FRET Imaging of Hemoglobin Concentration in Plasmodium falciparum-Infected Red Cells

Background

During its intraerythrocytic asexual reproduction cycle Plasmodium falciparum consumes up to 80% of the host cell hemoglobin, in large excess over its metabolic needs. A model of the homeostasis of falciparum-infected red blood cells suggested an explanation based on the need to reduce the colloid-osmotic pressure within the host cell to prevent its premature lysis. Critical for this hypothesis was that the hemoglobin concentration within the host cell be progressively reduced from the trophozoite stage onwards.

Methodology/Principal Findings

The experiments reported here were designed to test this hypothesis by direct measurements of the hemoglobin concentration in live, infected red cells. We developed a novel, non-invasive method to quantify the hemoglobin concentration in single cells, based on Förster resonance energy transfer between hemoglobin molecules and the fluorophore calcein. Fluorescence lifetime imaging allowed the quantitative mapping of the hemoglobin concentration within the cells. The average fluorescence lifetimes of uninfected cohorts was 270±30 ps (mean±SD; N = 45). In the cytoplasm of infected cells the fluorescence lifetime of calcein ranged from 290±20 ps for cells with ring stage parasites to 590±13 ps and 1050±60 ps for cells with young trophozoites and late stage trophozoite/ early schizonts, respectively. This was equivalent to reductions in hemoglobin concentration spanning the range from 7.3 to 2.3 mM, in line with the model predictions. An unexpected ancillary finding was the existence of a microdomain under the host cell membrane with reduced calcein quenching by hemoglobin in cells with mature trophozoite stage parasites.

Conclusions/Significance

The results support the predictions of the colloid-osmotic hypothesis and provide a better understanding of the homeostasis of malaria-infected red cells. In addition, they revealed the existence of a distinct peripheral microdomain in the host cell with limited access to hemoglobin molecules indicating the concentration of substantial amounts of parasite-exported material.     

Passive Ca2+ Transport and Ca2+-Dependent K+ Transport in Plasmodium falciparum-Infected Red Cells

Previous reports have indicated that Plasmodium falciparum-infected red cells (pRBC) have an increased Ca²⁺ permeability. The magnitude of the increase is greater than that normally required to activate the Ca²⁺-dependent K⁺ channel (K _Ca channel) of the red cell membrane. However, there is evidence that this channel remains inactive in pRBC. To clarify this discrepancy, we have reassessed both the functional status of the K _Ca channel and the Ca²⁺ permeability properties of pRBC. For pRBC suspended in media containing Ca²⁺, K _Ca channel activation was elicited by treatment with the Ca²⁺ ionophore A23187. In the absence of ionophore the channel remained inactive. In contrast to previous claims, the unidirectional influx of Ca²⁺ into pRBC in which the Ca²⁺ pump was inhibited by vanadate was found to be within the normal range (30–55 μmol (10¹³ cells · hr)⁻¹), provided the cells were suspended in glucose-containing media. However, for pRBC in glucose-free media the Ca²⁺ influx increased to over 1 mmol (10¹³ cells · hr)⁻¹, almost an order of magnitude higher than that seen in uninfected erythrocytes under equivalent conditions. The pathway responsible for the enhanced influx of Ca²⁺ into glucose-deprived pRBC was expressed at approximately 30 hr post-invasion, and was inhibited by Ni²⁺. Possible roles for this pathway in pRBC are considered. Received: 12 May 1999/Revised: 8 July 1999     

The Selective Staining of Red Blood Cells

A method for the selective staining of red blood cells is described. Material is fixed in 10% neutral formalin in .85% NaCl and imbedded in paraffin or celloidin. Sections 6-10 μ are stained 1-5 minutes in chromotrope 2R. Basophilic and the less strongly acidophilic elements are decolorized with 5% phosphotungstic acid in 95% ethyl alcohol. Red blood cells and other strongly acidophilic elements that may be present in the preparation retain the chromotrope 2R. A counterstain of methyl blue may be used for staining the decolorized basophilic elements. As a result, erythrocytes are stained red by the chromotrope 2R, and basophilic elements blue, by the methyl blue. Less strongly acidophilic elements, having little affinity for either primary or secondary dye, are colorless or gray.     

Adherence of Plasmodium falciparum-Infected Erythrocytes to Chondroitin 4-Sulfate

Adherence of Plasmodium falciparum-infected erythrocytes (PRBCs) to the microvascular endothelium of specific organs and consequent sequestration is believed to be responsible for the development of malaria pathology. A number of studies have shown that cell adhesion molecules expressed on the surface of endothelial cells mediate the adherence. Recent studies indicate that a subpopulation of PRBCs adhere to chondroitin 4-sulfate (C4S). This adhesion can be effectively inhibited by C4S oligosaccharides. In pregnant women, the placenta specifically selects C4S-adherent PRBCs, and thus these phenotypes multiply and sequester in the intervillous spaces. Over successive pregnancies, women develop a protective humoral response to the C4S-adhesion phenotype. Disruption of C4S-mediated PRBCs adhesion using either a C4S oligosaccharide mimetic or an antiC4S-adhesion vaccine can be an efficient strategy for the treatment of malaria caused by C4S-adherent P. falciparum.     

Cryopreservation of Sheep Red Blood Cells

The protection of sheep erythrocytes at freezing temperatures was investigated using glycerol, dimethylsulfoxide (DMSO), glucose and four different types of polyvinylpyrrolidone (PVP) as cryoprotective agents. Depending on type (molecular weight) and concentration good protection was obtained with PVP, whereas glycerol, DMSO and glucose were unsatisfactory. Recovery of cells after thawing was most successful when the cells had been frozen at a concentration of 1–2 × 109 cells/ml. No cells tolerated freezing at −20 °G. Best results were obtained when the cells were frozen directly in liquid nitrogen (−196°G).     

An in Vitro Assay for Sequestration: Binding of Plasmodium falciparum-Infected Erythrocytes to Formalin-Fixed Endothelial Cells and Amelanotic Melanoma Cells1

Erythrocytes infected with Plasmodium falciparum bind specifically to cultured endothelial cells and to a line of amelanotic melanoma cells. We have fixed endothelial cells and amelanotic melanoma cells in various ways and determined whether the fixed cells were still able to bind infected erythrocytes. Only cells fixed with 1.0-2.5% formalin in phosphate-buffered saline continued to bind infected erythrocytes as well as unfixed cells. The mechanism of binding to fixed and unfixed cells appeared to be identical for the following reasons. First, erythrocytes infected by parasite strains that bound to unfixed cells also bound to fixed cells while those that did not bind to unfixed cells did not bind to fixed cells. Second, immune serum that inhibited binding to unfixed cells also inhibited binding to fixed cells. Third, electron microscopy showed that knobs were the points of attachment between infected erythrocytes and both fixed and unfixed melanoma cells. Fixed cells gave reproducible results over at least 2 months. Thus, we have developed a simplified, reproducible assay for measuring binding of P. falciparum-infected erythrocytes to target cells.     

Temperature-Induced Catch-Slip to Slip Bond Transit in Plasmodium falciparum-Infected Erythrocytes

Plasmodium falciparum malaria-infected red blood cells (IRBCs), or erythrocytes, avoid splenic clearance by adhering to host endothelium. Upregulation of endothelial receptors intercellular adhesion molecule-1 (ICAM-1) and cluster of differentiation 36 (CD36) are associated with severe disease pathology. Most in vitro studies of IRBCs interacting with these molecules were conducted at room temperature. However, as IRBCs are exposed to temperature variations between 37°C (body temperature) and 41°C (febrile temperature) in the host, it is important to understand IRBC-receptor interactions at these physiologically relevant temperatures. Here, we probe IRBC interactions against ICAM-1 and CD36 at 37 and 41°C. Single bond force-clamp spectroscopy is used to determine the bond dissociation rates and hence, unravel the nature of the IRBC-receptor interaction. The association rates are also extracted from a multiple bond flow assay using a cellular stochastic model. Surprisingly, IRBC-ICAM-1 bond transits from a catch-slip bond at 37°C toward a slip bond at 41°C. Moreover, binding affinities of both IRBC-ICAM-1 and IRBC-CD36 decrease as the temperature rises from 37 to 41°C. This study highlights the significance of examining receptor-ligand interactions at physiologically relevant temperatures and reveals biophysical insight into the temperature dependence of P. falciparum malaria cytoadherent bonds.     

Transgenic Mosquitoes Expressing a Phospholipase A2 Gene Have a Fitness Advantage When Fed Plasmodium falciparum-Infected Blood

Background

Genetically modified mosquitoes have been proposed as an alternative strategy to reduce the heavy burden of malaria. In recent years, several proof-of-principle experiments have been performed that validate the idea that mosquitoes can be genetically modified to become refractory to malaria parasite development.

Results

We have created two transgenic lines of Anopheles stephensi , a natural vector of Plasmodium falciparum, which constitutively secrete a catalytically inactive phospholipase A₂ (mPLA₂) into the midgut lumen to interfere with Plasmodium ookinete invasion. Our experiments show that both transgenic lines expressing mPLA₂ significantly impair the development of rodent malaria parasites, but only one line impairs the development of human malaria parasites. In addition, when fed on malaria-infected blood, mosquitoes from both transgenic lines are more fecund than non-transgenic mosquitoes. Consistent with these observations, cage experiments with mixed populations of transgenic and non-transgenic mosquitoes show that the percentage of transgenic mosquitoes increases when maintained on Plasmodium -infected blood.

Conclusions

Our results suggest that the expression of an anti-Plasmodium effector gene gives transgenic mosquitoes a fitness advantage when fed malaria-infected blood. These findings have important implications for future applications of transgenic mosquito technology in malaria control.     

The Measurement of Sodium Concentration in Human Red Blood Cells

Experiments are described which indicate that iodinated human serum albumin underestimates the amount of extracellular sodium trapped in the packed layer of red blood cells, when cells and plasma are separated by centrifugation. Sucrose-¹⁴C also underestimates the amount of trapped extracellular sodium, but the difference between the percentages of sucrose-¹⁴C and extracellular sodium trapped is constant and independent of mean relative centrifugal force. It is concluded that human red blood cell sodium concentration can be measured with accuracy (a) if trapped plasma sodium is estimated with radioisotopes of sodium and a correction made for entry of sodium into the cells, providing cells and plasma can be separated rapidly; (b) by the use of sucrose as a standard plasma marker to derive the amount of trapped plasma sodium; (c) by washing the cells with sodium-free solutions. Reported values for red blood cell sodium concentration in healthy adults are critically reviewed.     

新型人工红细胞的制备

目的:接枝淀粉包裹血红蛋白制备新型人造红细胞的代替品。方法:利用油酸接枝淀粉,在超声条件下下自组装,包裹天然牛血红蛋白,并鉴定其物理化学及生物学性能。测定包封率、红外光谱分析(FTIR)、电镜观察形态学及粒径,测定P50和Hill系数。结果:人工红细胞呈圆球形,平均粒径250nm,包封率高,具有良好的携氧、释氧能力。结论:成功制备了人工纳米红细胞,为进一步临床应用提供了基础。     

Viscoelastic Transient of Confined Red Blood Cells

The unique ability of a red blood cell to flow through extremely small microcapillaries depends on the viscoelastic properties of its membrane. Here, we study in vitro the response time upon flow startup exhibited by red blood cells confined into microchannels. We show that the characteristic transient time depends on the imposed flow strength, and that such a dependence gives access to both the effective viscosity and the elastic modulus controlling the temporal response of red cells. A simple theoretical analysis of our experimental data, validated by numerical simulations, further allows us to compute an estimate for the two-dimensional membrane viscosity of red blood cells, ηmem2D ∼ 10⁻⁷ N⋅s⋅m⁻¹. By comparing our results with those from previous studies, we discuss and clarify the origin of the discrepancies found in the literature regarding the determination of ηmem2D, and reconcile seemingly conflicting conclusions from previous works.     

De Novo Generated Human Red Blood Cells in Humanized Mice Support Plasmodium falciparum Infection

Immunodeficient mouse–human chimeras provide a powerful approach to study host specific pathogens like Plasmodium (P.) falciparum that causes human malaria. Existing mouse models of P. falciparum infection require repeated injections of human red blood cells (RBCs). In addition, clodronate lipsomes and anti-neutrophil antibodies are injected to suppress the clearance of human RBCs by the residual immune system of the immunodeficient mice. Engraftment of NOD-scid Il2rg^-/- mice with human hematopoietic stem cells leads to reconstitution of human immune cells. Although human B cell reconstitution is robust and T cell reconstitution is reasonable in the recipient mice, human RBC reconstitution is generally poor or undetectable. The poor reconstitution is mainly the result of a deficiency of appropriate human cytokines that are necessary for the development and maintenance of these cell lineages. Delivery of plasmid DNA encoding human erythropoietin and interleukin-3 into humanized mice by hydrodynamic tail-vein injection resulted in significantly enhanced reconstitution of erythrocytes. With this improved humanized mouse, here we show that P. falciparum infects de novo generated human RBCs, develops into schizonts and causes successive reinvasion. We also show that different parasite strains exhibit variation in their ability to infect these humanized mice. Parasites could be detected by nested PCR in the blood samples of humanized mice infected with P. falciparum K1 and HB3 strains for 3 cycles, whereas in other strains such as 3D7, DD2, 7G8, FCR3 and W2mef parasites could only be detected for 1 cycle. In vivo adaptation of K1 strain further improves the infection efficiency and parasites can be detected by microscopy for 3 cycles. The parasitemia ranges between 0.13 and 0.25% at the first cycle of infection, falls between 0.08 and 0.15% at the second cycle, and drops to barely detectable levels at the third cycle of infection. Compared to existing mouse models, our model generates human RBCs de novo and does not require the treatment of mice with immunomodulators.     

Liver Accumulation of Plasmodium chabaudi-Infected Red Blood Cells and Modulation of Regulatory T Cell and Dendritic Cell Responses

It is postulated that accumulation of malaria-infected Red Blood Cells (iRBCs) in the liver could be a parasitic escape mechanism against full destruction by the host immune system. Therefore, we evaluated the in vivo mechanism of this accumulation and its potential immunological consequences. A massive liver accumulation of P. c. chabaudi AS-iRBCs (Pc-iRBCs) was observed by intravital microscopy along with an over expression of ICAM-1 on day 7 of the infection, as measured by qRT-PCR. Phenotypic changes were also observed in regulatory T cells (Tregs) and dendritic cells (DCs) that were isolated from infected livers, which indicate a functional role for Tregs in the regulation of the liver inflammatory immune response. In fact, the suppressive function of liver-Tregs was in vitro tested, which demonstrated the capacity of these cells to suppress naive T cell activation to the same extent as that observed for spleen-Tregs. On the other hand, it is already known that CD4+ T cells isolated from spleens of protozoan parasite-infected mice are refractory to proliferate in vivo. In our experiments, we observed a similar lack of in vitro proliferative capacity in liver CD4+ T cells that were isolated on day 7 of infection. It is also known that nitric oxide and IL-10 are partially involved in acute phase immunosuppression; we found high expression levels of IL-10 and iNOS mRNA in day 7-infected livers, which indicates a possible role for these molecules in the observed immune suppression. Taken together, these results indicate that malaria parasite accumulation within the liver could be an escape mechanism to avoid sterile immunity sponsored by a tolerogenic environment.     

Phosphate Compounds in Vertebrate Red Blood Cells

This review brings together and discusses the significance ofexisting information about water-soluble (small molecule) organicphosphate constituents of red blood cells in different vertebratespecies, with emphasis on 2,3 diphosphoglycerate (DPG), inositolpentaphosphate (IP₅) ATP and guanosine triphosphate (GTP), compoundswhich may play an important role in respiratory physiology bymodifying the affinity of hemoglobin for oxygen. Results onthe distribution and concentration of these compounds in redcells of vertebrate animals can be summarized as follows 1)DPG High in mammals (except cats and ruminants) Absent in crocodilianssquamata and fishes. High briefly in the bird embryo absentin adult. High briefly in turtle embryo low in juvenile lowto absent in adult 2 IP₅. High in birds. Absent in mammals,crocodilians squamata and fishes (with the exception of Arapaimagigas). Low in turtles 3 ATP Intermediate in mammals. High inbirds and turtles. Very high in squamata Intermediate to veryhigh in fishes. Low in crocodilians 4) GTP Very low in mammalsbirds, reptiles and amphibians (except for small pool in Ranatadpole). Low to very high in fishes.     

Theory of the Sphering of Red Blood Cells

A rigorous mathematical solution of the sphering of a red blood cell is obtained under the assumptions that the red cells is a fluid-filled shell and that it can swell into a perfect sphere in an appropriate hypotonic medium. The solution is valid for finite strain of the cell membrane provided that the membrane is isotropic, elastic and incompressible. The most general nonlinear elastic stress-strain law for the membrane in a state of generalized plane stress is used. A necessary condition for a red cell to be able to sphere is that its extensional stiffness follow a specific distribution over the membrane. This distribution is strongly influenced by the surface tension in the cell membrane. A unique relation exists between the extensional stiffness, pressure differential, surface tension, and the ratio of the radius of the sphere to that of the undeformed red cell. The functional dependence of this stiffness distribution on various physical parameters is presented. A critique of some current literature on red cell mechanics is presented.     

The Entry of Sodium into Human Red Blood Cells in Vivo

The kinetics of sodium, movement into human red blood cells has been studied in vivo with ²⁴Na. When human serum albumin^-131I is used to measure the percentage of plasma trapped in the packed red blood cells after centrifugation, approximately 30 % of red blood cell sodium is found to equilibrate immediately with plasma. It is concluded that this immediately exchangeable compartment of red blood cell sodium is an experimental artefact, associated with the use of labeled albumin for measuring plasma trapping. This immediately exchangeable fraction disappears when sucrose-¹⁴C is used to measure plasma trapping. The experimental results were examined by compartmental analysis, using an analogue computer. The results obtained, when plasma trapping was measured with sucrose-¹⁴C could be simulated by the use of models containing two compartments, arranged in series or in parallel. The errors of the techniques used and the possible physical basis for the results are discussed.     

Hemagglutination Inhibition of Passively Sensitized Red Blood Cells

It has been shown that sheep red blood cells sensitized with the polysaccharides of Haemophilus influenzae type B and pneumococcal types I, III, IV, VII, VIII, XII, and XIV respond readily in hemagglutination-inhibition testing and exhibit antigen inhibition at levels of 0.09 to 4.0 ng.     

Stretching and Relaxation of Malaria-Infected Red Blood Cells

The invasion of red blood cells (RBCs) by malaria parasites is a complex dynamic process, in which the infected RBCs gradually lose their deformability and their ability to recover their original shape is greatly reduced with the maturation of the parasites. In this work, we developed two types of cell model, one with an included parasite, and the other without an included parasite. The former is a representation of real malaria-infected RBCs, in which the parasite is treated as a rigid body. In the latter, where the parasite is absent, the membrane modulus and viscosity are elevated so as to produce the same features present in the parasite model. In both cases, the cell membrane is modeled as a viscoelastic triangular network connected by wormlike chains. We studied the transient behaviors of stretching deformation and shape relaxation of malaria-infected RBCs based on these two models and found that both models can generate results in agreement with those of previously published studies. With the parasite maturation, the shape deformation becomes smaller and smaller due to increasing cell rigidity, whereas the shape relaxation time becomes longer and longer due to the cell’s reduced ability to recover its original shape.