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R E Sinden

Publications and source records attributed to R E Sinden.

At least 19 recordsLinked to original sources
PubMed1992

Immunoprotection in mice susceptible to waning memory against the pre-erythrocytic stages of malaria after validated immunisation with irradiated sporozoites of Plasmodium berghei.

The induction of immunity by irradiated sporozoites has been a bench-mark of immunological protection against the malaria parasite. Herein we confirm that different mouse strains exhibit different susceptibilities to sporozoite-induced infection of Plasmodium berghei. We note, however, that after hepatic schizogony, early parasite growth in the blood demonstrates no strain preference between C57BL/6 and BALB/c mice. Sporozoite-susceptible C57BL/6 mice, although initially protected by irradiated sporozoite immunisation against a challenge of 10(3) live sporozoites, progressively lose this protection; a challenge with fewer sporozoites 2 months later elicits a blood infection. BALB/c mice treated in parallel remain protected. Analysis of the kinetics of blood parasitaemia (a measure of hepatic schizont burden) with waning protection shows clearly that immunocompetence remains, as indicated by a reduction in the effective exo-erythrocytic schizont load. This immunocompetence can be shown to be absolutely protective, given an appropriately low dose of viable infective sporozoites. We discuss the testable proposition that this elicitation of protective memory is a consequence either of 'unsaturated' threshold levels of recirculating immunoeffector CD8+ cells or of CD4 cell activation by nonviable sporozoites.

Animals↗
PubMed1992

Gamete development in Plasmodium berghei regulated by ionic exchange mechanisms.

Ionic regulation in the induction of exflagellation of Plasmodium berghei was investigated by culturing the parasites in various isotonic media. Of the salts tested, NaHCO3 exhibited the highest activity in inducing exflagellation, whereas KHCO3 showed no activity. In the absence of HCO3-, media containing monovalent cation (Na+, K+, Cs+, Rd+, choline+, lysine+, arginine+) and Cl- also induced exflagellation, but their activities were lower than that of NaHCO3. Anions of Br- or NO3- could be substituted with Cl-, whereas other anions such as I-, NO2-, SO4(2-), SCN-, H2PO4-, or HPO4(2-) failed to induce exflagellation, as did tetramethylammonium-Cl, CaCl2, MgSO4, MgCl2 and sucrose as well. These results suggest that the induction of exflagellation requires the presence of Na+ and HCO3- or monovalent, membrane-permeable cation and Cl- in the medium. Measurements of the efflux of H[14C]O3- or Cl- indicated that these anions were released from the cells into the NaCl or the NaHCO3 medium, respectively, probably by exchange in HCO3-/Cl-. Determination of intracellular ionic concentrations by electron microscopic X-ray microanalysis of cryopreserved specimens revealed that in the NaHCO3 medium, external Na+ (and probably HCO3-) enters the gametocytes by exchange with internal Cl- (and probably H+), whereas in Cl(-)-containing media, external unspecified cation and Cl- influx by exchange, probably with H+ and HCO3-. It is therefore suggested that two separate ion exchangers, i.e., Na(+)-dependent HCO3-(in)/Cl-(out) and nonspecific monovalent-cation-dependent Cl-(in)/HCO3-(out) exchangers, are involved in the induction of gametogenesis in P. berghei.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Plasmodium berghei: sporozoites are sensitive to human serum but not susceptible host serum.

Human complement was activated by rodent malaria, Plasmodium berghei, sporozoites through the alternative pathway, as revealed by C3 deposition on sporozoites using the fluorescent antibody technique. Sporozoites exposed to fresh human serum decreased in infectivity to HepG2 cells, but those exposed to heated or C3-deficient human serum showed normal infectivity to HepG2 cells. In contrast, C3 deposition was not observed on the sporozoites treated with mouse or rat serum even in the presence of specific polyclonal anti-sporozoite antibody. However, following treatment with trypsin (250 micrograms/ml), 81% of salivary gland sporozoites and 49% of oocyst sporozoites became reactive with mouse serum, and reactive sporozoites deposited mouse C3 on their surface in the presence of 30 mM EGTA and 1 mM Mg2+ without antibody. Concomitantly some sporozoites lost reactivity to anti-circumsporozoite protein monoclonal antibody. These results suggest that P. berghei sporozoites possibly express surface molecules that regulate the complement activation pathway of susceptible hosts but not of nonhosts, and that the putative structures consist of protease-sensitive molecule(s) which are closely associated with the circumsporozoite protein.

Animals↗
PubMed1991

Plasmodium berghei: ionic regulation and the induction of gametogenesis.

The role of ionic regulation in the induction of gametogenesis of Plasmodium berghei at 20 degrees C was investigated. A potent inhibitor of Na+/H+ exchange, amiloride, strongly inhibited exflagellation and subsequent ookinete formation induced by RPMI 1640 with 10% fetal calf serum at pH 8.0, whereas Na+ or K+ channel inhibitors, H(+)-ATPase inhibitors, and a protonophore had no significant effect. Amiloride-treated 'activated' microgametocytes synthesized DNA to levels consistent with the expected 8C, but failed to develop further. These results may suggest that an increase in intracellular pH induced by Na+/H+ exchange plays an important role in the induction of gametogenesis by cultivating at pH 8.0 and 20 degrees C. Cultivation at pH 8.0 and 37 degrees C did not induce the development, and microgametocytes remained as nonactivated forms, having the DNA content of 1.5C. By culturing at pH 7.3 and 20 degrees C, however, most of microgametocytes finished synthesis of DNA up to the 8C level, but ceased development at various stages. Additionally, exflagellation occurred in a simple medium composed of buffered saline with 10 mM glucose. Glucose was indispensable for exflagellation, presumably acting as an energy source. Exflagellation induced by this solution was also inhibited by amiloride. It is therefore suggested that the induction of microgametogenesis may be composed of two distinct mechanisms, one is a temperature-dependent DNA synthesis and the other is a pH-dependent control of developmental events leading to microgamete assembly and exflagellation.

Amiloride↗
PubMed1991

The development of exo-erythrocytic schizonts of Plasmodium berghei in vitro from gamma-irradiated and non-irradiated sporozoites: a study using confocal laser scanning microscopy.

Confocal scanning laser microscopy has been used to study the distribution of antigens expressed by the liver stages of Plasmodium berghei in cultured hepatoma cells. The 3-dimensional images obtained of intact parasites clearly show complex patterns of antigen expression not apparent when using conventional IFAT or immunoelectron microscopy. A liver-stage specific antigen (Pbl 1) was shown to be confined to the parasitophorous vacuole; the vacuole has extensive diverticulae extending into the host cell. Small parasites were detected for the first time in 'mature' cultures. These did not represent a distinct population, but the 'tail' of a broad continuum of parasite sizes. Irradiated sporozoites produce a transient population of slow-growing parasites which express a very limited range of antigens de novo in the invaded hepatoma cell. A comparison of the reactivity of normal EE parasites with anti-circumsporozoite antibody and with anti-Pbl 1 suggests that the former reagent may reliably be used to identify sporozoites invading host cells, but should not be used to determine the number of parasites that successfully undergo intrahepatic development. Anti-Pbl-1 indicates on 33% of invaded sporozoites identified by anti-CSP subsequently differentiate.

Animals↗
PubMed1991

Asexual blood stages of malaria modulate gametocyte infectivity to the mosquito vector--possible implications for control strategies.

In the rodent malarial parasite Plasmodium berghei sexual parasites are produced in a single major wave with maximal numbers between day 7 and day 16. Irrespective of their time of appearance during infection these sexual parasites are equally fertile in vitro. In contrast, in vivo infectivity to the mosquito is maximal at day 3-5 when gametocyte numbers are only 9% of the peak levels seen between days 7 and 16. Up to 96% of natural potential infectivity of gametocytes for the mosquito is therefore suppressed. The suppression is humoral, reversible and correlates with the appearance of an effective host response to the initial rapid increase in asexual parasitaemia. These data are consistent with published evidence which indicates that a reduction in parasitaemia may cause an increase in infectivity of gametocytes to the mosquito vector. Therefore the impact of strategies aiming to control asexual parasites is re-examined. Inefficient strategies might be predicted to increase and not suppress malaria transmission.

Animals↗
PubMed1991

Analysis of immunity induced by the affinity-purified 21-kilodalton zygote-ookinete surface antigen of Plasmodium berghei.

By using affinity-purified ookinete surface antigen from the rodent malaria parasite Plasmodium berghei, a transmission-blocking immunity was induced in mice. Groups of mice were immunized via different routes, with total quantities of antigen ranging from 0.5 to 40 micrograms (with or without Freund adjuvant). Vaccination by the intramuscular route with 20 micrograms of antigen in the absence of adjuvant and boosted once with the same amount of protein induced a total transmission blockade. Immunoblot analysis confirmed that immune sera invariably recognized Pbs21 antigen. The isotype and titer of the anti-Pbs21 immunoglobulin G (IgG) response was determined by enzyme-linked immunosorbent assay. The antibody isotype was predominantly IgG1. The concentration of specific anti-Pbs21 IgG reached a peak of 182.45 +/- 92.13 micrograms/ml by week 7 postimmunization and fell progressively to 38 micrograms/ml at week 34 (at which time the transmission was still inhibited by 98%).

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PubMed1990

An ultrastructural study of the exoerythrocytic schizonts of Plasmodium cynomolgi and P. knowlesi in Macaca mulatta.

Exoerythrocytic schizonts of Plasmodium cynomolgi and P. knowlesi were examined by electron microscopy in biopsy samples of primate livers. With maturity the parasitophorous vacuole membrane becomes highly sculptured by the addition of a discontinuous dense thickening, the distribution of which can be a distinguishing character between these two species. The parasitophorous vacuole membrane follows the contours of the parasite faithfully with a minimal surrounding vacuole. The marked destruction of the cytoplasm of the host hepatocyte by most of the parasites studied however gave the distinct, but erroneous, appearance of a large parasitophorous vacuole at the light microscope level. The mature parasite often exhibited a highly invaginated surface contour with the result that the cytoplasm of the host cell and parasite became intimately interdigitated, this interweaving is unlikely to be recognized in light microscopic studies.

Animals↗
PubMed1990

Possible roles of Ca2+ and cGMP as mediators of the exflagellation of Plasmodium berghei and Plasmodium falciparum.

The roles of Ca2+ and cyclic nucleotides as secondary, intracellular messengers for exflagellation of Plasmodium berghei and Plasmodium falciparum were investigated. Treatment with Ca2+ antagonists such as TMB-8 (an inhibitor of intracellular Ca2+ release) or W-7 (a calmodulin inhibitor) strongly inhibited exflagellation induced by alkaline medium at pH 8.0 whereas EGTA (a Ca2+ chelator) or nicardipine and nifedipine (Ca2+ channel inhibitors) had no effect. These results may indicate that mobilization of parasites' internal resources of Ca2+ is a prerequisite for exflagellation. Agents which increase cAMP levels did not induce exflagellation at the non-permissive pH of 7.3, and had no significant inhibitory effect at the permissive pH of 8.0. IBMX (cAMP/cGMP-phosphodiesterase inhibitor), however, enhanced exflagellation at pH 7.3, indicating the possibility that cGMP, but not cAMP, may be involved in the induction of exflagellation. Furthermore, cGMP or agents which increase cGMP levels such as nitroprusside (a potent activator of guanylate cyclase), enhanced exflagellation at pH 7.3, whereas N-methyl-hydroxylamine (guanylate cyclase inhibitor) inhibited the exflagellation at pH 8.0. From these results, it may be concluded that the induction of exflagellation requires both Ca2+ mobilization and an increase in cGMP levels.

Animals↗
PubMed1990

Sexual development in malarial parasites: gametocyte production, fertility and infectivity to the mosquito vector.

Using cloned lines of Plasmodium berghei producing mixed asexual and sexual (clone 234L) and purely asexual (clone 233L) parasitaemias, the courses of parasitaemia, gametocytogenesis, exflagellation, ookinete production in vitro and mosquito infectivity have been followed. For clone 234L mosquito infectivity is maximal at day 3 and has ceased by day 6 post-infection. Conversely, gametocytogenesis, exflagellation and ookinete production are at minimal levels at day 3 and rise to peaks between days 10 and 15 of infection (in TO mice infected with blood at mechanical blood passage 3). Sexual potential declines progressively with sustained mechanical passage (up to P14). Gametocyte conversion is highest early in infection and declines exponentially; however, upon each mechanical passage conversion is again raised but decreases more rapidly with succeeding passages. For practical mosquito transmission in the laboratory we consider P8 to be the useful limit for mechanical transmission for this parasite clone. Asexual parasite growth (virulence) is more rapid with increased mechanical passage inducing a rapid fall in haematocrit. By P14 the course of infection closely parallels that of the purely asexual clone 233L.

Animals↗
PubMed1990

Three non-repeated transmission blocking epitopes recognized in the 21 kD surface antigen of zygotes-ookinetes of Plasmodium berghei.

Two-site and competitive ELISA have been developed against a surface antigen of zygotes-ookinetes of Plasmodium berghei using monoclonal antibodies (MoAbs) which block transmission of the parasite to the mosquito. Three such MoAbs have been studied, each of which recognized a protein of an Mr 21 kD (Pbs21) using immunoblot techniques. The assays showed that there are at least 3 single B-cell epitopes expressed in Pbs21. One epitope recognized by MoAb 17.9 is conformation dependent and antibodies bound to it interfered with other MoAbs (12.1 and 13.1) each recognizing a different, apparently linear epitope. Glycosylation might not be relevant to the binding of any of the antibodies tested to their respective epitopes.

Animals↗
PubMed1990

An antigen specific to the liver stage of rodent malaria recognized by a monoclonal antibody.

Vaccines currently being evaluated against malaria are based on proteins derived from the blood, sporozoite and sexual stages. Antigens from the liver stage, which is now recognized as the major target of protective sporozoite induced immunity, have received comparatively little attention. This paper describes the generation of a monoclonal antibody (MoAb), which recognizes an antigen specific to the liver stage of the rodent malaria Plasmodium berghei. The antigen is expressed throughout liver stage development and appears to be localized to the parasitophorous vacuole membrane. The MoAb did not affect the growth of liver stages cultured in vitro nor could protection be demonstrated in vivo following passive transfer of the antibody.

Animals↗
PubMed1990

Survival and antigenic profile of irradiated malarial sporozoites in infected liver cells.

Exoerythrocytic (EE) stages of Plasmodium berghei derived from irradiated sporozoites were cultured in vitro in HepG2 cells. They synthesized several antigens, predominantly but not exclusively those expressed by normal early erythrocytic schizonts. After invasion, over half the intracellular sporozoites, both normal and irradiated, appeared to die. After 24 h, in marked contrast to the normal parasites, EE parasites derived from irradiated sporozoites continued to break open, shedding their antigens into the cytoplasm of the infected host cells. Increasing radiation dosage, which has previously been shown to reduce the ability of irradiated sporozoites to protect animals, correlated with reduced de novo antigen synthesis by EE parasites derived from irradiated sporozoites.

Animals↗
PubMed1990

The development and routine application of high-density exoerythrocytic-stage cultures of Plasmodium berghei.

Methods are reviewed for the culture of the exoerythrocytic stages of Plasmodium berghei wherein development reproducibly reflects growth observed in vivo in laboratory rodents. The combination of these methods with the culture of both asexual and sexual blood stages has allowed the completion of the entire vertebrate phase of malaria development in vitro. The development of new methods for high-density exoerythrocytic-stage culture combined with robust statistical analysis of parasite growth by morphological (light microscopy), or DNA probe methods now allows the critical and precise evaluation of chemotherapeutic or immunological treatments. These methods are illustrated by data obtained on pyrimethamine, primaquine and a hydroxynaphthoquinone. Some of the new avenues of research made feasible by the high-density cultures, e.g., direct immunization to produce monoclonal antibodies and biochemical studies are discussed.

Animals↗
PubMed1990

A liver-stage specific antigen of P. berghei identified by a monoclonal antibody.

Stage-specific immunity (to the sporozoite, the asexual blood-stages and the sexual stages of malaria) has been well documented and antigens from each stage are being tested for their potential as vaccine candidates. Recently it has become clear that the liver stage can also be the target of protective immune responses; however, only the circumsporozoite protein has been identified as a protective liver antigen. It is critical for vaccine evaluation and development to identify other liver antigens and assess their potential role in immunity. In this paper we describe a monoclonal antibody, which recognizes a liver-specific antigen of Plasmodium berghei (referred to as Pbl1). Passive immunization studies using this antibody suggest that it may influence the course of sporozoite-induced infections.

Animals↗
PubMed1989

Recent advances in the parasitology of malaria.

This review represents a highly personal view of only some of the advances made on the biology of malaria in the period 1986-1987. Progress has stemmed largely from new or refined techniques applied to the logical demonstration of eukaryotic principles; and more rarely from either new conceptual advances, or the revelation of critical differences in the biology of Plasmodium from that of other eukaryotes. Significant steps have been made in the recognition of the complexity of interaction between the parasite and both its vertebrate and invertebrate hosts. It is this very complexity of interaction which warns against the presumption that the successful application of any single control strategy is imminent.

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