Acquisition of IgG to ICAM-1-Binding DBLß Domains in the Plasmodium falciparum Erythrocyte Membrane Protein 1 Antigen Family Varies between Groups A, B, and C.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important malaria virulence factor. The protein family can be divided into clinically relevant subfamilies. ICAM-1-binding group A PfEMP1 proteins also bind endothelial protein C receptor and have been associated with cerebral malaria in children. IgG to these PfEMP1 proteins is acquired later in life than that to group A PfEMP1 not binding ICAM-1. The kinetics of acquisition of IgG to group B and C PfEMP1 proteins binding ICAM-1 is unclear and was studied here. Gene sequences encoding group B and C PfEMP1 with DBLß domains known to bind ICAM-1 were used to identify additional binders. Levels of IgG specific for DBLß domains from group A, B, and C PfEMP1 binding or not binding ICAM-1 were measured in plasma from Ghanaian children with or without malaria. Seven new ICAM-1-binding DBLß domains from group B and C PfEMP1 were identified. Healthy children had higher levels of IgG specific for ICAM-1-binding DBLß domains from group A than from groups B and C. However, the opposite pattern was found in children with malaria, particularly among young patients. Acquisition of IgG specific for DBLß domains binding ICAM-1 differs between PfEMP1 groups.

Natural and Vaccine-Induced Acquisition of Cross-Reactive IgG-Inhibiting ICAM-1-Specific Binding of a Plasmodium falciparum PfEMP1 Subtype Associated Specifically with Cerebral Malaria.

Cerebral malaria (CM) is a potentially deadly outcome of Plasmodium falciparum malaria that is precipitated by sequestration of infected erythrocytes (IEs) in the brain. The adhesion of IEs to brain endothelial cells is mediated by a subtype of parasite-encoded erythrocyte membrane protein 1 (PfEMP1) that facilitates dual binding to host intercellular adhesion molecule 1 (ICAM-1) and endothelial protein receptor C (EPCR). The PfEMP1 subtype is characterized by the presence of a particular motif (DBLß_motif) in the constituent ICAM-1-binding DBLß domain. The rate of natural acquisition of DBLß_motif-specific IgG antibodies and the ability to induce such antibodies by vaccination are unknown, and the aim of this study was to provide such data. We used an enzyme-linked immunosorbent assay (ELISA) to measure DBLß-specific IgG in plasma from Ghanaian children with malaria. The ability of human immune plasma and DBLß-specific rat antisera to inhibit the interaction between ICAM-1 and DBLß was assessed using ELISA and in vitro assays of IE adhesion under flow. The acquisition of DBLß_motif-specific IgG coincided with age-specific susceptibility to CM. Broadly cross-reactive antibodies inhibiting the interaction between ICAM-1 and DBLß_motif domains were detectable in immune plasma and in sera of rats immunized with specific DBLß_motif antigens. Importantly, antibodies against the DBLß_motif inhibited ICAM-1-specific in vitro adhesion of erythrocytes infected by four of five P. falciparum isolates from cerebral malaria patients. We conclude that natural exposure to P. falciparum as well as immunization with specific DBLß_motif antigens can induce cross-reactive antibodies that inhibit the interaction between ICAM-1 and a broad range of DBLß_motif domains. These findings raise hope that a vaccine designed specifically to prevent CM is feasible.

Mapping the Binding Site of a Cross-Reactive Plasmodium falciparum PfEMP1 Monoclonal Antibody Inhibitory of ICAM-1 Binding.

The virulence of Plasmodium falciparum is linked to the ability of infected erythrocytes (IE) to adhere to the vascular endothelium, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). In this article, we report the functional characterization of an mAb that recognizes a panel of PfEMP1s and inhibits ICAM-1 binding. The 24E9 mouse mAb was raised against PFD1235w DBLß3_D4, a domain from the group A PfEMP1s associated with severe malaria. 24E9 recognizes native PfEMP1 expressed on the IE surface and shows cross-reactivity with and cross-inhibition of the ICAM-1 binding capacity of domain cassette 4 PfEMP1s. 24E9 Fab fragments bind DBLß3_D4 with nanomolar affinity and inhibit ICAM-1 binding of domain cassette 4-expressing IE. The antigenic regions targeted by 24E9 Fab were identified by hydrogen/deuterium exchange mass spectrometry and revealed three discrete peptides that are solvent protected in the complex. When mapped onto a homology model of DBLß3_D4, these cluster to a defined, surface-exposed region on the convex surface of DBLß3_D4. Mutagenesis confirmed that the site most strongly protected is necessary for 24E9 binding, which is consistent with a low-resolution structure of the DBLß3_D4::24E9 Fab complex derived from small-angle x-ray scattering. The convex surface of DBLß3_D4 has previously been shown to contain the ICAM-1 binding site of DBLß domains, suggesting that the mAb acts by occluding the ICAM-1 binding surface. Conserved epitopes, such as those targeted by 24E9, are promising candidates for the inclusion in a vaccine interfering with ICAM-1-specific adhesion of group A PfEMP1 expressed by P. falciparum IE during severe malaria.

A novel domain cassette identifies Plasmodium falciparum PfEMP1 proteins binding ICAM-1 and is a target of cross-reactive, adhesion-inhibitory antibodies.

Cerebral Plasmodium falciparum malaria is characterized by adhesion of infected erythrocytes (IEs) to the cerebral microvasculature. This has been linked to parasites expressing the structurally related group A subset of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of IE adhesion ligands and to IEs with affinity for ICAM-1. However, recent evidence has cast doubt on both these associations, tempering hopes of the feasibility of developing a vaccine based on ICAM-1-binding PfEMP1. In this study, we report the identification of a domain cassette (DC) present in group A var genes from six genetically distinct P. falciparum parasites. The three domains in the cassette, which we call DC4, had a high level of sequence identity and cluster together phylogenetically. Erythrocytes infected by these parasites and selected in vitro for expression of DC4 adhered specifically to ICAM-1. The ICAM-1-binding capacity of DC4 was mapped to the C-terminal third of its Duffy-binding-like ß3 domain. DC4 was the target of broadly cross-reactive and adhesion-inhibitory IgG Abs, and levels of DC4-specific and adhesion-inhibitory IgG increased with age among P. falciparum-exposed children. Our study challenges earlier conclusions that group A PfEMP1 proteins are not central to ICAM-1-specific IE adhesion and support the feasibility of developing a vaccine preventing cerebral malaria by inhibiting cerebral IE sequestration.

Cytochrome b5 coexpression increases Tetrahymena thermophila Δ6 fatty acid desaturase activity in Saccharomyces cerevisiae.

Very-long-chain polyunsaturated fatty acids such as arachidonic, eicosapentaenoic, and docosahexaenoic acids, are important to the physiology of many microorganisms and metazoans and are vital to human development and health. The production of these and related fatty acids depends on Δ6 desaturases, the final components of an electron transfer chain that introduces double bonds into 18-carbon fatty acid chains. When a Δ6 desaturase identified from the ciliated protist Tetrahymena thermophila was expressed in Saccharomyces cerevisiae cultures supplemented with the 18:2(Δ9,12) substrate, only 4% of the incorporated substrate was desaturated. Cytochrome b5 protein sequences identified from the genome of T. thermophila included one sequence with two conserved cytochrome b5 domains. Desaturation by the Δ6 enzyme increased as much as 10-fold when T. thermophila cytochrome b5s were coexpressed with the desaturase. Coexpression of a cytochrome b5 from Arabidopsis thaliana with the Δ6 enzyme also increased desaturation. A split ubiquitin growth assay indicated that the strength of interaction between cytochrome b5 proteins and the desaturase plays a vital role in fatty acid desaturase activity, illustrating the importance of protein-protein interactions in this enzyme activity.

A fragile effect: The influence of episodic memory on delay discounting.

Delay discounting occurs when a reward loses value as a function of delay. Episodic future thinking (EFT) reliably decreases delay discounting. EFT may share cognitive features with recalling episodic memories such as constructive episodic simulation. We therefore explored whether recalling episodic memories also reduces delay discounting. In Experiment 1, participants wrote about episodic memories and recalled those memories before completing a delay discounting task. Episodic memories reduced delay discounting according to one commonly used delay discounting measure (area under the curve) but not another (using the hyperbolic model). Experiment 2 compared the effects of general and episodic memories. Neither general nor episodic memories significantly decreased delay discounting compared with a control "counting" condition, but episodic memories reduced delay discounting compared with general memories under some conditions. In Experiment 3, episodic memories did not decrease delay discounting compared with three other control conditions while EFT did. Experiment 3 therefore found that thinking must be both episodic and future orientated to reduce delay discounting. Together, these results suggest that episodic thinking is not sufficient to reliably decrease delay discounting, rather, features unique to episodic future thinking are required. Episodic memory might reduce delay discounting in some contexts, but this effect is small and fragile.

The better model to predict and improve pediatric health care quality: performance or importance-performance?

BACKGROUND: The perpetual search for ways to improve pediatric health care quality has resulted in a multitude of assessments and strategies; however, there is little research evidence as to their conditions for maximum effectiveness. A major reason for the lack of evaluation research and successful quality improvement initiatives is the methodological challenge of measuring quality from the parent perspective. PURPOSE: Comparison of performance-only and importance-performance models was done to determine the better predictor of pediatric health care quality and more successful method for improving the quality of care provided to children. APPROACH: Fourteen pediatric health care centers serving approximately 250,000 patients in 70,000 households in three West Central Florida counties were studied. A cross-sectional design was used to determine the importance and performance of 50 pediatric health care attributes and four global assessments of pediatric health care quality. Exploratory factor analysis revealed five dimensions of care (physician care, access, customer service, timeliness of services, and health care facility). Hierarchical multiple regression compared the performance-only and the importance-performance models. In-depth interviews, participant observations, and a direct cognitive structural analysis identified 50 health care attributes included in a mailed survey to parents(n = 1,030). The tailored design method guided survey development and data collection. FINDINGS: The importance-performance multiplicative additive model was a better predictor of pediatric health care quality. PRACTICE IMPLICATIONS: Attribute importance moderates performance and quality, making the importance-performance model superior for measuring and providing a deeper understanding of pediatric health care quality and a better method for improving the quality of care provided to children. Regardless of attribute performance, if the level of attribute importance is not taken into consideration, health care organizations may spend valuable resources targeting the wrong areas for improvement. Consequently, this finding aids in health care quality research and policy decisions on organizational improvement strategies.

Immunomagnetic Selection of Plasmodium falciparum-Infected Erythrocytes Expressing Particular PfEMP1 Variants.

Plasmodium falciparum expresses a broad range of proteins on the surface of infected erythrocytes (IEs), including members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. This protocol describes an immunomagnetic selection method using PfEMP1-specific antibodies to obtain a parasite clone homogenously expressing a particular PfEMP1 protein. The expression of the corresponding PfEMP1 is later tested by flow cytometry, and the selected parasites can be used for further analysis.

Receptor Affinity-Based Purification of PfEMP1 Proteins.

The virulence of Plasmodium falciparum is linked to the ability of infected erythrocytes (IEs) to bind a range of human receptors. This binding is mediated by a family of highly polymorphic proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 proteins are expressed on the surface of IEs and are composed of extracellular domains (NTS, CIDR, DBL), a transmembrane region and an acidic C-terminal segment. Subdomains of the extracellular N-terminal part of PfEMP1 molecules have been shown to bind specific receptors.In this chapter, we describe how to purify PfEMP1 proteins by a receptor affinity-based method. This includes how to prepare affinity columns and how to subsequently test the functionality of the purified PfEMP1 protein in an ELISA-based assay.

Affinity Purification of PfEMP1-Specific Antibodies from Human Blood.

Acquired immunity against Plasmodium falciparum infections relies heavily on IgG antibodies specific for PfEMP1 proteins expressed on the surface of infected erythrocytes. Purified human antibodies can be used, for example, to study the interactions between specific PfEMP1 proteins and receptors expressed by human endothelial cells, and to identify which IgG antibodies play a functional role in natural acquired immunity.This chapter describe how to affinity purify PfEMP1-specific human antibodies on an affinity column coupled with PfEMP1 protein. We include ELISA-based methods for identification of human plasma samples reactive against PfEMP1, and for testing of affinity purified IgG antibodies prior to their use in more advanced procedures.

Production of anti-PfEMP1 Polyclonal Antisera in Rats and Mice.

Plasmodium falciparum-infected erythrocytes (IEs) bind various host receptors via members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on the surface of the IEs. Antibody reagents are needed to investigate interactions between specific PfEMP1 proteins and receptors expressed by human endothelial cells. This protocol describes the production of rat and mouse polyclonal anti-PfEMP1 antibodies. Polyclonal antibodies are relatively easy to produce and have advantages compared to monoclonal antibodies (see Chapters 28 - 30 ) for some applications. An ELISA-based method to test the polyclonal antibodies before their use in more advanced procedures is also presented.

Analysis of Antibody Inhibition of PfEMP1 Binding by Competition ELISA.

Plasmodium falciparum express variant antigens on the surface of infected erythrocytes (IEs). These include proteins of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. PfEMP1 proteins mediate binding of IEs to various human endothelial receptors and induce antibodies response during natural infections. In this chapter, we describe a competition ELISA that is an assay for antibodies neutralizing binding of PfEMP1 proteins to their cognate receptor.

ICAM-1-binding Plasmodium falciparum erythrocyte membrane protein 1 variants elicits opsonic-phagocytosis IgG responses in Beninese children.

Members of the highly polymorphic Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on the surface of infected erythrocytes (IEs) are important virulence factors, which mediate vascular adhesion of IEs via endothelial host receptors and are targets of naturally acquired immunity. The PfEMP1 family can be divided into clinically relevant subgroups, of which some bind intercellular adhesion molecule 1 (ICAM-1). While the acquisition of IgG specific for ICAM-1-binding DBLß domains is known to differ between PfEMP1 groups, its ability to induce antibody-dependent cellular phagocytosis (ADCP) is unclear. We therefore measured plasma levels of DBLß-specific IgG, the ability of such IgG to inhibit PfEMP1-binding to ICAM-1, and its ability to opsonize IEs for ADCP, using plasma from Beninese children with severe (SM) or uncomplicated malaria (UM). IgG specific for DBLß from group A and B ICAM-1-binding PfEMP1 were dominated by IgG1 and IgG3, and were similar in SM and UM. However, levels of plasma IgG inhibiting ICAM-1-binding of group A DBLß of PFD1235w was significantly higher in children with UM than SM, and acute UM plasma induced a higher ADCP response than acute SM plasma.

Pellicular particles with spherical carbon cores and porous nanodiamond/polymer shells for reversed-phase HPLC.

A new stationary phase for reversed-phase high performance liquid chromatography (RP HPLC) was created by coating spherical 3 µm carbon core particles in a layer-by-layer (LbL) fashion with poly(allylamine) (PAAm) and nanodiamond. Unfunctionalized core carbon particles were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and Raman spectroscopy. After LbL of PAAm and nanodiamond, which yields ca. 4 µm core-shell particles, the particles were simultaneously functionalized and cross-linked using a mixture of 1,2-epoxyoctadecane and 1,2,7,8-diepoxyoctane to obtain a mechanically stable C(18)/C(8) bonded outer layer. Core-shell particles were characterized by SEM, and their surface area, pore diameter, and volume were determined using the Brunauer-Emmett-Teller (BET) method. Short stainless steel columns (30 × 4.6 mm i.d.) were packed and the corresponding van Deemter plots obtained. The Supporting Information contains a MATLAB program used to fit the van Deemter data. The retentions of a suite of analytes were investigated on a conventional HPLC at various organic solvent compositions, pH values of mobile phases, including extreme pH values, and column temperatures. At 60 °C, a chromatogram of 2,6-diisopropylphenol showed 71,500 plates/m (N/m). Chromatograms obtained under acidic conditions (pH 2.7) of a mixture of acetaminophen, diazepam, and 2,6-diisopropylphenol and a mixture of phenol, 4-methylphenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol, and 1-tert-butyl-4-methylphenol are presented. Retention of amitriptyline, cholesterol, and diazinon at temperatures ranging from 35 to 80 °C and at pH 11.3 is reported. A series of five basic drugs was also separated at this pH. The stationary phase exhibits considerable hydrolytic stability at high pH (11.3) and even pH 13 over extended periods of time. An analysis run on a UHPLC with a "sandwich" injection appeared to reduce extra column band broadening and gave best efficiencies of 110,000-120,000 N/m.

Plasmodium falciparum erythrocyte membrane protein 1 variants induce cell swelling and disrupt the blood-brain barrier in cerebral malaria.

Cerebral malaria (CM) is caused by the binding of Plasmodium falciparum-infected erythrocytes (IEs) to the brain microvasculature, leading to inflammation, vessel occlusion, and cerebral swelling. We have previously linked dual intercellular adhesion molecule-1 (ICAM-1)- and endothelial protein C receptor (EPCR)-binding P. falciparum parasites to these symptoms, but the mechanism driving the pathogenesis has not been identified. Here, we used a 3D spheroid model of the blood-brain barrier (BBB) to determine unexpected new features of IEs expressing the dual-receptor binding PfEMP1 parasite proteins. Analysis of multiple parasite lines shows that IEs are taken up by brain endothelial cells in an ICAM-1-dependent manner, resulting in breakdown of the BBB and swelling of the endothelial cells. Via ex vivo analysis of postmortem tissue samples from CM patients, we confirmed the presence of parasites within brain endothelial cells. Importantly, this discovery points to parasite ingress into the brain endothelium as a contributing factor to the pathology of human CM.

Antigenic and immunogenic evaluation of permutations of soluble hepatitis C virus envelope protein E2 and E1 antigens.

Yearly, about 1.5 million people become chronically infected with hepatitis C virus (HCV) and for the 71 million with chronic HCV infection about 400,000 die from related morbidities, including liver cirrhosis and cancer. Effective treatments exist, but challenges including cost-of-treatment and wide-spread undiagnosed infection, necessitates the development of vaccines. Vaccines should induce neutralizing antibodies (NAbs) against the HCV envelope (E) transmembrane glycoprotein 2, E2, which partly depends on its interaction partner, E1, for folding. Here, we generated three soluble HCV envelope protein antigens with the transmembrane regions deleted (i.e., fused peptide backbones), termed sE1E2 (E1 followed by E2), sE2E1 (E2 followed by E1), and sE21E (E2 followed by inverted E1). The E1 inversion for sE21E positions C-terminal residues of E1 near C-terminal residues of E2, which is in analogy to how they likely interact in native E1/E2 complexes. Probing conformational E2 epitope binding using HCV patient-derived human monoclonal antibodies, we show that sE21E was superior to sE2E1, which was consistently superior to sE1E2. This correlated with improved induction of NAbs by sE21E compared with sE2E1 and especially compared with sE1E2 in female BALB/c mouse immunizations. The deletion of the 27 N-terminal amino acids of E2, termed hypervariable region 1 (HVR1), conferred slight increases in antigenicity for sE2E1 and sE21E, but severely impaired induction of antibodies able to neutralize in vitro viruses retaining HVR1. Finally, comparing sE21E with sE2 in mouse immunizations, we show similar induction of heterologous NAbs. In summary, we find that C-terminal E2 fusion of E1 or 1E is superior to N-terminal fusion, both in terms of antigenicity and the induction of heterologous NAbs. This has relevance when designing HCV E1E2 vaccine antigens.

In vitro selection for adhesion of Plasmodium falciparum-infected erythrocytes to ABO antigens does not affect PfEMP1 and RIFIN expression.

Plasmodium falciparum causes the most severe form of malaria in humans. The adhesion of the infected erythrocytes (IEs) to endothelial receptors (sequestration) and to uninfected erythrocytes (rosetting) are considered major elements in the pathogenesis of the disease. Both sequestration and rosetting appear to involve particular members of several IE variant surface antigens (VSAs) as ligands, interacting with multiple vascular host receptors, including the ABO blood group antigens. In this study, we subjected genetically distinct P. falciparum parasites to in vitro selection for increased IE adhesion to ABO antigens in the absence of potentially confounding receptors. The selection resulted in IEs that adhered stronger to pure ABO antigens, to erythrocytes, and to various human cell lines than their unselected counterparts. However, selection did not result in marked qualitative changes in transcript levels of the genes encoding the best-described VSA families, PfEMP1 and RIFIN. Rather, overall transcription of both gene families tended to decline following selection. Furthermore, selection-induced increases in the adhesion to ABO occurred in the absence of marked changes in immune IgG recognition of IE surface antigens, generally assumed to target mainly VSAs. Our study sheds new light on our understanding of the processes and molecules involved in IE sequestration and rosetting.

Blood outgrowth endothelial cells (BOECs) as a novel tool for studying adhesion of Plasmodium falciparum-infected erythrocytes.

The lack of suitable animal models for the study of cytoadhesion of P. falciparum-infected erythrocytes (IEs) has necessitated in vitro studies employing a range of cell lines of either human tumour origin (e.g., BeWo and C32 cells) or non-human origin (e.g., CHO cells). Of the human cells available, many were isolated from adults, or derived from a pool of donors (e.g., HBEC-5i). Here we demonstrate, for the first time, the successful isolation of blood outgrowth endothelial cells (BOECs) from frozen stabilates of peripheral blood mononuclear cells obtained from small-volume peripheral blood samples from paediatric malaria patients. BOECs are a sub-population of human endothelial cells, found within the peripheral blood. We demonstrate that these cells express receptors such as Intercellular Adhesion Molecule 1 (ICAM-1/CD54), Endothelial Protein C Receptor (EPCR/CD201), platelet/endothelial cell adhesion molecule 1 (PECAM-1/CD31), Thrombomodulin (CD141), and support adhesion of P. falciparum IEs.

A good friend is hard to find: friendship among adolescents with disabilities.

We asked 27 Euro American teens ages 16 to 17 with developmental disabilities in Los Angeles to describe friendships. Eleven characteristics of friendship reported in the research literature (similarity, proximity, transcending context, companionship, reciprocity, mutuality, intimacy, support, trust/loyalty, conflict management, and stability) were mentioned by at least some teens. However, most teens focused on companionship, doing activities across contexts, similarity in interests/personality, sheer proximity, and stability. Gender did not influence number or types of themes reported. Teens with higher IQ/Vineland Communication scores mentioned more friendship themes and were less positive about their friendships. Most teens reported some satisfying friendships, and friendships between peers with developmental disability usually were more stable and positive than friendships with typically developing peers.