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R E Sinden

Publications and source records attributed to R E Sinden.

At least 37 records · Page 2Linked to original sources
PubMed2000

Systematic screening of Anopheles mosquito genomes yields evidence for a major clade of Pao-like retrotransposons.

We developed a degenerate PCR procedure to simultaneously amplify products from divergent retrotransposon families within the genomes of Anopheles mosquitoes. The procedure required cloning of multiple PCR products, but more than half of the clones subsequently sequenced were of retrotransposon origin. These included Copia-like and LINE retrotransposons, as well as the first Gypsy-like retrotransposons reported from mosquitoes. Furthermore, some Anopheles retrotransposon sequences showed similarity to the divergent Pao element from the silkmoth Bombyx mori. Phylogenetic analyses provided consistently strong bootstrap support (> 95%) for a major clade of Pao-like retrotransposons, which includes five mosquito sequences and the recently discovered Drosophila retrotransposons BEL and ninja. This appears to represent a new family of Pao-like LTR-retrotransposons distinct from the Copia and Gypsy families.

Amino Acid Sequence↗
PubMed2000

Anopheles arabiensis and An. funestus are equally important vectors of malaria in Matola coastal suburb of Maputo, southern Mozambique.

Transmission characteristics of malaria were studied in Matola, a coastal suburb of Maputo, the capital City, in southern Mozambique, from November 1994 to April 1996. The local climate alternates between cool dry season (May-October) and hot rainy season (November-April) with mean annual rainfall 650-850 mm. Saltmarsh and freshwater pools provide mosquito breeding sites in Matola. Malaria prevalence reached approximately 60% among people living nearest to the main breeding sites of the vectors. Plasmodium falciparum caused 97% of malaria cases, others being P. malariae and P. ovale. Potential malaria vector mosquitoes (Diptera: Culicidae) collected at Matola during daytime indoor-resting (n = 1021) and on human bait at night (n = 5893) comprised 12% Anopheles coustani Laveran (93% biting outdoors), 46% An. funestus Giles (68% biting indoors) and 42% An. gambiae Giles sensu lato (60% biting outdoors). All 215 specimens of An. gambiae s.l. identified genetically were An. arabiensis Patton. Anopheles funestus populations remained stable throughout the year, whereas densities of the An. gambiae complex fluctuated considerably, with An. arabiensis peaking during the rainy season. No concomitant rise in malaria incidence was observed. Human landing indices of An. funestus and An. arabiensis averaged 1.8 and 3.8 per man-night, respectively. Overall Plasmodium sporozoite rates were 2.42+/-1.24% in 2181 An. funestus and 1.11+/-1.25% in 1689 An. arabiensis dissected and examined microscopically. Mean daily survival rates were 0.79 for both vector species. Estimated infective bites/person/year were 15 An. funestus and 12 An. arabiensis. Biting rates were greatest at 2100-24.00 hours for An. funestus (68% endophagic) and 21.00-03.00 hours for An. arabiensis (40% endophagic). The entomological inoculation rate (EIR) declined sharply over very short distances (50% per 90m) away from breeding-sites of the vectors. Consequently, P. falciparum prevalence among Matola residents was halved 350 m within the town. Implications for the protective effectiveness of a 'cordon sanitaire' by residual house-spraying and/or the use of insecticide-treated bednets are discussed.

Animals↗
PubMed2000

The roles of the glycosylphosphatidylinositol anchor on the production and immunogenicity of recombinant ookinete surface antigen Pbs21 of Plasmodium berghei when prepared in a baculovirus expression system.

Malarial ookinetes express an immunodominant surface protein (P28) that is a priority candidate for the development of transmission-blocking vaccines. The full length P28 gene from Plasmodium berghei [Pbs21(1-213)] and a deletion construct [Pbs21(1-188)] encoding a protein that lacks the 25 C-terminal amino acids, including the glycosylphosphatidylinositol (GPI) anchor signal, were expressed in insect cells using baculovirus vectors. Pbs21(1-213) protein is strongly hydrophobic, found in the cytoplasm and on the surface of Spodoptera Sf21 cells, and in the culture medium. Pbs21(1-188) protein was largely found in the aqueous phase of the medium and in the cytoplasm of Sf21 cells, but was not detected on the cell surface. The presence of 25 C-terminal amino acids is therefore critical to the attachment of recombinant Pbs21 to the parasite plasma membrane. Mice were immunized subcutaneously or intramuscularly with affinity purified recombinant Pbs21(1-213), Pbs21(1-188) or native Pbs21 proteins. Following two immunizations, native Pbs21 induces higher titres when administered by either route, than the recombinant protein bearing an insect GPI anchor, which in turn is markedly more immunogenic than the recombinant polypeptide lacking a GPI anchor. When specific anti Pbs21 antibody titres exceeded 1 mg/ml all three antigens were capable of inducing transmission blockade > or = 90%, below 1 mg/ml blockade did not correlate with antibody concentration.

Animals↗
PubMed2000

Inhibition of the mosquito transmission of Plasmodium berghei by Malarone (atovaquone-proguanil).

Sera from patients treated with atovaquone-proguanil (Malarone) have previously been shown to inhibit the mosquito transmission of Plasmodium falciparum, though the inhibition was not complete and the effect declined 2 weeks after treatment. In marked contrast, the inhibition of transmission of P. berghei by human sera (fed to mosquitoes, with P. berghei gametocytes, via membrane feeders) from volunteers treated with atovaquone-proguanil was total up to day 28 post-treatment and still very significant at day 56. In view of the short half-lives of atovaquone and proguanil, this was unexpected, and further experiments, reported here, were undertaken. In contrast to the incomplete blockade of infectivity of P. falciparum by serum taken 4 days post-treatment, such serum was totally inhibitory against P. berghei at a 1:10,000 dilution, indicating a remarkable sensitivity of P. berghei and demonstrating an unusual difference between the two Plasmodium species in response to a drug. The inhibitory effect on P. berghei after day 4 was caused by atovaquone and mainly through blockade of development from ookinete to oocyst. Despite previous information on the rapid elimination of atovaquone by patients, the present data indicate that low concentrations of this drug may persist in the plasma for some weeks after treatment.

Animals↗
PubMed2000

Distinct roles for pbs21 and pbs25 in the in vitro ookinete to oocyst transformation of Plasmodium berghei.

We have developed an in vitro culture system for early sporogonic stages of Plasmodium berghei, which can be used to study developmental events normally taking place in the midgut of an infected mosquito. These include penetration of insect cells by the mature ookinete, transformation into oocysts and the early development of the latter, sustained through several rounds of nuclear division. The system, based upon co-culture of enriched ookinetes with several established insect cell lines, was used to study the development of mutant ookinetes lacking both the Pbs21 and Pbs25 surface proteins. Motility and entry of double knockout and Pbs21 single knockout ookinetes into the insect cells are normal, but the number of ookinetes successfully transforming into oocysts expressing the CSP protein are substantially reduced. Finally, using the yeast two-hybrid system we also show that Pbs25 has the capacity to homodimerise as well as to form heterodimers with Pbs21.

Aedes↗
PubMed1999

A single-chain antibody fragment specific for the Plasmodium berghei ookinete protein Pbs21 confers transmission blockade in the mosquito midgut.

Mouse monoclonal antibody 13.1 (mAb 13.1) directed against Pbs21, a 21-kDa sexual-stage surface protein of Plasmodium berghei, is known to inhibit oocyst development from gametocytes and ookinetes in the mosquito midgut. To examine the properties and potential uses of a single-chain antibody fragment (scFv) for blocking transmission of malaria parasites to mosquitoes, we have cloned and sequenced the genes encoding variable regions of the immunoglobulin heavy and light chains (V(H) and V(L)) of mAb 13.1. The V(H) and V(L) genes were assembled as an scFv gene, and expressed in a baculovirus expression system. Following purification of 13.1 scFv, Western blotting and inhibition ELISA assays confirmed that 13.1 scFv retained the binding specificity of the parent mAb 13.1 for Pbs21. Furthermore, 13.1 scFv bound to the surface of P. berghei ookinetes, and blocked oocyst development in the mosquito midgut by at least 93%, as assessed by oocyst counts in mosquitoes. We suggest that the 13.1 scFv gene could be useful not only in studying the mechanism of transmission blockade, but also in generating, by mosquito germline transformation, a model system to evaluate the production of mosquitoes refractory to malaria.

Amino Acid Sequence↗
PubMed1999

CTRP is essential for mosquito infection by malaria ookinetes.

The malaria parasite suffers severe population losses as it passes through its mosquito vector. Contributing factors are the essential but highly constrained developmental transitions that the parasite undergoes in the mosquito midgut, combined with the invasion of the midgut epithelium by the malaria ookinete (recently described as a principal elicitor of the innate immune response in the Plasmodium-infected insect). Little is known about the molecular organization of these midgut-stage parasites and their critical interactions with the blood meal and the mosquito vector. Elucidation of these molecules and interactions will open up new avenues for chemotherapeutic and immunological attack of parasite development. Here, using the rodent malaria parasite Plasmodium berghei, we identify and characterize the first microneme protein of the ookinete: circumsporozoite- and TRAP-related protein (CTRP). We show that transgenic parasites in which the CTRP gene is disrupted form ookinetes that have reduced motility, fail to invade the midgut epithelium, do not trigger the mosquito immune response, and do not develop further into oocysts. Thus, CTRP is the first molecule shown to be essential for ookinete infectivity and, consequently, mosquito transmission of malaria.

Animals↗
PubMed1999

The biosynthesis and post-translational modification of Pbs21 an ookinete-surface protein of Plasmodium berghei.

Radiolabelled methionine incorporation into synchronised Plasmodium berghei gametocytes or ookinete cultures, showed that Pbs21 is not synthesised in bloodstage parasites; synthesis was detected within three hours of induction of gametogenesis; synthesis was triggered at gametogenesis, not by fertilisation. We show native Pbs21 to be a hydrophobic membrane protein that was insensitive to cleavage by phosphatidylinositol phospholipase C (PI-PLC), but sensitive to alkaline hydroxylamine, and partially sensitive to glycosylphosphatidylinositol-dependent phospholipase D (GPI-PLD) and HNO2. 3H-myristic and palmitic acid, 3H-glucosamine and mannose incorporation indicated Pbs21 was acylated and glycosylated. Linkage of the acyl group was sensitive to HNO2, which released an acyl-phosphatidylinositol more hydrophobic than that released from P3 of Trypanosoma brucei. All these properties are consistent with the presence of a malaria-specific glycosylphosphatidylinositol (GPI) anchor. In contrast recombinant Pbs21 (rPbs21), expressed in Spodoptera frugiperda cells, was sensitive to both PI-PLC and GPI-PLD, consistent with the protein being modified by a different (S. frugiperda) GPI anchor. Brefeldin A blocked secretion of rPbs21 within a cytoplasmic reticular compartment. Following deletion of the putative GPI anchor addition site (amino acids 189 213), the protein was transported to the cell surface and secreted directly into the aqueous phase of the culture medium. Deletion of amino acids 205-213 disrupted Pbs21 processing, transport through the ER and distribution onto the cell surface. Deletion of amino acids 1-28 prevented transport of Pbs21 into the ER. This suggests that correct processing of the GPI anchor in the ER-Golgi network is essential for the successful secretion of the recombinant protein, which is additionally dependent upon an N-terminal secretory signal sequence.

Acylation↗
PubMed1999

Heterogeneous ribosome populations are present in Plasmodium berghei during development in its vector.

The genome of the rodent malaria parasite, Plasmodium berghei, contains two sets of variant ribosomal RNA (rRNA) genes, termed the A and S types, that are expressed predominantly during the vertebrate and mosquito stages of the parasite's development respectively. Using in situ hybridization, we have examined the transcriptional activity of the A- and S-type rRNA genes, and the switch in expression of the ribosome populations that occurs after parasite transmission to the mosquito. By detection of precursor rRNA molecules, we show that A-type rRNA transcription is downregulated throughout development in the mosquito, whereas the initiation of S-type rRNA transcription is linked to the proliferative phase of the oocyst. Mature A-type rRNA persists during development of the zygote into the ookinete/young oocyst. In contrast, mature S-type rRNA is first detectable in young oocysts and is subsequently present at high levels during further development of oocysts and sporozoites. These results demonstrate that the switch in transcription between the A- and S-type rRNA genes is developmentally regulated, taking place only as the parasite begins to proliferate in the mosquito. A-type ribosomes are therefore not only translationally active in the early stages of development in the mosquito, but are also crucial at this phase.

Animals↗
PubMed1999

Plasmodium differentiation in the mosquito.

The essential passage of the malarial parasite through a mosquito vector results in major population bottlenecks in parasite numbers. The volume of the bloodmeal ingested by the female mosquito is 1-2 microliters. This may contain from 1 to 10(5) gametocytes. Of these, it is normal for just 12 to become macrogametes; 5-6 become ookinetes, and 2 develop into oocysts 2-7 days later. Of the 16,000 sporozoites produced from these two oocysts just 10-20 are inoculated by the malaria-infected female mosquito each time she probes when taking a subsequent bloodmeal. These significant population bottlenecks suggest that parasite differentiation is severely constrained by the environment in the mosquito, and therefore by the interactions between the parasite and the vector. This review will describe parasite differentiation in the mosquito and try to highlight the more important interactions between the parasite, the bloodmeal and the mosquito, attempting to identify those interactions which are essential to parasite differentiation, and those where the mosquito may be mounting effective strategies against this important pathogen. The potential exploitation of these interactions as possible mechanisms for intervention will be discussed.

Animals↗
PubMed1999

Model systems to evaluate the use of transgenic haematophagous insects to deliver protective vaccines.

Insect vector control has proved an effective method for reducing the transmission of disease-causing organisms to human populations in many tropical countries. We are interested in employing direct genetic manipulation of insect vector genomes to use them in beneficial ways so as to have a profound and long-lasting effect on disease transmission. Our research focuses on assessing whether haematophagous insects may be used as a means to deliver protective proteins, such as an antimalarial vaccine, when they take a blood meal. The progress which has been made towards assessing this concept using a number of model systems is described.

Aedes↗
PubMed1998

Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito.

Malaria is transmitted from vertebrate host to mosquito vector by mature sexual blood-living stages called gametocytes. Within seconds of ingestion into the mosquito bloodmeal, gametocytes undergo gametogenesis. Induction requires the simultaneous exposure to at least two stimuli in vitro: a drop in bloodmeal temperature to 5 degrees C below that of the vertebrate host, and a rise in pH from 7.4 to 8.0-8.2. In vivo the mosquito bloodmeal has a pH of between 7.5 and 7.6. It is thought that in vivo the second inducer is an unknown mosquito-derived gametocyte-activating factor. Here we show that this factor is xanthurenic acid. We also show that low concentrations of xanthurenic acid can act together with pH to induce gametogenesis in vitro. Structurally related compounds are at least ninefold less effective at inducing gametogenesis in vitro. In Drosophila mutants with lesions in the kynurenine pathway of tryptophan metabolism (of which xanthurenic acid is a side product), no alternative active compound was detected in crude insect homogenates. These data could form the basis of the rational development of new methods of interrupting the transmission of malaria using drugs or new refractory mosquito genotypes to block parasite gametogenesis.

Animals↗
PubMed1998

Protection from Plasmodium berghei infection by priming and boosting T cells to a single class I-restricted epitope with recombinant carriers suitable for human use.

The desirability of inducing cytotoxic T cell responses to defined epitopes in humans has led to the development of a variety of recombinant delivery systems. Recombinant protein particles derived from a yeast retrotransposon (Ty) and the modified Ankara vaccinia (MVA) virus can deliver large epitope strings or even whole proteins. Both have previously been administered safely in humans. Immunization with recombinant Ty and MVA containing a single Plasmodium berghei class I-binding epitope provided 95% sterile protection against malaria in mice. The sequence of immunization, Ty followed by MVA, was critical to elicit high levels of IFN-gamma-producing cells and protection. The reciprocal sequence (MVA/TY) or homologous boosting was not protective. Both constructs (Ty and MVA) contain the H-2Kd-restricted pb9 CTL epitope from the circumsporozoite protein of P. berghei among a string of 8-15 human P. falciparum-derived CTL epitopes restricted through 7 common HLA alleles as well as widely recognized CD4 T cell epitopes. Thus, the novel recombinant Ty/MVA prime/boost combination with these constructs provides a safe alternative for evaluation for human vaccination against P. falciparum malaria.

Animals↗
PubMed1998

Transgenic expression of a mosquito-stage malarial protein, Pbs21, in blood stages of transformed Plasmodium berghei and induction of an immune response upon infection.

Pbs21 is a surface protein of the ookinete of Plasmodium berghei, which can induce a potent transmission-blocking immune response. Pbs21 is normally expressed only by parasite stages in the mosquito, i.e., female gametes/zygotes, ookinetes, and oocysts. However, the Pbs21 gene is transcribed in female gametocytes which circulate in the bloodstream of the host, where translation of the resulting mRNA is totally repressed. Episomal transfection has been used to investigate whether expression of Pbs21 protein could be achieved in blood stages of the parasite. By using plasmid pMD221, the complete mRNA-encoding region of Pbs21, flanked only by 218 nucleotides (nt) of its promoter region and 438 nt of its 3' region downstream from the polyadenylation site, was introduced into the blood stages of gametocyte-producing and non-gametocyte-producing clones of P. berghei. In both of these transformed parasite lines, Pbs21 protein was expressed in asexual trophozoites, schizonts, and, when present, in both male and female gametocytes. Hence, the flanking regions present are sufficient to allow transcription but lack the elements that exert natural control of sex- and stage-specific transcription. The mRNA and the protein expressed by transformed blood stages were indistinguishable from the wild-type forms by the criteria tested, and the protein was recognized by both conformation-dependent and conformation-independent monoclonal antibodies raised against native Pbs21. In mice infected with transformed non-gametocyte-producing parasites, a Pbs21-specific immune response was induced and characterized with respect to isotype (IgG2a/IgG2b) and quantity (11. 5 +/- 10 microg/ml) of antibody produced. However, as found in previous studies, these antibody levels were insufficient to inhibit development of the parasites in the mosquito. The ability to express mosquito midgut-stage antigens in blood-stage parasites will facilitate further investigations of molecular and immunological properties of these proteins.

Animals↗
PubMed1998

The differing impact of chloroquine and pyrimethamine/sulfadoxine upon the infectivity of malaria species to the mosquito vector.

Using serum or infected blood from Danish volunteers and Plasmodium falciparum-infected Mozambican patients, respectively, the impact of curative doses of chloroquine and pyrimethamine/sulfadoxine upon infectivity of P. falciparum to Anopheles arabiensis and An. gambiae or of P. berghei to An. stephensi was studied. Both treatments cleared circulating P. falciparum gametocytes within 28 days. Before this clearance, chloroquine enhanced infectivity to An. arabiensis, whereas pyrimethamine/sulfadoxine decreased infectivity. Patients harboring chloroquine-resistant parasites as opposed to -sensitive ones were 4.4 times more likely to have gametocytes following treatment. In contrast, pyrimethamine/sulfadoxine-resistant parasites were 1.9 times less likely to produce gametocytes. In laboratory infections using replicated P. berghei or P. falciparum preparations, serum from chloroquine-treated, uninfected, nonimmune volunteers enhanced gametocyte infectivity with increasing efficiency for 21 days following treatment, whereas pyrimethamine/sulfadoxine significantly suppressed infectivity. The observed enhancement in infectivity induced by the use of chloroquine combined with increased gametocytemias in chloroquine-resistant strains may in part explain the rapid spread of chloroquine resistance in endemic populations.

Animals↗
PubMed1997

Cloning and expression of the thrombospondin related adhesive protein gene of Plasmodium berghei.

Sporozoite recognition of host cells is a key step in the life-cycle of malaria parasites. Two sporozoite proteins have so far been characterized in some detail, the circumsporozoite protein (CS) and thrombospondin related adhesive protein (TRAP). We report here the cloning and expression of the TRAP gene homologue from Plasmodium berghei, PbTRAP. The PbTRAP gene encodes a protein of 606 amino acids having a deduced molecular mass of 66 kDa. The overall structure is clearly that of the TRAP family having a signal sequence followed by an integrin A domain, a sulphatide binding motif, followed by a proline based repeat before a transmembrane domain and helical cytoplasmic tail. The observed molecular mass is almost 50% larger than expected, this can be explained almost entirely by the abnormal behaviour in SDS-PAGE of the proline based repeat. As would be expected PbTRAP shows greatest similarity with the P. yoelli TRAP homologue sporozoite surface protein 2 (SSP2) than with PfTRAP, the TRAP gene from P. falciparum. The pattern of expression is similar to that of SSP2.

Amino Acid Sequence↗