Strain-specific immunity induced by immunization with pre-erythrocytic stages of Plasmodium chabaudi.

One of the most promising approaches in the efforts to produce a malaria vaccine involves the use of attenuated whole sporozoite immunizations. Attenuation may be achieved by the use of genetic modification, irradiation, chemical attenuation, or by the contemporaneous administration of antimalarial drugs that target only the erythrocytic stages of the parasite. Most research to date has focused on the efficacy of these approaches upon challenge with parasites homologous to those used for the initial immunizations. We, as have others, have previously shown that a component of the immunity achieved against the erythrocytic stages of the rodent malaria parasite Plasmodium chabaudi chabaudi is strain-specific, with a stronger immune response targeting the immunizing strain than genetically distinct strains. Here, we show that the immunity induced by infection with the pre-erythrocytic stages of these parasites, achieved via inoculation of sporozoites contemporaneously with mefloquine, also has a strain-specific component.

Evidence for strain-specific protective immunity against blood-stage parasites of Plasmodium cynomolgi in toque monkey.

We have conducted experiments to test the induction of strain-specific protective immunity against Plasmodium cynomolgi infections in toque monkeys. Plasmodium cynomolgi is closely related biologically and genetically to the human malaria parasite, P. vivax. Two groups of monkeys were immunized against either of two strains of P. cynomolgi, namely PcCeylon and Pc746, by giving two successive drug-cured infections with asexual blood-stage parasites of one or the other strain, 12-weeks apart. To test for strain-specific protective immunity these infection-immunized monkeys were challenged 8 weeks later with a mixture of asexual blood-stage parasites of both strains. A pyrosequencing-based assay was used to quantify the proportion of parasites that survived in the challenge infections. The assay was based on a SNP within the P. cynomolgi Merozoite Surface Protein-1 gene. Compared to their behaviour in nonimmunized monkeys, the growth of parasites of the homologous (immunizing) strain in mixed-strain challenge infections in the immunized monkeys were reduced relative to that of the nonimmunizing strain. These results indicate the development of blood infection-induced strain-specific protective immunity against P. cynomolgi in toque monkeys. The work prepares for using genetic analysis to identify target antigens of strain-specific protective immunity in this host and malaria parasite combination.

Malaria parasites can develop stable resistance to artemisinin but lack mutations in candidate genes atp6 (encoding the sarcoplasmic and endoplasmic reticulum Ca2+ ATPase), tctp, mdr1, and cg10.

Resistance of Plasmodium falciparum to drugs such as chloroquine and sulfadoxine-pyrimethamine is a major problem in malaria control. Artemisinin (ART) derivatives, particularly in combination with other drugs, are thus increasingly used to treat malaria, reducing the probability that parasites resistant to the components will emerge. Although stable resistance to artemisinin has yet to be reported from laboratory or field studies, its emergence would be disastrous because of the lack of alternative treatments. Here, we report for the first time, to our knowledge, genetically stable and transmissible ART and artesunate (ATN)-resistant malaria parasites. Each of two lines of the rodent malaria parasite Plosmodium chabaudi chabaudi, grown in the presence of increasing concentrations of ART or ATN, showed 15-fold and 6-fold increased resistance to ART and ATN, respectively. Resistance remained stable after cloning, freeze-thawing, after passage in the absence of drug, and transmission through mosquitoes. The nucleotide sequences of the possible genetic modulators of ART resistance (mdr1, cg10, tctp, and atp6) of sensitive and resistant parasites were compared. No mutations in these genes were identified. In addition we investigated whether changes in the copy number of these genes could account for resistance but found that resistant parasites retained the same number of copies as their sensitive progenitors. We believe that this is the first report of a malaria parasite with genetically stable and transmissible resistance to artemisinin or its derivatives.

Exploring the value of shiatsu in palliative care day services.

This qualitative study sought to evaluate the effects of shiatsu therapy on clients attending hospice day services. Eleven clients with advanced progressive disease received five therapy sessions each at weekly intervals. Data about the effects was collected through five unstructured interviews with each client. Four of these were conducted before, during, and shortly after the therapy regime, and the fifth was undertaken four weeks after treatment ended. All the interviews were tape-recorded, transcribed and subject to content analysis. The results of the analysis revealed significant improvements in energy levels, relaxation, confidence, symptom control, clarity of thought and mobility. These benefits were of variable duration - in some instances lasting a few hours but in others extending beyond the 5-week treatment regime. Action to ensure research trustworthiness included keeping research journals to provide an audit trail, conducting member checks and using peer debriefing. The study involved three overlapping cohorts of participants in a data collection period that took approximately 6 months.

The topoisomerases of protozoan parasites.

Protozoan parasites are responsible for a wide range of debilitating and fatal diseases that are proving notoriously difficult to treat. Many of the standard chemotherapies in use today are expensive, have toxic side effects and, in some cases have marginal efficacy because of the emergence of drug-resistant parasites. In the search for more effective treatments, protozoan topoisomerases are now being considered as potential drug targets, building on the clinical success of anticancer and antibacterial agents that target human and bacterial topoisomerases. In this review, Sandra Cheesman explores progress in this relatively new but potentially important field of research.

Investigating and implementing change within the primary health care nursing team.

Primary care is developing rapidly with significant impacts on the nursing team. Such changes have brought inter-professional team-working into sharper focus, particularly community care and collaborative working. This paper: examines the nursing roles within a general practice; describes the perspectives of service users; identifies areas of change; clarifies core and specialist skills; defines new roles among the primary health care nursing team; proposes a new model of working; and identifies appropriate education. The project was set in a general practice in south-west England and used an action research methodology. The objectives were to create a change in practice and to develop and refine existing theory to underpin nursing roles. Throughout the research regular team meetings allowed reflection and discussion about research findings and progress. Data were collected from multiple sources, including team workshops, patient focus group interviews, and individual interviews with GPs, practice managers and area managers. Reflective diaries and a patient survey were also used. The analysis of the quantitative and qualitative data collected from patients formed a basis for practice development and facilitated the team's reflection on the areas of change. Overall high satisfaction with services and care was expressed in the patient interviews and the questionnaire. The themes from the data highlighted areas important for patients and helped in shaping the new roles and responsibilities for team members. Regarding the team perspective, the data indicated many areas that could be considered for development. The community nursing team decided to concentrate on three key areas: child health, leg ulcer management, and cardiovascular health. The research concludes that action research presents some problems and challenges but is a useful approach to developing team-working in primary health care.

Plasmodium falciparum: stage-related expression of topoisomerase I.

The expression and activity of topoisomerase I (PfTopoI) has been examined during the intraerythrocytic stages of the Plasmodium falciparum life cycle. The promoter is inactive during the early ring stage and becomes active only during the later trophozoite and schizont stages. The PfTOP1 transcript starts to accumulate in the trophozoite stage parasite, decreasing again in the schizont stage. Using both stage-specific Western analysis and immunofluorescent assays we show that PfTopoI is present at low levels in rings and accumulates to approximately equal levels in the trophozoite and schizont stages. Experiments to determine the activity of PfTopoI, using a topoisomerase I relaxation assay, show that there is a low level of PfTopoI activity in both ring and trophozoite stages, but activity increases dramatically in the schizont stage. The PfTopoI activity can be inhibited by treatment with specific antiserum and by the type I topoisomerase-specific inhibitor camptothecin.

Katanin, a microtubule-severing protein, is a novel AAA ATPase that targets to the centrosome using a WD40-containing subunit.

Microtubule disassembly at centrosomes is involved in mitotic spindle function. The microtubule-severing protein katanin, a heterodimer of 60 and 80 kDa subunits, was previously purified and shown to localize to centrosomes in vivo. Here we report the sequences and activities of the katanin subunits. p60 is a new member of the AAA family of ATPases, and we show that expressed p60 has microtubule-stimulated ATPase and microtubule-severing activities in the absence of p80. p80 is a novel protein containing WD40 repeats, which are frequently involved in protein-protein interactions. The p80 WD40 domain does not participate in p60 dimerization, but localizes to centrosomes in transfected mammalian cells. These results indicate katanin's activities are segregated into a subunit (p60) that possesses enzymatic activity and a subunit (p80) that targets the enzyme to the centrosome.

Intraerythrocytic expression of topoisomerase II from Plasmodium falciparum is developmentally regulated.

The stage-specific relationship between promoter activity, transcript production, protein expression and enzyme activity has been investigated for the gene encoding Plasmodium falciparum topoisomerase II (PfTopoII). Nuclear run-on experiments have shown that the P. falciparum topoisomerase II gene (PfTOP2) promoter is active at low levels in ring stage parasites, but reaches high levels of activity as the parasites progress into trophozoite/schizont asexual stages. Steady-state PfTOP2 transcripts are present at low levels in rings, accumulate in trophozoites, but are completely undetectable in schizonts. An antiserum raised against the species-divergent carboxy-terminus of PfTopoII, which neutralised the decatenation activity in parasite extracts, was used to probe Western blots of ring, trophozoite and schizont stage parasite extracts. Relatively low levels of PfTopoII were seen in rings compared with those in trophozoite and schizont preparations. Parasite extracts were also used to compare the patterns of protein accumulation and enzyme activity at these stages. Complete decatenation of kinetoplast substrate DNA (KDNA) was found in schizont stages, very low levels of activity were observed in rings and trophozoites showed intermediate levels. These finding show that, as parasites progress towards the stages where DNA replication occurs, there is a concomitant increase in both topoisomerase II production and activity.

Yeast as a model system to study drugs effective against apicomplexan proteins.

Biochemical and genetic analyses are required to identify potential drug targets in apicomplexan parasites, but these studies have proved difficult in most parasite systems. We have developed methods based on expression of parasite proteins in the budding yeast, Saccharomyces cerevisiae, to rapidly screen drugs directed against particular parasite targets, to study the structure and function of these target molecules, and to identify mutations in the parasite genes that alter enzyme specificity or drug sensitivity. In this paper we outline the parameters that need to be considered to design yeast strains that function efficiently to assay function of parasite proteins. Basic protocols and methods are included. We detail some problems that might be encountered in the engineering of these yeast strains and suggest possible solutions.

Stage specific expression of proliferating cell nuclear antigen and DNA polymerase delta from Plasmodium falciparum.

Antisera raised against proliferating cell nuclear antigen (PfPCNA) and DNA polymerase delta (PfDNA Pol delta) have been used against extracts from synchronised parasites to show that both proteins accumulate in trophozoites and persist in schizonts. The steady-state transcripts from both PfPCNA and PfDNA Pol delta also accumulate at the trophozoite stage. However, nuclear run on analysis shows that, whereas PfDNA Pol delta promoter activity is absent in rings but present in trophozoites and schizonts, the PfPCNA promoter is active throughout the intraerythrocytic cycle. This suggests that mechanisms regulating the expression of these two genes may be different although their coordinated activity is required for DNA replication.

The gene encoding topoisomerase II from Plasmodium falciparum.

The gene for topoisomerase II has been isolated from genomic libraries of strain K1 of the human malarial parasite, Plasmodium falciparum. The sequence reveals an open reading frame of 4194 nucleotides which predicts a polypeptide of 1398 amino acids. There are apparently no introns. The sequence is present as a single copy which has an identity of 47.4% and a similarity of 65.4% with its human homologue. Sequences conserved in topoisomerase II from other species are present in Pftopoisomerase II but in addition it has two adjacent asparagine-rich insertions which are unique to it. We have also detected asparagine-rich regions in the gene for PfDNA polymerase alpha. The gene for Pftopoisomerase II has been localised to chromosome 14 and northern analysis reveals a transcript of 5.8 kb. Two independent antisera raised in mice against glutathione-S-transferase fusion proteins containing the amino terminal portion of the malarial protein detect a weak band on western blots at about 160kDa, the expected size of the protein. Use of the same antisera for immunofluorescence analysis suggests that the protein is present at all stages of intraerythrocytic growth of the parasite.

The gene encoding DNA polymerase alpha from Plasmodium falciparum.

The gene encoding DNA polymerase alpha from the human malaria parasite Plasmodium falciparum has been sequenced and characterised. The deduced amino acid sequence possesses the seven sequence motifs which characterise eukaryotic replicative DNA polymerases (I-VII) and four of five motifs (A-E) identified in alpha DNA polymerases. The predicted protein also contains sequences which are reminiscent of Plasmodium proteins but absent from other DNA polymerases. These include four blocks of additional amino acids interspersed with the conserved motifs of the DNA polymerases, four asparagine rich sequences and a novel carboxy-terminal extension. Repetitive sequences similar to those found in other malarial proteins are also present. cDNA-directed PCR was used to establish the presence of these features in the approximately 7kb mRNA. The coding sequence contains a single intron. The gene for DNAPol alpha is located on chromosome 4 and is transcribed in both asexual and sexual erythrocytic stages of the parasite.

Antifungal effects of fluconazole (UK 49858), a new triazole antifungal, in vitro.

Fluconazole is a novel triazole antifungal intended for oral treatment of superficial and systemic mycoses. In tests done in standard mycological media, the compound had minimal inhibitory concentrations against pathogenic Candida species that were usually in excess of 100 mg/l. By contrast, its 'relative inhibition factors' against Candida species (calculated from areas under the antifungal dose-response curves) were of the same order as those of other imidazole and triazole antifungal agents. Against pathogenic Aspergillus species and dermatophytes, the mean relative inhibition factors were the highest so far recorded for an azole antifungal, indicating a relatively weak inhibitory activity against these fungi. Fluconazole inhibited branching and hyphal development in C. albicans at concentrations as low as 10(-6) M (0.3 mg/l), but miconazole and ketoconazole were still active in these tests at concentrations 100 times lower than this. The new antifungal did not suppress ATP concentrations in C. albicans spheroplasts, in common with other weakly lipophilic azole antifungals. This overall poor activity of fluconazole in vitro corresponds badly with its high activity in animal models of mycoses in vivo, and provides more evidence for the unreliability of tests with azole antifungals in vitro as predictors of potential efficacy in vivo.

Suppression of ATP in Candida albicans by imidazole and derivative antifungal agents.

Several antifungal agents, at concentrations of 10 micrograms/ml, were shown to suppress ATP concentrations very rapidly in intact cells and spheroplasts of Candida albicans. The highest ATP-suppressing activity was shown by the highly lipophilic imidazole derivatives difonazole, clotrimazole, econazole, isoconazole, miconazole, oxiconazole and tioconazole, which all caused a reduction of cellular ATP content of more than 50% in 10 min. Relatively hydrophilic imidazole derivatives such as ketoconazole were essentially inactive in the test, as were the triazole derivatives fluconazole, ICI 153066, itraconazole and terconazole, and 5-fluorocytosine. Amphotericin B and terbinafine possessed intermediate ATP-suppressing activity, and the dose-response and pH-response curves for these compounds suggested their mechanism of ATP suppression differed from that of the active imidazole derivatives. ATP suppression by azole antifungals did not involve leakage of ATP from the cells and the effect was entirely abrogated by the presence of serum. Intact cells and spheroplasts of yeast-form and hyphal-form C. albicans were generally equally sensitive to ATP suppression, but stationary-phase cells of both morphological forms were less sensitive than exponential-phase cells. The extent of ATP suppression was significantly reduced in stationary-phase yeast cells of a C. albicans strain with known resistance to azole antifungals, but exponential-phase cells of resistant and susceptible strains were equally sensitive. The effect is tentatively ascribed to membrane damage caused directly by the antifungals.

Alterations in progesterone metabolism and luteal function in infertile women with endometriosis.

The concentrations of pregnanediol-3-glucuronide (PGD) and pregnanolone (PN) were measured in daily morning urine specimens from 66 infertile women (40 with varying degrees of endometriosis and 26 control subjects) and correlated with daily changes in basal body temperature (BBT) and with midluteal levels of serum progesterone (P). PN and BBT rose at midcycle in women with endometriosis, as expected, indicating secretion of some P at that time. However, PGD, the major endpoint of P metabolism, was delayed in its excretion. Endometrial biopsies were similarly delayed (out of phase) in women with endometriosis, and a significantly higher incidence of follicular luteinization was seen. It appears that while P secretion begins at midcycle, the bulk of P secretion is delayed, perhaps because of the process of follicular luteinization, and that a shortened functional luteal phase thus exists in women with endometriosis.