 ligation. Neutralizing antibody to TGF-beta does not reverse CTLA-4-mediated inhibition. Also, CTLA-4 ligation equally inhibits proliferation of wild-type, TGF-beta1(-/-), and Smad3(-/-) T cells. Further, CTLA-4 engagement does not result in the increased production of either latent or active TGF-beta by CD4(+) T cells. These results indicate that CTLA-4 ligation does not regulate TGF-beta production and that CTLA-4-mediated inhibition can occur independently of TGF-beta. Collectively, these data demonstrate that CTLA-4 and TGF-beta represent distinct mechanisms for regulation of T cell responses.     

Oxidative stress modulation and T cell activation

During the immune response T cell function is influenced by extrinsic factors, some of which lead to increased protein and DNA damage and are thought to play a role in age-related immune dysfunction. Damage is in part due to reactive oxygen species produced as a result of aerobic metabolism during a vigorous immune response, but in the in vitro models commonly used to study human immunity may also be due to culturing cells under hyperoxic conditions, i.e., in air. Reactive oxygen species (ROS) are ubiquitously generated but an imbalance between ROS production and protection against ROS may severely affect T cell activation. Controlling and modulating oxidative stress in the extracellular milieu may influence T cell signalling and activation. Here, we discuss the relevance of oxidative stress modulation to prevent T cell dysfunction. We draw attention to some technical, but critical, aspects of T cell culture under hyperoxic conditions.     

Excess hemoglobin digestion and the osmotic stability of Plasmodium falciparum-infected red blood cells

During their asexual reproduction cycle (about 48 hours) in human red cells, Plasmodium falciparum parasites consume most of the host cell hemoglobin, far more than they require for protein biosynthesis. They also induce a large increase in the permeability of the host cell plasma membrane to allow for an increased traffic of nutrients and waste products. Why do the parasites digest hemoglobin in such excess? And how can infected red cells retain their integrity for the duration of the asexual cycle when comparably permeabilized uninfected cells hemolyse earlier? To address these questions we encoded the multiplicity of factors known to influence host cell volume in a mathematical model of the homeostasis of a parasitized red cell. The predicted volume changes were subjected to thorough experimental tests by monitoring the stage-related changes in the osmotic fragility of infected red cell populations. The results supported the model predictions of biphasic volume changes comprising transient shrinkage of infected cells with young trophozoites followed by continuous volume increase to about 10% lower than the critical hemolytic volume of approximately 150 fL by the end of the asexual cycle. Analysis of these results and of additional model predictions demonstrated that the osmotic stability of infected red cells can be preserved only by a large reduction in impermeant solute concentration within the host cell compartment. Thus, excess hemoglobin consumption represents an essential evolutionary strategy to prevent the premature hemolysis of the highly permeabilized infected red cell.     

Nonimmune immunoglobulin binding and multiple adhesion characterize Plasmodium falciparum-infected erythrocytes of placental origin

The harmful effects of pregnancy-associated malaria (PAM) are engendered by the heavy sequestration of Plasmodium falciparum-parasitized RBCs in the placenta. It is well documented that this process is mediated by interactions of parasite-encoded variant surface antigens and placental receptors. A P. falciparum erythrocyte membrane protein 1 variant, VAR2CSA, and the placental receptor chondroitin sulfate A (CSA) are currently the focus of PAM research. A role for immunoglobulins (IgG and IgM) from normal human serum and hyaluronic acid as additional receptors in placental sequestration have also been suggested. We show here (i) that CSA and nonimmune IgG/IgM binding are linked phenotypes of in vitro-adapted parasites, (ii) that a VAR2CSA variant shown to bind CSA also harbors IgG- and IgM-binding domains (DBL2-X, DBL5-epsilon, and DBL6-epsilon), and (iii) that IgG and IgM binding and adhesion to multiple receptors (IgG/IgM/HA/CSA) rather than the exclusive binding to CSA is a characteristic of fresh Ugandan placental isolates. These findings are of importance for the understanding of the pathogenesis of placental malaria and have implications for the ongoing efforts to develop a global PAM vaccine.