Comparison between nerve conduction study and high-resolution ultrasonography with color doppler in type 1 and type 2 leprosy reactions.

OBJECTIVE: To analyze the role of high-resolution ultrasonography with color Doppler (HRUS with CD) to diagnose inflammatory activity (IA) in nerves of leprosy patients under type 1 (RT1) and 2 (RT2) reactions compared to Nerve Conduction Studies (NCS). METHODS: Leprosy patients with signs or symptoms suggestive of neuritis (RT1 and RT2) without corticosteroids use were selected. They were evaluated by NCS and subsequently by HRUS with CD. Subacute segmental demyelination and the presence of blood flow, respectively, were considered signs of IA. The two methods were compared for their ability to diagnose patients with leprosy reactions. RESULTS: A total of 257 nerves from 35 patients were evaluated. NCS and HRUS with CD diagnosed IA in 68% and 74% of patients, respectively. When both methods were used concomitantly, the diagnosis rate was 91.4%. HRUS with CD was particular helpful when there was minimal neurophysiological compromise in NCS or when motor potentials were not detected. CONCLUSION: HRUS with CD was able to detect leprosy reactions, especially when combined with NCS. It was especially useful in two opposite situations: nerves with only minor changes and those without motor response in NCS. SIGNIFICANCE: Our data shows the usefulness of HRUS and CD, similar to NCS, as a tool to diagnose leprosy reactions.

How does cot death prevention advice influence parents' behaviour?

OBJECTIVE: To explore parents' understanding of the recommended cot death prevention strategies, and to discuss what they are doing in practice. If there is a difference between knowledge and implementation measures, possible reasons for this will be considered. STUDY DESIGN: A qualitative study using thematic analysis, aimed at finding out attitudes and opinions of parents about cot death prevention measures. Twelve participants were interviewed from two disadvantaged communities in south Birmingham. RESULTS: Parents found that much of the cot death prevention advice they were provided with was conflicting and caused confusion. As such, many parents chose to follow advice from non-healthcare sources. Some parents were carrying out preventative measures but were not aware of the reason for doing it. Many felt they did not receive enough advice relating to cot death prevention. CONCLUSION: Cot death health promotion advice appears to be inadequate among patients from a deprived socioeconomic background. Some of these issues could be resolved with increased training addressing these matters.

Are trials in New World monkeys on the critical path for blood-stage malaria vaccine development?

The development of malaria blood-stage vaccines is gathering momentum: there are several new funding initiatives, one multiantigen formulation is currently being tested and at least one other blood-stage vaccine is expected to begin trials in 2001. However, there is no consensus over the best way to select which form of an antigen to take into clinical testing. There is thus a danger that less-effective vaccines might be tested in the field in the order of their availability, rather than merit. Here, we argue that first proving efficacy in the New World monkey challenge model would accelerate development.

Modifications in the CD36 binding domain of the Plasmodium falciparum variant antigen are responsible for the inability of chondroitin sulfate A adherent parasites to bind CD36.

Adhesion of mature Plasmodium falciparum parasitized erythrocytes to microvascular endothelial cells or to placenta contributes directly to the virulence and severe pathology of P falciparum malaria. Whereas CD36 is the major endothelial receptor for microvasculature sequestration, infected erythrocytes adhering in the placenta bind chondroitin sulfate A (CSA) but not CD36. Binding to both receptors is mediated by different members of the large and diverse protein family P falciparum erythrocyte membrane protein-1 (PfEMP-1) and involves different regions of the molecule. The PfEMP-1-binding domain for CD36 resides in the cysteine-rich interdomain region 1 (CIDR-1). To explore why CSA-binding parasites do not bind CD36, CIDR-1 domains from CD36- or CSA-binding parasites were expressed in mammalian cells and tested for adhesion. Although CIDR-1 domains from CD36-adherent strains strongly bound CD36, those from CSA-adherent parasites did not. The CIDR-1 domain has also been reported to bind CSA. However, none of the CIDR-1 domains tested bound CSA. Chimeric proteins between CIDR-1 domains that bind or do not bind CD36 and mutagenesis experiments revealed that modifications in the minimal CD36-binding region (M2 region) are responsible for the inability of CSA-selected parasites to bind CD36. One of these modifications, mapped to a 3-amino acid substitution in the M2 region, ablated binding in one variant and largely reduced binding of another. These findings provide a molecular explanation for the inability of placental sequestered parasites to bind CD36 and provide additional insight into critical residues for the CIDR-1/CD36 interaction.

Malaria: a sporozoite runs through it.

A recent study reveals new insights into the development of Plasmodium sporozoites, the infectious agents of malaria. These findings may lead to changes in the approach to malaria vaccines and novel interpretations of the mechanisms of immunity to malaria.

Characterization of a Plasmodium falciparum erythrocyte-binding protein paralogous to EBA-175.

A member of a Plasmodium receptor family for erythrocyte invasion was identified on chromosome 13 from the Plasmodium falciparum genome sequence of the Sanger Centre (Cambridge, U.K.). The protein (named BAEBL) has homology to EBA-175, a P. falciparum receptor that binds specifically to sialic acid and the peptide backbone of glycophorin A on erythrocytes. Both EBA-175 and BAEBL localize to the micronemes, organelles at the invasive ends of the parasites that contain other members of the family. Like EBA-175, the erythrocyte receptor for BAEBL is destroyed by neuraminidase and trypsin, indicating that the erythrocyte receptor is a sialoglycoprotein. Its specificity, however, differs from that of EBA-175 in that BAEBL can bind to erythrocytes that lack glycophorin A, the receptor for EBA-175. It has reduced binding to erythrocytes with the Gerbich mutation found in another erythrocyte, sialoglycoprotein (glycophorin C/D). The interest in BAEBL's reduced binding to Gerbich erythrocytes derives from the high frequency of the Gerbich phenotype in some regions of Papua New Guinea where P. falciparum is hyperendemic.

Molecular identification of Knops blood group polymorphisms found in long homologous region D of complement receptor 1.

Complement receptor 1 (CR1) has been implicated in rosetting of uninfected red blood cells to Plasmodium falciparum-infected cells, and rosette formation is associated with severe malaria. The Knops blood group (KN) is located on CR1 and some of these antigens, ie, McCoy (McC) and Swain-Langley (Sl(a)), show marked frequency differences between Caucasians and Africans. Thus, defining the molecular basis of these antigens may provide new insight into the mechanisms of P falciparum malaria. Monoclonal antibody epitope mapping and serologic inhibition studies using CR1 deletion constructs localized McC and Sl(a) to long homologous repeat D of CR1. Direct DNA sequencing of selected donors identified several single nucleotide polymorphisms in exon 29 coding for complement control protein modules 24 and 25. Two of these appeared to be blood group specific: McC associated with K1590E and Sl(a) with R1601G. These associations were confirmed by inhibition studies using allele-specific mutants. A sequence-specific oligonucleotide probe hybridization assay was developed to genotype several African populations and perform family inheritance studies. Concordance between the 1590 mutation and McC was 94%; that between Sl(a) and 1601 was 88%. All but 2 samples exhibiting discrepancies between the genotype and phenotype were found to be due to low red cell CR1 copy numbers, low or absent expression of some alleles, or heterozygosity combined with low normal levels of CR1. These data further explain the variability observed in previous serologic studies of CR1 and show that DNA and protein-based genetic studies will be needed to clarify the role of the KN antigens in malaria.

Efficacy of two alternate vaccines based on Plasmodium falciparum merozoite surface protein 1 in an Aotus challenge trial.

In an attempt to produce a more defined, clinical-grade version of a vaccine based on Plasmodium falciparum merozoite surface protein 1 (MSP1), we evaluated the efficacy of two recombinant forms of MSP1 in an Aotus nancymai challenge model system. One recombinant vaccine, bvMSP1(42), based on the 42-kDa C-terminal portion of MSP1, was expressed as a secreted protein in baculovirus-infected insect cells. A highly pure baculovirus product could be reproducibly expressed and purified at yields in excess of 8 mg of pure protein per liter of culture. This protein, when tested for efficacy in the Aotus challenge model, gave significant protection, with only one of seven monkeys requiring treatment for uncontrolled parasitemia after challenge with P. falciparum. The second recombinant protein, P30P2MSP1(19), has been used in previous studies and is based on the smaller, C-terminal 19-kDa portion of MSP1 expressed in Saccharomyces cerevisiae. Substantial changes were made in its production process to optimize expression. The optimum form of this vaccine antigen (as judged by in vitro and in vivo indicators) was then evaluated, along with bvMSP1(42), for efficacy in the A. nancymai system. The new formulation of P30P3MSP1(19) performed significantly worse than bvMSP1(42) and appeared to be less efficacious than we have found in the past, with four of seven monkeys in the vaccinated group requiring treatment for uncontrolled parasitemia. With both antigens, protection was seen only when high antibody levels were obtained by formulation of the vaccines in Freund's adjuvant. Vaccine formulation in an alternate adjuvant, MF59, resulted in significantly lower antibody titers and no protection.

The surface variant antigens of Plasmodium falciparum contain cross-reactive epitopes.

Plasmodium falciparum parasites evade the host immune system by clonal expression of the variant antigen, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Antibodies to PfEMP1 correlate with development of clinical immunity but are predominantly variant-specific. To overcome this major limitation for vaccine development, we set out to identify cross-reactive epitopes on the surface of parasitized erythrocytes (PEs). We prepared mAbs to the cysteine-rich interdomain region 1 (CIDR1) of PfEMP1 that is functionally conserved for binding to CD36. Two mAbs, targeting different regions of CIDR1, reacted with multiple P. falciparum strains expressing variant PfEMP1s. One of these mAbs, mAb 6A2-B1, recognized nine of 10 strains tested, failing to react with only one strain that does not bind CD36. Flow cytometry with Chinese hamster ovary cells expressing variant CIDR1s demonstrated that both mAbs recognized the CIDR1 of various CD36-binding PfEMP1s and are truly cross-reactive. The demonstration of cross-reactive epitopes on the PE surface provides further credence for development of effective vaccines against the variant antigen on the surface of P. falciparum-infected erythrocytes.

Expression of heterologous promoters in Lutzomyia longipalpis and Phlebotomus papatasi (Diptera: Psychodidae) cell lines.

To establish a transient expression system for genes introduced into sand fly cell lines, we tested the expression of the luciferase reporter gene under control of different promoters. Towards this end, we lipofected cell lines obtained from New and Old World sand flies, LL-5 from Lutzomyia longipalpis Lutz & Neiva and PP-9 from Phlebotomus papatasi Scopoli, respectively. The relative levels of luciferase expression were studied under control of Drosophila melanogaster Meigen heat shock protein 70 (hsp70), human cytomegalovirus, simian virus 40 or Junonia coenia (Hübner) densovirus (P9) promoters. The Drosophila heat shock protein 70 promoter, originating from insect genes, functioned as a strong promoter in both cell lines. Promoters from the different virus genes also were capable of driving transgene expression in both cell lines.

Classification of adhesive domains in the Plasmodium falciparum erythrocyte membrane protein 1 family.

The Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family of cytoadherent proteins has a central role in disease from malaria infection. This highly diverse gene family is involved in binding interactions between infected erythrocytes and host cells and is expressed in a clonally variant pattern at the erythrocyte surface. We describe by sequence analysis the structure and domain organization of 20 PfEMP1 from the GenBank database. Four domains comprise the majority of PfEMP1 extracellular sequence: the N-terminal segment (NTS) located at the amino terminus of all PfEMP1, the C2, the Cysteine-rich Interdomain Region (CIDR) and the Duffy Binding-like (DBL) domains. Previous work has shown that CIDR and DBL domains can possess adhesive properties. CIDR domains grouped as three distinct sequence classes (alpha, beta, and gamma) and DBL domains as five sequence classes (alpha, beta, gamma, delta, and epsilon). Consensus motifs are described for the different DBL and CIDR types. Whereas the number of DBL and CIDR domains vary between PfEMP1, PfEMP1 domain architecture is not random in that certain tandem domain associations--such as DBLalphaCIDRalpha, DBLdeltaCIDRbeta, and DBLbetaC2--are preferentially observed. This conservation may have functional significance for PfEMP1 folding, transport, or binding activity. Parasite binding phenotype appears to be a determinant of infected erythrocyte tissue tropism that contributes to parasite survival, transmission, and disease outcome. The sequence classification of DBL and CIDR types may have predictive value for identifying PfEMP1 domains with a particular binding property. This information might be used to develop interventions targeting parasite binding variants that cause disease.

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.

Disruption of the C-terminal region of EBA-175 in the Dd2/Nm clone of Plasmodium falciparum does not affect erythrocyte invasion.

EBA-175 is a Plasmodium falciparum micronemal protein that binds to sialic acid in the context of the peptide backbone of glycophorin A and has been implicated in sialic acid-dependent invasion of erythrocytes. The existence of an alternative invasion pathway has been suggested by the finding that the P. falciparum clone Dd2/Nm can invade sialic acid-depleted erythrocytes. To study the role of EBA-175 in this alternative pathway, we have generated Dd2/Nm clones expressing a truncated form of EBA-175 that lacks region 6 and the cytoplasmic domain. The protein still appears to be localized to the apical end in the vicinity of the micronemes, suggesting that region 6 and the cytoplasmic domain are not involved in EBA-175 trafficking to the micronemes. In these genetically modified clones, the level of truncated EBA-175 protein expression was greatly reduced. EBA-175-disrupted clones displayed normal rates of invasion of untreated and enzyme-treated human and animal erythrocytes, suggesting a lack of involvement of EBA-175 in this alternative invasion pathway.

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.

International union of pharmacology. XXII. Nomenclature for chemokine receptors.

Chemokine receptors comprise a large family of seven transmembrane domain G protein-coupled receptors differentially expressed in diverse cell types. Biological activities have been most clearly defined in leukocytes, where chemokines coordinate development, differentiation, anatomic distribution, trafficking, and effector functions and thereby regulate innate and adaptive immune responses. Pharmacological analysis of chemokine receptors is at an early stage of development. Disease indications have been established in human immunodeficiency virus/acquired immune deficiency syndrome and in Plasmodium vivax malaria, due to exploitation of CCR5 and Duffy, respectively, by the pathogen for cell entry. Additional indications are emerging among inflammatory and immunologically mediated diseases, but selection of targets in this area still remains somewhat speculative. Small molecule antagonists with nanomolar affinity have been reported for 7 of the 18 known chemokine receptors but have not yet been studied in clinical trials. Virally encoded chemokine receptors, as well as chemokine agonists and antagonists, and chemokine scavengers have been identified in medically important poxviruses and herpesviruses, again underscoring the importance of the chemokine system in microbial pathogenesis and possibly identifying specific strategies for modulating chemokine action therapeutically. The purpose of this review is to update current concepts of the biology and pharmacology of the chemokine system, to summarize key information about each chemokine receptor, and to describe a widely accepted receptor nomenclature system, ratified by the International Union of Pharmacology, that is facilitating clear communication in this area.

Molecular characterization of Plasmodium falciparum S-adenosylmethionine synthetase.

S-Adenosylmethionine (AdoMet) synthetase (SAMS: EC 2.5.1.6) catalyses the formation of AdoMet from methionine and ATP. We have cloned a gene for Plasmodium falciparum AdoMet synthetase (PfSAMS) (GenBank accession no. AF097923), consisting of 1209 base pairs with no introns. The gene encodes a polypeptide (PfSAMS) of 402 amino acids with a molecular mass of 44844 Da, and has an overall base composition of 67% A+T. PfSAMS is probably a single copy gene, and was mapped to chromosome 9. The PfSAMS protein is highly homologous to all other SAMS, including a conserved motif for the phosphate-binding P-loop, HGGGAFSGKD, and the signature hexapeptide, GAGDQG. All the active-site amino acids for the binding of ADP, P(i) and metal ions are similarly preserved, matching entirely those of human hepatic SAMS and Escherichia coli SAMS. Molecular modelling of PfSAMS guided by the X-ray crystal structure of E. coli SAMS indicates that PfSAMS binds ATP/Mg(2+) in a manner similar to that seen in the E. coli SAMS structure. However, the PfSAMS model shows that it can not form tetramers as does E. coli SAMS, and is probably a dimer instead. There was a differential sensitivity towards the inhibition by cycloleucine between the expressed PfSAMS and the human hepatic SAMS with K(i) values of 17 and 10 mM, respectively. Based on phylogenetic analysis using protein parsimony and neighbour-joining algorithms, the malarial PfSAMS is closely related to SAMS of other protozoans and plants.

Plasmodium falciparum domain mediating adhesion to chondroitin sulfate A: a receptor for human placental infection.

Malaria during the first pregnancy causes a high rate of fetal and neonatal death. The decreasing susceptibility during subsequent pregnancies correlates with acquisition of antibodies that block binding of infected red cells to chondroitin sulfate A (CSA), a receptor for parasites in the placenta. Here we identify a domain within a particular Plasmodium falciparum erythrocyte membrane protein 1 that binds CSA. We cloned a var gene expressed in CSA-binding parasitized red blood cells (PRBCs). The gene had eight receptor-like domains, each of which was expressed on the surface of Chinese hamster ovary cells and was tested for CSA binding. CSA linked to biotin used as a probe demonstrated that two Duffy-binding-like (DBL) domains (DBL3 and DBL7) bound CSA. DBL7, but not DBL3, also bound chondroitin sulfate C (CSC) linked to biotin, a negatively charged sugar that does not support PRBC adhesion. Furthermore, CSA, but not CSC, blocked the interaction with DBL3; both CSA and CSC blocked binding to DBL7. Thus, only the DBL3 domain displays the same binding specificity as PRBCs. Because protective antibodies present after pregnancy block binding to CSA of parasites from different parts of the world, DBL-3, although variant, may induce cross-reactive immunity that will protect pregnant women and their fetuses.