Plasmodium falciparum: gelatin enrichment selects for parasites with full-length chromosome 2. implications for cytoadhesion assays.

Waterkeyn, J. G., Cowman, A. F., and Cooke, B. M. 2001. Plasmodium falciparum: Gelatin enrichment selects for parasites with full-length chromosome 2. Implications for cytoadhesion assays. Experimental Parasitology 97, 115-118.

Role of phosphorylation of the cytoplasmic domain of the alpha(2)-macroglobulin receptor (LRP).

The low density lipoprotein receptor-related protein (alpha(2)MR/LRP) is a cell surface receptor which is present on most cells and tissues. We show that the 85 kDa subunit, containing the transmembrane region and cytoplasmic domain is phosphorylated in vivo. Comparison of the phosphorylation of the low density lipoprotein receptor (LDLR) with a chimeric receptor containing the cytoplasmic domain of the alpha(2)MR/LRP (LDLR/LRP) showed that phosphorylation is exclusive to the cytoplasmic domain. Staurosporine, a general kinase inhibitor, resulted in a 40% lowering of phosphorylation of LDLR/LRP, but did not give rise to measurable changes in its membrane traffic in MDCK cells. The role of phosphorylation on degradation of the receptor was studied using inhibitors of lysosomal and proteasomal degradation. These studies showed that LDLR/LRP was rapidly turned over by proteasomal degradation but that this turnover was also not a consequence of phosphorylation.

Targeted mutagenesis of Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) disrupts cytoadherence of malaria-infected red blood cells.

Adhesion of parasite-infected red blood cells to the vascular endothelium is a critical event in the pathogenesis of malaria caused by Plasmodium falciparum. Adherence is mediated by the variant erythrocyte membrane protein 1 (PfEMP1). Another protein, erythrocyte membrane protein-3 (PfEMP3), is deposited under the membrane of the parasite-infected erythrocyte but its function is unknown. Here we show that mutation of PfEMP3 disrupts transfer of PfEMP1 to the outside of the P.FALCIPARUM:-infected cell. Truncation of the C-terminal end of PfEMP3 by transfection prevents distribution of this large (>300 kDa) protein around the membrane but does not disrupt trafficking of the protein from the parasite to the cytoplasmic face of the erythrocyte membrane. The truncated PfEMP3 accumulates in structures that appear to be associated with the erythrocyte membrane. We show that accumulation of mutated PfEMP3 blocks the transfer of PfEMP1 onto the outside of the parasitized cell surface and suggest that these proteins traffic through an erythrocyte membrane-associated compartment that is involved in the transfer of PfEMP1 to the surface of the parasite-infected red blood cell.

Genomic distribution and functional characterisation of two distinct and conserved Plasmodium falciparum var gene 5' flanking sequences.

Approximately 50 highly diverse var genes distributed throughout the haploid genome of the malaria parasite Plasmodium falciparum code for PfEMP1 variants located on the surface of infected erythrocytes. PfEMP1 is involved in cytoadherence of parasitised red blood cells and undergoes antigenic variation through differential expression of var genes. Members of the var gene family are located in chromosome-internal positions on chromosomes 4, 7, 8 and 12, and in subtelomeric regions of all chromosomes. Here we show that there are two distinct and conserved types of 5' upstream regions (var17-type and 5B1-type) of var genes, and suggest that most subtelomeric var genes are flanked by a var17-type 5' upstream sequence. In contrast, 5B1-type 5' upstream are localised to chromosomes that have been shown to contain var genes within chromosome-internal regions. Transcriptional analysis using RT-PCR revealed that var genes flanked by either type of 5' upstream sequence are transcribed in in vitro cultured trophozoite stage parasites. In addition, we have shown that the 5' flanking sequences of four different var genes are able to drive transient expression of the cat reporter gene. Our results suggest that at least the minimal regulatory sequences required for transcription of var genes are conserved among both subgroups of the var gene family. Furthermore, these sequences provide new markers for the investigation of the chromosomal organisation of var genes.

Transfection of the human malaria parasite Plasmodium falciparum.

In the past few years, methods have been developed which allow the introduction of exogenous DNA into the human malaria parasite Plasmodium falciparum. This important technical advance known as parasite transfection, provides powerful new tools to study the function of Plasmodium proteins and their roles in biology and disease. Already it has allowed the analysis of promoter function and has been successfully applied to establish the role of particular molecules and/or mutations in the biology of this parasite. This review summarises the current state of the technology and how it has been applied to dissect the function of the P. falciparum genome.

Sequential nucleic acid and recombinant adenovirus vaccination induces host-protective immune responses against Taenia ovis infection in sheep.

Sheep were immunized with a protective recombinant antigen (45W) from the cestode parasite Taenia ovis using three different vaccine delivery systems, either alone or in different combinations. The DNA encoding 45W was cloned into the expression plasmid pcDNA 3 and an ovine adenovirus to create nucleic acid and recombinant viral vector vaccines, respectively. Sheep received two vaccinations with various combinations of these two delivery systems and/or purified recombinant 45W protein in a conventional vaccine formulation containing Quil A as adjuvant (protein/Quil A vaccine). Sheep receiving two inoculations of either the nucleic acid or the recombinant adenovirus alone, demonstrated only low levels of 45W-specific antibody. However, immunization with either nucleic acid or recombinant adenovirus primed animals to mount an enhanced immune response after a subsequent vaccination with the protein/ Quil A vaccine. The most striking result was that sheep initially immunized with the nucleic acid vaccine and boosted with the recombinant adenovirus, mounted IgG1 responses > 65 fold higher than those of sheep receiving either vaccine alone. The level of antibody in these sheep was commensurate with that observed in animals vaccinated twice with the protein/Quil A adjuvanted vaccine. In both cases, host-protection from experimental challenge infection with T. ovis was obtained.

Sequence analysis of a gene family encoding Taenia ovis vaccine antigens expressed during embryogenesis of eggs.

The 45W vaccine antigen of the cestode parasite Taenia ovis is a member of a small gene family estimated to comprise six members. All six genes were cloned and characterised. Nucleotide sequence analysis of the different family members identified a set of closely related genes exhibiting between 75.2 and 98.6% DNA sequence homology. Intron/exon structure for the various genes constituting the family was found to be conserved. Open reading frame analysis and pairwise alignments of predicted polypeptides from the various members of the family revealed that the encoded proteins share between 51.8 and 96.5% amino acid identity with the host-protective 45W antigen. Polymerase chain reaction (PCR) primers were designed which allowed amplification of individual family members in a transcript-specific manner. These were used to investigate gene expression in different developmental stages of the parasite by reverse-transcription polymerase chain reaction (RT-PCR). All members of the gene family were expressed during the T. ovis life-cycle. 45W gene family transcription correlates with embryogenesis of eggs and expression is increased in activated eggs (oncospheres). One family member, ToW3S, was expressed in the early larvae (cysticercus) stages. Expression of genes closely related to the 45W vaccine antigen has important implications for immune evasion under selection from the application of a 45W-based vaccine.

Nucleic acid vaccination of sheep: Use in combination with a conventional adjuvanted vaccine against Taenia ovis.

This report describes the use of a nucleic acid vaccine in a large outbred animal species both alone and in combination with a conventionally adjuvanted vaccine. The gene encoding a host-protective antigen (45W) from the sheep parasite Taenia ovis was cloned into the expression vector pcDNA3 and the resultant plasmid termed pcDNA3-45W. Eleven of 15 sheep injected either intramuscularly or intradermally with pcDNA3-45W mounted a serum antibody response to 45W which for both routes of injection was predominantly IgG1. However, the level of antibody elicited by the nucleic acid vaccine was low and repeated vaccinations did not boost the response. Injection of pcDNA3-45W into animals in which an immune response had previously been generated by vaccination with recombinant 45W using Quil A as adjuvant (rec45W vaccine), did not result in enhanced antibody levels. Initial vaccination with pcDNA3-45W and subsequently with the rec45W vaccine resulted in antibody levels significantly higher (P < 0.05) than those obtained in sheep which had only received the rec45W vaccine. This enhanced antibody response was predominantly of the IgG1 subclass (IgG1 : IgG2, 5 : 1) in animals injected with the nucleic acid vaccine by the i.m. route. Surprisingly, a second rec45W vaccination of these animals led to little or no increase in IgG1 levels and a 10-fold increase in IgG2 resulting in a predominance of 45W-specific IgG2 (IgG1 : IgG2, 0.25 1). These studies revealed that nucleic acid vaccination has efficacy, albeit limited, in the sheep and supports previous investigations which showed that antibody responses elicited by immunization are determined by both the route and mode of antigen delivery.

Stable transgene expression in Plasmodium falciparum.

Plasmid vectors designed to express transgenes and a selectable marker in Plasmodiumfalciparum were constructed. These consist of a selectable gene cassette comprising the Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene mutated to confer pyrimethamine resistance flanked by either Plasmodium chabaudi DHFR-TS or P. falciparum calmodulin promoter sequences and the P. falciparum histidine rich protein 2 3' region. Also, each vector includes a different expression cassette driven by various Plasmodium transcriptional control sequences. Initially, the chloramphenicol acetyl transferase (CAT) reporter gene was cloned into the expression site of two vectors, pCC6-CAT and pCC13-CAT, which were identical except for the orientation of the expression cassette with respect to the selectable gene cassette. Approximately 8-fold more CAT activity was detected when the direction of transcription of the expression cassettes was in a head to head, rather than a tail to head, orientation. Importantly, it was found that stable transfection could only be achieved when the gene cassettes were in the head to head direction suggesting that this orientation also has an effect on the level of expression of the selectable marker. All other plasmids were designed with the cassettes in a head to head orientation. With the exception of pCC6-CAT and a second vector pHC4-CAT, stable transfectants were obtained with each vector in which the CAT gene had been inserted into the expression cassette. This is the first time vectors for the stable expression in Plasmodium parasites of transgenes other than a selectable marker have been described.

Host-protective fragments and antibody binding epitopes of the Taenia ovis 45W recombinant antigen.

The protective efficacy of a recombinant Taenia ovis vaccine antigen, 45W, was compared in sheep vaccine trials with antigen expressed by the full length 45W cDNA and by incomplete copies of the cDNA. Vaccine, trials were also carried out using antigen expressed by a cDNA (45S) having a sequence similar, but not identical, to 45W. Stability of the 45W antigen expressed in Escherichia coli was found to be increased after deletion of cDNA sequence encoding 19 COOH-terminal amino acids. This truncated form of the antigen was designated 45WB/X. Vaccination of sheep with antigen expressed by 45W, 45WB/X, as well as full length 45W and 45S cDNAs, induced high levels of protection. Vaccination with antigen expressed by an incomplete copy of the 45S cDNA clone did not induce protection. Comparison of deduced amino acid sequences for these clones suggests that the host-protective epitope(s) of the 45W antigen occur on either or both of the 23 and 9 amino acid peptides at the amino and carboxyl termini of 45W, respectively. Antibody binding epitopes of 45W were investigated in ELISA using overlapping 9 amino acid peptides. Protection was found to correlate with the induction of antibody to two 9 amino acid peptides.

Characterization of the gene family encoding a host-protective antigen of the tapeworm Taenia ovis.

Genomic structure has been determined for a gene encoding a host-protective antigen of the parasitic platyhelminth Taenia ovis. An incomplete cDNA, known as 45W, encodes the protective antigen. Southern hybridisation experiments using 45W cDNA as a probe, revealed that the 45W gene was a member of a multigene family. Differential Southern hybridisation and rapid amplification of cDNA end (RACE) experiments were used to characterise the related genes, allowing the full-length coding region of the 45W encoded antigen to be determined. The gene family comprises a minimum of four members per haploid genome with each member showing varying degrees of 5' and 3' homology with respect to the 45W cDNA. A close homologue of the 45W gene, designated 45S, differed from 45W at 11 of 985 nt comprising the full-length mRNA. Sequencing of several independent RACE products for both 45W and 45S identified a cDNA which may be a product of homologous recombination between these genes, suggesting that the two genes may be alleles. Homologous recombination in genes which encode a host protective antigen such as 45W would provide a mechanism by which antigenic variants could arise.

[Functional radical neck excision. Evaluation of 10 years' experience. Apropos of 434 cases]. / Evidement fonctionnel du cou. Bilan d'une expérience de 10 ans. A propos de 434 cas.

A total of 434 functional évidements of neck were conducted in 313 patients, taking into account all primary localizations. Details of the procedure used are described while emphasizing the need for an évidement tending towards one as complete as a radical operation. Surgery was usually elective (89% of cases) but in 11% considered as essential. A high proportion of lymph nodes classified as no were, however, found to be invaded (22.8%, including 27.5% of these with capsule rupture). Despite this, glandular recurrence on the side of functional évidement was extremely low (less than 7%) even when recurrence was epithelial or involved a second localization. These findings emphasize the validity of functional évidement and confirm published data, known to be difficult to analyze. Although the value of this operation with respect to the cancer is well established, the actual functional interest, particularly in relation, to external branch of XI th nerve is debatable.