[Early-onset of primary intestinal lymphangiectasia. A case report and diet treatment]. / Linfangectasia intestinale primitiva ad esordio precoce. Descrizione di un caso e trattamento dietetico.

Primary intestinal lymphangiectasia is a rare disorder, characterized by hypoproteinemia due to obstruction of the intestinal lymphatic vessels and loss of lymph fluid in the gastrointestinal tract. The case of a 3-month old patient with protein-losing enteropathy due to a primitive intestinal lymphangiectasia diagnosed with duodenal histology is reported. The adapted formula was replaced by a formula enriched with medium-chain triglycerides (MCT) and the patient presented a clinical and biochemical improvement. The importance of an early diagnosis and the efficacy of treatment with MCT is stressed.

A novel ligand from Plasmodium falciparum that binds to a sialic acid-containing receptor on the surface of human erythrocytes.

Invasion of the merozoite form of Plasmodium falciparum into human erythrocytes involves multiple receptor-ligand interactions. The EBA175 protein of P. falciparum has been shown to be the ligand that binds to a sialic acid-dependent site on glycophorin A. We have identified a novel P. falciparum ligand, termed erythrocyte-binding antigen 140 (EBA140), that shares structural features and homology with EBA175. Subcellular localization of EBA140 suggests that it is located in the micronemes, the same localization as EBA175. EBA140 binds to a sialic acid-dependent receptor on the surface of human erythrocytes. Binding of EBA140 to this erythrocyte receptor is sensitive to neuraminidase and resistant to trypsin, proteinase K and pronase. The protease-resistant properties of the erythrocyte receptor suggests that it is not glycophorin A or C. Additionally, analysis of mutant erythrocytes from humans has shown that EBA140 does not bind glycophorin B. Interestingly, we have identified a parasite line that lacks the eba140 gene, suggesting that this protein is not essential for in vitro invasion. These results suggest that EBA140 may be involved in merozoite invasion using a sialic acid-dependent receptor on human erythrocytes.

An EBA175 homologue which is transcribed but not translated in erythrocytic stages of Plasmodium falciparum.

Plasmodia species can bind to the Duffy blood group antigen (Plasmodium vivax and P. knowlesi) or glycophorin A (P. falciparum) on human erythrocytes as receptors for the invasion of merozoites in the asexual life cycle. A number of proteins have been identified in P. vivax, P. knowlesi and P. falciparum that serve as parasite ligands for these interactions and this group of proteins form the erythrocyte binding protein (EBP) family. The availability of sequence data generated as part of the P. falciparum Genome Project has allowed the identification of other genes related to the known EBP family members. We describe the Psi EBA165 gene and show that it has four exons, a structure identical to that described for EBA175. Analysis using reverse transcriptase-polymerase chain reaction (RT-PCR) has shown that all introns are spliced and that this gene is transcribed. The predicted protein would have the same structure as EBA175 containing the F1/F2 domains, a cysteine-rich region followed by a predicted transmembrane region and a short cytoplasmic tail, but the coding region of Psi EBA165 contains frameshifts. It was possible that the frameshifts may be corrected in the transcript, or alternatively, a mechanism could operate that allowed the translation machinery to read through the frameshifts. Antibodies that recognise EBA165 fusion proteins could not detect this protein in the P. falciparum parasites tested. Additionally, it was possible to disrupt the Psi EBA165 gene without affecting the parasite's ability to invade and grow in erythrocytes. These results suggest that the Psi EBA165 gene is a transcribed pseudogene.

Plasmodium falciparum homologue of the genes for Plasmodium vivax and Plasmodium yoelii adhesive proteins, which is transcribed but not translated.

The 235-kDa family of rhoptry proteins in Plasmodium yoelii and the two reticulocyte binding proteins of P. vivax comprise a family of proteins involved in host cell selection and erythrocyte invasion. Here we described a member of the gene family found in P. falciparum (PfRH3) that is transcribed in its entirety, under stage-specific control, with correct splicing of the intron, but appears not to be translated, probably due to two reading frameshifts at the 5' end of the gene.

Identification of proteins from Plasmodium falciparum that are homologous to reticulocyte binding proteins in Plasmodium vivax.

Plasmodium falciparum infections can be fatal, while P. vivax infections usually are not. A possible factor involved in the greater virulence of P. falciparum is that this parasite grows in red blood cells (RBCs) of all maturities whereas P. vivax is restricted to growth in reticulocytes, which represent only approximately 1% of total RBCs in the periphery. Two proteins, expressed at the apical end of the invasive merozoite stage from P. vivax, have been implicated in the targeting of reticulocytes for invasion by this parasite. A search of the P. falciparum genome databases has identified genes that are homologous to the P. vivax rbp-1 and -2 genes. Two of these genes are virtually identical over a large region of the 5' end but are highly divergent at the 3' end. They encode high-molecular-mass proteins of >300 kDa that are expressed in late schizonts and localized to the apical end of the merozoite. To test a potential role in merozoite invasion of RBCs, we analyzed the ability of these proteins to bind to mature RBCs and reticulocytes. No binding to mature RBCs or cell preparations enriched for reticulocytes was detected. We identified a parasite clone that lacks the gene for one of these proteins, showing that the gene is not required for normal in vitro growth. Antibodies to these proteins can inhibit merozoite invasion of RBCs.

Apical membrane antigen 1 plays a central role in erythrocyte invasion by Plasmodium species.

Apical membrane antigen 1 (AMA1) is an asexual blood-stage protein expressed in the invasive merozoite form of Plasmodia species, which are the causative agent of malaria. We have complemented the function of Plasmodium falciparum AMA1 (PfAMA1) with a divergent AMA1 transgene from Plasmodium chabaudi (PcAMA1). It was not possible to disrupt the PfAMA1 gene using 'knock-out' plasmids, although we demonstrate that the PfAMA1 gene can be targeted by homologous recombination. These experiments suggest that PfAMA1 is critical, perhaps essential, for blood-stage growth. Importantly, we showed that PcAMA1 expression in P. falciparum provides trans-species complementation to at least 35% of the function of endogenous PfAMA1 in human red cells. Furthermore, expression of this transgene in P. falciparum leads to more efficient invasion of murine erythrocytes. These results indicate an important role for AMA1 in the invasion of red blood cells (RBCs) across divergent Plasmodium species.

Functional analysis of proteins involved in Plasmodium falciparum merozoite invasion of red blood cells.

Plasmodium falciparum causes the most lethal form of malaria in humans and is responsible for over two million deaths per year. The development of a vaccine against this parasite is an urgent priority and potential protein targets include those on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to transfect P. falciparum has enabled the construction of gain-of-function and loss-of-function mutants and provided new strategies to analyse the role of parasite proteins. In this review, we describe the use of this technology to examine the role of merozoite antigens in erythrocyte invasion and to address their potential as vaccine candidates.

Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria.

We have exploited the recently developed ability to trans- fect the malaria parasite Plasmodium falciparum to investigate the role of polymorphisms in the enzyme dihydropteroate synthase (DHPS), identified in sulfadoxine-resistant field isolates. By using a truncated form of the dhps gene, specific mutations were introduced into the endogenous gene by allelic replacement such that they were under the control of the endogenous promoter. Using this approach a series of mutant dhps alleles that mirror P.falciparum variants found in field isolates were found to confer different levels of sulfadoxine resistance. This analysis shows that alteration of Ala437 to Gly (A437G) confers on the parasite a 5-fold increase in sulfadoxine resistance and addition of further mutations increases the level of resistance to 24-fold above that seen for the transfectant expressing the wild-type dhps allele. This indicates that resistance to high levels of sulfadoxine in P.falciparum has arisen by an accumulation of mutations and that Gly437 is a key residue, consistent with its occurrence in most dhps alleles from resistant isolates. These studies provide proof that the mechanism of resistance to sulfadoxine in P.falciparum involves mutations in the dhps gene and determines the relative contribution of these mutations to this phenotype.

Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum.

Plasmodium falciparum causes the most severe form of malaria in humans. An important class of drugs in malaria treatment is the sulfone/sulfonamide group, of which sulfadoxine is the most commonly used. The target of sulfadoxine is the enzyme dihydropteroate synthase (DHPS), and sequencing of the DHPS gene has identified amino acid differences that may be involved in the mechanism of resistance to this drug. In this study we have sequenced the DHPS gene in 10 isolates from Thailand and identified a new allele of DHPS that has a previously unidentified amino acid difference. We have expressed eight alleles of P. falciparum PPPK-DHPS in Escherichia coli and purified the functional enzymes to homogeneity. Strikingly, the Ki for sulfadoxine varies by almost three orders of magnitude from 0.14 microM for the DHPS allele from sensitive isolates to 112 microM for an enzyme expressed in a highly resistant isolate. Comparison of the Ki of different sulfonamides and the sulfone dapsone has suggested that the amino acid differences in DHPS would confer cross-resistance to these compounds. These results show that the amino acid differences in the DHPS enzyme of sulfadoxine-resistant isolates of P. falciparum are central to the mechanism of resistance to sulfones and sulfonamides.

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.

Molecular cloning of a gene from Plasmodium falciparum that codes for a protein sharing motifs found in adhesive molecules from mammals and plasmodia.

Adhesion of Plasmodium to host cells is an important phenomenon in parasite invasion and in malaria-associated pathology. We report here the molecular cloning of a putative adhesive molecule from P. falciparum that shares both sequence and structural similarities with a sporozoite surface molecule from Plasmodium termed the thrombospondin-related anonymous protein (TRAP) and, to a lesser extent, with the circumsporozoite (CS) protein. The gene, which is present on chromosome 3 as a single copy, was termed CTRP for CS protein-TRAP-related protein. The full-length CTRP encodes a protein containing a putative signal sequence followed by a long extracellular region of 1990 amino acids, a transmembrane domain, and a short cytoplasmic segment. The putative extracellular region of CTRP is defined by two separated adhesive domains. The first domain contains six 210-amino acid-long homologous repeats, the sequence of which is related to the A-type domain found in adhesive molecules including the alpha subunits of several integrins and a number of extracellular matrix glycoproteins. The second domain contains seven repeats of 87-60 amino acids in length, which share similarities with the thrombospondin type 1 domain found in a variety of adhesive molecules. Finally, CTRP also contains consensus motifs found in the superfamily of haematopoietin receptors. Interstrain analysis of eight different parasite isolates revealed that CTRP does not show size polymorphism except in repetitive regions flanking potential adhesive domains.

A YAC contig map of Plasmodium falciparum chromosome 4: characterization of a DNA amplification between two recently separated isolates.

We have generated a physical map of Plasmodium falciparum chromosome 4 using yeast artificial chromosomes (YACs). The map is defined by a YAC contig spanning approximately 1.05 Mb, which has been restriction mapped to a resolution of 30 kb and is punctuated by 22 sequence-tagged sites. The physical information obtained has enabled us to compare and contrast the structure of chromosome 4 in detail between FCR3 and B8, two recently separated isolates of P. falciparum, leading to characterization of a novel chromosome polymorphism occurring in a subtelomeric region. Comparison of chromosomes 4 from 10 different isolates has shown that chromosome size polymorphisms are restricted to both subtelomeric regions. These analyses provide a high-resolution physical map that will be important to complement genetic analysis of this human pathogen.

Relationship between humoral response to Plasmodium falciparum merozoite surface antigen-2 and malaria morbidity in a highly endemic area of Papua New Guinea.

The prevalence and concentration of antibodies to merozoite surface antigen-2 (MSA-2) were measured in blood samples collected during a cross-sectional survey. Antibodies were measured by enzyme-linked immunosorbent assay using two recombinant proteins that closely approximated the full-length mature MSA-2 polypeptides expressed by the Plasmodium falciparum isolate FC27 and the cloned line 3D7 and that were representative of the dimorphic forms of MSA-2. Antibodies were also measured to a form of the 3D7 MSA-2 lacking the central repetitive sequences (d3D7). High antibody prevalence was observed to all three antigens: the overall prevalence of IgG to FC27, 3D7, and d3D7 was 91%, 90%, and 90%, respectively. The majority of individuals > or = 5 years of age had antibodies to both forms of MSA-2. The geometric mean antibody units increased with age with a plateau being reached by 15-20 years of age. There was a significant positive association of antibody prevalence with both the presence of the parasite and an enlarged spleen in children. This study provides the first evidence that antibodies against nonrepeat regions of MSA-2 are associated with fewer fever episodes and less anemia, both known to be indicators of malaria morbidity.

Primary structure and expression of the dihydropteroate synthetase gene of Plasmodium falciparum.

The enzyme dihydropteroate synthetase (DHPS) from Plasmodium falciparum is involved in the mechanism of action of the sulfone/sulfonamide group of drugs. We describe the cloning and sequencing of the gene encoding the P. falciparum DHPS enzyme and show that it is a bifunctional enzyme that includes dihydro-6-hydroxymethylpterin pyrophosphokinase (PPPK) at the N terminus of DHPS. The gene encodes a putative protein of 83 kDa that contains two domains that are homologous with the DHPS and PPPK enzymes of other organisms. The PPPK-DHPS gene is encoded on chromosome 8 and has two introns. An antibody raised to the PPPK region of the protein was found to recognize a 68-kDa protein that is expressed throughout the asexual life cycle of the parasite. We have determined the sequence of the DHPS portion of the gene from sulfadoxine-sensitive and -resistant P. falciparum clones and identified sequence differences that may have a role in sulfone/sulfonamide resistance.

Mapping the genetic locus implicated in cytoadherence of Plasmodium falciparum to melanoma cells.

Many lines of Plasmodium falciparum undergo a deletion of the right end of chromosome 9 during in vitro cultivation accompanied by loss of cytoadherence to melanoma cells. The deletion also results in loss of expression of PfEMP1, the putative cytoadherence ligand, suggesting that PfEMP1 or a regulatory gene controlling PfEMP1 expression is encoded in this region. Initially a library of short fragments highly enriched for the right arm of chromosome 9 was constructed in bacteriophage lambda. Clones from this library were obtained randomly by the polymerase chain reaction (PCR) technique, sequenced and used to screen a yeast artificial chromosome (YAC)-P. falciparum library by PCR so that the region could be cloned and physically mapped in detail. We have used probes from this region to demonstrate that clones derived from ITG2 have undergone a deletion of intermediate length on chromosome 9. This could explain the unusual stability of cytoadherence in these clones.

Towards a high-resolution map of the Plasmodium falciparum genome.

Until recently very little was known about the genome of Plasmodium falciparum. The situation has changed considerably with the advent of pulsed field gradient electrophoresis and yeast artificial chromosome technologies. It should now be possible to generate a high-resolution map within a few years. Here, Tony Triglia, Thomas Wellems and David Kemp review current knowledge.

A chromosome 9 deletion in Plasmodium falciparum results in loss of cytoadherence.

Many lines of Plasmodium falciparum undergo a deletion of the right end of chromosome 9 during in vitro culture accompanied by loss of cytoadherence and gametocytogenesis. Selection of cytoadherent cells from a mixed population co-selects for those with an undeleted chromosome 9 and the selected cells produce gametocytes. The deletion also results in loss of expression of PfEMP1, the putative cytoadherence ligand, suggesting that PfEMP1 or a regulatory gene controlling PfEMP1 expression and gametocytogenesis may be encoded in this region. We have isolated several markers for the deleted region and are currently using a YAC-P. falciparum library to investigate this region of the genome in detail.

A chromosome 9 deletion in Plasmodium falciparum results in loss of cytoadherence

Many lines of Plasmodium falciparum undrgo a deletion of the right end of chromosome 9 during in vitro culture accompanied by loss of cytoadherence and gametocytogenesis. Selection of cytoadherent cells from a mixed population co-selects for those with an undeleted chromosome 9 and selected cells produce gametocytes. The deletion also results in loss of expression of PfEMP1, the putative cytoadherence ligand, suggesting PfEMP1 or a regulatory gene controlling PfEMP1 expression and gametocytogenesis may be encoded in this region. We have isolated several markers for the deleted region and are currently using a YAC-P. falciparum library to investigate this region of the genome in detail