Q. D. Bickle

Locally Up-regulated Lymphotoxin α, Not Systemic Tumor Necrosis Factor α, Is the Principle Mediator of Murine Cerebral Malaria

Cerebral malaria (CM) causes death in children and nonimmune adults. TNF-α has been thought to play a key role in the development of CM. In contrast, the role of the related cyto-kine lymphotoxin α (LTα) in CM has been overlooked. Here we show that LTα, not TNFα, is the principal mediator of murine CM. Mice deficient in TNFα (B6.TNFα−/−) were as susceptible to CM caused by Plasmodium berghei (ANKA) as C57BL/6 mice, and died 6 to 8 d after infection after developing neurological signs of CM, associated with perivascular brain hemorrhage. Significantly, the development of CM in B6.TNFα−/− mice was not associated with increased intracellular adhesion molecule (ICAM)-1 expression on cerebral vasculature and the intraluminal accumulation of complement receptor 3 (CR3)-positive leukocytes was moderate. In contrast, mice deficient in LTα (B6.LTα−/−) were completely resistant to CM and died 11 to 14 d after infection with severe anemia and hyperparasitemia. No difference in blood parasite burden was found between C57BL/6, B6.TNFα−/−, and B6.LTα−/− mice at the onset of CM symptoms in the two susceptible strains. In addition, studies in bone marrow (BM) chimeric mice showed the persistence of cerebral LTα mRNA after irradiation and engraftment of LTα-deficient BM, indicating that LTα originated from a radiation-resistant cell population.

Efficacy of mebendazole and levamisole alone or in combination against intestinal nematode infections after repeated targeted mebendazole treatment in Zanzibar

OBJECTIVE To evaluate the efficacy of and resistance to mebendazole (500 mg) and levamisole (40 or 80 mg), alone or in combination, for the treatment of Ascaris lumbricoides, Trichuris trichiura and hookworm infections on Pemba Island - an area exposed to periodic school-based mebendazole treatment since 1994. METHODS A randomized, placebo-controlled trial was carried out in 914 children enrolled from the first and fifth grades of primary schools. Stool samples collected at baseline and 21 days after treatment were examined by the Kato-Katz technique to assess the prevalence and intensity of helminth infection. FINDINGS Efficacies of mebendazole and levamisole as single treatments against intestinal nematode infections were comparable with those in previous trials, but mebendazole treatment of hookworm infections gave significantly lower cure (7.6%) and egg reduction (52.1%) rates than reported in a study undertaken before the beginning of periodic chemotherapy (cure rate, 22.4%; egg reduction rate, 82.4%). Combined treatment with mebendazole and levamisole had a significantly higher efficacy against hookworm infections (cure rate, 26.1%; egg reduction rate, 88.7%) than either drug given alone. No difference in mebendazole efficacy was found in children who had been treated repeatedly compared with those who had not been treated previously. CONCLUSION The overall efficacy of mebendazole against hookworm infections after periodic chemotherapy is reduced. The efficacy of benzimidazoles in chemotherapy-based control programmes should be monitored closely. Combined treatment with mebendazole and levamisole may be useful as a tool to delay the development of benzimidazole resistance.

Immunological control of hepatotoxicity and parasite egg excretion in Schistosoma mansoni infections: stage specificity of the reactivity of immune serum in T-cell deprived mice

Within seven weeks of infection with 200 Schistosoma mansoni cercariae, T-cell deprived mice have been shown to suffer from extensive microvesicular damage to hepatocytes, and an inability to excrete parasite eggs at the same rate as comparably infected, immunologically intact controls. Administration of serum (CIS) from chronically infected, immunologically intact donors prevented the development of microvesicular cell damage and partially restored egg excretion rates in infected deprived mice. Serum pools obtained from mice injected either with intact S. mansoni eggs or with a homogenate of eggs emulsified in Freunds complete adjuvant (FCA) were as effective as CIS in preventing hepatotoxicity and restoring the rate of egg excretion in infected deprived recipients. The degree of protection of liver tissue afforded by immune sera could be monitored either by histopathological examination of liver sections or by estimation of serum transaminase concentrations, the results from both assays being generally in agreement. Sera from donor mice injected with cercarial or worm antigens in FCA were relatively inactive either in protecting against S. mansoni-induced liver damage or in reconstituting egg excretion rates in infected deprived mice. Serum from donor mice infected with 25 cercariae became hepato-protective between 49 and 53 days after infection of the donors, and the degree of hepatoprotective activity and egg excretion reconstituting capacity in the serum of 25 cercariae-infected donors was shown to increase between 8 and 16 weeks after infection. Increasing the size of infection of the serum donors to 100 cercariae gave only a marginal increase of hepatoprotective activity at 7 weeks when compared with serum donors infected with 25 cercariae for 7 weeks. Liver parenchymal cells of very heavily infected, immunologically intact mice were found to show microvesicular damage similar to that in livers of infected deprived mice, and administration of CIS to these normal mice was histopathologically protective. However, the elevated serum transaminase concentrations obtaining in the infected normal mice were not reduced to any extent by CIS. The results obtained from serum-reconstituted deprived mice are discussed in terms of the contribution they may make to a better understanding of the host-parasite relationship in immunologically intact mice.

Identification and partial purification of an antigen (ω1) from Schistosoma mansoni eggs which is putatively hepatotoxic in T-cell deprived mice.

T-cell deprived mice heavily infected with Schistosoma mansoni suffer from severe microvesicular damage to hepatocytes within seven weeks of infection. The damage can be prevented by administration of serum (CIS) obtained from mice chronically infected with S. mansoni or from mice immunized with intact or homogenized S. mansoni eggs. Reaction of serum samples from individual chronically infected mice in immunoelectrophoresis with S. mansoni egg homogenate has enabled the identification of at least 12 distinct immuno precipitation reactions. Precipitating antibody against one S. mansoni egg antigen, omega 1, has been detected in all mice with patent chronic infections, and anti-omega 1 antibody is the most concentrated of the precipitating anti-egg antibody species in pooled CIS. Pooled serum obtained from infected intact mice reacting predominantly against omega 1 was found partially to prevent the hepatotoxicity reaction on transfer to infected deprived mice. Serum samples from mice injected with egg homogenate fractionated either by preparative electrophoresis or by cation exchange chromatography, and containing antibodies reactive with antigen omega 1 in immunoprecipitation, were fully protective against liver cell damage induced by S. mansoni in deprived mice. Sera from mice immunized with other S. mansoni egg fractions, and which did not contain antibodies reactive with omega 1, were not hepatoprotective. Antigen omega 1 is compared and contrasted with other S. mansoni egg antigens that have been described.

Resistance against Schistosoma mansoni induced by highly irradiated infections: studies on species specificity of immunization and attempts to transfer resistance

Significant levels of resistance against Schistosoma mansoni challenge were developed by mice exposed to highly irradiated (20 krad.) cercariae of the homologous species (53-67%), whereas vaccination with S. bovis, S. haematobium or S. japonicum failed to confer significant levels of resistance (-5-12%), thus confirming the specificity of the immunizing procedure. Attempts to transfer resistance to naive recipients by injection of serum and of spleen or lymph node cells from donor mice vaccinated with highly irradiated cercariae were largely unsuccessful. However, significant levels of resistance could be transferred to mice by injection of serum from rabbits exposed to irradiated cercariae. Comparable levels of resistance were conferred by injection of serum at the time of challenge (34-69%) or 5-6 days later (31-56%). In contrast, sera from rabbits injected with soluble egg antigen or homogenized cercariae failed to confer protection upon recipient mice. Sera from vaccinated mice, vaccinated rabbits and antigen-injected rabbits all caused cell adherence to skin-transformed schistosomula but neither the level of adherence nor the serum titre correlated with the ability to confer protection to mice.

Schistosomes: challenges in compound screening.

Major progress in studying the biology of schistosomes had been achieved since the late 1960s with the successful laboratory cultivation of the parasites life cycle stages in the vertebrate (in vivo animal models) and snail hosts. This was followed by establishment of in vitro culture techniques for cultivation of the different life cycle stages to understand the mechanisms regulating the parasites growth, development, transformation, pathogenicity and survival, with prospects to develop and identify relevant candidate diagnostic, immunological and chemotherapeutic targets. Chemotherapeutic measures have been the mainstay in the control of schistosomiasis. The use of praziquantel, a relatively safe and orally administered drug, in targeted or mass treatment programmes had significantly reduced the prevalence of schistosomiasis in disease-endemic countries. However, with only one drug of choice for treatment, parasite resistance remains a major concern. Thus, new drug discovery against schistosomes cannot be overemphasised. Undoubtedly, this will require an integrated system that includes not only rational chemical synthesis and lead optimisation, but also appropriate drug screening strategies. This paper reviews the present state of in vitro and in vivo drug screening strategies against schistosomes. It also highlights the best practices for compound screening in the TDR-designated compound screening centres and details some of the challenges involved in in vitro and in vivo compound screening.

Analysis of Human Antibodies to Erythrocyte Binding Antigen 175 of Plasmodium falciparum

ABSTRACT Invasion of human erythrocytes by Plasmodium falciparummerozoites is a multistep process. For many strains of the parasite, part of this process requires that the erythrocyte binding antigen 175 (EBA-175) of the merozoite binds to sialic acid residues of glycophorin A on the erythrocyte surface, a receptor-ligand interaction which represents a potential target for inhibition by antibodies. This study characterizes the reactivity of naturally acquired human antibodies with four recombinant proteins representing parts of EBA-175 (region II, regions III to V, and the dimorphic C and F segment region) in populations in which the organism is endemic. Serum immunoglobulin G (IgG) recognizing the recombinant proteins is predominantly of the IgG1 and IgG3 subclasses, and its prevalence increases with age. In a large population study in The Gambia, serum positivity for IgG or IgG1 and IgG3 subclass antibodies to each of the EBA-175 recombinant antigens was not significantly associated with subsequent protection from clinical malaria. However, there was a trend indicating that individuals with high levels of IgG to region II may have some protection.

Immunization of mice with gamma-irradiated intramuscularly injected schistosomula of Schistosoma mansoni.

The parameters involved in the induction of resistance against Schistosoma mansoni by injection of irradiated, artificially transformed schistosomula were studied in mice. Single intramuscular injections of 500 schistosomula exposed to radiation doses in the range 2.3 to 160 krad. resulted in significant protection (in the range 20 to 50% as assessed by reduced worm burdens) against a challenge infection administered at intervals from 3 to 24 weeks post-vaccination. However, schistosomula irradiated with 20 krad. consistently resulted in better protection than those exposed to either higher or lower radiation doses despite the persistence of stunted adults from the infections irradiated with 2.3 krad. Vaccination with 40 krad. schistosomula resulted in significant protection in terms of reduced worm and tissue egg burdens and increased survival following lethal challenge. Varying the number of irradiated schistosomula, the frequency and route of their administration, the site of challenge and the strain of host all failed to enhance the level of resistance. However, percutaneously applied, irradiated cercariae were found to be more effective in stimulating resistance (60%) than intramuscularly injected, irradiated schistosomula (40%).

Vaccination of mice with a cocktail DNA vaccine induces a Th1-type immune response and partial protection against Schistosoma japonicum infection.

Several defined vaccine candidate antigens of Schistosoma japonicum have shown promise in large animal vaccination experiments. However, vaccination of mice in the laboratory with either single recombinant antigens or DNA encoding forms of the individual antigens has so far failed to induce significant protection against S. japonicum cercarial challenge infection as judged by worm reduction, although specific antibodies were generated. This is in contrast to the results achieved using radiation-attenuated vaccines which are highly protective. Even in large animal vaccination experiments, the protection levels obtained with single defined antigens were far below those achieved using the attenuated vaccines. One possible interpretation is that the immune responses induced by single antigen vaccination may not be strong enough to combat the challenging infection. We, therefore, carried out mouse vaccination experiments using a cocktail DNA vaccine comprising four DNA plasmids encoding four different S. japonicum antigens, Sj62, Sj28, Sj23 and Sj14-3-3, respectively. We, also investigated whether co-injection of the mouse IL-12 encoding plasmid with the cocktail DNA vaccine was able to enhance the Th1 responses and hence the protective immunity. Three intramuscular injections of the cocktail DNA vaccine induced a significant Th1-type cellular response with high level of IFN-gamma production by splenocytes upon in vitro stimulation with recombinant antigens. Importantly, significant IgG antibody responses were also induced against crude worm antigens. In two out of three experiments, significant resistance (34-37 and 44-45%, respectively) was demonstrated while another experiment did not show any protection against S. japonicum cercarial challenge infection. Co-injection of the IL-12 encoding DNA did not further enhance these responses, nor the level of resistance, compared with the cocktail DNA alone.

The induction of specific immunity against Schistosoma japonicum by exposure of mice to ultraviolet attenuated cercariae.

Mice can be partially protected against Schistosoma japonicum by prior exposure to ultraviolet (UV)-attenuated infections which fail to survive to the adult stage and produce no overt pathology in the host. Optimum resistance was induced by parasites exposed to 40 seconds of UV, significantly lower levels of resistance being stimulated by both shorter and longer exposures. No consistent relationship between the degree of resistance induced and the number of irradiated cercariae given could be demonstrated and equivocal results were obtained when comparing the efficacy of single and multiple vaccinations. Vaccinations with UV-attenuated cercariae given intraperitoneally (i.p.) were as efficacious as those given percutaneously but mice were as or more resistant to challenges given by the i.p. route, the possible reasons are discussed. There was no observed delay in the migration of the challenge, vaccinated mice being as resistant when perfused 6 or 3.5 weeks after challenge. Vaccination was species specific since mice exposed to either UV-attenuated S. japonicum cercariae or gamma-attenuated S. mansoni cercariae were resistant to homologous but not heterologous challenge.