Biomedical subjects

Lisa C Ranford-Cartwright

Publications and source records attributed to Lisa C Ranford-Cartwright.

10 recordsLinked to original sources

PubMed2006

An improved and highly sensitive microfluorimetric method for assessing susceptibility of Plasmodium falciparum to antimalarial drugs in vitro.

BACKGROUND: The standard in vitro protocol currently in use for drug testing against Plasmodium falciparum, based on the incorporation of the purine [3H]-hypoxanthine, has two serious drawbacks. Firstly it is unsuitable for the testing of drugs that directly or indirectly impact on purine salvage or metabolism. Secondly, it relies on the use of expensive radiolabelled material, with added issues concerning detection, storage and waste disposal that make it unsuitable for use in many disease-endemic areas. Recently, the use of fluorochromes has been suggested as an alternative, but quenching of the fluorescence signal by the haemoglobin present in cultures of Plasmodium falciparum-infected erythrocytes severely limits the usefulness of this approach. METHODS: In order to resolve this problem, a new PicoGreen-based procedure has been developed which incorporates additional steps to remove the interfering haemoglobin. The 50% inhibitory concentration (IC50) values of chloroquine and pyrimethamine against P. falciparum laboratory lines 3D7 and K1 were determined using the new protocol. RESULTS: The IC50 values of chloroquine and pyrimethamine against P. falciparum laboratory lines 3D7 and K1 determined with the new fluorescence-based protocol were statistically identical to those obtained using the traditional 3H-hypoxanthine incorporation method, and consistent with literature values. CONCLUSION: The new method proved to be accurate, reproducible and sensitive, and has the advantage of being non-radioactive. The improved PicoGreen method has the potential to replace traditional in vitro drug resistance assay techniques.

Animals↗

PubMed2006

Trypanosoma brucei: a survey of pyrimidine transport activities.

Purine uptake has been studied in many protozoan parasites in the last few years, and several of the purine transporters have been cloned. In contrast, very little is known about the salvage of preformed pyrimidines by protozoa, and no pyrimidine transporters have been cloned, yet chemotherapy based on pyrimidine nucleobases and nucleosides has been as effective as purine antimetabolites in the treatment of infectious and neoplastic disease. Here, we surveyed the presence of pyrimidine transporters in Trypanosoma brucei brucei. We could not detect any mediated uptake of thymine, thymidine or cytidine, but identified a very high-affinity transporter for cytosine, designated C1, with a K(m) value of 0.048+/-0.009 microM. We also confirmed the presence of the previously reported U1 uracil transporter and found it capable of mediating uridine uptake as well, with a K(m) of 33+/-5 microM. A higher-affinity U2 uridine transporter (K(m)=4.1+/-2.1 microM) was also identified, but efficiency of the C1 and U2-mediated transport was low. Pyrimidine antimetabolites were tested as potential trypanocidal agents and only 5-fluorouracil was found to be effective. This drug was efficiently taken up by bloodstream forms of T. b. brucei.

Animals↗

PubMed2005

Comparison of microsatellite and antigen-coding loci for differentiating recrudescing Plasmodium falciparum infections from reinfections in Kenya.

We have compared the ability of five Plasmodium falciparum microsatellites and three antigen-coding loci to differentiate recrudescence from reinfection. We used 133 pairs of P. falciparum-infected blood samples collected during in vivo drug efficacy trials from three sites in Kenya with different malaria endemicities. There were no significant differences between the marker subsets in their ability to discriminate recrudescences from new infections across the three sites. Overall, microsatellite loci revealed significantly higher expected heterozygosity and multiplicity of infection levels than antigen-coding loci. The mean expected heterozygosity across all loci in the three populations was significantly higher with microsatellites (0.70, 0.78 and 0.79) than antigen-coding loci (0.53, 0.60 and 0.62) for Mwea, Tiwi and Bondo areas, respectively. These observations can be explained by three non-exclusive hypotheses: (i) microsatellites are more polymorphic than antigenic loci; (ii) partially immune hosts remove certain parasites from infections on the basis of their antigenic alleles; and/or (iii) recombination occurs in vitro or in vivo with microsatellites.

Antigens, Protozoan↗

PubMed2005

Spreading the seeds of million-murdering death: metamorphoses of malaria in the mosquito.

Plasmodium spp. undergo a complex obligate developmental cycle within their invertebrate vectors that enables transmission between vertebrate hosts. This developmental cycle involves sexual reproduction and then asexual multiplication, separated by phases of invasion and colonization of distinct vector tissues. As with other stages in the Plasmodium life cycle, there is exquisite adaptation of the malaria parasite to its changing environment as it transforms within the blood of its vertebrate host, through the different tissues of its mosquito vector and onwards to infect a new vertebrate host. Despite the intricacies inherent in these successive transformations, malaria parasites remain staggeringly successful at disseminating through their vertebrate host populations.

Animals↗

PubMed2005

Do malaria ookinete surface proteins P25 and P28 mediate parasite entry into mosquito midgut epithelial cells?

BACKGROUND: P25 and P28 are related ookinete surface proteins highly conserved throughout the Plasmodium genus that are under consideration as candidates for inclusion in transmission-blocking vaccines. Previous research using transgenic rodent malaria parasites lacking P25 and P28 has demonstrated that these proteins have multiple partially redundant functions during parasite infection of the mosquito vector, including an undefined role in ookinete traversal of the mosquito midgut epithelium, and it has been suggested that, unlike wild-type parasites, Dko P25/P28 parasites migrate across the midgut epithelium via an intercellular, rather than intracellular, route. PRESENTATION OF THE HYPOTHESIS: This paper presents an alternative interpretation for the previous observations of Dko P25/P28 parasites, based upon a recently published model of the route of ookinete invasion across the midgut epithelium. This model claims ookinete invasion is intracellular, with entry occurring through the lateral apical plasma membrane of midgut epithelial cells, and is associated with significant invagination of the midgut epithelium localised at the site of parasite penetration. Following this model, it is hypothesized that: (1) a sub-population of Dko P25/P28 ookinetes invaginate, but do not penetrate, the apical surface of the midgut epithelium and thus remain within the midgut lumen; and (2) another sub-population of Dko P25/P28 parasites successfully enters and migrates across the midgut epithelium via an intracellular route similar to wild-type parasites and subsequently develops into oocysts. TESTING THE HYPOTHESIS: These hypotheses are tested by showing how they can account for previously published observations and incorporate them into a coherent and consistent explanatory framework. Based upon these hypotheses, several quantitative predictions are made, which can be experimentally tested, about the relationship between the densities of invading Dko P25/P28 ookinetes in different regions of the midgut epithelium and the number of oocyst stage parasites to which these mutant ookinetes give rise. IMPLICATIONS OF THE HYPOTHESIS: The recently published model of ookinete invasion implies that Dko P25/P28 parasites are greatly, although not completely, impaired in their ability to enter the midgut epithelium. Therefore, P25 and/or P28 have a novel, previously unrecognized, function in mediating ookinete entry into midgut epithelial cells, suggesting that one mode of action of transmission-blocking antibodies to these ookinete surface proteins is to inhibit this function.

Animals↗

PubMed2005

How do malaria ookinetes cross the mosquito midgut wall?

The route taken by malaria ookinetes to cross the midgut wall of the mosquito vector has been (and continues to be) controversial. In this article, we attempt to reinterpret and integrate the apparently conflicting observations of the past using the recently proposed Time Bomb model of ookinete invasion as a unifying framework. We argue that parasite entry into the midgut epithelium is always intracellular, occurs at sites where multiple adjacent host cells converge, and that parasite exit from the midgut epithelium can occur by either an intracellular or intercellular route, depending on the manner in which invaded host cells are extruded from the midgut wall. We also propose a novel hypothesis, the Cellular Treadmill model, to explain the movement of ookinetes through multiple adjacent midgut cells.

Animals↗

PubMed2002

Sexual differentiation and sex determination in the Apicomplexa.

Protozoan parasites of the phylum Apicomplexa have complex life cycles involving various types of asexual division that allow rapid proliferation of parasites within one or more hosts. Such replication is punctuated by obligate sexual differentiation that produces male and female gametocytes. These stages are transmissible to haematophagous vectors or are necessary ultimately to form resistant cysts that are released into the environment. This article examines the sexual differentiation of apicomplexan parasites as it relates to the timing of commitment and the mechanism of the switch from asexual proliferation to the development of male and female sexual stages.

Animals↗

PubMed2002

Sulfadoxine-pyrimethamine resistance in the rodent malaria parasite Plasmodium chabaudi.

We have studied resistance to sulfadoxine-pyrimethamine (S/P) in the rodent malaria parasite Plasmodium chabaudi. A stable S/P-resistant mutant, AS(50S/P), was selected by drug treatment of a clone, AS(PYR), already resistant to pyrimethamine. The sequences of the P. chabaudi dhfr and dhps genes were obtained and found to be identical in AS(50S/P) and AS(PYR), showing that resistance to S/P in AS(50S/P) was not due to additional mutations in either gene. AS(50S/P) was crossed with a drug-sensitive clone, AJ, and 16 independent recombinant progeny were obtained. These clones were phenotyped for their susceptibility to S/P and to sulfadoxine and pyrimethamine separately. Pyrimethamine resistance was invariably associated with S/P resistance, but no correlation was found between resistance to S/P and resistance to sulfadoxine. Quantitative trait locus analysis of the progeny with 31 chromosome-specific markers showed that mutant P. chabaudi dhfr, or one or more genes closely linked to it, was a major determinant of S/P resistance. In addition, the inheritance of genes on chromosomes 5 and 13 from the sensitive parent appeared to contribute to the level of resistance observed. These results demonstrate that the S/P resistance of the AS(50S/P) mutant of P. chabaudi does not involve mutation in dhps and is not due simply to a combination of two genes determining resistance to pyrimethamine and sulfadoxine separately.

Animals↗

PubMed2002

Detection of mutations in the Plasmodium falciparum dihydrofolate reductase (dhfr) gene by dot-blot hybridization.

There is a need for a specific, sensitive, robust, and large-scale method for diagnosis of drug resistance genes in natural Plasmodium falciparum infections. Established polymerase chain reaction (PCR)-based methods may be compromised by the multiplicity of P. falciparum genotypes in natural infections. Here we adopt a dot-blot method to detect point mutations at nucleotide 323 (residue 108) in the P. falciparum dihydrofolate reductase (dhfr) gene using allele-specific oligonucleotide probes. Serine (Ser) or threonine (Thr) at this position are associated with sensitivity to pyrimethamine while asparagine (Asn) is associated with resistance. The method combines PCR amplification and hybridization of amplified products with radiolabeled allele-specific probes. This technique is specific and sensitive; it detects parasitemia of less than 100 parasites/microl of blood, and can identify a minority parasite genotype down to 1% in a mixture. Analysis of P. falciparum isolates from Sudan, of known response to pyrimethamine, has demonstrated the sensitivity and specificity of the method and its ability to detect multiple genotypes in single infections. Furthermore, it has confirmed the association between pyrimethamine responses and dhfr alleles. The method has been successfully extended for analysis of other point mutations in dhfr at residues 51 and 59, which are associated with a high level of pyrimethamine resistance.

Animals↗

PubMed2002

Real-time quantitative PCR in parasitology.

Standard techniques for counting parasites are often time-consuming, difficult and inaccurate, and occasionally unpleasant. Real-time quantitative polymerase chain reaction has recently been applied to parasitology, specifically Plasmodium, Toxoplasma, Leishmania and Neospora. These techniques are truly quantitative, give results over a range of 6-7 orders of magnitude, are quick to perform and require no manipulations post-amplification. They can be used to count genome numbers and to study levels of gene expression. The advantages and limitations of existing thermocyclers and applicable detection systems are discussed here, and promising new developments are highlighted.

Animals↗