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Biomedical subjects

C I Newbold

Publications and source records attributed to C I Newbold.

At least 19 recordsLinked to original sources
PubMed2003

Reevaluation of flow cytometry for investigating antibody binding to the surface of Plasmodium falciparum trophozoite-infected red blood cells.

BACKGROUND: The acquisition of antibodies directed toward variant surface antigens (VSAs) expressed on the surface of the trophozoite-infected red blood cell is an important determinant of natural immunity to Plasmodium falciparum malaria. In recent years, flow cytometry has been used increasingly to investigate these responses, but few systematic assessments of this method are available in the published literature. METHODS: We developed a highly standardized experimental protocol and used parasites of the A4 laboratory clone, a monoclonal antibody to the VSA expressed by this clone (monoclonal antibody BC6), and a single pool of hyperimmune plasma to explore the parameters responsible for variations in VSA antibody responses measured by flow cytometry. RESULTS: Despite strenuous efforts to standardize our flow cytometric assay, we found marked variability in our assay readout, even between repeat experiments using identical antibody and parasite combinations. We found no remediable cause for much of this variability. However, we identified three major factors that we considered important contributors: antibody concentration, nonspecific antibody binding to uninfected red blood cells, and parasite agglutination. CONCLUSIONS: A number of potential pitfalls should be considered when designing and interpreting studies using this technique. In particular, we suggest that comparisons between assays conducted on different occasions can be made only through reference to carefully selected standards. We anticipate that a better appreciation of the factors that lead to assay variation will assist the design of improved experimental protocols.

Animals↗
PubMed2000

Mapping of the region of complement receptor (CR) 1 required for Plasmodium falciparum rosetting and demonstration of the importance of CR1 in rosetting in field isolates.

The malaria parasite Plasmodium falciparum induces a number of novel adhesion properties in the erythrocytes that it infects. One of these properties, the ability of infected erythrocytes to bind uninfected erythrocytes to form rosettes, is associated with severe malaria and may play a direct role in the pathogenesis of disease. Previous work has shown that erythrocytes deficient in complement receptor (CR) 1 (CR1, CD35; C3b/C4b receptor) have greatly reduced rosetting capacity, indicating an essential role for CR1 in rosette formation. Using deletion mutants and mAbs, we have localized the region of CR1 required for the formation of P. falciparum rosettes to the area of long homologous repeat regions B and C that also acts as the binding site for the activated complement component C3b. This result raises the possibility that C3b could be an intermediary in rosetting, bridging between the infected erythrocyte and CR1. We were able to exclude this hypothesis, however, as parasites grown in C3-deficient human serum formed rosettes normally. We have also shown in this report that rosettes can be reversed by mAb J3B11 that recognizes the C3b binding site of CR1. This rosette-reversing activity was demonstrated in a range of laboratory-adapted parasite strains and field isolates from Kenya and Malawi. Thus, we have mapped the region of CR1 required for rosetting and demonstrated that the CR1-dependent rosetting mechanism occurs commonly in P. falciparum isolates, and could therefore be a potential target for future therapeutic interventions to treat severe malaria.

Animals↗
PubMed2000

The mechanisms of parasite clearance after antimalarial treatment of Plasmodium falciparum malaria.

Studies were conducted to determine how malaria parasites are cleared from the blood after antimalarial treatment. Neither artesunate nor quinine decreased parasitized red cell deformability or increased antibody binding. In acute falciparum malaria, ring-infected erythrocyte surface antigen (RESA) was observed in erythrocytes without malaria parasites (RESA-red blood cell [RBC]), indicating prior parasitization. In uncomplicated malaria, RESA-RBC numbers increased significantly (P=.002) within 24 h of starting artesunate but rose much more slowly (7 days) after quinine treatment. In severe malaria, RESA-RBC increased significantly (P=. 001) within hours of starting artesunate but not with quinine treatment (P=.43). RESA-RBCs were not produced after drug treatment of malaria parasite cultures in vitro. Rapid malaria parasite clearance after treatment with artemisinin derivatives results mainly from the extraction of drug-affected parasites from host erythrocytes-presumably by the spleen. This explains why the fall in hematocrit after treatment of hyperparasitemia is often less than that predicted from loss of parasitized cells.

Animals↗
PubMed2000

Sulfated glycoconjugates enhance CD36-dependent adhesion of Plasmodium falciparum-infected erythrocytes to human microvascular endothelial cells.

A novel adhesive pathway that enhances the adhesion of Plasmodium falciparum-infected erythrocytes (IEs) to endothelial cells has been identified. The sulfated glycoconjugates heparin, fucoidan, dextran sulfate 5000, and dextran sulfate 500 000 caused a dramatic increase in adhesion of IEs to human dermal microvascular endothelial cells. The same sulfated glycoconjugates had little effect on IE adhesion to human umbilical vein endothelial cells, a CD36-negative cell line. The effect was abolished by a monoclonal antibody directed against CD36, suggesting that enhanced adhesion to endothelium is dependent on CD36. No effect was observed on adhesion to purified platelet CD36 cells immobilized on plastic. The same sulfated glycoconjugates enhanced adhesion of infected erythrocytes to COS cells transfected with CD36, and this was inhibited by the CD36 monoclonal antibody. These findings demonstrate a role for sulfated glycoconjugates in endothelial adherence that may be important in determining the location and magnitude of sequestration through endogenous carbohydrates. In addition, they highlight possible difficulties that may be encountered from the proposed use of sulfated glycoconjugates as antiadhesive agents in patients with severe malaria.

Animals↗
PubMed2000

Plasmodium falciparum-infected erythrocytes: agglutination by diverse Kenyan plasma is associated with severe disease and young host age.

The variant surface antigens (VSAs) of Plasmodium falciparum-infected red blood cells are potentially important targets of naturally acquired immunity to malaria. Natural infections induce agglutinating antibodies specific to the VSA variants expressed by the infecting parasites. Previously, when different parasite isolates were tested against a panel of heterologous plasma from Kenyan children, the proportion of plasma that agglutinated the parasites (the agglutination frequency [AF]) was highly variable among isolates, suggesting the existence of rare and prevalent variants. Here, the AF of 115 isolates from Kenyan children were compared. The results show that the AF of isolates causing severe malaria were significantly higher than those of isolates causing mild malaria; and AF decreased significantly with the increasing age of the infected child. We propose that parasites causing severe disease tend to express a subset of VSA variants that are preferentially associated with infections of children with low immunity.

Agglutination Tests↗
PubMed2000

Identification of a Plasmodium falciparum intercellular adhesion molecule-1 binding domain: a parasite adhesion trait implicated in cerebral malaria.

Binding of infected erythrocytes to brain venules is a central pathogenic event in the lethal malaria disease complication, cerebral malaria. The only parasite adhesion trait linked to cerebral sequestration is binding to intercellular adhesion molecule-1 (ICAM-1). In this report, we show that Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) binds ICAM-1. We have cloned and expressed PfEMP1 recombinant proteins from the A4tres parasite. Using heterologous expression in mammalian cells, the minimal ICAM-1 binding domain was a complex domain consisting of the second Duffy binding-like (DBL) domain and the C2 domain. Constructs that contained either domain alone did not bind ICAM-1. Based on phylogenetic criteria, there are five distinct PfEMP1 DBL types designated alpha, beta, gamma, delta, and epsilon. The DBL domain from the A4tres that binds ICAM-1 is DBLbeta type. A PfEMP1 cloned from a distinct ICAM-1 binding variant, the A4 parasite, contains a DBLbeta domain and a C2 domain in tandem arrangement similar to the A4tres PfEMP1. Anti-PfEMP1 antisera implicate the DBLbeta domain from A4var PfEMP1 in ICAM-1 adhesion. The identification of a P. falciparum ICAM-1 binding domain may clarify mechanisms responsible for the pathogenesis of cerebral malaria and lead to interventions or vaccines that reduce malarial disease.

Amino Acid Sequence↗
PubMed2000

A study of var gene transcription in vitro using universal var gene primers.

The polymorphic multigene family, var, encodes the variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), present on the surface of erythrocytes infected with the human malaria parasite, P. falciparum. PfEMP1 has been implicated in the pathology of malaria through its ability to bind to host endothelial receptors and uninfected erythrocytes. Understanding the relationship between host pathology, immune response and parasite variation is crucial, but requires a method of reliably detecting and differentiating all possible var genes. Several primer pairs used to date are biased and limited in their detection capacity. Here we describe a set of PCR primers that amplify the majority of var genes in the laboratory isolates 3D7 and A4, and appear to work equally well on all isolates tested. We use these universal primers to examine the relationship between var gene transcription as assessed by reverse transcriptase-PCR (RT-PCR) with that measured by Northern analysis of parasite RNA. Phenotypically selected young parasites have multiple transcripts detected by RT-PCR, but the full-length transcript appears to be homogeneous. In addition, we demonstrate that the choice of primers used for RT-PCR is crucial in data interpretation.

Amino Acid Sequence↗
PubMed2000

Intraerythrocytic polyubiquitin expression in Plasmodium falciparum is subjected to developmental and heat-shock control.

The polyubiquitin gene of the human protozoan parasite Plasmodium falciparum (PfpUB) was cloned and shown to be comprised of five tandem repeats of the ubiquitin open reading frame, present as a single copy on chromosome 12. The 1672 bp of PfpUB is interrupted at the 5' end by a single intron of 526 bp. PfpUB expression is developmentally regulated in intraerythrocytic stages with a marked increase in both steady-state transcript and polyubiquitin protein levels in late trophozoite stages. On response to heat shock, late stage parasites (late trophozoites and schizonts) have a slightly elevated PfpUB transcript level as well as readily observable increases in the amount of polyubiquitin and ubiquitin-conjugated proteins.

Amino Acid Sequence↗
PubMed2000

Genetic restriction of Plasmodium falciparum in an area of stable transmission: an example of island evolution?

To date, a high degree of polymorphism has been demonstrated at both the MSP1 and MSP2 loci in parasites from areas of stable malaria transmission. As a consequence, in such areas it is rare to find parasites of the same 2-locus genotype in more than 1 subject. We have studied MSP1 and MSP2 diversity in parasites collected from subjects with both symptomatic (n = 86) and asymptomatic (34) malaria living on the island of Santo, Vanuatu, an area of stable malaria transmission. Polymorphism at the MSP1 and MSP2 loci was considerably less than previously reported: only 5 MSP1 and 5 MSP2 alleles were detected and these showed no size variation within alleles. Santo is unique amongst the areas studied so far in that it is a small island at the limit of the malaria belt in the South Pacific. Thus, the evolution of the parasite population may have been affected by the small size and isolation of this island population. Moreover, limited parasite diversity may explain the unusually mild nature of Plasmodium falciparum disease on Santo. Islands have fascinated biologists for centuries and fuelled the advancement of evolutionary theory, since they are natural laboratories for the study of evolution. The simplicity of the Vanuatu P. falciparum population may facilitate the use and interpretation of sequence level analyses to address the mechanisms by which genetic diversity is generated and maintained in natural populations.

Adolescent↗
PubMed2000

Differential binding of clonal variants of Plasmodium falciparum to allelic forms of intracellular adhesion molecule 1 determined by flow adhesion assay.

Adhesion of Plasmodium falciparum-infected erythrocytes to the endothelial ligand intercellular adhesion molecule 1 (ICAM-1) has been implicated in the pathogenesis of cerebral malaria. Recently, a high-frequency coding polymorphism in the N-terminal domain of ICAM-1 (ICAM-1(Kilifi)) that is associated with susceptibility to cerebral disease in Kenya has been described. Preliminary static adhesion assays suggested that two different selected P. falciparum lines, ITO4-A4 (A4) and ItG-ICAM (ItG), have different properties of binding to the natural variant proteins ICAM-1 and ICAM-1(Kilifi). Using a flow adhesion assay system, we have confirmed differences between the two lines in binding of parasitized erythrocytes to the variant ICAM-1 proteins. Total adhesion of ItG-infected erythrocytes to ICAM-1 and ICAM-1(Kilifi) is greater than that of A4-infected erythrocytes, and erythrocytes infected by both parasite strains show reduced binding to ICAM-1(Kilifi). However, under these physiologically relevant flow conditions, we have shown differences between A4 and ItG strains in dynamic rolling behavior on ICAM-1(Kilifi). The percentage of erythrocytes infected with A4 that roll on both ICAM-1 and ICAM-1(Kilifi) is greater than that of those infected with ItG. Also, the rolling velocity of A4-infected erythrocytes on ICAM-1(Kilifi) is markedly increased compared to that on ICAM-1, in contrast to the rolling velocity of ItG-infected erythrocytes, which is similar on both proteins. These findings suggest that different parasite lines can vary in their avidity for the same host ligand, which may have important consequences for the pathophysiology of P. falciparum malaria.

Alleles↗
PubMed1999

Rifins: a second family of clonally variant proteins expressed on the surface of red cells infected with Plasmodium falciparum.

Many pathogens evade the host immune response or adapt to their environment by expressing surface proteins that undergo rapid switching. In the case of Plasmodium falciparum, products of a multigene family known as var are expressed on the surface of infected red cells, where they undergo clonal antigenic variation and contribute to malaria pathogenesis by mediating adhesion to a variety of host endothelial receptors and to uninfected red blood cells by forming rosettes. Herein we show that a second gene family, rif, which is associated with var at subtelomeric sites in the genome, encodes clonally variant proteins (rifins) that are expressed on the infected red cell surface. Their high copy number, sequence variability, and red cell surface location indicate an important role for rifins in malaria host-parasite interaction.

Animals↗
PubMed1999

Antigenic variation in Plasmodium falciparum: mechanisms and consequences.

In the past year, the major advances in malaria antigenic variation have been concerned with the transcription and switching of variant antigen genes, and the functional expression of regions of the major variant antigen. Also, new variant gene families have been discovered as a result of the Malaria Genome Project.

Animals↗
PubMed1998

Analysis of adhesive domains from the A4VAR Plasmodium falciparum erythrocyte membrane protein-1 identifies a CD36 binding domain.

The A4VAR is a variant antigen expressed by a clonal line that binds CD36 and intercellular adhesion molecule-1, ICAM-1. We have cloned and sequenced the extracellular domain coded by the A4var gene. To probe the relationship between A4var expression and parasite adhesion to ICAM-1, var mRNA and protein expression were analyzed in an enriched population of A4 parasites that displayed higher ICAM-1 binding. By Northern analyses, A4var was the predominant var message and antisera raised against a recombinant A4VAR protein reacted with the majority of infected erythrocytes, reinforcing previous conclusions that A4VAR binds ICAM-1. A4VAR contains five Duffy-binding like (DBL) domains, and two cysteine-rich interdomain regions (CIDR) domains. DBL and CIDR domains from A4VAR were expressed in mammalian cells to determine which regions mediate binding to CD36 and ICAM-1. Using several different binding assays, the A4VAR CIDR1 was the only domain found to bind CD36. In contrast, the same assays were unable to identify the ICAM-1 binding domain in A4VAR. This is the first time that each of the DBL and CIDR domains from a Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) have been systematically expressed and tested for binding. These results confirm that CIDR1 is sufficient to bind CD36 without any apparent contribution from other domains.

Animals↗
PubMed1998

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